Immunobiology (MCDB) 430/530: Fall 2012 Biology of the Immune System

Lectures in KBT 1214, MWF 9:25 - 10:15 AM Information for Students

Who should take this course? This course is intended to provide a detailed survey of current knowledge of the immune system for advanced undergraduates with a serious commitment to biology and for graduate students in Immunobiology and related programs. Prior knowledge of cell biology and biochemistry is absolutely necessary; Biology (MCDB) 300a provides a good basis. Students who believe that they have equivalent knowledge may take the course, but prior discussion with the course organizer is required. Course content: The course consists of lectures, tutorials, review sessions, and assigned reading material. We provide a syllabus with an outline of the individual lectures. Each lecture is accompanied by assigned reading material. In addition, the students are expected to know the experimental systems (techniques) routinely used to study the immune system as detailed below. All handouts and reading assignments plus supplemental items of interest can be found at the Yale website http://classesv2.yale.edu. Grades: The course grade is based on a mid-term exam (30% of the grade), take-home questions (20% of the grade), participation in tutorial sessions (10% of the grade), and a final exam (40% of the grade). The questions are designed to test the understanding and application of key immunological concepts. Reasoning as well as recall is required. You will also participate in small group tutorial sessions in which primary research articles and questions will be presented and discussed. These sessions will be oriented to discussion and analysis of primary literature, as well as review of the information provided in the lectures. Teachers: The course is taught by the faculty of the Department of Immunobiology. Three Teaching Fellows will assist in the class. They will lead the tutorial as well as the review sessions. Textbook: "Janeway’s Immunobiology” by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC.), 8th Edition, 2011 is required for this course. This textbook is available at the Barnes & Noble Bookstore on Broadway. Reading Material: In addition to material covered in the lecture, the assigned readings in “Immunobiology. The Immune System in Health and Disease” are required and will be covered on the examinations. Given the problem solving nature of the course, the students will be expected to know the experimental systems used to study the immune system. These include immunochemical techniques (ELISA, immunoblotting, immunohistochemistry, immunoprecipitation, anti-immunoglobulins, use of antibodies to isolate and identify proteins and genes, monoclonal antibodies); cell isolation techniques (flow cytometry, FACS); manipulation of the immune system (adoptive transfer, cell depletion in vivo, gene knockout, transgenic mice; and analysis of response (RNA expression, microarrays, proliferation, etc.). Information about these experimental procedures is found in Appendix I of the textbook. Course Director: Carla Rothlin, Ph.D. TAC, S624, Phone: 737-4679 carla.rothlin@yale.edu Teaching Fellows: Will Khoury-Hanold Omotooke Arojo Tianxia Guan william.khoury-hanold@yale.edu omotooke.arojo@yale.edu tianxia.guan@yale.edu

MCDB 430/530

Lecture Schedule & Syllabus Fall 2012

August 29 Why Study Immunology? Rothlin August 31 Structure of the Immune system I Rothlin September 3: Labor Day (no class) September 5 Structure of the Immune System II Rothlin September 7 Receptors and Signaling I Rothlin Evening Tutorial 1: Primary literature discussion – Methods in immunology (week September 3-7) September 10 Innate Immune System Iwasaki September 12 Pattern Recognition Receptors Medzhitov September 14 Complement, NK Cells and Acute Phase Responses Medzhitov September 17 Innate Immunity to Bacteria, Fungi and Parasites Medzhitov September 19 Innate Anti-viral Immunity Medzhitov Evening Tutorial 2: Primary literature discussion – Innate Immunity (week September 17-21) September 21 Structure and Genetics of MHC Molecules Cresswell September 24 MHC Class I Restricted Antigen Processing Cresswell September 26 MHC Class II Restricted Antigen Processing Cresswell September 28 Dendritic Cells and their Functions/CD1 Molecules Cresswell Evening Tutorial 3: Primary literature discussion – Antigen presentation (week October 1-5) October 1 B & T Cell Receptors: Structure and Classes Schatz October 3 Immunoglobulin and TCR Gene Structure & Rearrangement Schatz October 5 B & T Cell Development Schatz October 8 Positioning, Maturation & Trafficking of Lymphocytes Pereira October 10 Receptors and Signaling II Rothlin Evening Tutorial 4: Evening review session for midterm exam (week October 8-12) October 12 MIDTERM EXAM (on materials covered up to Oct. 8th lecture) October 15 Innate Control and Initiation of Adaptive Immune Responses Craft October 17 T Cell Priming and Effector Cell Differentiation I Craft October 19 T Cell Priming and Effector Cell Differentiation II Craft October 22 Effector Molecules (Function and Signaling) Craft

Tuesday October 23rd –RECESS BEGINS at 5:30 PM Monday, October 29th – CLASSES RESUME

October 29 Primary B Cell Responses Shlomchik October 31 Mechanisms of Somatic Hypermutation Shlomchik and Isotype Switch and Their Consequences November 2 Humoral Immune Responses Shlomchik Evening Tutorial 5: Primary literature discussion – Cell-mediated immunity (week October 29-2) November 5 B & T Cell Memory Shlomchik November 7 Vaccination Iwasaki November 9 Inflammation - Response to Infection Iwasaki Evening Tutorial 6: Primary literature discussion – Humoral immunity (week November 5-9) November 12 Mucosal Immunity Iwasaki November 14 Transcriptional Regulation in the Immune System Chi November 16 Primary and Acquired Immunodeficiencies Meffre Evening Tutorial 7: Primary literature discussion – Immunity and infection (week November 12-16)

Friday, November 16 – RECESS BEGINS at 5:30 PM Monday, November 26 – CLASSES RESUME

November 26 Allergy Medzhitov November 28 Peripheral Tolerance Herold November 30 Autoimmunity Herold December 3 Transplantation and Cancer Immunology Herold Evening Tutorial 8: Primary literature discussion – The immune system in health and disease (week Dec. 3-7) December 5 Unresolved Questions in Immunology Medzhitov December 7 Review session for final exam (in class) The TFs

Friday, December 7 – CLASSES END Thursday, December 13 – FINAL EXAMS BEGIN Tuesday, December 18 – FINAL EXAMS END

Evening Tutorial Schedule & Required Readings

Evening tutorials are held throughout the semester. These tutorials focus on a discussion of primary literature, but also allow for review of class material, as well as students’ questions. Tutorial grades (10% of the final grade) are based on participation and attendance. Readings will be posted on Classes*v2 prior to each tutorial. Evening Tutorial 1: Week of September 3 - 7: Methods in immunology tutorial Required reading: Janeway’s Immunobiology. 8th Edition. Appendix I: The Immunologist’s Toolbox Evening Tutorial 2: Week of September 17 - 21: Innate immunity tutorial Required reading: Sander et al. Detection of prokaryotic mRNA signifies microbial viability and promotes immunity. Nature (2011) vol. 474(7351). pp385-9. Evening Tutorial 3: Week of October 1 - October 5: Antigen presentation tutorial Required reading: Maric et al. Defective antigen processing in GILT-free mice. Science (2001) vol. 294(5545). pp. 1361-5. Evening Tutorial 4: Week of October 8 - October 12: Review for Midterm No required reading. Review your class notes and bring questions. Evening Tutorial 5: Week of October 29 - November 2: Cell mediated immunity tutorial Required reading: Barber, et al. Restoring function in exhausted CD8 T cells during chronic viral infection Nature (2006). vol. 439(7077). pp. 682-87. Evening Tutorial 6: Week of November 5 - November 9: Humoral immunity tutorial Required reading: Gatto, D, et al. Guidance of B cells by the orphan G protein-coupled receptor EBI2 shapes humoral immune responses. Immunity (2009). vol. 31(2). pp. 259-69. Evening Tutorial 7: Week of November 12 - November 16: Immunity & infection tutorial Required reading: Macpherson, et al. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Science (2004). vol. 303(5664). pp. 1662-65. Evening Tutorial 8: Week of December 3 - December 7: The immune system in health and disease tutorial Required reading: Boitard, et al. Acceleration of type 1 diabetes mellitus in proinsulin 2–deficient NOD mice. Journal of Clinical Investigation (2003) vol. 111(6). pp. 851-57.

Take-Home Question Schedule

Throughout the semester, there will be four graded take-home questions, each worth 5% of the final grade. Answers are to be typed and must not be longer than one page (one inch margins and 12 point “Times” font). If, for any reason, a student cannot attend class on the day that a take-home question is due, her or his answer may be emailed to their Teaching Fellow. However, every student must submit his or her answers by the end of the lecture (10:15 a.m.) on the due date. Answers are considered late beginning right after class on the due date and will have two points deducted per day late. Take-Home Question 1:

Posted on September 19th Due end of class September 26th

Take-Home Question 2: Posted October 3rd Due end of class October 10th

Take-Home Question 3: Posted October 31st Due end of class November 7th

Take-Home Question 4: Posted November 30th Due end of class December 7th

Why Study Immunology?

August 29

Instructor: Carla V. Rothlin In this introductory lecture I will introduce the topics to be covered by this course, course structure, grading schemes, and introduce the teaching faculty who are responsible for covering each topic. I will also discuss the importance of the immune system in health and disease. Sit back, relax and enjoy!

Structure of the Immune System I

August 31

Instructor: Carla V. Rothlin Immunology is the study of mechanisms used to defend our body from invasion by microorganisms. The purpose of the immune system is to recognize invading microorganisms and to contain the infection or clear the microorganisms from the body. Therefore, the immune response consists of two main activities: recognition and effector functions. Mechanisms of microbial recognition in vertebrates Microorganisms with the potential to cause diseases are called “pathogens”. There are four kinds of pathogens: bacteria, viruses, fungi and parasites. It is important to recognize that the pathogenicity of a particular microorganism depends on the fitness of the host. For example, certain microorganisms are only pathogenic in immuno-compromised individuals. These are known as opportunistic pathogens. In vertebrates, there are two types of immune recognition, referred to as innate (evolutionarily ancient host defense system) and adaptive (vertebrate host defense system).

• Innate immune recognition operates by non-clonal, germline encoded receptors that recognize conserved and invariant features of microorganisms known as pathogen-associated molecular patterns (PAMPs).

• Adaptive immune recognition operates by the clonal selection of individual lymphocytes expressing specific antigen receptors. The genes encoding antigen receptors are assembled through somatic recombination from gene segments in the germ line, allowing the generation of a diverse repertoire of receptors.

While the innate immune system operates early in a response to infection, the adaptive immune response requires time to develop. Importantly, innate and adaptive immunity do not operate independently of each other. The innate immune response is needed to initiate and direct adaptive immunity. Conversely, adaptive immunity produces signals that stimulate and modulate innate immune responses. Adaptive immunity amplifies the protection provided by the innate immune response and provides future immunity to the same pathogen (memory/adaptive immunity). Mechanisms of host defense Multicellular organisms defend themselves against pathogens in multiple ways. The mammalian immune response can be classified in three phases: immediate innate responses, induced innate responses and adaptive immunity. The first one consists of physical and chemical barriers (i.e.: physical barriers formed by epithelial surfaces, mucus, changes in pH, anti-microbial peptides). The innate and adaptive responses are briefly described below. The innate immune response. Invasion by a microorganism leads to a quick response by the innate immune system. The innate immune system includes complement proteins, phagocytes (macrophages, neutrophils, and dendritic cells), and natural killer cells. These components of the innate immune system contribute both to recognition of the pathogen and to effector responses. Phagocytes express various types of receptors including those for pathogen recognition (pattern recognition receptors; PRRs) and for complement proteins to aid in pathogen clearance. Phagocytes become activated upon interaction with the pathogen. Activated phagocytes secrete small proteins called cytokines that act by binding receptors on target cells, altering the behavior of the target cell. One class of cytokines is called chemokines that have chemoattractant properties. Phagocytes releasing chemokines can recruit additional cells to the area of infection to help in clearance. Activation of the innate immune response leads to the induction of adaptive immunity: some activated phagocytes mature and express molecules that will activate lymphocytes of the adaptive immune system. The adaptive immune response. The adaptive immune response is mediated by the activation of lymphocytes. Lymphocytes are divided into two classes (T and B lymphocytes) with different recognition receptors and function. For recognition, B cells express cell surface immunoglobulins, whereas T cells express T cell receptors. B cells

recognize regions of antigen (epitope) that is displayed on the surface of antigens. Once B cells are activated, they divide and differentiate into plasma cells that secrete a soluble form of their surface immunoglobulin receptor. These secreted immunoglobulins (antibodies) can circulate throughout the body and bind to the pathogen, contributing to its clearance. In contrast, T cells recognize epitopes (small stretch of amino acids) that are often buried inside the antigen. Once activated T cells divide and differentiate into effector T cells, they display various activities mediated by the release of specific cytokines. Most lymphocytes live in specialized lymphoid tissues or organs or can be found recirculating in the blood under normal condition. Once pathogens enter, the lymphocytes with specific receptors will divide and migrate to the site of infection to combat the pathogens. The induction of adaptive immune responses begins when the antigen presenting cells that are activated during the innate response (e.g.: dendritic cells) mature and interact with lymphocytes in specialized lymphatic tissues or organs. The mature dendritic cells, which are no longer phagocytic, carry the pathogen to the lymphoid tissues via lymphatic vessels to be recognized by lymphocytes. Microorganisms grow and survive in different niches once they have invaded. The adaptive immune system uses different effector responses to protect against the four classes of pathogen. B cells, by secreting antibodies can eliminate pathogens found extracellularly but not intracellularly. For those pathogens that reside and multiply in cells, there are specialized subsets of T cells. T cells can be divided into two major classes, CD4 T cells and CD8 T cells. CD8 T cells kill virally infected cells. CD4 T cells can be further sub-divided into various types, such as Th1, Th2, Th17, TFH and regulatory T cells that release unique panels of cytokines. For example, Th1 cells activate macrophages and help eliminate bacteria living intracellularly, whereas Th2 cells induce antibody secretion and immunity against large parasites. This specialization in T cell function allows the immune system to respond to a wide variety of microorganisms, while maintaining a balanced immune response. How is self-reactivity avoided? A variety of mechanisms ensure that the immune system is tolerant of itself. First, the innate immune system discriminates self from non-self by recognizing conserved molecular patterns associated with pathogens. Lymphocytes of the adaptive immune system recognize and respond to pathogens, but only when instructed to do so by the innate immune system. Furthermore, lymphocytes do not respond to self-components because clones of lymphocytes with receptors that bind to self- molecules are either eliminated, fail to mature, or fail to be activated. Also, a subset of T cells, known as regulatory T cells is fundamental for restraining auto-reactive immune responses. Reading: "Janeway’s Immunobiology", by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 1.

Structure of the Immune System II

September 5

Instructor: Carla V. Rothlin Hematopoietic stem cells and lineage commitment The cellular subsets of peripheral blood are constantly replenished by the process of hematopoiesis, as most blood cells have a limited life span. Hematopoiesis is a complex developmental process through which pluripotent stem cells in mammalian fetal liver and subsequently neonatal and adult bone marrow give rise to all terminally differentiated cells of the blood. Stem cells constitute a very small fraction of cells in the bone marrow. They are morphologically distinct from other cells and do not express lineage specific cell surface markers (Lin-). However these early cells express the antigen Thy 1 and Sca-1 (stem cell antigen, a member of the Ly6 family). Hematopoiesis gives rise to erythrocytes, granulocytes, macrophages, platelets and lymphocytes. Infusion of bone marrow stem cells into a lethally irradiated animal can repopulate all of these cell populations, indicating the highly proliferative and differentiation capacity of stem cells. The production and the maturation of the differentiated cell types from pluripotent precursors is regulated by hematopoietic growth factors. Examples of such regulators include erythropoietin, GM- CSF, SCF and various cytokines. In addition to influencing differentiation, these regulators also control the functional activity of the cells by eliciting intracellular responses mediated by cell surface receptors. The maturation of stem cells into different cell types is accompanied by the expression of lineage specific genes. Certain transcription factors have been shown by targeted deletion to be essential for particular lineages of cells. Examples include: GATA-1 (erythroid), GATA-2 (erythroid, myeloid, lymphoid), Ikaros (lymphoid), Oct-2 (B lymphocyte), etc. Cells of the immune system White blood cells consist of neutrophils, lymphocytes, monocytes, eosinophils, mast cells, and basophils. Lymphocytes are the primary cell of the adaptive immune system. Lymphocytes consist of T cells, B cells, and natural killer cells. Resting T and B cells are small, non-phagocytic cells. They can be found as naïve cells if they have not interacted with antigen or effector/memory cells if they have interacted with antigen. The other types of white blood cell are important in various processes, such as engulfing and destroying microorganisms and presenting antigens. Lymphocytes develop in primary lymphoid tissues The primary lymphoid tissues contain microenvironments that support the production of mature lymphocytes from progenitors. Three important events occur in these sites: 1) the expression and selection of cells with antigen-recognition receptors; 2) the expression of all surface molecules needed for activation and differentiation; and 3) the expression of cell surface molecules needed for recirculation and localization in tissues in the periphery. B lymphocytes develop in the bone marrow, while in birds they originate in a specialized organ, the Bursa of Fabricius. In all vertebrates, T cell precursors migrate from the bone marrow and further develop in the thymus, a thoracic organ anterior to the heart. The thymus is seeded by progenitor cells without immunological specificity; and produces clonally diverse, immunocompetent T cells. Since they manufacture lymphocytes, the bone marrow, avian Bursa and thymus are termed primary lymphoid organs. Lymphocytes in body fluids Naïve B and T cells circulating in the blood traverse the lymph nodes and the spleen. In the human, a milliliter of blood contains around 2-4 x 106 lymphocytes, more than half of them T cells. Naïve lymphocytes enter lymph nodes via a specialized structure called the high endothelial venules (HEV). Activated or memory lymphocytes extravasate from the vasculature, and enter inflamed tissues; they then percolate through intercellular spaces, enter the lymphatic vessels, and travel towards lymph nodes. Most T cells continuously recirculate between blood, tissues, and the secondary lymphoid organs (lymph nodes and spleen). The route of recirculation differs depending on the activation state of the lymphocyte. However, a specialized subset of large non-recirculating pool of T cells is found in the epithelia of the gut, skin, respiratory and reproductive systems. Some of these T cells appear to be independent of the thymus.

Lymphocytes are activated in secondary lymphoid tissues Secondary lymphoid tissues foster interactions between antigen presenting cells and lymphocytes. The major secondary lymphoid organs are: spleen, tonsils, appendix, lymph nodes and Peyer’s patches. Naïve T and B cells first encounter antigens in the secondary lymphoid organs and become activated. This activation includes the clonal selection and expansion of lymphocytes expressing specific antigen receptors. They divide and migrate out of the secondary lymphoid organs via peripheral blood and into the site of infection. The architecture of the secondary lymphoid tissues fosters interaction between antigen and lymphocytes. The secondary lymphoid tissues have specialized mechanisms to collect antigen and antigen reactive lymphocytes. The structure of the secondary lymphoid tissues varies depending on its function. Lymph nodes drain (via lymphatic vessels) extracellular fluid (lymph) from most tissues of the body as a means of collecting antigen. In addition, antigen presenting cells carrying the pathogen-associated antigens migrate from the peripheral tissues to the lymph nodes via the lymphatic vessels. The spleen filters the blood collecting antigens that enter the vasculature. The mucosa associated lymphoid tissues (including Peyer’s patches) collect antigens crossing the mucosal epithelia. Lymphocytes enter secondary lymphoid organs from the blood via specialized mechanisms designed to recruit naïve lymphocytes (high endothelial venules or HEV). Lymphocytes migrate to site of infection (the battleground) Activated lymphocytes leave the secondary lymphoid tissues and travel to the infected tissues via the blood to clear the pathogen. Based on early innate immune responses, the microvasculature at the site of infection is specialized to recruit activated lymphocytes and other cells important in defense. Reading: "Janeway’s Immunobiology", by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 1.

Receptors & Signaling I

September 7

Instructor: Carla V. Rothlin Immune cells are able to sense changes in the extracellular environment and respond accordingly. How are these external stimuli sensed? How is the recognition of these stimuli transduced into specific cellular responses in a timely fashion? In this first lecture we will discuss the basic features of signal transduction, the process by which extracellular signals interact with their cognate receptors leading to the activation of biochemical cascades that result in a cellular response. 1) Signal transduction is initiated upon recognition of the extracellular stimulus (agonist) by its cognate receptor. Receptors can be classified according to their cellular localization and shared mechanism of action into relatively few functional families, including but not limited to the following ones: Cell surface receptors

• Receptor protein kinases: have intrinsic enzymatic activity and exert their regulatory effects by phosphorylating diverse effector proteins. Most receptor protein kinases phosphorylate tyrosine residues in their substrates (e.g.: growth factor receptors, such as Kit and FLT3, which are expressed on developing lymphocytes). A few receptor protein kinases phosphorylate serine or threonine residues, such as the TGF-b receptor.

