Foundation Unit Immunology

Adaptive Humoral immunityAdapted from Andrea Crawley’s lecture

Dr. Lauren SegalLecturer, University of Ottawa,

Department of Community PediatricsAllergist and Clinical Immunologist, MD, FRCPC

Describe the structure of antibodies. Discuss antibody gene organization and expression during B cell ‐

development. Describe T cell independent and T cell dependent antibody responses. ‐ ‐ Recognize how antibody diversity is established. Describe isotype switching and the various classes of antibodies and

their functions. Describe the different functional roles of antibodies (opsonisation,

neutralizing, etc). Describe the activation and regulation of the adaptive immune system. Describe the different hypersensitivity reactions (Types I – IV).

Objectives

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How many of you have died from overwhelming infection?

How many of you have even skinned you knee?

Why do we have an immune system?

• To protect the host from infections • HOW?

– 1) Blocks the entry of the pathogen before it can overwhelm the host

– 2) Attenuates and localizes the infection to one area of the body

– 3) Controls and eliminates the infection prior to its overwhelming the host

Your body’s immune system

<---------------------------------------Innate immunity-------------------

Overview of the immune system

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A) Starfish cannot see out of their “legs” B) Only dogs have a circulatory system C) Starfish make better pets D) Only dogs have adaptive immunity

Which of the following is true?

Players in the adaptive immune system

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Describe the structure of antibodies Discuss antibody gene organization and

expression during B cell development. ‐

Antibodies

Structure of an antibody

Antigen binding sites

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An antibody has 4 chains•2 light chains•2 heavy chains•Chains are connected by disulfide bonds

Structure of an antibody

Antigen binding sites

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The top part of the antibody is called the variable region (Fab)•Each B cell secretes antibodies with a different Fab•The Fab recognizes antigen (epitope)•The Fab is the area of the antibody with the greatest genetic diversity•The Fab is responsible for the breadth of our B cell repertoire (the range of antigens our B cells can recognize)

The bottom part is called the constant region (Fc)•This region is responsible for the effector function of the antibody•Eg: will it be a BCR, IgG, IgA, IgM, IgE, IgD, etc

Functional Parts of an Antibody

The Fab part binds antigen –both Fab fragments have the same specificity.

The Fc part has distinct functions not related to antigen binding but related to effector function.

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There are five classes of antibodies:

Each class, or isotype of antibody has a different Fc region

The 5 isotypes are IgG, IgA, IgM, IgE and IgD

The different Fc region carries out different biological functions, which are distinct from antigen binding.

IgG, IgD and IgE are monomeric

IgA is a dimer IgM is a pentamer

The 5 Antibody Classes (or isotypes)

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Isotypes, Allotypes and Idiotypes

Definitions:

Isotype:•Different Fc•Different type of Ab•IgG, IgA, IgM, IgE, IgD

Allotype:•Difference between species•Most differences in Fc region.

Idiotypes•Different specificities of the antibody molecules.•Eg: each idioptye recognizes a different part of or different antigen

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Antibody Functions: NEUTRALIZATION: Preventing infection

Virus particles or toxins trigger an immune response. The antibodies produced inhibit re-infection

Bacteria trigger an immune response. The antibodies produced inhibit re-infection

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Antibody FunctionsNEUTRALIZATION : protection from toxins + viruses

Toxins can also be neutralized•Prevents the re-binding to the cells receptors•Neutralized toxins are phagocytized and digested by phagocytes.

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OPSONIZATION & Complement activation protection from bacteria

Opsonization + Complement activation•Antibody binding the bacteria marks the pathogen for phagocytosis•Phagocytosis occurs either with or without complement

• Without complement, Fc portion of Ab triggers ingestion• With complement, complement triggers ingestion

• Complement bind to surface of the bacteria• Phagocytes recognise complement on the surface of bacteria• Antibody helps activate classical complement cascade

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IgG binds antigen (virus) on the surface of an infected call NK (Natural Killer) Cells have receptors for the FC portion of the IgG on them When the Fc receptors on the NK cells meet a virally infected cell coated or “flagged”

with IgG, the NK kills the infected cell “Kiss of death”

Antibody Function:Antibody Dependent Cell-Mediated Cytotoxicity (ADCC)

NK Cell Activated NK

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Recognize how antibody diversity is established

