Some Chapter 10 study questions: What are the major steps required to generate robust B cell activation? What cell types are involved? How do B cell homing properties change over the course of B cell activation? What mediates this process? What critical events occur during formation of a B cell immunological synapse with a helper T cell? What is the function of the synapse? What is the basis of the hapten-carrier effect? What is the role of the germinal center? What cell types are present there? How do antibodies act by neutralizing immunity, by fixing complement, and by antibody-dependent cellular cytotoxicity (ADCC)? Which antibodies are typically multimeric? What are the special functions of IgE?

Chapter 10 Humoral Immunity Hepatitis C virus coated with neutralizing antibodies (green) Dean Tantin, PhD Department of Pathology Division of Microbiology & Immunology University of Utah JMRB 5200K Lecture - Oct. 9, 2014 B cell (blue) interacting with a helper T cell

1.Activation of naïve (mature) B cells 2.Effector functions of antibodies a)Neutralization b)Opsonization (coating) to enhance phagocytosis c)Activation of complement system d) Specialized functions of IgE Outline for today

Figure 8.39 YOU ARE HERE Development of B lymphocytes

Where this happens: Spleen (blood Ag) Lymph nodes (skin, mucosal) GALT (intestinal)

Atlas of Blood Cells Follicular B cells: circulate between secondary lymphoid organs (2-3X/day). Most never encounter antigen and die. Some proteins on surface: BCR (antigen recognition), CXCR5 (homing) and BAFF receptor (survival). a fibroblast…

Figure 10.2 BCR MHC-II Cytokine receptor Strong, high-affinity responses, esp to protein antigens Broad, abundant low- affinity antibodies against things like polysaccharides

Figure 10.2 Figure 7.23 BCR MHC-II Cytokine receptor

Complement augments BCR signal transmission CD19: B cell-specific Ig superfamily member Associates with CR2/CD21 Augments BCR signaling

Complement augments BCR signal transmission Complement receptor-deficient mice fail to mount an efficient immune response

Response of B cells with BCR plus costimulation Activate T H cells Receive T H signals

Figure 10.5

Receptor- mediated endocytosis of antigen MHC-II

Figure 10.5 Antigen processing and presentation T cell recognition of antigen MHC-II

Figure 10.5 Full-blown B cell activation. GC reaction. Plasma/memory differentiation MHC-II

Concept: B and T lymphocytes respond to Ag separately and migrate into proximity through the action of chemokines, then physically interact.

Can we visualize this in a REAL situation? YES! DC can also display Ag to B cells B cells express TLRs T cells: CD28, CD40L B cells: B7-1/2, CD40

Gunzer, et al. Blood 104: 2801 Transgenic mouse expressing Added immunoglobulin genes: Because of allelic exclusion, the endogenous Ig genes do no rearrange. As a consequence, the mouse produces only 1 type of immunoglobulin. PROVIDES WHAT A SCIENTIST NEEDS--EXPERIMENTAL CONTROL. Normally only 1 in B cells recognize a specific antigen

A T cell–B cell pair in cultured cells. B cells labeled green. Time is 15 seconds per frame and approximately 80 minutes total observation. Gunzer, et al. Blood 104: 2801

T–B cell pair migrating at the interface of the T-cell zone and a B-cell follicle of the inguinal lymph node in a living mouse 4 hours after injection of T cells. T cells stained red and B cells green. Time is 45 seconds per frame and approximately 50 minutes total observation. Gunzer, et al. Blood 104: 2801

Figure 10.3 “Immunological Synapse”

MHC/peptideICAM-1Overlay A term stolen from neurobiology Between T helper cell and APC ICAM-1 B7 CD40 MHC II/peptide TCR CD4 CD28 CD40 ligand “Immunological Synapse”

Figure 10.6 More on the immunological synapse…

Some helper T cells migrate towards the follicle (they now express CXCR5) From the T cell perspective…

Figure 10.5 Antigen processing and presentation T cell recognition of antigen MHC-II These can be different epitopes. The BCR doesn’t even have to recognize protein antigens.

Concept: B and T lymphocytes respond to Ag separately and migrate into proximity by the action of secreted chemokines, then physically interact. “The Hapten-Carrier Effect” --Exemplar: 2,4-Dinitrophenol Haptens ONLY generate responses when coupled to large carriers Generation of secondary/memory responses: The animal must be immunized with the same carrier conjugate (the carrier effect). Memory T cells: to the CARRIER Memory B cells: the the HAPTEN

Figure 1.18 Most CSR and all SHM takes place in germinal centers

Figure Boundary of mantle zone defines a lymphoid follicle that contains a germinal center GC=dark zone + light zone Dark zone – site of proliferation and AID-mediated diversification Light zone – site of selection Red cells are T FH M  gobble up the apoptotic B cells Most CSR and all SHM takes place in germinal centers

Figure Dark zone: proliferation/ generation of variability Light zone: FDCs are presenting antigen…

Figure Dark zone: proliferation/g eneration of variability Light zone: FDCs are presenting antigen… …only the cells best able to take up Ag and present to follicular helper T cells survive!