• Protein kinase–associated receptors: lack the intracellular enzymatic domains but, in response

to agonists, bind to or activate distinct protein kinases on the cytoplasmic face of the plasma membrane. Receptors of this group include multiple cytokine receptors and T and B-cell antigen receptors.

• Ligand-gated ion channels: are receptors for several neurotransmitters that form agonist-

regulated ion-selective channels in the plasma membrane that convey their signals by altering the cell's membrane potential.

• G-protein coupled receptors (GPCRs): interact with distinct heterotrimeric GTP-binding

regulatory proteins known as G proteins. G proteins are signal transducers that regulate multiple effectors including enzymes such as adenylyl cyclase, phospholipase C, phosphodiesterases, and plasma membrane ion channels selective for Ca2+ and K+.

Intracellular receptors, e.g.: nuclear receptors, NOD like receptors. 2) Recognition of the external stimulus by the receptor provides the first message in signal transduction. This event is followed by the propagation and amplification of the signal through the engagement of intracellular signaling pathways. Classical examples of intracellular signaling pathways involve:

• Activation of G proteins. G proteins are composed of a GTP-binding a subunit, which confers specific recognition by receptor and effector, and an associated dimer of b and g subunits that can confer both membrane localization of the G protein (e.g., via myristoylation) and direct signaling (e.g.: activation of inward rectifier K+ (GIRK) channels). GPCRs respond to agonists by promoting the binding of GTP to the G protein a subunit. Activation of the Ga subunit by GTP allows it to both regulate an effector protein

and drive the release of Gbg subunits, which can, in addition to regulating their own group of effectors, reassociate with GDP-liganded Ga, returning the system to the basal state. Common effector proteins of G-proteins include phosphodiesterases, adenylyl cyclases, phospholipases, and ion channels that upon activation permit the release of second messenger molecules such as cyclic-AMP (cAMP), cyclic-GMP (cGMP), inositol triphosphate (IP3), diacylglycerol (DAG), and Ca2+.

• Activation of small GTPases. Receptor signaling activates guanine-nucleotide exchange factors (GEFs),

which bind to small GTPases in their inactive state and displace GPD. This allows GTP to bind in its place, leading to the activation of the small GTPases and the recruitment of downstream effectors. GTPases such as those from the Ras family, play major roles in the regulation of gene expression and the rearrangement of the actin cytoskeleton. Overtime, the intrinsic GTPase activity of these proteins leads to the hydrolysis of GTP to GDP and their inactivation.

• Activation of the MAP kinase pathway. A complex pathway that consists of a cascade of three protein

kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAP kinase (MAPK). The elucidation of the modular MAPK cascade with its three consecutive protein kinases has had immediate implications for understanding signal amplification and switching, and most importantly for nuclear signaling, because the terminal MAPK, once activated, can migrate into the nucleus, leading to the activation of transcription factors.

• Nuclear Translocation of Transcription Factors. A classical example of this transcytoplasmic signaling is

the JAK/STAT system, in which ligand binding to a cytokine receptor activates the associated JAK family protein Tyrosine kinases, which leads to the activation of the STAT transcription factors.

• Phospholipid and ion-based signaling. In the canonical phosphoinositide pathway, activation of

phosphatidylinositol phospholipase C results in the hydrolysis of phosphatidylinositol bisphospate (PIP2) and the generation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 acts as a second messenger to release Ca2+ from intracellular stores, thus triggering a program of Ca2+-activated events. DAG binds and activates signaling proteins, such as Ras-GEF and PKC. These events are fundamental to antigen receptors signaling pathway and will be reviewed in the lecture Receptors and Signaling II.

3) Activation of signaling pathways is tightly regulated by a variety of molecular mechanisms that efficiently terminate the signaling response. These include:

• Receptor Desensitization. This mechanism, commonly observed in ligand-gated ion channels, results from the prolonged exposure of the receptor to the stimulus. This leads to conformational changes in the receptor, resulting in receptor “desensitization”, a state in which the receptor can still recognize the stimulus but is unable to transduce the signal.

• Receptor Internalization. In addition to desensitization, receptors can be internalized. This is a common feature in GPCR signaling. Endocytosed GPCRs are sorted from endosomes to lysosomes and degraded, a process important for signal termination. Within endosomes, some GPCRs can be dephosphorylated and efficiently recycled back to the cell surface in a resensitized state in which the receptors are competent to signal again. Thus, GPCR trafficking has critical functions not only in signal termination but also in receptor resensitization.

• Phosphatases. As a significant proportion of signaling events depend on protein phosphorylation, protein phosphatases (e.g.: Tyrosine phosphatase SHP1) play a key role in shutting down signaling pathways.

• Ubiquitin-mediated degradation. Covalent addition of one or more molecules of the small protein Ubiquitin targets proteins to proteosomal or lysosomal degradation.

Reading: Text: "Janeway’s Immunobiology", by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2012, 8th Edition, Chapter 7. Goodman & Gilman's “The Pharmacological Basis of Therapeutics”, Laurence L. Brunton, John S. Lazo and Keith L. Parker, 11th Edition, Chapter 1.

Innate Immune System

September 10 Instructor: A. Iwasaki The innate immune system is an evolutionarily ancient form of host defense. While the adaptive immune system is found only in vertebrates, the innate immune system exists in all animals and plants. The innate immune systesm provides the first line of defense against pathogens. Innate immune response can be activated immediately upon infection and helps maintain pathogens prior to the induction of adaptive immunity. Innate immune system consists of several host defense modules, each designed to protect from different types of infectious challenges. These include:

Epithelial barriers (skin and mucosal epithelia) Phagocytes (macrophages and neutrophils) Anti-microbial peptides and proteins Complement system and acute phase proteins NK cells and plasmacytoid dendritic cells Mast cells, eosinophils and basophils

Each of these host defense modules can be directly activated by the receptors of the innate immune system upon recognition of different classes of invading pathogens. Different infections induce different combination of host defense modules. For example, bacterial infections will typically activate epithelial defenses, phagocytes, antimicrobial peptides and proteins and the complement system. The activation of host defense modules is coordinated by different cytokines, including TNF, IL-1, IL-6, and chemokines, including MCP-1, IL-8 and KC. Many infections are successfully handled by the innate immune system. When innate host defenses are insufficient, however, the adaptive immune response is generated to provide a powerful and antigen specific immunity to pathogens. Reading: Text: "Janeway’s Immunobiology", by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 2 and 3. Review Articles: Medzhitov, R. Recognition of microorganisms and activation of the immune response. Nature. 2007 Oct 18:449(7164):819-826.

Pattern Recognition Receptors

September 12 Instructor: R. Medzhitov The major distinguishing feature of innate immunity is the mechanism of pathogen recognition. Innate immune recognition is mediated by non-clonal, germline-encoded receptors that recognize conserved molecular patterns associated with pathogens. These are referred to as Pathogen-Associated Molecular Patterns (PAMPs). The receptors that recognize PAMPs are called Pattern Recognition Receptors (PRRs). PAMPs recognized by PRRs represent the major targets of innate immune recognition and have several characteristics in common: i) PAMPs are relatively invariant structures shared by large groups of microorganisms. This property of PAMPs

allows a limited number of germline-encoded receptors of the host to recognize a wide variety of microorganisms.

ii) PAMPs are produced only by microbes and not by the host organism. In other words, PAMPs are chemically distinct from any structure synthesized by the host cells. This property allows for self/non-self discrimination by the innate immune system.

iii) PAMPs are conserved molecules essential for the survival of the microbes. Recognition of PAMPs, therefore, prevents generation of 'escape mutants' because such mutations are lethal for the microbes.

The most familiar examples of PAMPs are bacterial LPS and lipoteichoic acids, and fungal glucans. The best-characterized family of PRRs is the Toll-like receptor (TLR) family. TLRs play a crucial role in the immune system. TLRs function as sensors of microbial infection. TLRs recognize conserved microbial molecular structures (PAMPs) and trigger activation of host defense responses. There are about dozen or so TLRs in mammalian species. TLR specificities cover almost entire spectrum of microbial pathogens. Thus, TLR4 is specific for LPS of gram-negative bacteria; TLR2 – for lipoteichoic acids of gram-positive bacteria, as well as for lipoproteins found in all bacteria; TLR5 recognizes bacterial flagellin; TLR3 is specific for double stranded RNA – a common product of viral replication, TLR7 and TLR9 recognize viral RNA and DNA, respectively. Upon activation by their microbial ligands, TLRs induce signaling pathways that activate NF-kB and MAP kinases. All TLRs share a conserved cytoplasmic domain called TIR domain, because it is also found in the members of the IL-1 receptor family. There are 4 TLR signaling adapters that also contain TIR domain – MyD88, TIRAP, TRAM, and TRIF. Different TLRs utilize different combination of the adapters to induce both overlapping and distinct subsets of cellular responses. The canonical pathway shared by all TLRs includes MyD88, serine/threonine kinases IRAK1 and IRAK4, ubiquitin ligase TRAF6, as well as several MAP3 kinases that function downstream of TRAF6 and activate NF-kB and MAP kinases. Activation of these signaling pathways leads to induction of inflammatory responses, innate host defense responses, as well as induction of adaptive immune responses. Thus TLRs represent a crucial component of the host defense system. They detect infection and activate the host immune responses. In addition to TLRs, several other families of pattern recognition receptors (PRRs) have been recently characterized, including Nod-like receptors, Dectins, and RIG-I/MDA-5. Nod1 and Nod2 are intracellular receptors that detect fragments of bacterial peptidoglycans. RIG-I and MDA-5 are also intracellular receptors, but they detect the presence of viral RNA in infected cells. Dectin is a transmembrane receptor specific for beta-glucans – major components of fungal cell walls. Collectively, these receptors detect majority of pathogens and trigger activation of host defense responses, including inflammatory responses. In addition, innate immune recognition can lead to activation of adaptive immunity.

Reading: Text: "Janeway’s Immunobiology", by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 2 and 3. Review Article: Ishii KJ, Koyama S, Nakagawa A, Coban C, Akira S. Host innate immune receptors and beyond: making sense of microbial infections. Cell Host Microbe. 2008 Jun 12;3(6):352-63.

Complement, NK cells and Acute Phase Proteins

September 14 Instructor: R. Medzhitov The innate immune system utilizes a diverse set of mechanisms to combat infection. Early response to infection includes the induction of local inflammation. The inflammatory cytokines IL-6 and IL-1 can also act at a distance and trigger expression of a variety of genes in the hepatocytes. These genes encode so-called acute phase proteins, which are secreted by lives into circulation. The concentration of the acute phase proteins in circulation increases markedly. The function of acute phase proteins is to help combat the infection at the system level. Some acute phase (C-reactive protein, surfactant proteins, mannan binding lectin) function as opsonins – they bind to the pathogen’s surface and facilitate pathogens uptake by phagocytes. These same proteins also activate the complement cascade. Other acute phase proteins have direct antimicrobial activity. For example, bacteriocidal permeability increasing protein (BPI) can bind to the cell walls of gram negative bacteria and destabilize the bacterial membranes. Yet other acute phase proteins regulate the coagulation cascades. The complement system plays a critical role in host defense against a variety of extracellular pathogens. Complement has two functions – lysis of microbial cells and opsonization of microbial cells to promote their elimination by phagocytes. Thus the complement and the phagocytic systems (as well as the acute phase response) are functionally linked. There are several pathways of complement activation, depending on the mechanism of pathogen recognition. The classical pathway of complement activation is triggered by antibodies bound to the pathogens’ surface. The alternative pathway is activated constitutively, but selectively inhibited on host cells, thus allowing for specific attack on pathogens. Finally, the lectin pathway is activated by the acute phase protein mannan binding lectin (MBL). The function of MBL is reminiscent of the antibodies, in that it binds to pathogens’ surface and activates the protease cascade much like the antibodies do. Reading: Text: "Janeway’s Immunobiology", by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 2 and 3. Review Articles: Janeway, CA Jr, Medzhitov, R., Innate immune recognition. Annu Rev Immunol. 20: 197-216. 2002.

Innate immunity to Bacteria, Fungi and Parasites

September 17 Instructor: R. Medzhitov The innate immune system uses a number of mechanisms to protect against bacterial, fungal and parasitic infections. In the case of bacteria and fungi, the main host defenses rely on the function of phagocytes: macrophages and neutrophils. These cells are endowed with powerful antimicrobial defenses, including ROS and NO generating enzymes, anti-microbial peptides and lysosomal enzymes. Phagocytosis and killing by neutrophils and macrophages is the main form of defense against bacteria and fungi. The function of phagocytes is aided by the complement system through opsonization. In addition, complement has direct anti-microbial activity. Epithelial cells also play an important role in antibacterial and anti-fungal defense, in part through the production of potent anti-microbial peptides (defensins) and production of mucus. Defense against multicellular parasites, including helminthes (parasitic worms) is mediated by eosinophils and mucosal epithelia. Eosinophils produce several toxic molecules that can directly target parasites. Mucosal epithelial produce mucins that help prevent parasite entry and promote their expulsion. Mast cells produce several inflammatory mediators that help orchestrate anti-parasitic defenses and promote parasite expulsion through their effects on smooth muscles and endothelium. Text: "Janeway’s Immunobiology", by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 2 and 3. Review Articles: Willment JA, Brown GD. C-type lectin receptors in antifungal immunity. Trends Microbiol. 2008 Jan;16(1):27-32.

Innate Anti-viral Immunity

September 19 Instructor: R. Medzhitov Viruses represent the simplest forms of pathogens. Viruses are obligate intracellular pathogens that can be roughly categorized into DNA and RNA viruses. Viruses contain genetic information within the capsid, and possess surface proteins that bind to and fuse with host cell receptors. Upon entry into the host cells, viruses synthesize proteins, replicate genomes and assemble using host cell machineries. Innate Immunity to Viruses By far the most important innate immune mechanisms against viruses involve the type I interferon system. Type I interferons can be secreted by most cells of the body, and upon binding to the receptor, induce a myriad of antiviral genes that inhibit productive replication of viruses. Recent studies have elucidated the mechanisms by which viral PAMPs can trigger the innate IFN production pathways. Innate recognition of viral PAMPs occurs by two distinct mechanisms depending on the cell types. In most cells, ssRNA virus infection is detected by a cytosolic sensor, as retinoic acid inducible gene I (RIG-I) and melanoma differentiation associated gene 5 (mda-5). In contrast, plasmacytoid dendritic cells (pDCs) use the Toll-like receptors (TLR) 7 and 9 to detect ssRNA and dsDNA viruses, respectively. This form of recognition occurs within the lysosome instead of in the cytosol, as both TLR7 and TLR9 are localized in this compartment. The key cell types involved in innate clearance of viruses and virus-infected cells include pDCs and Natural killer (NK) cells. The pDCs, upon sensing viruses by TLR7 and TLR9 in the lysosomes, secrete large amounts of type I IFNs and IL-12. NK cells are capable of recognizing and lysing cells that lack surface MHC class I molecules. Complement can also deposit onto the virus surface and neutralize or lyse the virus. IFN-I is induced upon infection in most cell types and triggers expression of over hundred anti-viral proteins which interfere with every aspect of viral infection cycle. When the action of these anti-viral proteins is insufficient, the most efficient anti-viral defense is to get rid the infected host cell. Natural killer (NK) cells do just that and play an important role in the anti-viral host defense. NK cells express a number of receptors that can either activate or inhibit their function. The activating receptors recognize molecules expressed on the surface of infected cells and induce the cytotoxic response directed at the unwanted infected cell. The inhibitory receptors recognize ligands that are constitutively expressed on normal healthy cells and inhibit the cytotoxic response against these cells. The combination of the activating and inhibitory ligands generally determines whether or not a given cell would be eliminated by NK cells. Reading: Text: "Janeway’s Immunobiology", by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 2 and 3. Review Articles: Lanier LL. NK cell recognition. Annu Rev Immunol. 2005;23:225-74.

Structure and Genetics of MHC Molecules

September 21 Instructor: P. Cresswell 1. MHC class I molecules are composed of two subunits. The large subunit (44kDa) is a transmembrane

glycoprotein which is the actual product of the MHC-linked gene. The small subunit, β2-microglobulin (β2m), is the product of an unlinked gene and has a molecular weight of 12kDa.

2. MHC class II molecules are also composed of two non-covalently associated subunits, called α and β. Both are

transmembrane glycoproteins and both are products of genes within the MHC. 3. Both human and mouse MHC class I and class II genes have a similar organization at the intron/exon level, with

individual exons encoding the leader sequence, the defined extracytoplasmic domains, and the transmembrane regions. Additional exons encode the cytoplasmic regions.

4. Class I and class II molecules can be regarded as similar structures. In class I, the membrane proximal domain

of the large subunit is homologous to Ig-constant region domains, as is β2m. In class II, the membrane proximal domains of both the α and β-subunits are Ig-like. For class I, the first and second domains of the large subunit fold to generate a cleft or groove between two a-helices, in which bind small peptides of between 8 and 10 amino acids. The membrane distal domains of the α and β-subunits of class II generate a similar peptide-binding groove, but the peptides bound are longer and more variable in length.

5. The class I heavy chain and the two class II subunits show allelic variability in amino acid sequence. Structural

studies show that the variable residues generally line the peptide binding groove. As a result of this variability, the peptide binding groove of each allele has an affinity for a different subset of peptides, with preferred interacting amino acids, called anchor residues, at fixed positions in the peptide sequence. The T-cell receptor recognizes a surface consisting of residues in the a-helices and surface exposed residues in the associated peptide.

6. The MHC contains multiple class I and class II genes. The "classical" class I genes are HLA-A, B, and C in the

human, H2-K, D, and L in the mouse. The β2m gene is not in the MHC. In the mouse there are multiple additional class I genes. These encode molecules which have a limited tissue distribution, generally in hematopoietic cells. Some of these have been suggested to have an antigen processing function. One, H-2M3 from the Qa region, has a role in presenting N-formylated peptides derived from bacteria. Three other human class I genes, HLA-E, F and G, have also been defined.

Genes encoding class II α and β-subunits are both present in the MHC, encoding three different ab dimers in

humans (HLA-DR, DQ and DP) and two different ab dimers in the mouse (I-A and I-E). Individual αβ pairs (e.g., I-Ab and I-Ek) are encoded by very tightly linked genes. The invariant chain gene (see below) is not in the MHC.

7. Genes not obviously related to antigen processing are present in the MHC. These include genes encoding

complement components (C2, C4, Factor B), genes encoding various enzymes (e.g., 21-hydroxylase), the genes for tumor necrosis factors, and genes encoding heat shock proteins (Hsp70).

8. Genes relevant to antigen processing functions are also tightly linked to the MHC. These include two genes

(TAP.1 and TAP.2) encoding a peptide transporter involved in transferring peptides derived from cytosolic proteins into the endoplasmic reticulum where they bind class I molecules, and a gene encoding tapasin, which facilitates MHC class I peptide binding. Also two genes, LMP.1 and LMP.2, encode subunits of a cytosolic macromolecular protease called the proteasome, which is involved in generating peptides in the cytosol. Also in the MHC are two genes, DMA and DMB, which encode a molecule structurally similar to class II molecules

called H2-M or H2-DM in mice and HLA-DM in humans. These genes are required for efficient class II-restricted antigen processing.

Reading: Janeway’s Immunobiology, Garland Science, Ltd., 2012, 8th Edition, Chapter 6, 217-230.

MHC Class I Restricted Antigen Processing

September 24

Instructor: P. Cresswell MHC class I-restricted antigen processing 1. Animals infected with a virus generate virus-specific, class I-restricted CD8-positive cytotoxic T-lymphocytes

(CTL). Many of these CTL prove to be specific for viral proteins that are only expressed intracellularly, e.g., SV40 T-antigen (nuclear) or influenza virus matrix protein (cytosolic).

2. Target cells expressing the appropriate class I allele are not killed unless they are: a) infected with the virus,

or; b) incubated with a defined short (8-10 amino acid) peptide which corresponds to a specific sequence in the particular viral protein recognized by the CTL. Thus, peptide can bind to class I molecules at the cell surface in vitro and generate a class I-peptide complex that the CTL can recognize. However, the normal mechanism which generates the class I-peptide complex involves: a) proteolysis of the cytosolic viral protein; b) transport of the peptide into the endoplasmic reticulum (ER) of the cell; c) association of the peptide with the class I molecule and; d) transport of the class I peptide complex to the cell surface.

3. Proteolysis of cytosolic proteins is predominantly mediated by the proteasome, a multisubunit protease which,

in Antigen Presenting Cells (APCs) or cells treated with interferon-g, incorporates two subunits encoded in the MHC, called LMP.2 and LMP.7, and a third, called MECL-1. Such proteasomes are often called immunoproteasomes.