Discuss antibody gene organization and expression during B-cell development

Objective

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Overview of antibody genetic changesVDJ

rearrangement establishes antibody diversity----------Somatic

hypermutation enhances

antigen binding affinity of antibody-----------

Class switch recombination

changes the isotype, hence the function

Antibody Diversity : our amazing antibody repertoire

• Every person could produce antibodies to almost any foreign antigen – This does NOT happen in certain immunodeficiencies– Repertoire exists before contact with antigen or immunization

• The repertoire is dictated by our DNA (thus differs for every person)– Diversity is generated by genetic recombination from a limited number of genes at the immunoglobulin locus.– We can each make 1012 possible different B-cell clones!

• This happens while maintaining tolerance to self– In autoimmune conditions, this self tolerance fails

• This generation of diversity occurs through the maturation of the B cell.– In bone marrow: Pre-B cells: rearrange variable (V), diversity (D), and joining (J) genes

for primary antibody repertoire. – In the periphery: Mature B cells, when activated by antigen recognition, refine their

antibodies by affinity maturation (somatic hypermutation) as they differentiate into plasma or memory cells.

• Non diversity-related genetic change to antibody: – Isotype switch of antibody by RNA splicing happens at the same time as affinity maturation.

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The Ig Locus – V(D)J (variable region) and constant region genes

• Light and heavy chains contain a variable region, which binds to antigen• 200 V genes, 27 DHeavy genes, 6 J genes • The light chain variable region contains only V and J genes• The heavy chain variable region contains V and D and J genes• The light chain is coded by 1 of 2 alleles λ or κ, each with different V and J genes

• Constant regions in the heavy chain are coded by • Cδ for IgD, Cμ for IgM, Cγ for IgG, Cα for IgA, Cε for IgE

The answer lies in the structure of the immunoglobulin locus.

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Ig Gene Rearrangement VDJ Recombination

In the bone marrow:The VDJ recombination events occur during B-cell development in the absence of antigen.

Later, during an immune response in a lymph node: -B cells meet antigen-Somatic hypermutation in the variable region improves antibody binding affinity -Isotype switching changes antibody to a different isotype by splicing genes in the constant region (blue).

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Incredible Antibody Diversity

Diversity comes from the number of possible combinations of•Different V light chain segments•Different V heavy chain segments•Could then be combined to make about 1.9 x 106 different antigen-binding sites.

Extra diversity introduced at because of how the segments are joined •Increases possibilities by 3 x 107

Total possible clones ~5x1013

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Joining of D and J segments in the variable region generates

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Sources of diversity

Combination of different V(D)J genes together

An enzyme called RAG cuts the hairpin, adding P-nucleotides

An enzyme called TdT adds on extra N- nucleotides

Then strands pair

Then DNA is repaired

Non-Productive Gene Rearrangement Causes B-cell loss

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B-Cell maturation and regulation of the adaptive immune system. Where do B cells mature? What are the steps that lead to maturation?

Objective

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B Cell Maturation – creation of the BCR

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• B cells develop from stem cells called committed lymphoid progenitors

• They go through several developmental stages in the bone marrow• During this time, they develop their B cell receptor (BCR) by putting

together the B cell receptor’s heavy chain and light chain

B Cell Maturation – creation of the BCR

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• During the Pro-B cell phase• Heavy chain gene rearrangement occurs

• During the Pre-B cell stage• The heavy chain joins with a surrogate light chain and Ig and Ig

polypeptides -> all together, these form a pre-B cell receptor (pre-BCR) • Pre-B Cell Receptor

• Sends a signal into the cell = essential for further B cell development• But can’t yet recognize antigen.

B Cell Maturation – creation of the BCR

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• Once the light chain is rearranged, mature IgM is expressed on the cell surface

• The B cell can now recognize antigen• Then they are released into circulation as immature B cells

B cell Maturation – What is VDJ rearrangement?

At each stage of B cell development, the genes that make up the BCR look differentTo make the heavy chain:•First D segment joins with J (early pro B cell stage)•Then V joins with DJ (late pro-B stage) Same thing happens for the light chain (though it is only made of a V and a J)•V segment joins with J (small pre-B cell stage)Thus, an immature B cell has both heavy (VDJ) and light chain (VJ) genes rearranged

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B Cell Maturation – negative and positive selection

What else happens in the bone marrow before the B cells can leave? •Negative selection: If the IgM on the immature B cells recognize self antigens, they are deletedThen the naïve B cells (never encountered antigen) migrate to lymph nodes•There they encounter foreign (non-self) antigen -> get activated•Activated B-cells will expand (make clones) and become plasma and memory B cells.