1.Activation of naïve (mature) B cells 2.Effector functions of antibodies a)Neutralization b)Activation of complement system c)Opsonization (coating) to enhance phagocytosis d)Antibody-dependent cellular cytotoxicity (ADCC) d) Ch.14 brief preview: specialized functions of IgE Outline for today

1.Humoral immunity is mediated by secreted antibodies a. Therefore they can act at a distance from the site of production b. Action in, e.g., blood, mucosal surfaces, gut, lung 2.Antibody functions are triggered by Ag binding to Antibody V regions 3.Most blood protective antibodies made by long-lived plasma cells in BM Figure 4.1 Overview of humoral immunity Fc receptor/compl ement binding

Figure 10.1 Secreted antibodies have multiple effector functions

1.Activation of naïve (mature) B cells 2.Effector functions of antibodies a)Neutralization b)Activation of complement system c)Opsonization (coating) to enhance phagocytosis d)Antibody-dependent cellular cytotoxicity (ADCC) d) Ch.14 brief preview: specialized functions of IgE Outline for today

Neutralization of microbes and their toxins Figure 10.26, Hepatitis C virus coated with neutralizing antibodies (green) Prevent microorganisms and their toxic products from binding to host cells Requires only antigen- binding (V) region of the antibody (i.e., passive mechanism) Neutralizing activity can be mediated by any secreted antibody isotype

Neutralization of microbes and their toxins Figure Prevent microorganisms and their toxic products from binding to host cells Requires only antigen- binding (V) region of the antibody (i.e., passive mechanism) Neutralizing activity can be mediated by any secreted antibody isotype

Neutralization of microbes and their toxins in the gut by IgA Figure 10.22

Many of our best vaccines stimulate production of neutralizing antibodies

1.Activation of naïve (mature) B cells 2.Effector functions of antibodies a)Neutralization b)Activation of complement system c)Opsonization (coating) to enhance phagocytosis d)Antibody-dependent cellular cytotoxicity (ADCC) d) Ch.14 brief preview: specialized functions of IgE Outline for today

The classical pathway is initiated by C1 Figure 2.17

The classical pathway is initiated by bound IgM/IgG Interaction with C1 through constant regions Fc receptor/compl ement (C1) binding Figure Blue: IgM (L), IgG (R) Yellow: C1q Green/purple: C1r/s

1.Activation of naïve (mature) B cells 2.Effector functions of antibodies a)Neutralization b)Activation of complement system c)Opsonization (coating) to enhance phagocytosis d)Antibody-dependent cellular cytotoxicity (ADCC) d) Ch.14 brief preview: specialized functions of IgE Outline for today

Antibody-mediated opsonization and phagocytosis of microorganisms Phagocytes bind to microbes coated with specific antibodies. IgG is particularly good at this. IgG binding enhances phagocytosis and killing. The process of coating microbes with IgG to enhance phagocytosis and bacterial killing is called opsonization. IgG is bound by Fc  RI, expressed on different phagocytes. Fc: crystallizable fragment,  : IgG, R1: receptor 1 (Fc  RI=CD64).

Figure Not shown (well) here or in 10.34: clustering of Fc receptor is obligatory Complement receptors (e.g., CR1) can further enhance

1.Activation of naïve (mature) B cells 2.Effector functions of antibodies a)Neutralization b)Activation of complement system c)Opsonization (coating) to enhance phagocytosis d)Antibody-dependent cellular cytotoxicity (ADCC) d) Ch.14 brief preview: specialized functions of IgE Outline for today

Figure NK cells bind to antibody-coated target cells by Fc  RIII (=CD16). Fc  RIII is low affinity (requiring multivalent interactions). NK-mediated target cell destruction. Monomeric IgG does not activate NK cells. Once again, clustering is obligatory.

1.Activation of naïve (mature) B cells 2.Effector functions of antibodies a)Neutralization b)Activation of complement system c)Opsonization (coating) to enhance phagocytosis d)Antibody-dependent cellular cytotoxicity (ADCC) d) Ch.14 brief preview: specialized functions of IgE Outline for today

Figure Ig Class Switch Recombination (CSR) is instructed Unlike V(D)J, which is largely random CSR is initiated by CD40L:CD40 + appropriate cytokine

1.IgE Structure a) Like IgM, has an extra C H 4 domain in the constant region. b) Binds Fc  RI with very high affinity. 2.IgE Properties a) IgE does not fix complement. b) The least common serum Ig because it binds tightly to Fc receptors on basophils and mast cells in tissue even before interacting with antigen. c) IgE plays a role in parasitic helminth diseases. Since serum IgE levels rise in parasitic diseases, measuring IgE levels is helpful in diagnosing parasitic infections. Binding of eosinophils to IgE-coated helminths results in killing of the parasite. d) As a consequence of its binding to basophils and mast cells, IgE is also involved in allergic reactions. Binding of the allergen to the IgE on the cells results in the release of mediators (such as histamine) that result in allergic symptoms.

Figure Very rapid (seconds) mast cell degranulation following IgE crosslink Because of the high affinity, IgE-coated mast cells sit there in your tissues…like a loaded gun.

Some Chapter 10 study questions: What are the major steps required to generate robust B cell activation? What cell types are involved? How do B cell homing properties change over the course of B cell activation? What mediates this process? What critical events occur during formation of a B cell immunological synapse with a helper T cell? What is the function of the synapse? What is the basis of the hapten-carrier effect? What is the role of the germinal center? What cell types are present there? How do antibodies act by neutralizing immunity, by fixing complement, and by antibody-dependent cellular cytotoxicity (ADCC)? Which antibodies are typically multimeric? What are the special functions of IgE?