4. Transport of peptides into the ER is mediated by a heterodimeric transporter. Both subunits of this transporter

(TAP.1 and TAP.2) are encoded in the MHC. The TAP transporter is a member of a structurally homologous family of transporters that use ATP-hydrolysis to translocate small molecules across membranes. After translocation into the ER, peptides may be “trimmed” at the N-terminus by ER Amino Peptidases (ERAAP in mice, ERAP1 and ERAP2 in humans) to facilitate binding to class I molecules.

5. TAP molecules physically associate with newly synthesized "empty" class I molecules in the ER, with a third

protein, tapasin, serving as a bridge. Two additional proteins are also associated; the chaperone calreticulin and the thiol oxidoreductase, ERp57. ERp57 is permanently disulfide-linked to tapasin. These proteins constitute the Peptide Loading Complex, or PLC, which facilitates peptide binding. After peptide binds, the "loaded" class I molecules dissociate from the PLC and are transported to the cell surface where they are available for T-cell recognition.

6. Note that all cytosolic proteins can feed into this system. Thus in normal circumstances class I molecules are

full of peptides derived from normal cell proteins. Virus-derived peptides generated in infected cells are superimposed on this background of normal host peptides.

7. Certain viruses (e.g., Herpes simplex virus I and II, human cytomegalovirus) have developed strategies that

interfere with MHC class I peptide loading, presumably to protect the cells they infect from recognition by CD8-positive T-cells.

Reading: Text: Janeway’s Immunobiology, Garland Science, Ltd., 2012, 8th Edition, Chapter 6, 202-217.

Primary Literature: The International HIV Controllers Study, The major genetic determinants of HIV-1 control affect HLA class I peptide presentation. Science 330: 1551-1557, 2010.

Wearsch, P. and Cresswell, P. The quality control of MHC class I peptide loading. Current Opinion in Cell Biology: 20:1-8, 2008.

MHC Class II Restricted Antigen Processing

September 26 Instructor: P. Cresswell MHC class II-restricted antigen processing

1. CD4-positive T-cells respond to class II-positive cells which have internalized protein antigens. Internalization can occur by phagocytosis (in dendritic cells and macrophages) or by endocytosis. Inhibitors of lysosomal proteolysis (e.g., chloroquine, which neutralizes the normally acidic lysosome) inhibit antigen processing and subsequent presentation to T-cells. Internalization followed by proteolysis of the antigen is essential. For antigens that contain disulfide bonds a lysosomal thiol reductase, GILT, facilitates protein unfolding and peptide generation. Like for class I, peptides corresponding to specific short segments of the antigenic proteins can directly bind class II molecules at the cell surface in vitro and can stimulate T-cells.

2. The biosynthesis of class II molecules is important for proper function. Class II α and β subunits associated

with a third glycoprotein, the invariant chain, immediately upon biosynthesis in the ER. The invariant chain is trimeric and associates with three αβ dimers to form a nonameric structure. Invariant chain regulates antigen processing in two ways. First, it prevents class II molecules from binding unfolded proteins or peptides in the early stages of biosynthesis. Second, it contains a cytoplasmic targeting signal that drives class II-invariant chain complexes to the endocytic pathway. Here the invariant chain is removed by proteolysis, freeing the class II peptide-binding groove to associate with peptides generated from internalized protein antigens. Class II-peptide complexes are then transported to the cell surface where they can be recognized by T-cells.

3. The B-cell/T-cell interaction is critical for making antigen-specific antibody responses. The Ig antigen receptor

on the surface of B-cells acts to enhance presentation of specific protein antigens to CD4-positive T-cells by binding to the antigen and internalizing it. The specifically endocytosed antigen is degraded and peptides derived from it presented on the surface in association with class II molecules. Recognition of this peptide-class II complex by a CD4-positive T-cell induces activation of the B-cell which can differentiate into a plasma cell secreting antibodies specific for the antigen. B-cells are up to 10,000 times more efficient at processing and presenting the antigen for which they are specific than are non-specific B-cells.

4. Mutant cells lacking DM molecules, a class II-like αβ dimer, cannot generate normal class II-peptide

complexes. They accumulate class II molecules associated with a single peptide (CLIP), derived from the invariant chain. DM functions by catalyzing the displacement of CLIP and facilitating the binding of the normal complement of peptides derived from internalized proteins. In B-cells, some dendritic cells, and in thymic epithelial cells, DM function is down-regulated by its association with another class II-like molecule, DO, which inhibits DM function. The DO α and β subunits are also encoded in the MHC.

5. Note that, as in the class I system, even in the absence of pathogenic proteins the class II molecules are still

occupied with peptides. These are usually derived from membrane proteins or internalized serum proteins undergoing lysosomal degradation.

Reading: Text: Janeway’s Immunobiology, Garland Science, Ltd., 2012, 8th Edition, Chapter 6, 202-217. Primary Literature: Sadegh-Nasseri, S. et al., The convergent roles of tapasin and HLA-DM in antigen presentation. Trends in Immunology 29: 141-147, 2008.

Dendritic Cells and Their Functions/CD1 Molecules

September 28 Instructor: P. Cresswell Dendritic cells

Dendritic cells are the “first line of defense” in the antigen presenting cell world. In peripheral tissues they are highly active in macropinocytosis and phagocytosis, allowing efficient non-specific uptake of soluble and particulate protein antigens. At this stage, they have high levels of intracellular class II molecules. Activation of dendritic cells by innate immune mechanisms, e.g., by exposure to bacterial lipopolysaccharides, causes their maturation to a stage with high expression of surface class II-peptide complexes. At this stage they migrate to lymph nodes. Thus, dendritic cells are thought to perform antigen processing in peripheral tissues while antigen presentation to antigen-specific CD4-positive T-lymphocytes occurs in the lymph nodes. Dendritic cells are also the major antigen presenting cell type capable of cross-presentation. This is a mechanism whereby extracellular antigens can be internalized by endocytosis or phagocytosis and peptides derived from them presented by MHC class I molecules to CD8+ T cells. Recent data suggests that recruitment of ER membrane to the phagosome may contribute to this process by introducing a mechanism for protein transfer to the cytosol and by making the MHC class I peptide loading machinery accessible to peptides from the internalized antigens. Subsets of dendritic cells have been described and one of them, in the mouse the CD8a-positive dendritic cell, is the primary cross-presenting subset.

CD1 molecules

Certain Antigen Presenting Cells, including dendritic cells, express an additional type of antigen presenting molecule. These are CD1 molecules. They are not the products of MHC genes but are homologues of the classical class I molecules, forming similar dimers with β2m. In humans there are five types of CD1, called CD1a, CD1b, CD1c, CD1d and CD1e. Mice only express CD1d. The crystal structures of CD1b and CD1d molecules reveal a binding groove deeper and more hydrophobic than that of the MHC class I molecules.

Work in humans has found that cytotoxic T-cells specific for Mycobacterium tuberculosis or Mycobacterium leprae are restricted by CD1 molecules, but it is not CD1-peptide complexes that are recognized. Instead, mycobacterial lipids are bound by CD1 molecules and are recognized by these T-cells. Current evidence suggests that the CD1-lipid complexes are generated in the endocytic pathway and that saposins, co-factors involved in lysosomal lipid degradation, catalyze lipid binding to CD1 molecules. CD1d molecules are detected by a T cell subset referred to as NKT cells, which recognize CD1d complexes containing self lipids and which play an important role linking innate and adaptive immunity.

Reading: Text: Janeway’s Immunobiology: Garland Science, Ltd., 2012, 8th Edition, Chapter 9, 342-353. Primary Literature: Segura E. and Villadangos J. A., Antigen presentation by dendritic cells in vivo. Current Oopinions in Immunology, 21:105-110. 2009. Barral D.C and Brenner M.B., CD1 antigen presentation: how it works. Nature Reviews in Immunology, 7: 929-941, 2007.

The B Cell and T Cell Receptors: Structure & Classes

October 1 Instructor: D. Schatz

The Antibody, or Immunoglobulin Antibodies are molecules that are either present in a membrane bound form on the surface of B cells or are secreted by B cells into body fluids. Antibodies initiate their biological functions by binding to antigen with high affinity, non-covalent interactions. The binding of antibodies to foreign molecules results in their inactivation by effectively targeting the foreign molecules for clearance by other mechanisms such as phagocytosis or complement. All antibody molecules are similar in overall structure. They have a common core that is comprised of 2 heavy and 2 light chains. The two heavy chains are joined to one another by disulfide bonds, as are the light chains to the heavy chains. Treatment with proteases separates the antigen binding domain (Fab) from the effector domain (Fc). Both the heavy and light chains contain a repeated 110 amino acid motif known as the immunoglobulin domain. The N-terminal domains of both heavy and light chains are variable in sequence, while the remaining domains are relatively invariant. The variable domains are known as V domains or regions and the constant domains are known as C domains or regions. The domains at the N-terminal end of one heavy and light chain make up the antigen binding site and therefore each monomeric antibody contains two such binding sites. The constant domain of the heavy chain determine the isotype of the antibody and therefore the functional characteristics of the antibody molecule. The structure of immunoglobulins has been elucidated using X-ray crystallography. The 3-dimensional structure of antibodies reveals that it consists of three globular domains linked by a flexible hinge region, giving it an overall Y shape. Each immunoglobulin domain consists of two layers of polypeptide chains (which are β-pleated sheets) linked by a disulfide bond, to form a barrel structure. The variable region of each antibody chain contains three regions of hypervariability and the hypervariable regions of the heavy and light chain together form the antigen binding site. Therefore the hypervariable regions are known as complementary determining regions (CDRs). Antigens bind to antibodies through non-covalent interactions that can be disrupted by high salt, detergents or extremes of pH. The affinity of binding of a particular antibody for its antigen depends on the multiplicity and strength of these interactions.

Antibody classes Antibody molecules come in subclasses defined by the H chain. The use of different heavy chain constant regions produces antibodies of differing molecular weight, degree of oligomerization, anatomical location, and biological function. These distinct forms of antibody are termed isotypes. The isotypes found in mammals are (more on this in a later lecture): IgM: has an extra domain formed by two extra β-barrels at the C termini of the H chains; these allow pentamerization to form a high avidity structure with 10 combining sites. IgM antibodies dominate early in immune responses. Much low-affinity serum Ig is IgM. IgG: a monomeric, divalent form of antibody which predominates in serum, and is the main component of late and memory systemic responses. In mammals there are subclasses of IgG antibodies with different biological properties, e.g., some fix complement, some don't. Most high-affinity serum Ig is IgG. IgA: a dimeric, tetravalent form of antibody in which two Ig units are joined by a "secretory piece" which is a separate protein. The major function of IgA is to protect mucosal surfaces and it is the main antibody secreted into the gut, respiratory track, milk etc.

IgE: a monomeric antibody produced in response to macroscopic parasites such as worms. Triggers severe inflammatory reactions by binding to a specific IgE receptor on mast cells; crosslinking of this receptor results in mast cell degranulation with release of histamine, etc. Involved in many human allergies, e.g., to grass pollen, or fungal spores. IgD: found on the membranes of mature B cells, involved in B cell activation. Minute quantities in serum, probably without a function there.

Antigenic epitopes on antibody molecules Immunoglobulin molecules may be immunogenic in unrelated species, in other members of the same species, between genetically identical individuals of an inbred strain, or in autoimmunity within one individual. Antibodies may recognize epitopes on the distinct H chains of Ig isotypes; these are anti-ISOTYPE antibodies, e.g., goat anti-mouse IgM. Antibodies may recognize epitopes encoded by allelic differences between antibodies of the same isotype of unrelated members of the same species; these are anti-ALLOTYPE antibodies. Antibodies may recognize epitopes on the combining site, which are distinct for antibodies of different specificity; these are anti-IDIOTYPE antibodies.

The T Cell Receptor T cells recognize antigen as processed fragments in association with MHC molecules, which is fundamentally different from B cells which recognize only the antigen. However, since T cells recognize specific antigens, and their specificity is clonally distributed, it is not surprising that T cell receptors (TCRs) are similar to antibody molecules. TCRs were first identified by raising monoclonal antibodies against T cell clones. The antibodies immunoprecipitated a heterodimer with two disulfide linked chains, termed the α and β chains. Alpha β T cells: Most T cells have alpha beta TCRs on their surface (>95%) and these alpha beta T cells account for the major functional classes of T cells (helper T cells and cytotoxic T cells). γδ T cells: A smaller population of T cells has a TCR on its surface composed of gamma and delta chains. The γδ TCR is probably structurally similar to the Alpha β TCR. The functional significance of gamma deltaT cells is less understood. TCR vs. BCR: The sequence of the alpha, beta, gamma and delta chains reveal that they are similar to the chains of the immunoglobulin molecule, with the overall structure of the TCR being analogous to an Fab region of the immunoglobulin molecule. Each chain of the TCR contains a variable N-terminal region and a constant C-terminal region. The variable regions contain hypervariable regions (positioned much as they are in immunoglobulin V regions) that are functionally analogous to the CDRs of immunoglobulin chains. Of particular note is that the diversity of the CDR3 region of TCRs is much greater than the diversity of CDR1 or 2. As discussed in detail in the reference below, this reflects the fact that CDR3 makes critical contacts with the antigenic peptide while the other two CDRs are primarily responsible for contacting the MHC molecule. The 3-dimensional structure of the alpha-beta TCR has been determined, and the structure shows that the overall architecture is very similar to that of immunoglobulins although surprisingly the Cα domain does not form an immunoglobulin-like domain. TCR and BCR signaling complexes: Immunoprecipitation of the TCR from T cells in non-ionic detergents precipitates a complex of associated proteins that are known as the CD3 complex. The CD3 complex consists of delta, theta, ε, ζ chains. The CD3 proteins are invarient and their function is to allow signal transduction upon

activation of T cells. In like manner, Ig molecules on the surface of B cells are non-covalently associated with two signal transduction proteins known as Iga and Igb. MHC class I or II restricted, CD8 or CD4 expressing T cells: Of critical importance to the interaction between the TCR and peptide:MHC are the CD4 and CD8 co-receptors found on the surface of T cells. CD4 is a single chain polypeptide that interacts with MHC class II molecules, while CD8 is a disulfide linked heterodimer that binds to MHC class I molecules. CD4 positive T cells recognize antigen presented by MHC class II molecules, while CD8 positive T cells recognize antigens presented by MHC class I molecules. Like CD3, the function of CD4 and CD8 co-receptors is in sending signals to the interior of the T cell: when the TCR:CD3 complex and the co-receptor bind to the same MHC:peptide molecule, they send a much more potent signal than if TCR or co-receptor alone is involved in the binding.

Reading: Text: "Janeway’s Immunobiology", by K. Murphy (Garland Science), 2011, 8th Edition, Chapter 4 and Chapter 5, sections 5.12, 5.13, and 5.16. **This is a very structure-oriented topic and pictures are worth a thousand words; please carefully study the beautiful diagrams in the book.** Review Article: Williams, A.F. and Barclay, A.N. The immunoglobulin superfamily-domains for cell surface recognition (1988). Annual Review of Immunology 6:381-405. Primary literature: Saphire, E.O., Parren, P.W., Pantophlet, R., Zwick, M.B., Morris, G.M., Rudd, P.M., Dwek, R.A., Stanfield, R.L., Burton, D.R., and Wilson, I.A. (2001). Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design. Science 293, 1155-1159.

Immunoglobulin and TCR Gene Structure and Rearrangement

October 3 Instructor: D. Schatz The problem: how to encode the millions of different immunoglobulin (Ig) and T cell receptor (TCR) molecules that are made by B and T cells; the dogma of "one gene/one polypeptide" won't work due to the limited number of genes in the genome. The answer: Ig and TCR genes are assembled from component gene segments, and diversity is generated combinatorially.

Ig and TCR Genes The variable region gene exon is assembled from gene segments called V (variable), D (diversity) and J (joining). For each antigen receptor locus, these gene segments and the constant (C) region all lie on a single chromosome. The Ig heavy chain locus (Igh) and TCRb and TCRd loci contain V, D, and J gene segments while the two Ig light chain loci (Igk and Igl) and the TCRa and TCRg loci contain only V and J gene segments. Antigen receptor loci can be very large (spanning as much as several megabases). The random rearrangement of V, D, and J segments is guided by short, flanking DNA sequences that all V, D, and J genes have in common (recombination signal sequence, or RSS). See figure at bottom of the notes for this lecture for diagrams of the loci. Generation of antigen receptor diversity The diversity of Igs is generated by four main processes, the first three of which are shared by TCRs. Gene segment combinatorial diversity results from the presence in the germline of multiple different copies of each type (e.g., V, D, J) of gene segment, and different combinations of these gene segments can be used in different rearrangement events, Junctional diversity, which is particularly concentrated in the third hypervariable region (CDR3 region), is introduced at the joints between the different V, D, and J segments as a result of addition and subtraction of nucleotides in the recombination process. Non-germline encoded (N-) nucleotides can be added by the enzyme terminal deoxynucleotidyl transferase (TdT), while exonucleases delete nucleotides. Such added/subtracted nucleotides often disrupt the reading frame of the coding sequence beyond the joint (called nonproductive rearrangements), leading to a nonfunctional protein, and hence junctional diversity is achieved only at the expense of considerable wastage. A third source of diversity, chain pairing, is also combinatorial, arising from the many possible different combinations of heavy- and light-chain V regions (or TCRa chain and TCRb chain) that pair to form the antigen-binding site in these molecules. Somatic hypermutation is the fourth source of diversity; it affects only Ig genes and is considered in a later lecture. Example: diversity in Igh and Igk Combinatorial diversity: One V, one D and one J are used to assemble the Igh gene: with, say, 100 Vs, 13 Ds and 4 Js, there are 5200 different V-D-J combinations (100x13x4). The Igk locus contains about 150 Vs and 4 Js (no Ds), for a combinatorial potential of 600 different genes. Junctional diversity dramatically increases the number of possible proteins encoded by these loci (to more than a billion for Igh, 100,000 for Igk). Chain pairing: Assuming that each Igh chain can pair with each Igk chain to form an antibody, the theoretical number of possible Igh-Igk pairs is well over one trillion.

V(D)J recombination V, (D) and J gene segments are joined by a site-specific recombination reaction known as V(D)J recombination. This reaction takes place in two phases. In the first, the recombination machinery binds to the RSSs flanking the two participating gene segments and then cuts the DNA immediately adjacent to each gene segment, resulting in a

chromosome that has been cut in two places. In the second, the four free DNA ends are processed and joined to form two new junctions (see diagram): a coding joint (the fusion of the two coding elements, like V and J—rectangles in the diagram) and a signal joint (the fusion of the two RSSs—triangles in the diagram). Two V(D)J recombination events are required to assemble the Igh, TCRb, and TCRd loci (D-to-J and V-to-DJ) and one event (V-to-J) for the other loci.

+

Inversion

Deletion

The first phase of V(D)J recombination (DNA binding and cleavage) is performed by the RAG1 and RAG2 proteins. RAG1 and RAG2 are expressed only in developing lymphocytes and are both essential for V(D)J recombination. The second phase of V(D)J recombination (end processing and joining) is performed by a group of ubiquitously expressed DNA repair proteins together with TdT (mentioned above).

Class Switch Recombination (Isotype Switching) Immunoglobulins can be made in several different forms, or isotypes (e.g., IgM, IgD, IgG, IgA, and IgE); this structural variation is generated by linking different heavy-chain constant regions to the same Igh variable region. The CH regions are encoded in separate genes located downstream of the V genes at the heavy-chain locus. Initially only the first (most 5') of these genes, Cm, is expressed in conjunction with an assembled V gene. However, during the course of an antibody response activated B cells often switch to express a different downstream CH gene by a process of somatic recombination known as class switch recombination (CSR) or isotype switching; CSR is unlike V(D)J recombination in several ways. To reiterate, the same VH exon can associate with different CH genes in the course of an immune response.

Membrane and secreted forms of Ig molecules Immunoglobulins of all heavy-chain isotypes can be produced either in secreted form or as a membrane-bound receptor. The membrane forms of all isotypes are monomers comprised of two light and two heavy chains; IgA and IgM polymerize (into dimers and pentamers, respectively) only when they are secreted. In its membrane-bound form the immunoglobulin heavy chain has a hydrophobic transmembrane domain at the C-terminus which anchors it to the surface of the B lymphocyte. This transmembrane domain is absent from the secreted form (antibody), whose C-terminus is a hydrophilic secretory tail. The two different C-termini (transmembrane and secreted) of the heavy chains are encoded in separate exons, and production of the two forms is achieved by alternative RNA processing.

Regulation of V(D)J Recombination V(D)J recombination is regulated at several levels. 1) RAG1 and RAG2 are expressed only in developing lymphocytes and hence V(D)J recombination can only occurs in these cells; 2) Through the control of the "accessibility" of the DNA substrates. RAG1/RAG2 can only bind to DNA substrates that are in an "open" chromatin structure. Such "opening up" of the chromatin is facilitated by activating histone modifications as well as transcription of the unrearranged gene segments—so called "germline" transcripts; 3) Through the control of long range chromosome "looping"; gene segments that are far apart on the chromosome will only recombine if they are brought into close proximity.

