Basic Immunology Chapter 7: Humoral Immune Responses I. Phases and Types of Humoral Immune Responses A. Intro 1.

Nave B lymph express 2 classes of membrane antibodies: IgM and IgD that function as receptors for antigens a. Activated by antigens binding to membrane Ig b. Activation of B lymph results in proliferation of antigen specific cells (clonal expansion) c. Plasma cells actively secrete antibodies and are effector cells of humoral immunity. 2. Antibodies secreted in response to microbial antigen have the same specificity as that of the surface receptors on nave B cells that recognize the antigen 3. Heavy-chain isotype switching during differentiation, B cells may being to produce AB of different heavy chain isotype, which mediate different effector functions and specialized to combat different types of microbes 4. Affinity maturation repeated exposure to a protein antigen results in production of antibodies with increasing affinity for the antigen a. Leads to production of Abs with improved capacity to bind to neutralize microbes and toxins B. Antibody responses to different antigens are classified as T dependent or T independent 1. T dependent protein antigens and the antibody response to these antigens a. Protein antigens are processed in antigen presenting cells (APCs) and recognized by helper T lymphocytes 2. T independent polysaccharides, lipids and other nonprotein antigens stimulate Ab production w/o involvement of helper T cells a. Ab show little heavy chain isotype switching ad affinity maturation C. Different subsets of B cells responds preferentially to protein and non protein antigens 1. Majority of b cells are follicular b cells a. Make up bulk of Tdependent Ab response to protein antigens and give rise to long lived plasma cells 2. Marginal zone B cells and B-1 Cells responds to nonprotein antigens a. Make predominantly IgM responses D. Ab responses to the first and subsequent exposures to an antigen, called primary and secondary responses, different quantitatively and qualitatively 1. Amt of Ab produced after first encounter with an antigen (primary immune response) are smaller than amt produced after repeated immunization (secondary immune response) a. With protein antigens, secondary response also who increased heavy chain isotype switching and affinity maturation because repeated stimulation by antigen leads to increase in number and activity of helper T lymphocytes Stimulation of B Lymphocytes by Antigen A. Humoral immune responses are initiated when antigen-specific B lymphocytes in the spleen, lymph nodes, and mucosal lymphoid tissues recognize antigens 1. B lymphocytes specific an antigen use their membrane-bound Ig receptors to recognize antigen directly without any need for processing a. Triggers signaling pathways that initiate B cell activation b. Also requires signals in addition to antigen recognition B. Antigen-Induced Signaling in B Cells 1. Antigen induced clustering of membrane Ig receptors triggers biochemical signals that are transduced by receptor associated signaling molecules a. Ig receptor mediated signal transduction requires cross linking of two or more receptor molecules b. Receptor crosslinking occurs when tow or more antigen molecules in a aggregate bind to adjacent membrane Ig molecule of a B cell 2. Signals initiated by antigen receptor cross linking are transduced by recepotr associated proteins a. Membrane IgM and IgD are noncovalently associated with IgAlpha and IgBeta to form B cell receptor (BCR) complex b. IgAlpha and IgBeta have immunoreceptor tyrosine based activated motifs (ITAMs)

II.

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When antigen receptors of B cell are clustered, the tyrosine in the ITAMs are phosphorylated by kinases d. Phosphotyrosines recruit Syk tyrosine kinase which phosphorylates tyrosine residues on adaptor proteins that recruit a number of downstream signaling molecules e. Net result is activation of transcription factors that switch on the expression of genes involved in cell proliferation and differentiation C. Role of Complement Proteins and other innate Immune Signals in B Cell activation 1. B lymphocytes express a receptor for a protein of the complement system that provides signals for activation of these cells a. Complement system collection of plasma proteins that are activated by microbes and antibodies attached to microbes and function as effectors of host defense b. B lymphocytes express complement receptor type 2 (CR2) which binds C3d (fragment of complement protein) c. B cells specific for microbes antigens recognize antigen by Ig receptors and simultaneously recognize the bound C3d via the CR2 receptor (greatly enhances antigen dependent activation) 2. Microbial products also directly influence B cell activation a. B lymph express toll like receptors (TLRs) b. TLR engagement b microbial products triggers avtiavting signals that work in concert with signals from antigen receptors D. Functional Consequences of B Cell activation by Antigen 1. B Cell activation initiates the proliferation and differentiation of the eland prepares them to interact with helper T lymphocytes a. Enter cell cycle and being to proliferate b. Response is greatest when antigen is multivalent, cross-links many antigen receptors and activates complement strongly i. Seen with T-independent antigens 2. Protein antigens induce signals in b lymphocytes that lead to important changes in cells that enhance ability to interact with helper T lymphocytes a. Increased expression of receptors for chemokines that are produced in the T cell zones of lymphoid organs b. Activated B cells migrate out of follicles and toward the helper T cells c. B Cells can interact with helper T cells that have been activated by the same antigen presented to nave T cells by dendritic cells Function of Helper T Lymphocytes in Humoral Immune Responses to Protein Antigens A. Process of T-B Cell Interaction and T cell dependent antibody responses 1. CD4+ helper T cells and b cells activated by protein antigen in different regions of lymphoid organ and migrate toward each other 2. T and Cells initially interact outside the follicles 3. Initial antigen specific T-B cell interaction consists of 2 phases a. B cells process and present antigen to T cells b. Previously activated helper T cell express CD40 ligand and secrete cytokines, which act on B cells to initiate proliferation and differentiation to plasma cells i. Early Ab response, antibody secretion by plasma cells and some degree of isotype switching, occurs in these extrafollicular foci 4. Some activated B cells migrate back into the follicle, accompanied by helper T cells mean to develop into follicular helper T cells (TFH cells) a. In response to signals from TFH cells, B cells begin to proliferate, forming an organized structure called a germinal center, and the proliferation germinal center B cells under extensive somatic mutation of the antibody gene variable regions and Ig heavy chain isotype switching b. High affinity B cells are selected in germinal center, result in production of high affinity antibodies c. Germinal center reaction also results in long lived plasma cells and memory b Cells B. Activation and Migration of Helper T cells 1. Helper T cells that have been activated by dendritic cells migrate toward the b cell Zone and interact with antigen-stimulated B lymphocytes in parafolicular zones of the peripheral lymphoid organs a. Initial activation of T cells requires antigen recognition and co stimulation i. Antigens typically bound to class II MHC molecules of APCs in the T cell rich zones

c.

