(9/18) Campara Lecture: Lymphocyte Activation: B Cell Humoral Immunity: Naive B Cells pick up extracellular Ag, process it, proliferate and differentiate into activated B Cells. Differentiation produces either Memory Cells or Plasma Cells. Plasma Cells engage in antibody production, of which venture to bind Ag.

- Plasma Cells: Programmed to only produce antibodies, they do not carry any surface antibody.

- Memory Cells: These are only produced in the non-splenic areas. Once made, they go to invade the tissue where the antigenic source was first encountered and stay there for their lifetime. Once their surface antibody IgD is attenuated, IgM predominates.

- The Splenic Marginal Zone is the largest lymphoid organ. In this organ, B Cells are short-lived, stay there die there

- CD20: This surface protein is on every mature B cell, and is the drug target of Retuximab.

B Lymphocyte Development- 10-15% remain circulating, otherwise at lymphoid organs - In the bone marrow, naïve B cell will select the BCR to display on its surface. It functions as

the Ag recognition unit in the form of a membrane bound antibody (Ig). As a Naïve B Cell, Ig may be IgD or IgM. (M>D, mature). B Cell has ~1000 identical BCRs on its surface all for the same Ag. The specificity arises from random gene rearrangements while in the bone marrow.

- BCR: o Fab: The variable domain, serving as the Ag binding site o Fc: The constant domin, responsible for modulating immune activity

- Nothing happens without the Coreceptor Complex- modulates BCR signal transduction o CD21: Binds opsonized Ag particle (at C3d), surface Ab recognizes and attaches to it o CD19: Responsible for the signal transduction

Naïve B Cell Activation - The chemical structure of the Ag critically influences the type of immune response produced

o Thymus-dependent (TD) Ag: Protein Ag that requires activation guided by CD4+ Th Cells o Thymus-independent (TI) Ag: Polymeric Ag that crosslink the BCR, directly activating the B Cell

- The location of naïve B Cell activation also determines the type of immune response o Lymph Node Germinal Center (TD) o MALT (TD) o Splenic Marginal Zone (TI)

(TD) Ag Activation of B Cells: Anatomically restricted to the germinal centers of lymph nodes + MALT. 3 Signals - (1): BCR Encounters Ag (2) Costimulatory Signal (3) Cytokine Signal

o Response is delayed 5-15 days, and generally involves producing more than 1 Ig Isotype (M, G, E, A) o *The Ab produced are specific for more than one epitope of the Ag. o *Memory Cells are generated (for a faster future secondary response, 3-7 days)

- (1) B Cells recognize Ag in its native form as a soluble protein. B Cell will ingest the entire protein, process it into peptides to prepare it for presentation to T Cell. The peptides are placed into the open grooves of MHC II

o The Ag-peptide to which the TCR binds may not necessarily be identical to the protein BCR binds, but the TCR does need to be specific for at least one of the peptide fragments derived from the degradation

- (2) Costimulatory Signals from B7 and CD40 to the T Cell CD28 and CD40L respectively produces an activation storm in the Th cell. IL-4, IL-5 (B Cell growth hormones) are produced.

o Decision to become activated or becoming inert depends on amount, avidity, and timing of the interaction and the degree of costimulation. Requires properly timed interaction of CD4+, otherwise the B Cell may become inert or tolerant to the Ag

o B7/CD8: Activates the T Cell o CD40/C40L: Critical specifically for B Cell activation = Interaction promotes up-regulation of B7 to

enable APC efficiency, promotes B Cell proliferation (clonal expansion), promotes somatic hypermutation (affinity maturation), allows for Ab “class switch”, and allows memory cell formation. For Th cells, it promotes the secretion of IL-4/5 cytokines to promote growth and Ab production of B Cell

- (3) IL-4 and IL-5 are secreted by the Th Cell, interaction and signal transduction occurs through the B Cell Cytokine Receptor to encourage growth and proliferation.

- Th-B Cell Linked Recognition Phenomenon: Both Th and B Cell must be activated and interact for Ab production.

- Primary TD Ag B Cell Activation: Each cell growth cycle takes 12 occurs, goes on for 7 days. After 7 days, there are 20k B Cell clones with identical Ag-specific Ig receptor. After proliferation, start effector functionà 2000 Ab per second. Cell dies in a week

o IgM is first, arriving mostly after day 5. IgG dominants after, nearing day 15 - Secondary TD Response: Must encounter same epitope. Memory B Cells have higher affinity for Ag, due to

somatic hypermutation! o IgG in this situation is first and most dominant.

- à The body drives towards IgG production, as it is the most potent for removing pathogenic source, and it is longer-lived.

(TI) Ag Activation of B Cells: Occurring in the marginal zone of the spleen, it does not require Th cell help - This is a restricted response that only occurs in the spleen. Since the spleen is the equivalent of a blood filter, it is

able to produce a rapid response within 2 days of Ag encounter. It produces short-lived plasma cells that only produce IgM. It does not develop a memory

- TI-1 Ag: Mitogens, activate multiple B Cell clones to produce Ab (polyclonal response) o Signal 1: BCR binds polymeric Ag (mitogen). This occurs non-specifically o Signal 2: Toll-like receptors respond to mitogen (pattern recognition receptor) à cell division, Ab

production - TI-2 Ag: Have multiple repeating identical epitopes. à Results in crosslinking of multiple BCR

o Signal 1: BCR binds Ag o Signal 2: Clustering of BCR

- WHY IS THIS A THING? This sounds inefficient and weak compared to TD Ag Activation, however this is essential in that it is capable of recognizing non-protein Ag. Plus, we can get an activated B cell in absence of CD4+ à immediate response!

o That being said… This reaction produces low-affinity IgM antibodies, no affinity maturation stage and no class switching. Also, limited ability to generate memory cells

As a result of TD B Cell activation, 3 key transcription factors are produced: NFAT, NFkB, and AP-1, of which promote the transcription of several genes involved in proliferation, synthesis of Ig, and cytokine receptors B Cell Maturation Events [TD]

- Class Switching: Changes the class/function of the Ab but not Ag specificity [Fab = unchanged. Fc = Changed] - Somatic Hypermutation à Affinity maturation. BCRs are fine tuned to have increased affinity to Ag, thus, Fab

region is changed - Differentiation into Ab producing memory B Cells

Antibody Isotypes - IgG: The most prevalent (85%) Ig that is very easily distributed throughout the body. It has the longest half-life of

all the Ig (23 days), and is most commonly found in the serum and intercellular fluid o Function: Complement activation, opsonization (IgG only! – in spleen), cross placenta, agglutination

- IgM: The second most abundant (5-10%), with a half-life of 5 days, it is located in the serum. Pentameric o Function: Complement activation, agglutination, neutralization

- IgA: In external secretions (like breast milk). Has odd wishbone-like structures - IgE: Triggers the release of histamine from basophils and mast cells - IgD: NOBODY KNOWS. Found on B Cell surface though

Negative Regulation: Ab Feedback: There are 3 mechanisms by which the humoral response is downregulated - Ab eliminates Ag source à No further stimulation - Sufficient soluble Ab-Ag complexes have been generated à Inhibit B Cell activation - Clustering of membrane Ig with Fc receptor on surface of B Cell à Sends inhibitory signal to terminate B

activity