Reading: Text: "Janeway’s Immunobiology", by K. Murphy (Garland Science), 2011, 8th Edition, Chapter 5, pp 157-173. Review Article: Gellert, M. (2002). V(D)J recombination: RAG proteins, repair factors, and regulation, Annu Rev Biochem 71, 101-132.

Primary literature: Chien, Y.H., Gascoigne, N.R., Kavaler, J., Lee, N.E., and Davis, M.M. (1984). Somatic recombination in a murine T-cell receptor gene. Nature 309, 322-326.

Figure Legend Schematic representation of the murine antigen receptor loci. Gene segments are depicted as white rectangles, but pseudogenes have been omitted. Enhancers and promoters are represented by gray circles and rectangles, respectively, and 12-RSSs and 23-RSSs as white and black triangles, respectively. Constant regions are depicted as single rectangles, with no attempt made to indicate individual exons. The two gray rectangles within the Igk locus represent the two start sites for Jk sterile transcripts (one of which is the KI/KII element). Some Vk genes, and Vd5 and Vb14 are known to rearrange by inversion. Va and Vd gene segments are interspersed. Not drawn to scale. Taken from Hesslein, D. G., and Schatz, D. G. (2001). Factors and forces controlling V(D)J recombination, Adv. Immunol. 78, 169-232. Ig and TCR Loci

VH(100-1000) DH(13) JH

V!(100-300)

CH(µ,",#,$,%)

J! C!

C&1

V%/"(100)

V#5

V'(50) V'14C'2J'2(6)J'1(6) D'2C'1D'1

D" J" C" V"5 J%(61) C%

J#1 C#1 C#2 J#2 V#1.2 V#1.1 J#4C#4V#1.3V#2V#4V#3

PDQ52 Eiµ 3'Eµ

Ei! 3'E!

E&2-4 E&3-1

PD'1 E'

E%E"

KI/KII

BEAD-1TEA

E#1 E#3 E#2

J&1C&3J&3V&1C&2J&2V&XV&2

IgH

Ig!

Ig&

TCR'

TCR%/"

TCR#

LCR

HsA

B Cell and T Cell Development

October 5 Instructor: D. Schatz

Overview The overall goal of lymphocyte development is to efficiently generate lymphocytes that have functional receptors and that are not self-reactive. Lymphocytes arise from hematopoietic stem cells in the bone marrow. The HSC gives rise to more differentiated progenitors that either migrate to the thymus, where they develop into T cells, or remain in the bone marrow, where they generate B cells. The local environment—defined by so-called stromal cells—provides the signals (e.g., cytokines such as IL-7) that induce proper differentiation and also that guide lymphoid cells through subsequent steps. Thymocyte development takes three weeks to complete. The first week is spent expanding slowly in the outer cortex of the thymus, the second in rearranging their receptor genes and undergoing positive selection and negative selection, and the third week as maturing thymocytes in the medulla of the thymus. B lymphocytes undergo a similar differentiation process in the bone marrow. The first phase of lymphocyte development is concerned with the generation of functional antigen receptor genes by V(D)J recombination, which creates “in-frame” joints that encode the desired protein 1/3 of the time. TCRs and BCRs have two chains encoded by two separate loci. Rearrangements occur on only one locus at a time. If the rearrangement is successful, the developing lymphocyte will express the protein product of the first locus to rearrange (either TCRb or the IgH locus), along with another invariant protein that mimics the TCRa or IgL chain. Cells that express this “pre-receptor” receive signals that cause them to divide a few times and move to the next stage of differentiation. Cells that do not express the pre-receptor can continue to rearrange (i.e. on the other of the two chromosomal alleles). If neither allele forms a productive rearrangement, the cell dies. The progeny of cells that did successfully rearrange then go on to rearrange the other locus, and if successful, they will express a complete TCR or BCR, again allowing them to proceed in differentiation. Once the B and T cell receptor gene rearrangements have occurred, the receptor must be tested for whether it is harmful (i.e. anti-self) and useful (i.e. can be stimulated by Ag). This occurs via two processes termed negative and positive selection. Negative selection mainly involves interaction by the antigen receptor with antigens presented in the microenvironment in which they have undergone their development, the thymus for T cells and the bone marrow for B cells. Any immature lymphocyte that is strongly stimulated by a self-antigen is prevented from further development before it becomes fully functional. This is called negative selection. Since T cells need to see self MHC:self peptide complexes in order to be stimulated, they will only be useful if they have some affinity for self MHC. Cells are tested for this in the thymus during interactions with thymic epithelial cells in the cortex. T cells that receive a weak but definite signal are selected for survival—so called positive selection. Cells that are not positively selected die while those that are selected also undergo the negative selection process. B cells also undergo both positive and negative selection, but in this case negative selection comes first. Positive selection of B cells is much less well understood. Finally, lymphocytes that pass both negative and positive selection turn off expression of RAG1 and RAG2 and migrate, under the influence of chemokines and “homing receptors” to their respective locations in the peripheral organs, through which they continue to recirculate.

Step 1: Igh or TCRb gene rearrangement in pro-B or pro-T cells In both the T and B cell lineages, the first rearrangement is in the genes that contain V, D, and J gene segments (TCRb locus in T cells, the Igh locus in B cells), in a cell termed either a pro-T or pro-B cell. The earliest pro-T and pro-B cells turn on expression of RAG1 and RAG2 and perform D-to-J recombination. They then perform V-to-DJ recombination. When recombination occurs "in frame" (i.e., produces a continuous reading frame that can be translated into the full length TCRb or Igh protein), these molecules are expressed on the cell surface with surrogates which stand in for the TCRa or Ig light chain protein (which the cell can't make yet because it hasn't assembled these genes yet). These receptors, called the pre-T cell receptor and the pre-B cell receptor, drive enlargement of the cell and its rapid expansion through cell division, so that one properly rearranged locus can generate at least 100 identical progeny. As they finish this burst of proliferation, the cells are called pre-T or pre-B cells. Note: pro-T cells are also referred to as "double negatives" because they do not express CD4 or CD8; pre-T cells are also referred to as "double positives" because they express both CD4 and CD8.

Step 2: IgL or TCRa gene rearrangement in pre-B or pre-T cells The second phase of gene rearrangement involves genes that lack D gene segments (TCRa locus or Ig light chain loci--k or l), and thus can occur multiple times. This is particularly true of T cells, in which the TCRa locus consists of large arrays (~60 gene segments) in which V to J joining can occur several times. This means that most cells that have expanded from the original V—D—J rearrangement are provided with a functional receptor.

Step 3: Selection Positive selection: Once cells express a functional receptor, the suitability of that receptor can be tested. There is negative selection against self-reactive lymphocytes (see below) as well as positive selection for T cells that are going to be functional in the host. Positive selection of T cells selects for cells whose receptors bind some self-antigen weakly but detectably, which is critical to achieve MHC-restriction of recognition by T cells. The positive selection signal is critical for T cell survival and continued development. It occurs at the CD4+/CD8+ (“double positive”) thymocyte stage and is mediated by interactions with MHC I/II+ thymic epithelial cells. T cells which do not recognize a ligand remain developmentally arrested and continue to rearrange their TCRα chains, thus potentially replacing one protein chain with another; if the cell still fails to make a positively-selectable receptor, it will die. Cells which are positively selected by MHC I recognition mature into CD8+ (and CD4-) T cells while those selected by MHC II mature into CD4+ (and CD8-) T cells. Positive selection of B cells is less well understood. Negative selection: Developing B or T cells whose receptors bind self-antigens too well either die (in the case of T cells in the thymus) or undergo developmental arrest (B cells in the bone marrow). Such developmentally arrested B cells, like the T cells that have failed positive selection, will continue to rearrange their light chains and may make a new Ig receptor (a process called "receptor editing"). If the new receptor is not self-reactive, development will proceed, else the cell will die by apoptosis. In any case, both the T cell and B cell populations are thereby purged of self-reactive cells. In order for T cells to be negatively selected, they must see their cognate peptides displayed by antigen presenting cells (such as dendritic cells and macrophages) within the thymus. Many of these antigens are expressed only in certain cell types not present in the thymus (for example, insulin, which is produced by cells of the pancreas). Thymic antigen presenting cells express a gene called autoimmune regulator (AIRE) that enables many such proteins to be expressed at low levels in thymus.

Numbers In the mouse, cortical thymocytes are created at a rate of 20-40 x 106/day, yet only 1-2 x 106 leave the thymus each day. The rest must therefore die in the thymus, almost certainly as a result of negative selection or a failure to receive a positive selection signal (called "death by neglect"). About 80% of the B cells that are produced in the bone marrow fail to find a proper survival "niche" in the periphery and hence die quickly (≈ 3 days). The other 20% become mature naïve B cells and live for about two months. Why some B cells are selected into the long-lived pool and others are not is not understood, but this has been termed B cell "positive selection". A mouse contains about 108 mature peripheral B cells, about 2% (2 x 106) of which die each day. Mouse bone marrow produces about 107 new B cells per day; the 20% that are "positively selected" to become long lived mature B cells replace those that die each day. Reading: Text: "Janeway’s Immunobiology", by K. Murphy (Garland Science), 2011, 8th Edition, Chapter 5, pp 162-173; Chapter 8, pp 275-316. Review Article: Schebesta, M., Heavey, B., and Busslinger, M. (2002). Transcriptional control of B-cell development, Curr. Opin. Immunol. 14, 216-223. Primary Literature : He, X., He, X., Dave, V.P., Zhang, Y., Hua, X., Nicolas, E., Xu, W., Roe, B.A., and Kappes, D.J. (2005). The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment. Nature 433, 826-833.

Positioning, Maturation & Trafficking of Lymphocytes

October 8 Instructor: J. Pereira I. Overview.

Lymphocytes are continuously moving within and between lymphoid organs since early stages of their differention. Their movement is largely controlled by three types of transmembrane receptors: G protein-coupled receptor, integrins and selectins. These family of receptors recognize specific extracellular signals and adhesion molecules that guide and retain lymphocytes in specific locations during their development in primary lymphoid organs, and maturation in secondary lymphoid organs. This lecture explains how lymphocyes are organized within primary and secondary lymphoid organs, and why such compartmentalization is important for the immune system to function apropriately.

II. Thymus Structure and Thymocyte Positioning During Development

a. DN (CD4-CD8-) cells develop in the cortex and subcapsular region. b. DP (CD4+CD8+) thymocytes are positively selected at the cortico-medullary junction. c. SP (CD4+ or CD8+) lymphocytes are negatively selected in the medulla. d. Non-autoreactive SP-lymphocytes egress via thymic blood vessels at the cortico-medullary junction. Egress is mediated by S1P1 expressed on lymphocytes, and its ligand S1P, which is abundant in blood.

III. B Cell Development in bone marrow niches. a. CXCR4 is fundamental for bone marrow (BM) derived hematopoiesis. Retention of hematopoietic cells in the BM requires CXCR4 and its ligand CXCL12. b. The bone marrow cellular organization is poorly understood. It is characterized by two distinct compartments: parenchymal tissue composed by developing hematopoietic cells, progenitors, stromal cells, adipocytes, osteoblasts, osteoclasts, and chondrocytes; and by a network of small sinusoids that perfuse the marrow and anastomose into large collecting and central sinusoid. In late stages of B cell deleopment, immature B lymphocytes become distributed between parenchyma and sinusoids before exiting into the spleen for further maturation c. In the parenchyma, immature B lymphocytes are negatively selected against membrane-bound self-antigens.

IV. Secondary lymphoid organs (SLOs). a. SLOs include Spleen (Sp), Lymph Nodes (LN), Peyer’s Patches (PP), Mucosal-associated lymphoid tissue (MALT) such as bronchial (BALT), nasal (NALT) and gut (GALT), peritoneal and pleural cavity “milky spots”. Spleen is divided in two compartments: white pulp and red pulp. b. Main functions: i. to filter antigens from body fluids; ii. bring together antigen, antigen-presenting cells and antigen-specific lymphocytes iii. support lymphocyte activation and differentiation events c. SLOs are mainly populated by B and T lymphocyte subsets, and smaller numbers of antigen-presenting cells (e.g. interdigitating dendritic cells (IDCs), follicular dendritic cells (FDCs), macrophages), and stromal cells. d. The peripheral B cell compartment is made by B1 cells (mostly in the peritoneal cavity), B2 cells (often called follicular B cells), and marginal zone B cells (MZB, found in the mouse spleen, and in human spleen and LNs). In mice, MZB are located at the interface between Red and White pulp, are exposed to incoming blood, and respond vigorously to blood-borne pathogens. B1 cells populate body cavities exposed to potential pathogens. Both MZB and B1 cells respond faster than B2 cells to antigens of microbial origin (e.g. LPS). e. B2 lymphocytes reside in follicles. FDCs predominate at the center follicle. Specialized follicular stromal cells pave the entire follicle. f. T lymphocytes are distributed in the T cell zone around central arterioles. IDCs, and T-zone stromal cells also populate t-zone.

V. Chemokines and Integrins.

a. Chemokines are chemoattractant proteins: these are the “scents” that guide cells to specific locations. b. Chemokines are categorized structurally by location of cysteine residues (CC, CXC, C, CX3C). c. Chemokine receptors belong to the G protein-coupled receptor family and are characterized by 7 transmembrane domains that serpentine through the membrane. Induction of chemotaxis depends predominantly on receptor coupling to Gαi proteins. d. Chemokines play two fundamental roles: i. lymphoid or homeostatic chemokines organize the lymphoid organ compartments: CCL19 and CCL21 and their shared receptor CCR7; CXCL13 and its receptor CXCR5; and CXCL12 and its receptor CXCR4. ii. proinflammatory: RANTES, MCP-1, IL-8 e. Integrins are obligate heterodimers composed of an alpha and beta subunits both of which containing a single transmembrane domain. f. Integrins can be in an inactive (non-sticky) and active (sticky) states. Integrin activation requires GPCR signaling. g. Integrins play two basic functions: i. attachment of the cell to the extracellular matrix (ECM) ii. signaling from ECM to cell h. Lymphocytes mainly express 3 integrin heterpdimers: α4β1, αLβ2, and α4β7.

VI. Cues organizing SLOs: Integrins, Chemokines and receptors.

a. B and T lymphocytes require integrins (αLβ2 and α4β1) for entry into splenic white pulp cords. b. B cells require CXCR5 for homing into follicles; FDCs and follicular stromal cells express the CXCR5 ligand, CXCL13 (a.k.a. BLC – B Lymphocyte Chemoattractant). c. CCR7, and its ligands CCL19 and CCL21 produced by T-zone stromal cells guide cells to the T-zone. d. T cells migrate within the T-zone with an average speed of 12µm/min. As many as 5000 T cells can interact with a single DC per hour. e. B cells move within follicles at about 6µm/min where they survey for antigen displayed on FDCs. d. MZB cells utilize S1P1 and CXCR5 to position at the interface between the red (S1P is abundant in blood) and white pulp (CXCL13 is abundant in white pulp follicles). e. Retention of MZB cells in the MZ also depends on integrins αLβ2 and α4β1 and their ligands ICAM1 and VCAM1, respectively. f. MZB and B1 cell development requires Gαi coupled CXCR5 and CXCL13.

V. Entry in SLOs a. Entry in LNs occurs through specialized blood vessels called High Endothelial Venules (HEVs). HEVs express vascular addressins (GlyCAM-1, PECAM-1, PNAd); recognized by L-selectin expressed on lymphocytes. This event slows down lymphocytes circulating in blood. b. Lymphocyte entry also requires αLβ2 and α4 integrins, and integrin ligands (VCAM-1, ICAM-1 and -2, MadCAM) expressed on HEVs. Activated integrins bring rolling lymphocytes to full arrest and allow transendothelial migration and movement into the lymphoid tissue. c. Entry in the spleen is not via HEV but through terminal open arterioles. Cells are in fact released into the red pulp. e. CCR7, CCL19 and CCL21 are required for T cell entry in LNs; while CCR7, CXCR4, CCL19, CCL21, and CXCL12 promote B cell entry in LNs. B cell homing to PPs is dependent upon CXCR5 and CXCL13.

VI. Exit from SLOs a. Egress of B and T lymphocytes requires S1P1 expression, and a gradient of its ligand S1P. S1P is kept at low concentrations in lymphoid organs by the S1P-degrading enzyme S1P lyase, whereas blood and lymph contain high concentrations of S1P. Egress from spleen occurs via blood; LN exit is via the lymphatics. b. Current model hypothesizes that the time spent in lymphoid organs is dictated by a “tug of war” between lymphoid organ retention cues (e.g. CXCL13, CCL19, CCL21) and egress promoting cues (S1P at exit sites).

c. Lymphoid organ shutdown and blocked egress. Activated lymphocytes rapidly up-regulate CD69 that counteracts S1P1 function. This prevents activated T and B lymphocytes from exiting SLOs during an immune response.

Reading: Text: C.A. Janeway, Jr., P. Travers, M. Walport and M. Shlomchik. Immunobiology: The immune system in health and disease, Garland Science, Ltd., 2012, 8th Edition, Chapter 8. Review articles: Cyster, JG. “Chemokines, S1P and Cell migration in lymphoid organs.” 2005. Annu. Rev. Immunol

Receptors & Signaling II

October 10

Instructor: Carla V. Rothlin In this lecture we will review signaling pathways that are central to lymphocytes’ biology. Antigen receptor signaling The capacity of T cells and B cells to specifically recognize and respond to antigens is fundamental to the activation of the adaptive immune system. B-cell and T-cell antigen receptors are made up of variable antigen-binding chains (immunoglobulin chains in the B-cell receptor and TCRa:b chains in the T-cell receptor) that lack intrinsic signaling activity. Therefore, they are associated with invariant proteins that possess the capacity to activate downstream signaling pathways. Surface immunoglobulins are associated with two polypeptides termed Iga and Igb, while the TCRa:b heterodimer is associated with three CD3 chains (g, d, e) and two z chains. Assembly of the antigen-binding chains with these accessory proteins not only provides adequate signaling capacity, but also allows the correct transport of the complex to the plasma membrane. Optimal signaling also requires co-aggregation of co-receptors such as CD19 in B-cells, and CD4 or CD8 molecule in T-cells. Aggregation leads to the activation of tyrosine kinases that are associated with these cell surface molecules. The tyrosine kinases that have been found associated with antigen receptors include the Src family members: blk, fyn and lyn for the B-cell receptor, and fyn and lck for the TCR. In addition two soluble tyrosine kinases, syk in B-cells and ZAP-70 in T-cells are recruited to the signaling complex upon activation. Activation of these tyrosine kinases and the subsequent phosphorylation of downstream targets initiates a signaling cascade that culminates in the activation of the transcription factors NFkB, NFAT and AP-1 to induce specific gene expression, leading to cell proliferation and differentiation. Cytokine Receptor induced JAK/STAT signaling pathway Type I and Type II cytokine receptors are typically non-covalently associated with protein tyrosine kinases of the Janus kinase (JAK) family. This family is formed by four members: Jak1, Jak2, Jak3 and Tyk2. Activation of cytokine receptors leads to the trans-phosphorylation and consequent activation of the associated JAKs. The activated JAKs then phosphorylate their associated cytokine receptors on specific tyrosine residues that generate binding sites for the transcription factors known as signal transducers and activators of transcription (STATs). Recruitment of the STATs to the activated cytokine receptor brings them to proximity to the activated JAKs, which induce STAT phosphorylation. This leads to a conformational change that allows for STATs homo or heterodimerization, their translocation to the nucleus and induction of gene expression. Death receptor induced signaling pathway Programmed cell death or apoptosis plays a key role in the regulation of the immune function. In particular, it has an important role in the termination of the immune responses and removal of potentially autoreactive lymphocytes. Apoptosis can be induced by extracellular ligands, through the activation of death receptors (extrinsic pathway). Apoptosis can also occur through the activation of an intrinsic pathway that involves the release of cytochrome c from the mitochondria upon exposure to a noxious stimulus. Common to both pathways is the activation of specialized proteases called caspases. The death receptors (e.g.: Fas and TNFR-I) are members of the large TNF receptor family and contain a conserved cytoplasmic domain known as death domain (DD). Activation of the death receptors by their cognate ligands leads to clustering of the death domains and the recruitment of specific adaptor proteins. For example, activation of the Fas receptor leads to the recruitment of FADD (Fas-associated via death domain). FADD contains a death effector domain (DED) that allows for the recruitment of the initiator caspase, pro-caspase 8. Pro-caspase 8 is self-activated by proteolytic cleavage and subsequently released from the receptor complex. This step is followed by the activation of effector caspases leading to a series of events that are hallmarks of apoptosis, such as the enzymatic fragmentation of chromosomal DNA.