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Putting it all together…• In the lymph node Naïve B cells

meet antigen (Ag)• If the BCR (IgM) recognizes the

antigen it gets activated• In the lymph node there will

also be helper T cells • The activated B cell “shows”

the T cell what it has found• If the T cell recognizes what the

B cell is showing it and decides that it is a foreign antigen, it “tells” the B cell to expand and differentiate

• B cells will expand and differentiate into plasma and memory B cells

• Memory B cells = remember for next time

• Plasma cells secrete antibodies

Describe isotype switching

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Isotype switch

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Ig isotype doesn’t change the specificity of antibody binding site (i.e. variable region)Switch only happens once B cell meets antigen. Process is regulated by a switch region in the DNA located upstream of the constant region of the heavy chain Switch happens thanks to many enzymes also involved in DNA repair

Immature B cell expresses only IgM on the cell surface (exit bone marrow) Naïve B cells: express low affinity IgM and IgD (circulating looking for antigen) After B cell is activated by antigen

IgM is secreted Affinity maturation of variable region genes occurs Isotype switching of heavy chain constant gene regions occurs

Different isotypes for different stages

When B cell encounters antigen in lymph node -> B cells are activated They then become either

Plasma cells (secrete antibody) OR memory B cells (long term immunological memory)

The cytokine milieu is the signal that governs this switch T cells will secrete different “flavours” or mixes of cytokines to tell the B cells what to secrete T helper cell, either Th1 or Th2, each secretes a different mix of cytokines these cytokines direct B

cell development

Isotype Switch

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Cytokines Dictate the Isotype Production

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IL-4 leads to IgG and IgE production (TH2)IFN-γ leads to IgG production (TH1) TNF-α leads to IgG and IgA production (TH1)They will also inhibit the production of other isotypes

How the isotype switch happens

Immature Naïve B cells express only IgM (a.k.a the B-cell receptor)

Mature Naïve B cells express low affinity IgM and IgD on the cell membrane

Maturing B cells “loop out” DNA containing constant regions after contacting antigen

This leads to isotype switch

Specific functions of different Ig isotypes

• B cell receptor:– Is membrane-bound IgM– Activation and maturation of the B cell

• Antibody Dependant Cellular Cytotoxicity (IgG)• Immediate hypersensitivity (IgE)• Antigen “marking” for opsonization (IgG> IgA, IgM)• Agglutination of clumping of antigen (IgM)• Activation complement cascade (IgM & IgG> IgA)

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10% of antibodies are IgM IgM - Expressed as the first

antigen receptor on immature B-cells.

When secreted, IgM is a pentamer, containing a J (joining) chain

Lots in the small intestine Function:• Fixation and activation of

complement (main fcn)• Agglutination (clumping)

which makes a pathogen more likely to be phagocytized.

• Neutralization• Opsonization

Isotype Functions of IgM

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1% of all antibodies are IgD

They are co-expressed on naïve B-cells.

Function: unknown

Isotype Functions of IgD

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Isotype functions of IgA

• IgA is a dimer• Two constant regions are attached by a J (joining) – chain• Functions:

• Neutralization (Main Fcn) -> quiet housekeeper• Opsonization• Fixation and activation of complement

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Isotype functions of IgA

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Quiet housekeeper of mucosa

IgA is secreted as a dimer into the GI tract

Transported across the gut epithelial cell by a secretory component called the poly-Ig receptor

IgG is a monomer 4 subtypes: IgG1, IgG2, IgG3, IgG4 The predominant antibody in the circulation The only Ig that can cross the placenta providing passive immunity to baby Immunologist’s favourite : critical for intact humoral immune function Functions:

Fixation and activation of complement Targeting cells to be killed by NK cells (ADCC) Neutralization (toxins and viruses) Opsonisation bacteria for easy phagocytosis

Isotype Function of IgG

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Allergist’s favourite Circulating IgE binds to mast

cells via the Fc receptor Allergens and antigens cause

cross-linking of IgE’s on mast cells

Leads to mast cell degranulation releasing Histamine Leukotrienes Prostaglandins

Responsible for Hives Positive skin tests Allergic rhinitis IgE mediated anaphylaxis Response to parasites

Isotype Function of IgE

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Where are different isotypes found in the body?

