B Cell Activation and Antibody Production Lecture 15

Overview of B Cell Development, Activation, Antibody Production

B Cell Antigens

B Cell Responses to Thymus- Dependent Antigens (T Cell- Dependent Antibody Responses)

Primary and Secondary Antibody Responses

Phases of the Humoral Immune Response

T Cell Epitope B Cell Epitope Antigen A T-Dependent Antigen Must Contain Both B and T Cell Epitopes LINKED RECOGNITION

(B Cells) (T Cells) Follicles

Activation of B Cells by Antigen and Complement 1. Biochemical Signals 2. Endocytosis of Antigen

Antigen Recognition Phase of T-Dependent Antibody Response

Interactions of B Cells with Helper T Cells Initial ContactT-B Conjugate TEM Picture Note the broad area of membrane contact between B and T Cells. B Cell T Cell

Helper T Cell-Dependent Activation Of B Lymphocytes

B-Cell Activation by Thymus-Dependent Antigens Cytokines Linked Recognition C’R

Activated B Cells (Following Interaction with T H Cells Extra-follicular SiteFollicle Antibody Secreting Cells Germinal Center Antibodies

Late Events in T Cell-Dependent Antibody Responses-Germinal Center Reaction Affinity Maturation –Somatic Hypermutation Generation of Memory B Cells Affinity Maturation –Somatic Hypermutation Generation of Memory B Cells

Somatic Hypermutation and Affinity Maturation of Antibodies Affinity maturation is the process that leads to increased affinity of antibodies for a particular antigen as a result of somatic mutation in the Ig genes followed by selective survival of B cells producing the antibodies with the highest affinity

Affinity Maturation in Antibody Responses

Selection of High Affinity B Cells in Germinal Center

Phases of the Humoral Immune Response to T-Dependent Antigen

Anatomy of Humoral Immune Responses

Antibody Isotype Switching

Isotype Switching Under the Influence of Helper T Cell-Derived Cytokines

Mechanism of Ig Isotype Switching

CD4 T Cell-Dependent Effects in Antibody Responses Memory B Cell Development Isotype Switching Affinity Maturation Memory B Cell Development Isotype Switching Affinity Maturation

Thymus- Independent Antigens

B-Cell Activation by Thymus-Independent and Dependent Antigens Most TI antigens are polyvalent and induce maximal Crosslinking of membrane Ig on B cells, without a Need for T cell help.

Features of Antibody Responses to T-Dependent and T-Independent Antigens

Antibody Response to T-Dependent Antigens Role of Helper T Cells –Cytokines –CD40/CD40L interactions Isotype Switching –Switch Recombination –Cytokines and Isotypes Affinity Maturation –Somatic hypermutation –Selection for B cells which produce High Affinity Antibodies Memory B Cells Role of Helper T Cells –Cytokines –CD40/CD40L interactions Isotype Switching –Switch Recombination –Cytokines and Isotypes Affinity Maturation –Somatic hypermutation –Selection for B cells which produce High Affinity Antibodies Memory B Cells

Antibody Effector Functions

Effector Functions of Antibodies

Neutralization of Microbes by Antibodies

Neutralization of Toxins by Antibodies

Opsonization of Microbes by Antibodies

Antibody-Dependent Cellular Cytotoxicity (ADCC)

Functions of Complement

Complement-Mediated Lysis of E. coli Alive Killed

Cellular Interactions in Immune Responses

The Immune Response: A Summary

WHY can immune system recognize so many different epitopes?? Antibody heavy and light chains are composed of gene segments Variable regions are unique A limited variety of constant region sequences are used They must be rearranged into functional genes before they can be transcribed

p. 106

Organization of Ig genes Germline DNA- gene segments surrounded by noncoding regions These are rearranged to form functional genes Light chains- V, J and C segments Heavy chain- V, D, J, C V regions rearrange first A single V can rearrange to more than one C

Multigene families  or In humans: 40 V , 5 J , 1 C  Similar number of genes in humans; this is rare in mice Heavy-chain gene families are similar but more complex (D segment) CH regions formed from exons

One of many possible combinations p. 111

Heavy chain DNA D-J and V-DJ rearrangements must occur separately On a mature B cell, both mIgM and mIgD are expressed on the cell surface

How does rearrangement occur? Each V, D and J is flanked by RSS (Recombination signal sequences) Mechanism is controlled by RAG-1 and RAG-2 proteins and an enzyme TdT If any of these proteins is defective no mature B cells can form; nor T cells

p. 112

“Junctional flexibility” contributes to diversity But not all rearrangements are “productive” p. 115

B cells are diploid and contain chromosomes from both parents However, heavy chain genes are rearranged from only one chromosome, as are light chain genes. Therefore, any one B cell will contain one V H and one V L (antigen specificity) How? Allelic exclusion (Yancopoulos and Alt, 1986)

Model for allelic exclusion p. 116

Generation of antibody diversity (why are there so many possible antigen combining sites?)

Multiple germline gene segments In human germline: 51 V H, 27 D, 6 J H 40 V , 5 J  30 V, 4 J

Combinatorial V-J and V-D-J joining 57 V X 27 D X 6 J= 8262 possible combinations for VDJ joining 40 V X 5J = 200 possible V  120 possible V 8262 X ( ) = 2.64 X 10 6 possible combinations Without taking into account other sources of diversity

Junctional flexibility in V-J or V-D-J junction Additional nucleotides added at junctions (P or N addition), if a single-stranded region is created during the joining process Somatic hypermutation mutations occur AFTER rearrangement tends to occur in CDR regions affects antigen affinity (tends to increase): “affinity maturation” occurs in B but not T cells

Class switching After antigen stimulation heavy-chain DNA can rearrange so VDJ joins to any isotype Cytokines help determine the isotype IgG2a or IgG3 (mice): IFN-  IgM: IL-2, IL-4, IL-5 IgE: IL-4

p. 122

Membrane-bound or secreted? Alternative splicing, p. 124

Mature B cells express both mIgM and mIgD No switch site between C  and C  The VDJC  C  contains 4 polyadenylation sites mIgM or mIgD can be generated depending on which polyadenylation site is used

Regulatory elements of transcription Promoters Enhancers Gene silencers Gene rearrangement brings enhancers close to the promoter they influence

Why aren’t Igs produced in B cells? In T cells a protein may bind to the  -enhancer and prevent V-J joining Arrangement of immunoglobulin genes (and formation from exons) and greatly facilitated formation of genetically engineered antibodies