Reading: Text: "Janeway’s Immunobiology", by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2012, 8th Edition, Chapter 7.

Innate Control and Initiation of Adaptive Immune Responses

October 15

Instructor: J. Craft MAIN POINTS 1. T CELL TYPES: There are two primary types of T cells, CD4 and CD8 T cells, that recognize small peptides presented on MHC class II and class I, respectively. CD8+ T cells see antigen presented by the ubiquitous MHC class I molecules and induce the death of any cell that they detect as infected, whereas CD4+ T cells recognize antigen presented by MHC class II molecules that are restricted in their distribution in the periphery. CD4 T cells serve many roles in the immune response. 2. PHASES OF THE T CELL RESPONSE: The T cell response can be characterized by three distinct stages (1) Activation and Clonal Expansion, (2) Contraction (i.e., death of the activated T cells) and (3) Formation of memory T cells. 3. INNATE RECOGNITION of INFECTION by DENDRITIC CELLS PERMITS T CELL ACTIVATION: Tissue resident dendritic cells (DCs) sense the invading pathogens at the site of infection and become “activated”. This switches them into professional APCs that upregulate lymph node (LN) homing receptors and causes them to migrate into the draining LNs. 4. ANAMTOMY OF THE FIRST KISS: Naïve T cells specific for a given virus are very rare and present at very low frequency in the LNs. The T cells become activated in the draining lymph nodes when they encounter their cognate antigen on the dendritic cells that have migrated into the lymph nodes from the infected tissue. Expression of chemokine receptors on T cells and DCs regulate their localization in the lymphoid organs to ensure they find each other. OVERVIEW All adaptive immune responses are mediated by the activation and clonal expansion of antigen-specific T and B lymphocytes. This is followed by their differentiation into effector cells that, upon encountering their specific antigen in the periphery, quickly respond to eliminate infected cells and pathogen replication. The effector functions expressed by antigen-specific T cells differ depending on the type of infection at hand and, in accordance, the types of signals elicited by the innate immune system. Following removal of the pathogen, the majority of the activated T cells die, but a minority persist as long-lived memory T cells. These memory T cells can mount rapid secondary responses to reinfection and help to protect against reoccurring disease and illness.

NaiveActivation/

ExpansionContraction Maintenance

of Memory

days post infection

1 5 8 15 30 60

CD8 T cell response

Virus

In this lecture, we will review concepts that were introduced earlier, such as the role of tissue dendritic cells in bringing antigens to the local lymph nodes, as well as the importance of the trafficking of lymphocytes through the lymph nodes in search of their specific antigen. Dendritic cells acquire antigen in the peripheral tissues, especially in sites of infection, where they reside as immature, highly phagocytic cells with little ability to activate T cells. The encounter with pathogens, via specific receptors of the innate immune system that bind common molecules on the surfaces of pathogens (called pathogen recognition receptors (PRRs)) induce signal transduction events that lead to their secretion of Type I IFNs, antimicrobial peptides and chemokines to attract other leukocytes to the site of infection. It also causes the DCs to mature into cells that have lost the ability to take up antigen, but have gained the ability to present antigens to T cells. They also begin to express new molecules that cause their migration to the local lymph nodes (such as CCR7) and increase expression of both classes of MHC molecules and co-stimulatory molecules CD80 and CD86 to activate both CD4+ and CD8+ T cells against the pathogen. In summary, the specific recognition of pathogen associated molecular patterns (PAMPs) by the tissue resident DCs and Mφ has two very critical effects for immunity—the first is that this causes the innate immune cells to directly release cytokines and other molecules that will directly attack and kill microbes. The second is that it switches a DC from a “tolerogenic” DC into an “immunogenic” DC, which upregulates expression of lymph node homing receptors, MHC:antigen complexes, and costimulatory molecules for T cell activation. Regulation of this switch in DC function is critical to prevent autoimmunity as it is the primary measure that ensure T cells are only activated when a pathogen is present and requires a T cell response for its removal. T cells re-circulate actively from the blood into the lymph nodes, via binding to endothelial cells by adhesion molecules, and subsequent migration through the endothelial cell barrier. Homing to lymph nodes and other secondary lymphoid tissues is also mediated by specific chemokines, produced by endothelial cells, stromal cells and dendritic cells that bind receptors on T cells. Once in these secondary lymphoid organs, naïve T cells then survey the surfaces of dendritic cells for their specific antigen. In the vast majority of cases, the lymphocyte does not encounter its specific antigen, although it does receive weak signals through its receptor that allow it to survive. However, in those rare instances when recognition occurs, after pathogen invasion, the T cell ceases to re-circulate and sets up stable interactions with the antigen-presenting dendritic cell, mediated initially by pairs of cell adhesion molecules. This leads to formation of an immunological synapse, which is a highly organized structure of proteins between the adhesion of a DC and an antigen-specific T cell. Reading: Text: Janeway’s “Immunobiology” by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 9 (9-1 through 9-9) and Chapter 3 (3-13 through 3-19). Primary Literature: Kearney ER, Pape KA, Loh DY, Jenkins MK. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity. 1994 Jul; 1(4):327-39.

T Cell Priming & Effector Cell Differentiation I

October 17

Instructor: J. Craft MAIN POINTS 1. ORGANIZATION and DURATION of an IMMUNOLOGICAL SYNAPSE: The inner C-SMAC and outer P-SMAC and can persist for more than 7hrs. 2. T CELL PRIMING: THE 3 SIGNAL HYPOTHESIS: Effector T cell clonal expansion and differentiation is governed by three major signals: Signal 1(antigen:TCR), signal 2 (costimulation) and signal 3 (effector-determining inflammatory cytokines). OVERVIEW The immunological synapse is composed of Central-SMAC (SupraMolecular Adhesion Complex) that contains the TCR/Ag:MHC, C4 or CD8 coreceptors, costimulatory receptors and signaling molecules. The Peripheral-SMAC contains adhesion molecules such as LFA-1 and talin. The immunological synapse directs the movement of the MTOC (microtubule organizing center) to the site of contact. Recent evidence suggests that the first T cell division is likely to be asymmetric leading the unequal distribution of molecules that influence effector T cell differentiation. As the T cells are engaged in a tight adhesion with the DCs, they receive 3 types of critical signals that are necessary for T cell activation and differentiation into effector T cells. The first is the direct recognition of

antigen (MHC-peptide complexes) by the T Cell Receptor (TCR) that leads to its activation. The second signal comes from costimulatory ligands expressed by activated DCs that activate costimlatory receptors on T cells and enhance TCR signaling. Several types of costimulatory ligand:receptor pairs exist, (B7:CD28, CD70:CD27, 41BBL:41BB, CD40L:CD40, ICOSL:ICOS). The 3rd signal is inflammation, in which inflammatory cytokines produce by the innate immune DCs, NK cells and macrophages help to skew the differentiation of the T cells to develop the appropriate type of effector functions to fight the present infection. Such inflammatory cytokines are IL-12, IFNγ, IL-4, IL-6+TGFβ, IL-21. Collectively, signal 1 (antigen), signal 2 (costimulation) and signal 3 (inflammation) invoke signal transduction, leading to production of the T cell growth factor IL-2 and synthesis of its receptor, events leading to clonal expansion and eventually to clonal differentiation into effector T cells. If T cells do not receive all 3 signals or only interact with the DC very briefly, the T cells will either become anergic, die or will persist in an undifferentiated state with little-to-no effector functions. Note, that all 3 signals are dependent on

the proper sensing of PAMPs and activation of the innate immune cells. Thus, the innate immune system ultimately determines whether a T cell response will be triggered or not. The activation of T cells is balanced by internalization of TCR, expression inhibitory receptors such as CTLA4, TGF-β and activation induced cell death (AICD) to ensure activated T cells to not grow out of control.

Bystander

innate immune cell

Reading: Text: Janeway’s “Immunobiology” by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 9 (9-10 through 9-31). Primary literature: Monks CR, Freiberg BA, Kupfer H, Sciaky N, Kupfer A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature. 1998 Sep 3; 395(6697):82-6.

T Cell Priming & Effector Cell Differentiation II

October 19

Instructor: J. Craft MAIN POINTS 1. Activated T cells leave the LN and go to sites of inflammation/ infection 2. TYPES of EFFECTOR and MEMORY T CELLS PRODUCED: CTLs, TH1, TH2, TH17, TFH and TREG 3. The different types of T cells are formed in response to particular innate immune cytokines that “specify” the appropriate type of T cell to form according to the pathogen that is infecting the host. These cytokines turn on key transcription factors of T cell differentiation that then induce the T cells to adopt particular traits to fight the infection. OVERVIEW Upon activation, T cells leave the APC and downregulate the LN homing molecules and depart into the peripheral tissues via the blood. The simultaneously upregulate chemokine receptors that detect inflammatory chemokines such as CCL5, CCL9, CCL10 produced at the sites of infection to enable their recruitment to these sites. They also migrate to most peripheral tissues including liver, lung, and gut. These activated T cells divide at an amazingly fast rate (every 4-6 hrs) and they upregulate telomerase to permit the cells to divide a massive number of times (~15-20 times within a 5-7 days). Many other changes occur in the activated T cells as they begin to express new effector molecules, adhesion receptors and many other genes. CD8 T cells recognize antigen presented on MHC class I (a ubiquitously expressed molecule) and differentiate into cytotoxic T lymphocytes (CTLs) that can kill infected cells upon direct contact by delivering cytotoxic granules or Fas ligand. When CD4+ T cells recognize antigen they can differentiate into a variety of different types of cells that perform specific functions depending on the nature of the infection or stimulus at hand: (i) Viral and intracellular bacterial infections induce the formation CTLs and TH1 CD4 T cells to enable direct killing of infected cells and intracellular pathogens. CTLs secrete the antiviral cytokine IFNγ and can kill on contact any cell in the body that harbors a cytosolic pathogen. CD8 kill cells via the expression cytolytic molecules called perforins and granzymes. Th1 CD4 T cells are also induced during these types of infections and secrete IFNγ, which activates Mφ to become highly anti-microbial. TH1 cells also help CD8 T cell activation and activate B cells to produce IgG2a and IgG2b. Both CTLs and TH1 cells express Fas ligand and can kill cells expessing the death receptor, Fas. (ii) Infection of helminths (worms) and certain parasites and extracellular bacteria induce a TH2 CD4 T cell response. A Th2 response is mainly aimed at destroying large, extracellular pathogens and thus, Th2 cells primarily produce IL-4, IL-5 an IL-13 to activate eosinophils, basophils, mast cells and macrophages to attack and phagocytose the large foreign bodies. In addition, these cytokines cause activated B cells to produce IgE. (iii) Some types of extracellular bacterial infections can also induce TH17 responses, which via production of IL-17 and IL-22 lead to the recruitment of neutrophils to eliminate the bacterium. (iv) Other types of T cells that need to be discussed are the formation of follicular helper T cells (TFH) which migrate into the B cell follicle to help B cells divide, class switch and mutate. Most regulatory T cells (TREGS) form in the thymus and suppress the responses of any “escapee” CD8 or CD4 T cells in the periphery. But during some infections, TREGS can also be induced to balance the activation of T cells to reduce tissue destruction and immunopathology.

The differentiation of these different lineages of T cells is determined by key transcription factors (TFs) that are induced by the appropriate types of inflammatory cytokines. This is how signal 3 (inflammation) controls the type of T cells produced to ensure that the right type of T cell is produced for the right type of infection. These major TFs are shown in the figure.

Figure from Anton M. Jetten. Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism. Nuclear Receptor Signaling (2009) 7, e003.

IL-2

IFN-

TNF

IL-4

IL-5

IL-13

IL-10

TGF- Anti-Viral

Anti-Bacterial

(intracellular)

Activate M

Parasite Infections

Allergy (IgE)

Reading: Janeway’s “Immunobiology” by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 9 (9-10 through 9-31). Primary literature: Szabo SJ, Kim ST, Costa GL, Zhang X, Fathman CG and Glimcher LH. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell. 2000 Mar 17; 100(6):655-69.

Effector Molecules – Function & Signaling

October 22

Instructor: J. Craft MAIN POINTS 1. The effector mechanisms used to clear an infection depend on the infectious agent. Pathogens have devised many ways to evade the attack of a T cell. 2. The molecules are precisely delivered to target cells and only upon TCR recognition (ON-OFF-ON) OVERVIEW As T cells differentiate into effector cells they become armed with the appropriate weapons that aide in pathogen eradication. Through millions of years of evolution, our T cells have become quite adept at producing the molecules that the pathogens we encounter in our environments are most vulnerable too. Likewise, through their own evolutionary process, the pathogens have devised numerous ways to circumvent or avoid these attacks. Sometimes they even use proteins similar to our own as decoys to their advantage. In general, T cells produce 3 main category of molecules: 1. Cytokines-

a. Interleukins- T and B cell factors that act as growth factors (IL-2) or modulate other lymphocytes (IL-4, IL-5, IL-21) such as B cells

b. Interferon gamma (IFNγ )- anti-viral factor, stimulator of Mφ. Type I interferons (IFN-α/β) produced by DCs, Mφ and infected cells are also anti-viral.

c. Cytokines can be membrane bound or secreted. They can work locally and for short periods of time. Secreted cytokines can act on the cell that produced them (autocrine) or on another cell (paracrine).

2. Chemokines and vasodilators- CCL3, 4, 5, TNFα

3. Cytotoxic Molecules- directly kill infected cells (or other antigen bearing cells such as APCs)

a. Perforin/ Granzymes are stored in granules in the T cells as preformed proteins that can be immediately expelled upon TCR triggering.

b. Fas Ligand (expressed by T cell)/ Fas (expressed on Target cell)

T cells use extreme stringency and precision to direct the effector molecules to the infected cells and avoid uninfected cells. By reorienting the MTOC to the immunological synapse, there is polarized delivery of the effector molecules to the target cells. The TCR is still required for release of effector molecules, however, now the T cells are interacting with infected cells that are not necessarily professional APCs (don’t receive costimulation). The T cells quickly shut off synthesis of these proteins in the absence of TCR engagement. Thus, these potential harmful proteins are only being made when there is antigen and infected cells present. This occurs in an ON-OFF-ON type of mode. Consequences of sloppiness would be excessive immunopathology and disease or even death of host.

Cytotoxic Functions of Effector Cells Granzyme/ Perforins: Enter cells and activate Caspase cascade that induces rapid apoptosis of target cell. Viruses and other pathogens have created clever ways of inhibiting effector molecule function to keep the infected cell alive and functioning and some of these will be discussed. Fas/Fas Ligand- activates Caspase cascade in target cell upon receiving FAS Ligand signal from effector cell.

Resolution of Immune Response: After pathogen clearance the macrophages “clean up” and engulf dead cells and debris. The effector T cells begin to die (death by neglect) and a few survive to become long live d memory T cells. Memory T cells can rapidly expand and exert effector functions to more rapidly combat secondary infection. Formation of these memory T cells are critical to long-term immunity and the ultimate goal of vaccination. Reading: Text: Janeway’s “Immunobiology” by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 9 (9-10 through 9-31) and Chapter 7 (7-19 through 7-22). Primary literature: Macchi P, Villa A, Giliani S, Sacco MG, Frattini A, Porta F, Ugazio AG, Johnston JA, Candotti F, O'Shea JJ, et al. Mutations of Jak-3 gene in patients with autosomal severe combined immune deficiency (SCID). Nature. 1995 Sep 7;377(6544):65-8.

Primary B Cell Immune Responses

October 29 Instructor: M.Shlomchik Overview: B cells recognize intact antigens (Ags). Most B cells are in spleen, lymph nodes, and blood. They encounter and respond to Ags that have reached these compartments. Upon binding Ag, and upon interaction with activated T cells, B cells undergo proliferation, differentiation and migration to constitute the primary B cell immune response. There are defined sequences of events and these occur in defined microanatomical locations within spleen or lymph node. B cell trafficking and splenic architecture B cells traffic through blood, exiting in the marginal sinus from where they migrate to the B cell follicle, where most splenic B cells reside. (Some B cells remain resident and nonrecirculating in the marginal zone, just outside the marginal sinus, not shown below). B cells in the follicle recirculate to blood occasionally.

central arteriole

1° B cell follicle Germinal center

marginal sinus T zone

(pe ria rter iolar

lymphoid sheath-

PALS)

Upon encounter with Ag, B cells stop migrating and arrest at the border of the T and B zone, where T cells and B cells interact and begin proliferating. This is accomplished in part by changes in chemokine receptor expression that control where B cells migrate. Expression of CXCR5, which allows B cells to migrate to follicles, is reduced. Expression of CCR7, which is the receptor for chemokines secreted in the T cell zone, is increased, allowing B cells to arrest near the T cell zone. In addition, a chemokine receptor (for the newly identified ligands termed “oxy-sterols”), called EBI-2 is expressed, enabling the B cells to migrate outward from the center of the follicle.

Molecular nature of the T-B interaction There is a complicated interplay between T and B cell, involving T cell recognition of processed peptide Ag fragments that are present on the B cell’s MHC Class II molecules, as well as a variety of other molecular pairs that interact. These pairs include CD40L and CD40, CD80/86 molecules and CD28, and ICOS (inducible costimulator) and ICOS-ligand. In addition, cytokines are released by activated T cells that have effects on both B and T cells. These include IL2, IL4, IL5, IL6, IL21 and IFN-γ. B cells can also secrete cytokines that may affect themselves and T cells; the roles of B cell cytokine secretion are not well understood yet. The details of this T-B interaction have been covered elsewhere in the course as they are typical of any T cell-Antigen Presenting Cell (APC) interaction, such as a T-dendritic cell interaction. B cells can be activated by certain antigens even without T cell help Although typically, the entire B cell activation and differentiation program is highly dependent on T cell signals, one exception is certain highly repetitive antigenic surfaces or antigens that also contain B cell mitogens; these are said to be relatively “T-cell independent”. These so-called T-independent responses can be divided into two types: TI-1 and TI-2. TI-1 responses are elicited by molecules with inherent mitogenic ability for B cells, such as bacterial lipopolysaccharide or nucleic acids that bind TLR7, 8 or 9. TI-2 responses on the other hand are elicited by highly repetitive antigenic structures such as bacterial polysaccharide cell wall or certain viral coat structures. It is thought that the very high amount of cross-linking that these latter antigens provide to the BCR are sufficient to bypass the need for T cell derived signals. Typically, these TI responses undergo limited isotype switching and they do not form germinal center and memory B cells, processes that will be described below. T-dependent B cell activation and differentiation at the T-B border Proliferation is the initial consequence of Ag and T cell encounter. Small clones of B cells, each descended from an initial Ag-specific B cell accumulate at the border of the T-B zones. After this initial encounter, they migrate to the border of the T cell zone and the red pulp, where they continue to proliferate. Differentiation and migration are the next steps. Although most B cells at this stage will continue to express IgM isotype, some cells will begin the process of isotype switch, and may express IgG. (Isotype switch will be covered in detail in the next lecture). Some B cells, responding to signals from T cells that include cytokines such as IL4, IL5, IL6, and IL21, become antibody-forming or “plasmablasts”, which continue to divide and differentiate at the same time. These cells and increase the production of Ig secreted mRNA by 100 to 1000-fold. They will also lose expression of many of the surface molecules that characterize B lineage cells—they become mini-factories for Ab secretion. Most of these cells live only a few days, but some will migrate to the red pulp areas of the spleen, where they stop dividing and can live for several weeks. Other B cells along with some T cells will migrate away from the T zone-red pulp border—at this point downregulating expression of EBI-2—into the center of the B cell follicles. Here they will continue proliferating and will form the “germinal center” (GC). The transcriptional repressor factor Bcl6 is essential for the formation of GCs. Bcl6 is turned on in nascent GC B cells, where it controls the expression of a large number of genes, causing the B cells to adapt a unique “GC” phenotype and suppressing their ability to become plasma cells. Interestingly, the same factor is required for T cells to enter the GC, and these too take on a unique identity as a “T follicular helper” cell. Recent data indicates that this transcription factor is expressed very early on by the cells that will go on to become GC B cells and T follicular helper cells and that this factor controls expression of genes essential for GC B and T cell function. Germinal Center Overview Architecture: “Germinal” means “growing”. Germinal centers (GC) are comprised of highly proliferative B cells (mainly) along with some T cells. They evolve in the “B cell zones” or “follicles” of lymphoid organs such as spleen and lymph node. In the follicles are other cells called follicular dendritic cells which are unusual, nonphagocytic dendritiform cells with uncertain but intriguing function (see below). As the initial proliferating cells generate more progeny, the germinal center grows and tends to push out surrounding non-proliferating B cells. In the GC three areas can thus be discerned: a “dark zone” (DZ) with many proliferating cells and which is located closer to the T cell zone, a “light zone” (LZ) which is less densely packed and contains many FDCs and most of the T cells, and a surrounding marginal zone that includes the surrounding B cells.