IgG the most prevalent antibody in the plasma. It is the only Ab that can cross the placentaIgM is found in all areas but is it is most concentrated in the small intestineIgA is found in the mucosaIgE is predominantly found in the epithelium (skin and gut) bound to mast cells

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Fc Receptors responsible for isotype function

• Fc Receptors are expressed on immune cell surfaces• When the Fc portion of the antibody binds to the receptor it starts a signaling

cascade that triggers the cell to do something (the function of the isotype)• Functions include

• Mast cell degranulation (IgE) • Cytotoxicity (IgG) • Phagocytosis • Opsonization

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T cell Independant and Dependent Antigens

• B cells bind specific antigen via their B cell receptor (BCR)• T cell independent antigens

• Can activate B cells on their own (without T cell help) • Minority of antigens• These antigens are usually large and can cross-link multiple Ig on the B-cell surface

• T cell dependant antigens• Require T cells (TH) CD4 positive cells to activate the B cell

• Most antigens are T cell dependant• Only this type leads to formation of antigen-specific memory B cells: long term memory• Interaction with T-cells induces isotype switch

• Whether the cells become memory B cells depends on the type of the antigen they encounter

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T1 T cell independent antigens intrinsically activate B cells to proliferate and differentiate without T cell stimulation

They also stimulate B cells independently of their BCR specificity.

TI-1 antigens activate B-cells by their Toll Like Receptors, called B cell Mitogens

Ex: Antigen such as Lipopolysacharide (LPS) The concentration of the antigen determines

the B cell response At high concentrations

Elicit a polyclonal response Non-specific antibody response

At low concentrations Elicit a specific antibody response

Type 1 T cell independent antigens

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Large highly repetitive epitopes of encapsulated bacteria (Bacterial polypeptides)

They must cross-link a critical number of receptors

This triggers cross activation of receptors because BCRS are all clustered at the surface

They trigger proliferation and differentiation of B cells and antibody production

Can only activate mature B cells Kids < 2 can’t respond well to

polysaccharide antigens (certain vaccines) because most of their B cells are immature

Type 2 T cell Independent antigens

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If a neighbouring cell produces cytokines (like a dendritic cell producing BAFF) B cells can class switch and produce IgG

Type 2 T cell Independent antigens

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T cell dependant antigens

Lets take the example of a virus particle = antigen1)Virus binds to BCR2)Virus-BCR binding leads to endocytosis of whole complex3)Virus gets broken up in the B cell4)Little pieces of virus bind to MHC class II molecules 5)MCH class II molecules bearing little bits of the virus get expressed on the B cell surface6)If T cell receptors “match” or recognize the virus bit: MHCII complex, the B cell gets activated

Match of MHCII:Ag and TCR is called “signal 1”

It’s the first of three signals needed to activate the B cell to become a memory B cell

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T cell dependant antigens

• Signal 3• Once the T helper cell receives signal 1+2,

it will secrete cytokines• These cytokines bind cytokine receptors

on the B cell• All three signals are needed to activate B

cell• Signal 1+2+3 lead to

• Activation of B cells • Proliferation of the B cell clone• Maturation of the B cells.

• Signal 2 : co-stimulatory molecules• The second signal needed is the CD40Ligand

molecule on the T cell• It will bind CD40 on the B cell• The absence of CD40 or CD40 ligand leads to

immunodeficiency

T cell dependant antigens: Memory cell development

When the B cell encounters an T cell dependant antigen there is •Clonal expansion•Affinity maturation•Isotype switching

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Memory B-cell formation

• Cytokines secreted from T helper cell tell B cell which isotypes to secrete (isotype switching)

• Signal 1+2+3 also lead to somatic hypermutation and lead affinity maturation

• Affinity maturation:• B cells whose variable region of the IgM

mutated to bind antigen poorly (with low affinity) die via apoptosis (T cells can’t see them well)

• B cells whose IgM mutated to bind antigen well (with high affinity) will survive (T cells bind them strongly)

• A strong bond with a T cell causes the T cell to stimulate the B cell to become either a plasma cell or a memory B cell

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Activated B cells become either plasma cells or memory B-cells.

