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 10Humoral Immunity

Hepatitis C virus coated with neutralizing

antibodies (green)

Dean Tantin, PhDDepartment of Pathology

Division of Microbiology & ImmunologyUniversity of Utah

JMRB 5200K7-3035

dean.tantin@path.utah.eduLecture - Oct. 9, 2014

B cell (blue) interacting with a helper T cell

1. Activation of naïve (mature) B cells2. Effector functions of antibodies

a) Neutralizationb) Opsonization (coating) to enhance phagocytosisc) Activation of complement systemd) Specialized functions of IgE

Outline for today

Figure 8.39

YOUARE

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

Cytokinereceptor

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

Cytokinereceptor

Complement augments BCR signal transmissionCD19:•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 TH cells

Receive TH signals

Figure 10.5

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 expressingAdded 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 104-105 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/peptide ICAM-1 Overlay

• A term stolen from neurobiology• Between T helper cell and APC

ICAM-1B7CD40MHC II/peptide

TCRCD4CD28CD40 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 CARRIERMemory B cells: the the HAPTEN

Figure 1.18

Most CSR and all SHM takes place in germinal centers

Figure 10.12

• 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 TFH

• M gobble up the apoptotic B cells

Most CSR and all SHM takes place in germinal centers

Figure 10.13

• Dark zone: proliferation/ generation of variability

• Light zone: FDCs are presenting antigen…

Figure 10.13

• Dark zone: proliferation/generation 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 cells2. Effector functions of antibodies

a) Neutralizationb) Activation of complement systemc) Opsonization (coating) to enhance phagocytosisd) 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/complement binding

Figure 10.1

Secreted antibodies have multiple effector functions

1. Activation of naïve (mature) B cells2. Effector functions of antibodies

a) Neutralizationb) Activation of complement systemc) Opsonization (coating) to enhance phagocytosisd) 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, 10.27

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 10.28

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 cells2. Effector functions of antibodies

a) Neutralizationb) Activation of complement systemc) Opsonization (coating) to enhance phagocytosisd) 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/complement (C1) binding

Figure 10.29

Blue: IgM (L), IgG (R)

Yellow: C1q

Green/purple: C1r/s

1. Activation of naïve (mature) B cells2. Effector functions of antibodies

a) Neutralizationb) Activation of complement systemc) Opsonization (coating) to enhance phagocytosisd) 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 FcRI, expressed on different phagocytes. Fc: crystallizable fragment, : IgG, R1: receptor 1 (FcRI=CD64).

Figure 10.33

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 cells2. Effector functions of antibodies

a) Neutralizationb) Activation of complement systemc) Opsonization (coating) to enhance phagocytosisd) Antibody-dependent cellular cytotoxicity (ADCC)d) Ch.14 brief preview: specialized functions of IgE

Outline for today

Figure 10.36

• NK cells bind to antibody-coated target cells by FcRIII (=CD16). FcRIII 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 cells2. Effector functions of antibodies

a) Neutralizationb) Activation of complement systemc) Opsonization (coating) to enhance phagocytosisd) Antibody-dependent cellular cytotoxicity (ADCC)d) Ch.14 brief preview: specialized functions of IgE

Outline for today

Figure 10.15

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 Structurea) Like IgM, has an extra CH4 domain in the constant region.b) Binds FcRI with very high affinity.

2. IgE Propertiesa) 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 10.37

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?