Clonal Expansion Initiation: Initial seeding of the GC comes from cells that were originally activated at the T-B zone interface. Although GC are ultimately comprised of the descendents of only a few cells, the initial seeding may be by 50 to 100 cells, since most are unsuccessful in competition and selection. Proliferation: B cells divide as quickly as every 6 to 8 hrs. in a GC. Initial proliferation may occur in the “dark zone” while cells in the “light zone” proliferate but at a somewhat lower rate. Exactly how cells migrate between dark and light zone is not entirely clear but most likely cells recirculate within their respective zones, with a net flow of cells from the DZ to the LZ, where most differentiation takes place. In mouse spleen GC responses begin about 5-6 days after immunization, peak at day 10 to 14 and are mainly over by 4-8 weeks. Events in the GC: Four important events occur in the GC: a) clonal expansion and differentiation; b) somatic mutation, in which point mutations are introduced into the V region; c) cellular selection; and d) isotype switching.

The last three will be covered in the next lecture. Figure 2: Diagram of the B cell immune response, with emphasis on the GC reaction. Fate of cells in the GC: Cells that survive in the GC can have at least three fates: memory cell differentiation (which means the cell becomes long-lived, stops dividing and probably leaves the GC); plasma cell differentiation (which means the cell begins making and secreting more Ig, stops dividing, and leaves the GC probably for the bone marrow); or apoptosis (which is probably the result of failure to get sIg or T cell signals, perhaps due to deleterious somatic mutations or just low affinity). These will be dealt with in the upcoming lecture on memory. Reading: Text: "Janeway’s Immunobiology", by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 10 pp. 387 – 407.

*ant igen

plasma cell

differentiation

light zone

dark zone

memory B cell

differentiation memory B cell

maintenance

mutation, isotype switch

and differentiation

migration to

follicle

Primary B cellfollicle

Follicular

Dendritic

cell (FDC)

T-B zone interface(outer PALS)

Late GerminalCenter

CD4+

T cell

B cell

Primary literature: Kerfoot, S.M., G. Yaari, J.R. Patel, K.L. Johnson, D.G. Gonzalez, S.H. Kleinstein, and A.M. Haberman. 2011. Germinal center B cell and T follicular helper cell development initiates in the interfollicular zone. Immunity. 34:947-60. Okada, T., M. Miller, I. Parker, M. Krummel, M. Neighbors, S. Hartle, A. O'Garra, M. Cahalan, and J. Cyster. Antigen engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLOS Biology 3 (6): e150, 2005. Jacob, J., G. Kelsoe, K. Rajewsky and U. Weiss. Intraclonal generation of antibody mutants in germinal centres. Nature 354: 389-392,1991.

Mechanisms of Somatic Hypermutation and Isotype Switch and Their Consequences

October 31

Instructor: M. Shlomchik Introduction: B cells are unique in the capability to modify their antigen receptors in response to activating signals. As already discussed, the V region can undergo point mutations (and in some animals gene conversion) in the germinal center. Activated B cells can also rearrange the DNA in the IgH constant region leading to a switch in the isotype expressed. In this lecture, we will cover how both mutation and switch are controlled and induced and the molecular mechanisms of both of these processes. We will also briefly cover the biological impact of somatic hypermutation on the germinal center reaction. The biological impact of isotype switch will be covered in the next lecture on humoral immune responses. Somatic Hypermutation Control of mutation: Somatic mutation is not a constitutive process in B cells; it is turned on only after a period of B cell activation. Somatic hypermutation takes place in germinal centers and until recently this was thought to be the exclusive site of mutation. However, recent discoveries indicate that mutation can also occur in chronic immune responses that occur in other areas of lymphoid and even non-lymphoid tissue. This raises the question of what signals turn the mutation process on in B cells. The answers are not entirely clear, but CD40 signals can do this, as can other signals that may include simultaneous BCR, TLR and cytokine signaling. It is likely that signals through the BCR are absolutely required, as nonspecific activators do not appear to induce mutation. Mechanism: General features of SHM: Somatic mutations are introduced into V regions of dividing B cells at the rate of approximately 0.5 mutations per cell per generation. They are mainly single point mutations, with a predilection for transitions (purine-> purine and pyrimidine -> pyrimidine). This rate is approximately 107 times higher than the background mutation rate in normal mitosis. Though we won’t discuss it much, in some species like chickens and rabbits, there is less somatic point mutation and instead a process of gene conversion in which small regions of one gene are replaced by the sequences of a nearby homologous but different gene via a process of unequal recombination. Location and targeting: Mutations occur only in a limited area from a few hundred bp downstream of the promoter region extending for about 1kb, but never reaching the downstream constant region. How mutation is so specifically targeted is not totally clear, but sequences in or near the immunoglobulin transcriptional enhancers are important. Transcription is also required for mutation to take place, although experiments have shown that any promoter that controls transcription can support mutation, not just the V region promoter. The final appearance of mutation is likely a balance between the introduction of mutations and the efficiency of their repair, as correction of mutations is an ongoing process in all cells. Indeed, recent data indicates that SHM occurs in many genes in a B cell, albeit at substantially lower rates than in the V region (10-100 fold lower). In some cases, these mutations are repaired and their existence was only revealed when repair pathways were genetically disabled. The basis for relative targeting of the SHM machinery is still not clear, nor is it clear why some genes have efficient mutation repair and others not. Nonetheless, mutations outside of V regions can promote cellular transformation and indeed have been implicated in B cell lymphomas. Enzymology of hypermutation: Several years ago, a gene was discovered that is absolutely required for somatic hypermutation to occur. This gene was called Activation Induced Deaminase (AID), so named because the gene had strong homology to a family of enzymes that are know to modify RNA (RNA editing enzymes) by deamination. The discovery led to two theories of how AID might work. In the first theory, AID modifies an RNA so that it now

encodes an enzyme that can carry out or otherwise induce somatic hypermutation—this is the “indirect” model. The second theory, which is now widely accepted, is that AID itself is a DNA-modifying enzyme that directly acts as a mutator. Evidence in favor of this model includes that AID in fact does modify DNA in vitro and also in E. coli. Moreover, it seems to be a cytidine deaminase that converts C to U, which in turn helps to explain the higher frequency of transition type mutations (particularly at G=C base pairs). The initial lesions created by AID in the direct model are repaired in a variety of ways by DNA repair enzymes and polymerases, leading in turn to a variety of mutations. These include uracil deglycosylase, which removes U base pairs as a mechanism of repairing lesions that have had a base removed. Mutations at adjacent A//T base pairs, which are not direct targets of AID, seem to occur via error-prone repair involving especially DNA poymerase eta. Other repair enzymes like XRCC2 promote gene recombination (leading to gene conversion). Indeed, AID is critical for gene conversion. Perhaps most remarkably, it has been known since the discovery of AID that it is required also for isotype switching, leaving the problem of how one gene can be involved in such seemingly disparate molecular processes. A summary of how AID might work was published by Neuberger and colleagues:

Figure 1: DNA deamination model of immunoglobulin gene diversification. For details, see text. KU70/80 are non- homologous end-joining proteins. AP endonuclease, apyrimidic endonuclease; DNA-PK, DNA-dependent protein kinase; dRPase, deoxyribophosphodiesterase; V, variable. (From Petersen-Mahrt et al., Nature 418, 99 - 104 (04 July 2002))

Isotype switch Control of switching: Cells initially express IgM, but “switch” to expressing non-IgM (e.g. IgG or IgA) isotypes. Isotype switch is driven by cytokines and T cells, along with CD40 signals. Specific cytokines tend to promote specific switches. In the mouse: Cytokine Type of switch

IL4, IL21 IgG1 and IgE

IFN-γ, IFN-α IgG2a

TGF-β IgA

These cytokines work in part by inducing transcription from promoters that lie just upstream of each of the Ig C region genes, just 5’ to the “switch region” (see below). This transcription is essential, as mutant switch loci that lack the promoter region do not undergo switching. Transcription may “open” the locus to recombination enzymes and for alignment with other switch regions. It may also play a mechanistic role in the actual switch rearrangement, as will be discussed: Molecular mechanism: DNA rearrangement leads to the deletion of intervening C regions and the juxtaposition of a new C region near the VDJ join (fig. 2). Upstream (5’) of every constant region lies a stretch of DNA known as the “switch region”. This DNA contains many repeats of a few short sequences, in particular G=C-rich sequences such as GAGCT and GGGGGT. Via unknown mechanisms (but possibly in part because they are preferred targets of AID, see below), these short sequences promote nonhomologous recombination between switch regions of different isotypes. For example, the switch region upstream of IgM might recombine with the switch region of IgG3. The intervening DNA is deleted. Now the cell will express IgG3. Both transcription (i.e. accessibility) of the downstream switch and C region, as well as a specific (but as yet uncharacterized) enzymatic machinery are required for the switch DNA rearrangement to take place. AID is thought to play a role by targeting DNA repair mechanisms to the switch sites. This is even more difficult to explain at this point than somatic hypermutation. However, as seen in the figure above, it is thought that AID targets the DNA in switch regions that is particularly G=C rich (and thus has many cytidine residues for deamination). With deglycosylation of the U bases, a lesion is created that could lead to a single or double stranded break in the DNA, thus setting the stage for switching. The pseudohomology between the repeat sequences shared by all of the switch regions may help in aligning the two regions to be joined, but this is not totally clear. As with somatic hypermutation, switching also requires transcription through the region to be switched. Recent evidence suggests that transcription serves to create regions of single stranded DNA, possibly with unique secondary structure that are preferred targets for AID. (A similar mechanism may apply in targeting somatic hypermutations, which are known to be preferentially introduced into certain sequence motifs.)

Figure 2: Isotype switching involves recombination between specific signals. Repetitive DNA sequences that guide isotype switching are found upstream of each of the immunoglobulin constant-region genes, with the exception of the δ gene. Switching occurs by recombination between these repetitive sequences, or switch signals, with deletion of the intervening DNA. The initial switching event takes place from the µ switch region; switching to other isotypes can take place subsequently from the recombinant switch region formed after µ-γ switching. S, switch region. (reproduced from Janeway text, fig. 3.26) Selection The overall result of somatic hypermutation is that among clonal descendents, nearly every cell is different from every other cell. Clones evolve, and indeed the GC can be thought of as a miniature evolution system that occurs in real time. All mutations can be divided into replacement (R) meaning that the base substitution leads to an amino acid substitution in the resulting protein—or silent (S), meaning that no amino acid substitution occurs. Only R mutations will influence the fate of the cell. Mutations can be classified according to where in the V region coding sequence they occur: complementarity determining region or CDR (i.e. part of the protein that contacts antigen) and framework region or FR (part that generally does not contact antigen but maintains the overall immunoglobulin structure). How mutations affect the cell’s fate: Many mutations (perhaps about 1/2) in FR might destroy the structure of the overall molecule. These mutations will

be selected against (“negative selection”) and cells that have them undergo apoptosis. Some mutations in the CDR might also destroy Ag-binding and would be selected against. But some are likely to improve binding to the Ag, increasing the affinity of the B cell. Cells with these mutations will be positively selected and preserved in the GC. Negative and positive selection can be inferred from the ratio of replacement type (R) to silent type (S) mutations (R/S ratio). In the absence of any selection, this ratio is about 3, which is determined by the codon translation table in that most mutations do cause an amino acid substitution. Only such R mutations change the cell’s phenotype and thus can be selected. So if there is negative selection—for example in a FR—the R/S ratio will be lower, since some R mutations will be eliminated. Conversely, if there is positive selection, some R mutations will be enriched and the ratio will be higher. Such skewing of R/S ratios are actually seen in real Abs. The average R/S ratio in FR regions is about 1.5, meaning that about 1/2 of all R mutations are selected against. Negative selection is thus a frequent event, and indeed many B cells die in the GC. The consequence of this is that clonal expansion is not at the ideal, exponential rate, but follows a much slower, more linear rate. How does selection occur in the GC?: Presumably, the affinity of a BCR determines the strength or frequency of positive signals a cell gets: higher affinity cells will get more/better signals. This can result in either more proliferation or less death or both among the higher affinity cells. Alternatively, higher affinity cells may be better able to capture antigen and present it to T follicular helper cells, and thereby gain survival or proliferative signals from the T cells. Reading: Text: "Janeway’s Immunobiology", by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 5 pp. 173-186. Reviews: Di Noia, J.M., and M.S. Neuberger. 2007. Molecular mechanisms of antibody somatic hypermutation. Annu Rev Biochem 76:1-22. Liu, M. and D.G. Schatz. 2009. Balancing AID and DNA repair during somatic hypermutation. Trends Immunol. 30:173-81. Primary literature: Di Noia, J., and M.S. Neuberger. 2002. Altering the pathway of immunoglobulin hypermutation by inhibiting uracil- DNA glycosylase. Nature 419:43-48. Hershberg, U., M. Uduman, M.J. Shlomchik, and S.H. Kleinstein. 2008. Improved methods for detecting selection by mutation analysis of Ig V region sequences. Int Immunol. 20:683-94.

Humoral Immune Responses

November 2 Instructor: M. Shlomchik Overview: Humoral responses are important to protect the extracellular spaces from pathogens. They prevent the spread of established infections and, in immune animals, prevent the establishment of new infections. Since antibody titers are long-lasting in immune animals (i.e. those that have already recovered from infection) antibodies protect mucosal surfaces and the blood from new infections. Antibodies are secreted by plasma cells, which are the terminally differentiated products of activated B cells. During the course of an immune response, and under the direction of signals from T cells, B cells rearrange their DNA to allow secretion of isotypes other than IgM. These other isotypes have unique properties and effector functions. Effector functions of Abs are dictated by the Fc portion of the Ab, which in turn controls whether the Ab can bind to certain receptors for the Fc portion (FcR’s), activate or “fix” complement, and also to which compartments the Ab molecule itself gains access (for example, IgM is too large to diffuse into most tissues). The effector functions of Abs are in turn linked to the cells that express FcR’s, which are diverse and mediate a variety of effects upon FcR binding of Abs. Most of these effects are mediated by cells that can be classified as part of the innate immune system, such as NK cells or macrophages, Similarly, other effector functions of Abs depend on the soluble complement system, another element that also functions in the innate immune system (see earlier lectures by Dr. Medzhitov). Thus the effects of Abs provide yet another link between the adaptive and innate immune system. The effector functions of Abs Antibodies carry out their effects via: a) neutralization, b) opsonization and c) activation of complement. To enter cells, viruses and intracellular bacteria bind to specific molecules on the target cell surface. Antibodies that bind to the pathogen can prevent this and are said to neutralize the pathogen. Neutralization by antibodies is also important in preventing bacterial toxins from entering cells. Opsonization is the process of coating the surface of a pathogen with Ab or complement fragments (i.e. C3b), which in turn enhances phagocytosis by cells, such as macrophages, that bear receptors for the Fc part of antibodies and for complement fragments. Complement activation mediated by antibody binding to the surface of a pathogen can lead to both the covalent binding of fragments of C3 and C4 to the pathogen— in a form of opsonization—but also to the activation of the terminal pathways of complement, which can result in lysis, particularly of eukaryotic pathogens.

Ab isotypes have different properties and effector functions Antibodies of different isotypes are adapted to function in different compartments of the body. Via isotype switching, the progeny of a single B cell can produce antibodies, each with the same combining site, yet expressing a variety of different Ig isotypes. IgM: The first antibodies to be produced in a humoral immune response are always IgM. These early IgM antibodies are produced before B cells have undergone somatic hypermutation and therefore tend to be of low affinity. IgM molecules, however, form pentamers whose 10 antigen-binding sites can bind simultaneously to multivalent antigens such as bacterial capsular polysaccharides. This compensates for the relatively low affinity of the IgM monomers. However, as a result of the large size of the pentamers, IgM is mainly found in the blood and, to a lesser extent, the lymph. The pentameric structure of IgM makes it especially effective in activating the complement system. IgG, IgA, and IgE: These Ab molecules are smaller in size and diffuse easily out of the blood into the tissues. Although IgA can form dimers, IgG and IgE are always monomeric. The affinity of the individual antigen-binding sites for their antigen is therefore critical for the effectiveness of these antibodies, and most of the B cells expressing these isotypes have been selected for increased affinity of antigen-binding in germinal centers. IgG is the principal isotype in the blood and extracellular fluid, whereas IgA is the principal isotype in secretions, the most important being those of the mucus epithelium of the intestinal and respiratory tracts. Whereas IgG efficiently opsonizes pathogens for engulfment by phagocytes and activates the complement system, IgA is a less potent opsonin and a weak activator of complement. This distinction is not surprising, as IgG operates mainly in the body tissues, where accessory cells and molecules are available, whereas IgA operates mainly on epithelial surfaces where complement and phagocytes are not normally present, and therefore functions chiefly as a neutralizing antibody. IgE antibody is present only at very low levels in blood or extracellular fluid, but is bound avidly by high affinity FcR’s specific for IgE (Fc R) on mast cells. Mast cells are found just beneath the skin and mucosa, and along blood vessels in connective tissue. When Ag is bound by this mast-cell associated IgE, it triggers mast cells to release powerful chemical mediators that induce reactions, such as coughing, sneezing, and vomiting, that can expel infectious agents, as will be discussed below. The distribution and main functions of antibodies of the different isotypes are summarized here (from Janeway text):

It is critical that soluble Ab molecules, which circulate at mg/ml concentrations, do not activate the complement cascade, as this would lead to uncontrolled inflammation. Instead, the ability of Abs to activate complement is controlled by a requirement for Ab to be bound to a surface. In the case of IgG, there must be several molecules in proximity, because C1q must bind at least two IgG Fc regions in order to be activated and start the cascade. A single molecule IgM, which is already a pentamer, can activate C1q, but only when that IgM is bound to a surface and undergoes a conformational change allowing C1q activation. As described earlier in the course, complement activation leads to binding of C3b and C4b to nearby protein structures, resulting in a form of opsonization. It also leads to the release of soluble inflammatory mediators. These two outcomes of complement activation lead to more efficient removal of immune-complex-bound pathogens, via complement receptors (CRs) that are on red blood cells as well as on a variety of immune system cells, including macrophages and dendritic cells. Complement coated pathogens are also especially potent at stimulating B cells, which also have CRs, and are much more sensitive to BCR crosslinking when the CRs are also bound. FcR’s mediate the destruction of antibody coated pathogens and result in activation of macrophages, NK cells and dendritic cells Although high affinity Abs can neutralize, they do not by themselves remove pathogens from the body, nor are they capable of destroying many kinds of pathogens. Complement activation is one adjunct for antibody-mediated protection. Another is a series of receptors for the Fc portion of Abs (FcRs). These are on the surface of many hematopoietic cell types, including: macrophages, dendritic cells, neutrophils, NK cells, B cells; and mast cells, eosinophils and basophils which all have receptors for IgE (see above). A key feature of FcRs for IgG is that they generally bind multivalent IgG that is part of an immune complex much better than they bind free IgG; this may be

due to affinity as well as steric reasons. Thus only IgG that is complexed to Ags will effectively activate these receptors. In the case of the phagocytic cell types (macrophages, dendritic cells and neutrophils), binding of FcRs activates the cells (for example to undergo oxidative burst) and in particular to enhance the phagocytosis of the bound IgG-coated pathogen or molecule. Phagocytosis in turn leads to destruction of the pathogen as well as enhanced presentation of its antigenic peptides to T cells. Cell types like NK cells are not phagocytic but instead are triggered to release stored mediators, like cytokines; perforin and granzyme B (that mediate cell lysis); and inflammatory agents, when their FcR’s are bound. The cells are also activated to undergo new protein synthesis again leading to subsequent secretion of cytokines. This process is referred to as antibody-dependent cell-mediated cytotoxicity (ADCC). A summary of the properties of the different types of FcRs is given here (FYI, from Janeway text; don’t memorize it!):

Reading Text: "Janeway’s Immunobiology", by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 10 pp. 408 - 424. Primary literature: Nimmerjahn, F., and J. Ravetch. Divergent Immunoglobulin G subclass activity through selective Fc receptor binding. Science 310: 1510 -1512, 2005.