Plasma cells are the cells that secrete most of the antibodies in response to infection.

They migrate to the bone marrow

Memory B cells are the cells that survive for many years and are ready to react with force upon re-infection. They lead to long lasting immunity

Both types of cells retain the changes they acquired such as high affinity antibodies and isotype switch.

B-cell memory

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The immune response - Immunization

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Type I-IV hypersensitivities

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Allergic conditions Allergic rhinitis Food, drug and venomous insect allergy Asthma Atopic Dermatitis Anaphylaxis

IgE mediates reactions Antigen must be soluble to bind receptors on

mast cells Effector cell is mast cell

Type I Hypersensitivity

Type II hypersensitivity• Antibody-mediated cytotoxicity

– Antibodies from immune response bind antigens on surface of patients cells

– Antigens may be self antigens or adsorbed onto patient cells (eg: drug or pathogen bound to cells)

– Antigens on cell surface are recognized by macrophages or dendritic cells

– These cells induce a B cell response and B cells make IgG to the antigen

– IgG binds antigen and targets cells for destruction – 2 types of type 2 hypersensitivities

• Macrophages or NK cells will kill the cell that is “tagged” for destruction by IgG

– Recognized via their Fc receptors– Antibody dependant cell mediated cytotoxicity (ADCC)

• Or complement cascade will be triggered and destroy the cell – Antibody mediated cytotoxicity

Peakman Fig. 10.10

Type II hypersensitivity• Antibody mediated cytotoxicity

– Eg: ABO blood incompatibility• Different antigens on transfused RBC recognized as different than patient’s cells• IgG produced against these cells• RBC bound with IgG get targeted by complement system and cytotoxic

lymphocytes destroyed (hours to days after transfusion)

– Eg: Goodpasture’s syndrome• Type 4 collagen in basement membrane of lung and kidney attacked by

autoimmune IgG

– Eg: Autoimmune Hemolytic Anemia• RBC are attacked by autoimmune (anti-self) IgG

– Eg: Grave’s disease• Autoantibodies bind to TSH receptors

Type III hypersensitivity• Anitgen-antibody complexes called immune complexes

– Small complexes form – > Complexes get deposited in vessels, serosa (pleura,

pericardium, synovium) and glomeruli – > WBC recognize these complexes via Fc Receptor – > Tissue injury– Large complexes are cleared w/o bystander injury

Type III hypersensitivity

• Immune complex reaction / vasculitis• Eg: Serum Sickness• Eg: Arthus reaction

– Maurice Arthus in 1903 noted when he repeatedly injected horse serum into rabbits this developed

– Why? Patient (or rabbit) is sensitized to the antigen (horse serum) after repeated injections

– These IgGs directed against horse serum bind the horse serum the next time it’s injected and clump together

– Small clumps deposit in vessels– Activate complement and neutrophils -> inflammation– Area of rabbits skin gets swollen and red

• Eg: Large local reaction 4-12 hrs after Td booster (rare)

Type IV hypersensitivity:• Delayed-type hypersensitivity (DTH)

– Takes 2-3 days to develop– Effector T-cell-mediated (not antibody mediated)

• Macrophages take up antigen and present it on their MCH molecules• Helper T cells recognize the antigen in MCH complex• Helper T cells secrete cytokines (IL2, IFN-γ) that cause local inflammation• Cytotoxic T cells kill target cells on contact• Macrophages produce enzymes that cause inflammation

Type IV hypersensitivity:• Delayed-type hypersensitivity (DTH)• Ex. TB (tuberculin) skin test

– Inject tuberculin (antigen) into dermis– Induration 2-3 days later indicates TB exposure

• Contact Dermatitis (Poison Ivy, Nickel)– Delayed formation of bullae and dermatitis

• Diabetes Mellitus Type I– Pancreatic Beta cell proteins destroyed

• Hashimoto’s thyroiditis– Thyroglobulin antigen is the target, leads to hypothyroidism

References

• Janeway’s Immunobiology, 7th edition, Garland Science, Edited by Keneth Murphy, chapters 9 + 10

• I VERY HIGHLY recommend the first 39 pages (chapter 1: Basic concepts in Immunology) of Janeway’s Immunobiology for a SUPERB overview of immunology. – One of the most elegant concise and friendly

overviews of immunology I have seen