B and T Cell Memory

November 5 Instructor: M. Shlomchik Introduction It has been known for a long time that people who recover from a particular infection often are not susceptible to reinfection by the same infectious agent. Jenner was perhaps the first to intentionally take advantage of this phenomenon to protect naive individuals. He invented vaccination, in which cowpox infection (a recoverable disease) was deliberately induced and later protected against fatal smallpox. This form of specific enhanced immunity to reinfection occurs because of immunologic memory. Memory in the immune system is defined functionally. A system shows memory when, compared to the first response, a second or later response is characteristically different: usually stronger, faster and qualitatively different. Memory is a property of the immune system as a whole, but since the system is composed of cells, one expects a cellular basis for memory. This could work in a number of (nonexclusive) ways: clonal expansion of Ag-specific cells, differentiation to a more "potent" type of cell, or increased longevity of a cell and/or its clonal descendants. B cell memory responses are qualitatively different from primary responses in three main ways: the antibody formed is IgG or IgA (not IgM), the affinity for the antigen is higher, and the V genes of cells participating in a secondary response are somatically mutated. Similarly, memory T cell responses occur faster, with higher amounts of cytokine production and secretion of “polarized” cytokines such as IL-4 and IFN-g without delay. Memory cell development The precursors of memory B cells form mainly in the germinal center (GC). Of the many cells which initially respond to antigen and enter the GC, only a small subset are retained as memory cells. This makes sense, since if all responding cells were retained, an animal would quickly fill up with memory cells and have no "room" left for new cells to respond to new antigens. Memory B cells are reactivated upon reexposure to the Ag. This is termed the secondary immune response. Secondary response Abs have higher affinity than primary response Abs, a phenomenon termed "affinity maturation". Presumably, higher affinity cells (possibly created through beneficial somatic mutations) are selected in some way to become memory cells and this underlies the affinity maturation. These cells could be better competitors for antigen or might receive a different or stronger signal to differentiate because of their higher affinity. We do not know which is true, nor do we know whether all high affinity cells become memory cells. However, we do believe that the GC reaction is necessary for the formation of high affinity memory cells, and treatments that block the GC reactions, such as blocking the CD40-CD40L interaction by infusing anti-CD40L Abs into mice, will block memory formation. The requirements for the formation of a memory T cell are less clear; they probably do not need a GC reaction, but instead an interaction with a professional APC such as a dendritic cell. Memory T cells seem to need a period in which there is little Ag exposure in order to form memory cells—too much persistent Ag or too much inflammation drives T cell differentiation towards an effector phenotype rather than a memory phenotype. How to track down a memory cell: Insight has been gained into the identity of memory cells by the study of cells specific for a particular Ag after the animal has been immunized with that Ag. These cells are too infrequent to observe in unimmunized animals but can be seen at low frequency in immunized animals due to clonal expansion following Ag exposure. For example: sequencing of V genes of such expanded cells isolated during a late primary or secondary immune response led to the discovery that these are somatically mutated. Similar studies showed that these cells are also isotype switched. B cells that had already responded to Ag (“secondary cells”) were also characterized by the expression or lack of certain surface molecules such as CD44 (elevated) and J11D (reduced). This approach is a descriptive one and doesn't really prove that these "secondary cells" are really the precursors to long-lived memory cells. To prove this, one must isolate putative memory precursors and do a functional assay. This has been easiest to do for the cell surface markers. For example, the IgG positive, IgM negative cells contain memory precursors though we don’t know that all IgG positive cells are memory precursors. Another approach is to use a property associated with memory cells as a surrogate marker for those cells. For example: memory cells carry

mutated Ig V regions. If we could know where mutations occur, this might be a site where memory cells are formed. This reasoning was one way in which the GC was implicated as the site of memory B cell development. Property Naive Activated (GC) Effector (plasma

cell) Memory (?)

IgM/sIgD (frequency of + cells) +++ +/- +/- IgG (frequency of + cells) - ++ +++ Secrete Ig +/- + +++ +/- Lifespan (without division) 6-8 wk Hours Weeks to months Indefinite?

Surface Markers

MHC Class II + ++ +/- + CD80 +/- +/- - ++ (subset) PD-L2 (mouse only) - + - ++ (subset) CD27 (human only) - +/- +++ ++ Bcl-2 (survival protein) + +/- ++ Fas (death receptor) + ++ +/- + CXCR4 (chemokine receptor) + + ++ ? CXCR5 ++ + +/- ++ Table 1. Surface marker (phenotype) comparison among naïve, activated, effector and memory B cells. Are Memory cells long-lived in the absence of continuous antigen stimulation? The answer to this question would seem to be an automatic "Yes". But it needn't be so for two reasons: first, the cells themselves may not be long-lived even though the clone from which they descend is. Memory cells thus may divide every so often yielding two memory daughters. Second, although it seems likely that antigen goes away after a period of time, there is good evidence that the body can store it in special cells (follicular dendritic cells) for long periods. In addition, replicating antigens (especially certain viruses) may remain at low levels which are inconsequential for the host but which maintain memory. Memory B cells and T cells both probably exist for long times and at stable overall levels without undergoing cell division. However, it is possible to observe memory cells dividing infrequently. The signals that stimulate this division and the consequences are unclear. It appears that cytokines are important for maintaining T cells, principally IL-15 and IL-7; without these, memory CD8 responses appear to decay. In fact, developing memory T cells can be distinguished by their expression of the IL-7 receptor, while effector cells that are destined to “burn out” do not upregulate this cytokine receptor.

The role of cytokines in maintaining B memory cells has yet to be elucidated. However, it is known that the TNF-family cytokine called BAFF (B Cell Activator of the TNF Family), which is required to maintain the survival of naïve B cells, is not needed to maintain memory B cells.

Are Memory cells heterogeneous? Memory cells have to carry out multiple functions including providing a variety of effector functions upon secondary challenge (see below) as well as to renew the memory cell compartment. If all of the memory cells became terminally differentiated effectors (i.e. plasma cells or effector T cells) upon Ag reexposure, this would actually deplete memory and impair any subsequent responses. These diverse requirements are probably dealt with by heterogeneity among memory cells—not all are created equally. Among memory B cells, the discovery of surface proteins that mark memory cells revealed, via multiparameter flow cytometry, that not all memory cells expressed all markers. For example, the diagram below shows that PD-L2 and CD80 expression defines distinct subsets of murine memory B cells. Emerging data indicates that these different memory B cell subsets carry out different functions in a secondary immune response. In the T cell compartment, heterogeneity is seen in terms of expression of surface proteins that control migration. Memory T cells are also heterogeneous in their ability to make various cytokines upon restimulation.

IgG1 memory cells specific for the hapten NP were generated in a transfer and immunization model, then identified by flow cytometry, which was also used to measure expression of CD80 and PD-L2. Property Naive Activated/ Effector Memory TCR expression +++ +/- +++ Cytokine secretion - +++ +/- Ag recognition for maintenance/activation

Self-MHC MHC+peptide No MHC

Cytokine polarization Not Evolving Definite Lifespan (without division) 6-8 wk Hours to days Indefinite? Surface Markers LFA-3/CD58 + ++ ++ CD44 + +++ ++ L-selectin (CD62L) ++ ++ +/-

(heterogeneous) Bcl-2 (survival protein) + +/- ++ Fas (death receptor) + ++ + IL-7 receptor ++ - +++ CD45RA ++ ++ + CD45RO + ++ +++ CD69 - ++ - CXCR5 +/- ++ (subset) ? CCR7 ++ ++ - or + (two

subsets) Table 2. Surface marker (phenotype) comparison among naïve, activated, effector and memory T cells. Secondary immune responses Much less is known about the nature of the secondary immune response than the primary. We do know, as mentioned, that Ab responses are more prompt, are of higher affinity, and are isotype switched and that T cell responses feature prompt induction of cytokines and/or cytotoxicity. But the cellular and microanatomic basis of this are less clear. Secondary immune responses seem to be highly variable, depending on the timing, Ag dose and nature of the Ag. There are several key differences with primary responses: a) higher B or T cell precursor frequency; b) more sensitive “memory” differentiated B or T cells; c) non-limiting Ag-specific T cell help; d) preexisting Ab. The degree to which all of these are present also influences the nature of the secondary response. In general, secondary responses are characterized by large bursts of extra-follicular B and T cell proliferation, early differentiation into plasma cells at these sites, and a paradoxically attenuated GC response. It is not even clear if, generally, the B cells that seed secondary response GC’s are memory cells or “new” primary B cells; in some circumstances it does appear that memory B cells can undergo a second round of somatic mutation, but there is

clearly early clonal expansion without further mutation as well. In any case, the consequence is a vigorous and high affinity IgG and effector T cell immune response that functions well to protect the host against reinfection. Reading: Text: Janeway, C., Travers, P., Walport, M. and Shlomchik, M. Immunobiology: The immune system in health and disease, 6th ed. Garland Science, Ltd., 2005. Sections 10:22-10:26. Reviews: Kalia, V., Sarkar, S., Gourley, T.S., Rouse, B.T., and Ahmed, R. (2006). Differentiation of memory B and T cells. Current opinion in immunology 18, 255-264. Shlomchik, M.J., and Weisel, F. (2012). Germinal center selection and the development of memory B and plasma cells. Immunol. Rev. 247, 52-63. Articles: Maruyama, M., K.P. Lam, and K. Rajewsky. 2000. Memory B-cell persistence is independent of persisting immunizing antigen. Nature. 407:636-642. Seddon, B., P. Tomlinson, and R. Zamoyska. 2003. Interleukin 7 and T cell receptor signals regulate homeostasis of CD4 memory cells. Nat Immunol. 4:680-686. Scholz, J.L., Crowley, J.E., Tomayko, M.M., Steinel, N., O'Neill, P.J., Quinn, W.J., 3rd, Goenka, R., Miller, J.P., Cho, Y.H., Long, V., et al. (2008). BLyS inhibition eliminates primary B cells but leaves natural and acquired humoral immunity intact. Proc Natl Acad Sci U S A 105, 15517-15522.

Vaccination

November 7 Instructor: A. Iwasaki True co-evolution between pathogen and host can be found when a “carrier” state is produced, whereby the pathogen causes no undo harm to the host to maximize its replication and spread. When a pathogen jumps hosts, illness, disease and death can result. The ultimate goal of vaccination therefore is to prevent the viruses and bacteria that cause disease in humans and other animals from infecting the host. They do so by generating long-lived, protective memory T cells that provide cell-mediated protection and remove infected cells or pathogens, and memory B cells and long-lived plasma cells that provide humoral immunity. There are 3 major ways in which vaccines can work: (1) prophylatic— these are the most common form of vaccination and are preventive vaccines to protect the host prior to exposure to the pathogen (2) therapeutic—these are vaccines designed to treat individuals already infected with the pathogen (common for chronic viral infections). (3) herd immunity— this is where individuals in a population are protected from the pathogen without direct vaccination because the frequency of vaccinated people is so high that spread of infection and risk of exposure drops to such a significant degree that they are, in essence, protected. Many critical elements need to be encompassed by a “good” vaccine. It must be safe with minimal side effects and protective against the live pathogen when encountered naturally. It needs to provide sustained and long-lived immunity. For example, small pox and yellow fever vaccines have demonstrated protection for more than 50 years in humans. A protective vaccine needs to produce neutralizing antibody to prevent immediate infection and contain spread. Sometimes “sterilizing” immunity is met by some vaccines that have extremely potent antibody responses. Likewise protective T cells should also be induced, but surprisingly to date, very few vaccines have been formulated in a way that rely only on T cell responses. An ideal vaccine would also be low cost and biologically stable for shipping and storage and have an ease of administration (i.e., a single dose). Many types of vaccines are being used today with success or are in clinical trials. The most effective vaccine is always a live, attenuated virus because it will mimic the natural infection the best. But, these are not available for many viruses. Killed viruses are also commonly used as these are very safe. Other ways of achieving “protection” from pathogens is to target the molecules that actually cause disease. For instance, certain bacteria, such as Vibrio cholerae, cause disease by secreting toxins. For such pathogens, generating blocking antibodies to toxins, but not the bacteria, is a viable strategy. Despite the great advances made in the field of vaccine research, much needed vaccines for major killers, HIV-1, malaria and TB, are lacking. We will discuss some of the obtacles of vaccine development against these agents, and what immunologists might do to tackle these problems in the future. Reading: Janeway’s “Immunobiology” by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 16 (16-19 through 16-31). Pulendran B, Ahmed R. Nat Immunol. 2011 Jun;12(6):509-17. Immunological mechanisms of vaccination.

Inflammation – Response to Infection

November 9 Instructor: A. Iwasaki Inflammation is a complex response to infection and injury that destroys the infecting agent and restores the injured tissues. The inflammatory response includes the following physiological components: vasodialtion, increased vascular permeability, recruitment and activation of neutrophils, and fever. Molecular mediators of inflammation include pro-inflammatory cytokines, such as TNF, IL-1 and IL-6, acute phase response proteins, plasma proteases (complement and coagulation protease cascades), lipid mediators, such as prostaglandins and leukotrienes, neuropeptides and amines (histamine and serotonin), as well as nitric oxide and reactive oxygen species (ROS). The cellular component of the acute inflammatory response includes activation of neutrophils, monocytes and macrophages, eosinophils, platelets and endothelial cells. The inflammatory response to infection is designed to optimize the elimination of the invading pathogen. This response is triggered most commonly by TLRs activated by infectious agents. TLRs in turn trigger production of inflammatory cytokines, most importantly TNF, IL-1 and IL-6. In particular, IL-1b processing requires the formation of inflammasome complex in the cytosol. These cytokines orchestrate the inflammatory response at the local and systemic levels. A typical inflammatory response is initiated by macrophages resident in peripheral tissues. Pathogens activate TLRs on macrophages, which leads to production of inflammatory cytokines and chemokines. Collectively, this results in local changes in endothelium (vasodialtion and increased vascular permeability, expression of adhesion molecules), which promotes recruitment of leukocytes to the site of infection. Inflammasomes NOD-like receptors (NLRs) are pattern recognition receptors that reside in the cytosol. Although the functions of many of the NLRs are largely unknown, several NLRs play a key role in the activation of caspase-1 by forming a multi-protein complex known as the ‘inflammasome’. Caspase-1 is an essential mediator of inflammatory response through its capacity to cleave and generate active forms of IL-1b and IL-18. Inflammasomes are induced by sensing cellular stress, damage or bacterial products. Fever TNF, IL-1 and IL-6 are called endogenous pyrogens because they case fever. They do so by inducing the sythesisi of prostaglandin E2 (PGE2) by the enzyme cyclooxygenase-2 (COX2), expression of which is induced by these cytokines. PGE2 act on hypothalamus resulting in increase in heat production by brown fat and increased vasoconstriction decreased in the loss of excess heat through the skin. Fever is beneficial to the host because most pathogens grow better at lower temperatures. Acute phase response TNF, IL-1 and IL-6 also act on the hepatocytes to induce the acute phase response. Liver cells start to produce acute phase proteins, which include surfactant proteins (which promote phagocytosis in the lung), MBL (binds to carbohydrate moiety on pathogens and induce opsonization), C-reactive protein (binds to phosophocholine portion of bacterial and fungal cell walls for opsonization and C’ activation). Regulation of inflammation Although inflammatory response is an essential component of host defense, it can be highly detrimental if it becomes excessive, persistent of systemic. Indeed, dysregulated inflammatory response leads to a wide variety of pathological conditions and is normally prevented by several anti-inflammatory mechanisms. The so-called anti-inflammatory cytokines, IL-10 and TGF-beta, are particularly important for the negative regulation of inflammation.

Reading: Review Articles: Nathan C. Points of control in inflammation. Nature. 2002 Dec 19-26;420(6917):846-52. Text: "Janeway’s Immunobiology", by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 3.

Mucosal Immunity

November 12 Instructor: A. Iwasaki Infection in real life With the exception of vector-borne diseases, all pathogens must enter the host through the mucosal surfaces. Consequently, mucosal surfaces are lined with mucus and other physical barriers that act as a first line of defense against pathogens. In addition, mucosal tissues have developed unique features not found in other types of tissues to 1) combat pathogens, 2) induce rapid immune responses, 3) provide a reservoir of effector and memory cells just beneath the surface epithelial cells for rapid protection, 4) prevent unnecessary Th1 immunity to maintain the physiological functions of the mucosal tissues. First, to combat pathogens, mucosal lining contains specialized cells that secrete antimicrobial peptides into the lumen. Second to induce rapid immune responses, one can find organized lymphoid structures just beneath the mucosal lining. Third, to provide the most efficient form of protection, pathogen specific T and B cells are found within the disorganized lymphoid tissues of the lamina propria. Forth, mucosal tissues contain specialized dendritic cells and regulatory T cells that suppress Th1 immune to harmless antigens to maintain the physiological functions of the organs (respiratory, digestive, reproductive…etc.). Two types of mucosal lining; Type I: Mucosal Surfaces Covered by Simple Epithelia Type I mucosal surfaces are covered by simple epithelia of one cell–layer thickness. These surfaces are represented by those that cover the small and large intestine, the upper female reproductive tract, and the pseudostratified epithelia of the respiratory tract. The primary role of these surfaces is to perform absorptive, respiratory, excretory, and reproductive functions that are vital to the life of the host. Common features of type I mucosal surfaces include the presence of mucus-secreting cells (goblet cells) and the expression of pIgR on the basolateral surface of the

epithelia. The pIgR binds to polymeric IgA (pIgA) secreted by the plasma cells in the LP and exports the pIgA transepithelially, releasing the secretory IgA (SIgA) into the lumen. Type II: Mucosal Surfaces Covered by Stratified Epithelia Type II mucosal surfaces are covered by stratified squamous epithelia (keratinocytes), which share many common features with the skin. The main function of type II mucosa is to provide a physical barrier, and the keratinocytes that cover these surfaces do not have absorptive or respiratory functions. Type II mucosal surfaces are devoid of MALT structures but are drained by regional lymph nodes. There are no pIgR on the type II mucosal surface and thus the dominant protective immunoglobulin at these surfaces is IgG. FcRn is expressed by vaginal epithelium and can transport antibodies from the tissue and back into the tissue. Unique Components of the Mucosal Immune System Innate Immunity Mucosal epithelial cells and Paneth cells produce a variety of antimicrobial peptides (defensins, cathelicidins, bactericidal/permeability increasing protein) and bacteriolytic enzymes (lysozyme, group IIA phospholipase A2) that protect mucosal surfaces and crypts containing intestinal stem cells against invading microbes. Another important cell type that provides early non-specific immune responses are the intraepithelial lymphocytes. These lymphocytes detect non-classical MHC I molecules induced by infection, injury or stress and destroy infected cells. Immune Inductive Sites Organized lymphoid tissue (inductive sites)

Tonsil, Peyer's patches, appendix Follicle Associated Epithelium (FAE)

The FAE is a specialized epithelial layer that cover the dome region of the Peyer’s patch, appendix, and tonsils. FAE contains M cells capable of transporting luminal antigens across by transcytosis.

Effector Immune Reservoir Diffuse lymphoid tissue of the lamina propria contain effector and memory T and B cells that provide immediate protection against invasion by pathogens. In the gut and lung, IgA is the most important isotype that provides protection against pathogens, and IgA secreting plasma cells can be found in the lamina propria of these tissues. IgA provides its effector functions in multiple ways;

• Prevention of bacterial colonization • Prevention of viral adherence • Neutralization of bacterial toxins • Prevention of intracellular spread of microbes within the epithelial cells

Common mucosal immune system The concept of the common mucosal immune system (CMIS) was pioneered by Bienenstock and McDermott in 1978, who proposed that the immune system of the mucosal tissues is somehow connected, based on the observation that mucosal sensitization at one site provide primed cells selectively to other mucosal sites. These observations were originally described for gut induction leading to bronchus effector functions. This concept has been the main force behind the efforts to develop “mucosal vaccines” in which immunogen delivery is targeted to various mucosal surfaces. Since the original description of the CMIS, many studies have now confirmed this theory and provided molecular mechanisms for the homing pattern of effector lymphocytes. Further, other studies have provided evidence that distinct branches exist within the CMIS. Cell migration mechanisms behind the CMIS have been elucidated recently. Certain chemokines, such as CCL25 and CCL28 are expressed by the small and large intestinal epithelial cells and are known to recruit CD8 T cells expressing the receptors CCR9 and CCR10, respectively.

Immune Regulation Mucosal tissues are constantly exposed to tremendous amounts of exogenous antigens that are either resident or ingested/inhaled. Mounting protective immune responses against harmful pathogens while preventing excessive responses to harmless Ags (including commensal bacteria) is the difficult task achieved by the mucosal immune system to maintain homeostasis (a disease-free state). When soluble protein antigen is given orally in the absence of an adjuvant, the animal becomes unresponsive to that antigen upon secondary encounter. This phenomenon is known as “oral tolerance”. The mechanism of oral tolerance is not completely understood, but it likely involves mucosal dendritic cells and regulatory T cells that suppress Th1 immune responses to antigens. The loss of tolerance in the intestinal mucosa can result in inflammatory bowel diseases. Questions Q1: How do we distinguish between commensal bacteria and pathogenic bacteria? Reading: Text: “Janeway’s Immunobiology." Kenneth Murphy. 2011, 8th Edition, Chapter 12. Review Article: Iwasaki, A. (2007) Mucosal dendritic cells. Annual Review of Immunology 25:381-418 Primary Literature: Macpherson AJ, Uhr T. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Science. 2004 Mar 12;303(5664):1662-5.

Transcriptional Regulation in the Immune System

November 14

Instructor: T. Chi The development and function of the immune system are controlled to a large extent at the level of transcription. Consequently, transcription defects underlie diverse immunological disorders, from the autoimmune disease IPEX to the immune deficiency disease DiGeorge’s syndrome, while transcription factors constitute effective targets for immune therapies (consider, for example, how cyclosporin A and corticosteroids work). Developmental cues are known to direct lineage commitment and differentiation by controlling transcription. Similarly, immune responses to pathogens and other stimuli rely heavily on transcriptional regulation in the responding cells, with the optimal adaptive immune responses achievable only after further transcription-mediated differentiation of the responding naïve lymphocytes into effector/memory cells. There are 5 general principles underlying transcriptional responses to environmental signals, which is best understood in the context of antigen-induced differentiation of naïve CD4 cells into effector cells: 1) Signaling molecules directly activate latent transcription factors such as NFAT and Stat1 that are activated by TCR signals and IFNγ, respectively. The latent factors are often constitutively localized in the cytoplasm until activation, when they move to the nucleus to regulate target genes. 2) The most important target genes turned on by the latent factors are “master transcription factors” specifying cell identity/critical immune functions, such as T-bet and GATA3, which are necessary and sufficient for directing the differentiation of naïve CD4 cells into Th1 and Th2 cells, respectively. 3) Environmental signals are transient, and so can be the corresponding transcriptional responses they elicit. However, sometimes, the transient signals can cause irreversible or long-lasting changes in gene function that persist after the termination of the signaling events, as if cells can “remember” things of the past. This cellular memory, typically called epigenetic memory, is at the heart of lineage differentiation and immunological memory, the latter being a special form of lineage differentiation. 4) Epigenetic memory is achieved by two mechanisms: a. Trans-mechanism, in which the induced transcription factors auto-regulate their own expression after the termination of the transient signals. b. Cis-mechanism, which involves chromatin. Chromatin is a focal point of transcription regulation, because incorporation of DNA into chromatin inhibits access of transcription factors. Chromatin structure is tightly regulated in part by covalent modifications of DNA and histones, and such modifications, once established, can sometimes be sustained and propagated to daughter cells independently of the initiating signals. 5) Epigenetic memory, just as our brains’ memory of our experiences, is not hardwired or coded in the DNA sequence, but rather is a functional state of genes. Although this state is stable, it is also intrinsically plastic, which underlies phenomena such as trans-differentiation, retrodifferentiation and epigenetic reprogramming. For example, various CD4 cell subsets (Th1, Th2, Th17 and Tregs) are interconvertable to some extents, which has important clinical implications. Questions: The effects of transient signals on target gene functions can be reversible or irreversible. What are the biological “purposes”, and what might be the underlying mechanisms?

Reading: Text: “Janeway’s Immunobiology”, by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 8-17 to 8-19 (pages 308-11) Primary Literature: O’Shea, J. & Paul, W., Mechanisms Underlying Lineage Commitment and Plasticity of Helper CD4+ T Cells. Science (2010) vol. 327. pp. 1098-1102.

Primary and Acquired Immunodeficiencies

November 16

Instructor: E. Meffre Primary immunodeficiencies are a set of rare diseases usually resulting from defects in a single gene. Symptoms often occur during the months/first years of life and affect diverse components of the immune systems. As a consequence, patients suffer from recurrent infections that can be life threatening. On a scientific aspect, the analysis of primary immunodeficiencies provides unique opportunities to determine the impact of gene mutations on the development and/or function of the immune system in humans. We will present in this lecture a group of primary immunodeficiencies and discus their impact on T, B, NK and other antigen presenting cell functions. Severe Combined Immunodeficiency diseases (SCID) SCIDs are a set of primary immunodeficiencies that are diagnosed during the first year of life. These life-threatening diseases usually affect several arms of the immune system. A classification of these diseases is based on the presence of T, B and NK cells in the patient’s blood. We will review three major kinds of SCID:

- Those resulting from defects of in the components of the V(D)J recombination machinery. In this case, early T and B cell precursors are unable to recombine their specific receptor genes (T cell receptor and immunoglobulin genes, respectively). As a consequence, patients have no or very few T and B cells.

- Those characterized by an absence of T cells and resulting from either T cell specific gene mutations (CD3 component) or cytokine receptors. Interestingly, we will discuss the case of the common g chain gene mutation, which in humans results in an absence of T cells and normal B cell counts whereas similar mutations result in an opposite phenotype in mice (no B cells and normal T cell numbers).

- Those resulting from mutations in genes encoding B cell receptor components and inducing specific blocks in B cell development

Primary immunodeficiencies affecting the maintenance of tolerance Patients with primary immunodeficiencies often suffer from paradoxical autoimmune syndromes. Indeed, while patients are usually unable to mount proper immune reactions against pathogens, it is very common to identify specific immune responses targeting the patient’s own body. In this section, we will present a few syndromes such as IPEX and APECED, which are characterized by the impaired generation of regulatory T cells. Indeed, these cells are very important for the establishment and/or maintenance of peripheral tolerance. Primary immunodeficiencies affecting late B cell development and the generation of memory cells There are many examples of primary immunodeficiencies affecting the production of isotype switched B cells and the production of gammaglobulins. Actually, the first reviewed case of primary immunodeficiency was reported in 1952 and affected a boy who displayed no gammaglobulins in his serum. It took more than forty years before gene mutations associated with this syndrome called X-linked agammaglobulinemia (XLA). More classical forms of such defects are called HyperIgM syndromes and still allow IgM secretion but impair IgG and IgA production. We will review in this section the different types of mutations that can affect the production of memory B cells and gammaglobulin secretion. They will be segregated in two main categories: -Those resulting from intrinsic B cell defects and characterized by gene mutations encoding component of the isotype switching machinery (AID deficiency) -Those characterize by defects extrinsinc to B cells. In these cases, the lack of expression of molecules expressed on T cells and essential to activate B cells such as CD40 ligand result in the first identified form of hyperIgM syndrome. -Finally, we will report on common variable immunodeficiency diseases (CVID), which are the most common of all immunodeficiencies and whose gene defects are mostly unknown, demonstrating that we have still a lot to learn from the analysis of primary immunodeficiencies.

Reading: Primary Literature: Cunningham-Rundles, et al. “Molecular Defects in T and B cell Primary Immunodeficiency disease.” Nat Rev Immun (2005). vol 5. pp. 880-892

Allergy

November 26 Instructor: R. Medzhitov Although Th2 immunity characterized by IL-4, IL-13 and IgE exists to provide protective immunity to parasites, in the industrialized countries where parasite infection is rare, Th2 responses to innocuous antigens cause allergic diseases. Antigens that evoke Th2 cells that drive an IgE response are known as “allergens”. Most human allergens are relatively small proteins that are inhaled or ingested in very small quantities. They tend to have enzymatic activity (cystein protease) and are stable and soluble. Once taken up into the airway mucosa, the inhaled allergen can be engulfed by the lung resident dendritic cells. Food allergens that are ingested survive the acidic environment of the stomach and reach the small intestine where they are taken up by dendritic cells. However, the precise mechanism by which the allergens induce Th2 immunity by dendritic cells is unknown. Cellular mediators of allergy Mast cells Mast cells express high affinity FceR on their cell surface. IgE can bind to the FceR in the absence of bound antigen. There are two main types of mast cells in the body, those associated with vascularized connective tissues (connective tissue mast cells) and those found in submucosal layers of the gut and lung (mucosal mast cells). In allergic individual, all of these mast cells have prebound IgE on their FceR, and depending on the route of allergen encounter, causes different diseases. Inhaled allergens trigger lung mucosal mast cells to release their granules and cause increased mucus production, contraction of bronchial smooth muscle leading to asthma. Ingestion of allergens triggers activation of the gut mucosal mast cells inducing contraction of intestinal smooth muscle (vomiting) and outflow of fluid into the intestine (diarrhea). Systemic encounter of allergen (via the blood stream) triggers general release of histamine by the connective tissue mast cells leading to anaphylaxis and even death. Basophils Like mast cells, basophils also constitutively express FceR and can be recruited to the site of IgE-triggered reactions via inflammatory lipid mediators cytokines and chemokines. Basophils can amplify the response stated by mast cells. Basophils release histamine and other substances toxic to parasites but also to the host. Recent studies indicated that basophils, but not dendritic cells, can prime Th2 responses to parasites and allergens. Eosinophils The eosinophils are found in tissues within the connective tissue beneath the mucosal epithelium. Upon activation, eosinophils release contents of preformed granules containing many toxic substances (peroxidase, collagenase, neurotoxins leukotrienes) that are intended to kill parasites but are also very toxic to the host. Thus, the activation of the eosinophils is tightly regulated at the level of 1) total cell number, 2) location and 3) threshold of activation. Unlike mast cells and basophils, eosinophils only express surface FceR upon activation by Th2 cytokines and chemokines. Predisposition to allergy The prevalence of atopy in the industrialized countries is on the rise in recent years. Atopic individuals possess higher levels of IgE and eosinophils in circulation. Genetic factors Several genetic loci have been identified to be associated with atopy.

• Chr. 5: cluster of Th2 genes (IL-3, IL-4, IL-5, IL-9, IL-13, GM-CSF), β2-adrenergic receptor, • Chr. 6: HLA-DR • Chr. 11: Encodes the β subunit of FceR.

Environmental factors • Early exposure to infectious diseases reduce the prevalence of allergy (“Hygiene hypothesis”) • Pollution

Allergic diseases Asthma Skin allergy (urticaria, chronic eczema) Food allergy, Celiac disease Treatment of allergy Desensitization: shift the preexisting Th2 response to Th1 Antihistamine: block histamine H1 receptor Steroids: anti-inflammatory Blockade of chitinase? Reading: Text: "Janeway’s Immunobiology", by Kenneth Murphy (Garland Science, Taylor & Francis Group, LLC), 2011, 8th Edition, Chapter 13. Primary Literature: Kay AB. Allergy and allergic diseases N Engl J Med. 2001 Jan 4;344(1):30-7. and N Engl J Med. 2001 Jan 11;344(2):109-13.

Peripheral Tolerance

November 28 Instructor: K. Herold

1. Definition of tolerance: The lack of an immune response to an antigen. In the case of self tolerance it refers

to the state in which an immune response against self antigens is not generated under normal conditions in spite of the fact that MHC molecules normally are binding self antigens. A failure of self tolerance results in autoimmunity.

2. Mechanisms of tolerance – over view – note that it is difficult to precisely define mechanisms for an immune response that doesn’t happen! a. Lymphocyte repertoire must be diverse. b. Generation of a diverse repertoire will lead to production of T and B cell receptors that are reactive with

self. c. Mechanisms needed to eliminate most autoreactive cells at the time and location of development – this is

referred to as central tolerance. d. In the event that central tolerance is not complete, for example for antigens that are not expressed in the

central compartments at the time of development, mechanisms are needed in the periphery to contain the immune response or to divert it from a pathogenic response = peripheral tolerance.

2. B cell tolerance

a. somatic hypermutation of Ig will generate self reactive T cells. b. Fate of autoreactive B cells depends on the strength of the BCR signal:

1. Central B cell tolerance in the Bone marrow: a. Death by apoptosis (clonal deletion b. Receptor editing

2. Outside of the bone marrow 3. Immunologic ignorance

3. T cell tolerance

a. Central Tolerance i. Tolerance limited to antigens expressed in the thymus

ii. The AIRE gene promotes the expression of several genes in the thymus. Proteins are expressed in the medullary thymic epithelial cells. Leads to negative selection.

iii. APECED = autoimmune polyendocrinopathy candidiasis ectodermal dystrophy b. In the periphery

1. Ignorance 2. Immune privileged sites: eye, testes, uterus, brain, 3. Lack of costimulation or negative costimulation 4. Regulation/regulatory T cells

a. Naturally occurring Tregs b. Foxp3: Foxp3 can reprogram CD4+ T cell function. It can inhibit NFAT activation at the IL-

2 promoter preventing transcriptional activation of the IL-2 gene. Inhibits AP-1 RORγt – a transcription factor involved in Th17 cell differentiation. Mutations are associated with IPEX (immune dysfunction/polyendocrinopathy/enteropathy/ enteropathy/X-linked. The identification of the molecular basis of the IPEX syndrome was a key finding in understanding the role of Foxp3 in regulatory T cells.

c. Adaptive Tregs: T cells that do not emerge from the thymus as regulatory T cells can acquire regulatory function. A number of different phenotypes have been described. Some of these cells also express Foxp3. Cells that produce IL-10, TGF-beta, and other cytokines been described.

d. Antigen specificity of Tregs? Treg function is not antigen specific c. Oral tolerance: mechanisms of tolerance at the gut mucosal surface

4. Cytokines

a. Effects of cytokines in the environment b. IL-4, IL-5, IL-10 c. TGF-β

5. Environmental tolerance (Cobbold, Waldmann, et al)

a. Bystander suppression b. Infectious tolerance c. Civil service model

Reading: Text: “Janeway’s Immunobiology." Kenneth Murphy, Paul Travers and Mark Walport. 8th Edition, Chapter 15 and Chapter 8 section 8-19 Review Article: Goodnow CC, Sprent J, Fazekas de St Groth B, Vinuesa CG. Cellular and genetic mechanisms of self tolerance and autoimmunity. Nature. 2005 Jun 2;435(7042):590-7.

Autoimmunity

November 30 Instructor: K. Herold 1. Autoimmunity

A. Autoimmunity is the ultimate example of loss of tolerance to self: horror autotoxicus. Therefore consideration of the causes and treatments of these diseases is closely linked to understanding of mechanisms that maintain tolerance. Please refer to these mechanisms discussed in the lecture on tolerance.

B. Autoimmune diseases affect every organ system. C. The immune nature of several diseases now identified as autoimmune was based on the finding of

autoantibodies but in most cases, the precise immunologic mechanisms are not certain. In certain cases cellular mechanisms are thought to be predominant mediators but studies of immune interventions in man have suggested that both humoral and cellular arms of the adaptive immune response are involved.

2. The initiation of autoimmune diseases, hence loss of self tolerance, involves a combination of environmental and genetic factors

A. Rates of concordance in identical twins B.

C. There are examples of specific genetic mutations that lead to autoimmune diseases i. Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy

ii. Scurfy/IPEX: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked disease c. The major genetic determinants of autoimmunity are genes within the major histocompatibility complex i. Examples of MHC-disease relationships ii. Mechanisms that may account for the relationship d. Other genetic loci, some of which are associated with immune responses, have also been associated with autoimmune diseases, e.g. IL-23R, CTLA-4, etc. e. Molecular mimicry is one postulated mechanism to account for the relationship between environmental events and autoimmunity – specific examples f. Innate immune responses may also be involved in initiation of autoimmune responses. Indeed,

targets of autoimmune responses such as DNA can activate TLRs (e.g TLR9). g. Certain drugs can also initiate autoimmunity. In certain cases (e.g. anti-CTLA-4 antibodies) this can occur through known mechanisms.

3. Adaptive immune responses are involved in autoimmune diseases

A. Role of B cells: as antibody producing cells, as antigen presenting cells B. Pathogenic autoreactive T cells can clearly cause autoimmune disease in animal models and are found in

human autoimmune diseases C. Unstable Tregs? D. Chronicity is a feature of autoimmune diseases:

i. development of tertiary lymphoid organs as sites of activation of adaptive immune responses,

ii. modification of self proteins as a mechanism of generating new epitopes, iii. intra and intermolecular spreading of the immune response

4. Examples of specific autoimmune diseases: Most organ specific autoimmune diseases are T cell mediated. Examples include:

a. Type 1 diabetes (as an example): MHC association, antigens, animal models, cellular mechanisms b. Rheumatoid arthritis c. Graves’ disease d. Multiple sclerosis But others are mediated by autoantibodies. Examples include: d. Autoimmune hemolytic anemia e. Myasthenia gravis 5. Therapies for autoimmune diseases: anti-T cell/anti-B cell/anti-cytokine/blockade of cell migration. Tolerance is the ultimate goal. Reading: Text: “Janeway’s Immunobiology." Kenneth Murphy, Paul Travers and Mark Walport. 8th Edition, Chapter 15. Review Articles: Bettelli E. and Kuchroo, VK et al. Induction and effector functions of T(H)17 cells. Nature. 2008 Jun 19;453(7198):1051-7. Bluestone JA, Herold K, Eisenbarth G. Genetics, pathogenesis and clinical interventions in type 1 diabetes. Nature. 2010 Apr 29;464(7293):1293-300.

Transplantation & Cancer Immunology

December 3 Instructor: K. Herold Cancer Immunology

1. Mechanisms whereby tumors avoid immune recognition a. Failure to present antigen including loss of Class I MHC expression b. Failure to activate T cells c. Poor access to the tissue d. Failure to express antigen: immune selection of antigen loss variants e. Inhibitory factors: TGF-β, recruitment of regulatory T cells

2. Immune surveillance may normally prevent the outgrowth of variant cells: role of NK cells, CD8+ T cells 3. Tumor antigens:

a. Tumor rejection antigens may arise by point mutations in self proteins b. Proteins selectively expressed in tumors are candidate tumor rejection antigens c. However, most tumor antigens are peptides that are over-expressed in tumor cells but area also

expressed at lower levels in normal tissues d. These proteins are generally presented by Class I MHC molecules

4. Paradox –T cells infiltrate a tumor but do not destroy the tumor - tumor infiltrating lymphocytes (TILs) 5. Enhancing immunogenicity to tumor antigens and cells

a. Immunization with tumor specific antigens/Adoptive transfer of dendritic cells pulsed with peptides

b. Adjuvant e.g. BCG c. Costimulation enhancers e.g. B7-1 d. Blocking negative signals: anti-CTLA-4

6. Biologics against antigens expressed on tumor cells e.g. Rituximab (anti-CD20 mAb), HER-2/Neu 7. Therapy with engineered effector cells.

Transplant Immunology and Immune therapy 1. Grafts

a. Autologous b. Syngeneic: same strain Allogeneic: different strain, same species c. Xenogeneic: different species

2. Classification of Rejection: Graft rejection is mediated by T cells. It can be transferred with T cells.

a. Hyperacute: preformed antibodies against a graft – often against blood group or polymorphic MHC antigens.

b. Acute i. Alloreactivity occurring days and weeks after transplantation.

c. Chronic: i. Alloreactivity can occur months to years after transplantation and is associated with

gradual loss of graft function. ii. Chronic organ rejection is caused by inflammatory vascular injury to the graft.

2. Presentation of graft antigens to the host a. Direct: recognition of donor cells by recipient’s T cells b. Indirect: Recognition of processed antigens of the donor by recipients APC’s

Drugs to treat rejection and autoimmunity:

a. Glucocorticoids b. Calcineurin inhibitors c. Janus kinase inhibitors d. Rapamycin e. Cytotoxic drugs:

i. Azathioprine: ii. Cyclophosphamide

iii. Mycophenolate mofetil

f. Examples of Biologics: Human Abs vs humanized abs vs murine abs vs other animals i. Anti-lymphocyte serum

ii. Nataluzimab binds to α4β1 (on central memory and effector T cells) and α4β3 integrins

iii. Rituximab (anti-CD20) iv. Campath (anti-CD52) v. Anti-CD3

vi. CTLA4Ig vii. Anti-Cytokines: e.g. Infliximab (anti-TNF) Etanercept (the p75 TNF receptor on a Fc

portion of IgG, anti-IL-1, anti-IL-6, g. Adoptive immune therapy with regulatory T cells?

Reading: Text: “Janeway’s Immunobiology." Kenneth Murphy, Paul Travers and Mark Walport. 8th Edition, Chapter 15. Review Articles: Peggs KS, Quezada SA, Allison JP. Cancer immunotherapy: co-stimulatory agonists and co-inhibitory antagonists. Clin Exp Immunol. 2009 Jul;157(1):9-19. Weiner LM, Dhodapkar MV, Ferrone S. Monoclonal antibodies for cancer immunotherapy. Lancet. 2009 Mar 21;373(9668):1033-4

Unresolved Questions in Immunology

December 5 Instructor: R. Medzhitov Despite tremendous advances in understanding the function of the immune system, there are many fundamental questions that remain unresolved. Why some infections are so common? Why some infections are so deadly? Why the majority of symptoms of infectious diseases are caused by the immune system itself? Why do we react to allergens? We now know a fair amount of details about activation of the immuen response, and yet we still cannot design successful vaccines for most pathogens. What parameters dictate the generation of a protective immune response? Does the immune system play a role outside of host defense from infections? These and other questions of fundamental scientific and medical importance will be discussed in this lecture.