Directed migration of activated B and t cells toward one another depends on changes in the expression of certain chemokine receptor i. T cells reduced expression of CCR7, which recognizes chemokines produced in T cell zones, and increases expression of CXCR5, which promotes migration into B cell follicles ii. B cells increase CCR7 expression iii. Migrated toward one another and meet C. Presentation of Antigens by B Lymphocytes to Helper T cells 1. B lymphocytes that bind protein antigens by their membrane Ig receptors endocytose antigens, process them in endosomes, and display class II MHC-associated peptides for recognition by CD4+ helper T cells a. B cells recognize the native epitopes i. Also express costimulators (B7 molecules) that play a role in T cell activation b. Helper T cells recognize the peptide fragments of the antigens D. Mechanisms of Helper T Cell-Mediated Activation of B Lymphocytes 1. Helper T lymph that recognize antigen presented by B cells express CD40 ligand (CD40L) and secrete cytokines, which activate the antigen-specific B Cells a. Process of helper T cell mediated B lymph activation is analogous to process of T cell mediated macrophage activated in cell mediated immunity b. CD40L binds to CD40 on B lymph, which delivers signals to B cells that stimulate proliferation (clonal expansion) and the synthesis and secretion of Abs i. Ensures only T and B lymph in physical contact engage in productive interactions c. Cytokines bind to cytokine receptors on B lymph and stimulate more B cell proliferation and Ig production E. Extrafollicular and Germinal Center Reactions 1. Initial T-B interaction results in production of low levels of Abs, plasma cells, and few memory cells 2. Helper T cells express high levels of CXCR5 which draws T cells into the adjacent follicles a. Follicular helper T cells (TFH cells) T cells that migrate into follicles b. Dependent on costimulators ICOS of the CD28 family c. TFH cells may develop from TH1, TH2, and TH17 and may secrete cytokines such as IFN-gamma, IL-4, or IL-17. Most secrete cytokine IL-21 3. Few of the activated b cells from extrafollicular focus migrate back into follicle and begin to divide rapidly in response to signals from TFH cells a. Germinal center region containing proliferating B cells b. Germinal center B cells undergo extensive isotype switching and somatic mutation of Ig genes i. Highest affinity B cells are the ones selected at the end of germinal center reaction to differentiate into memory B cells and long lived plasma cells F. Heavy-Chain Isotype (Class) Switching 1. Helper T cells stimulate the progeny of IgM and IgD expressing B lymph to produce antibodies of different heavy chain isotypes a. Different antibody isotypes perform different functions IgM IgG subclasses IgE IgA IgD Signal IFN-gamma IL-4 Cytokines produced in mucosal tissues (TGF-beta, BAFF) Principal effector -Complement -Fc receptor -Defense against -Mucosal immunity Antigen receptor functions activation dependent helminthes (transport of IgA for nave B -Antigen receptor phagocyte helminthic parasites through epithelia): lymphocytes for nave B response too large to be IgA is secreted into lymphocytes -Complement phagocytosed. IgE lumens of GI and -Secreted form is activation coat parasites and respiratory tract pentamer -Neonatal recruit eosinophils, where it neutralizes immunity this which release their microbes and type of Ab can granule contents, toxins cross placenta which include -Secreted form in -Neutralization of proteins that kill dimer microbes and helminthes toxins -Mast cell

b.

-Opsonization of antigens for phagocytosis by macrophages and neutrophils

degranulation (Immediate hypersensitivity): Fc receptors on mast cells bind IgE with high affinity causing mast cell to release many mediators. Basis of allergic disease

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V.

2. Heavy-chain isotype switching is induced by combination of CD40L-mediated signals and cytokines a. Ig heavy chain locus - rearranged VDJ gene adjacent to first constant region cluster (C) b. MRNA produced by spliced VDJ exon to C exon in initially transcribed RNA and this mRNA is translated to produce heavy chain, which combines with light chain c. Signals from CD40 and cytokine receptors stimulate transcription through one of the constant regions downstream of C i. Intron 5 of each constant region has a conserved nucleotide sequence called the switch region ii. When downstream constant region becomes transcriptionally active, the switch region of 5 of C recombines with switch region 5 of that downstream constant region, and the intervening DNA is deleted d. Activation-induced deaminase (AID) is induced by CD40 converts cytosine in DNA to uracil i. Causes removal of the Us and creation of nicks in DNA, leading to ds DNA breaks ii. When dsDNA breaks in two switch regions are brought together and repaired, the intervening DNA is deleted and rearranged VDJ exon that was originally close to C may be brought immediately upstream of the constant region of a different isotype e. Process is called switch recombination result is that B cells begins to produce a new heavy chain isotype (determined by C region the antibody) with the same specificity as that of the original B-cells (specificity determined by rearranged VDJ exon) 3. Cytokines produced by helper T cells determine which heavy-chain isotype is produced by influencing which heavy chain constant-region gene participates in switch recombination G. Affinity Maturation 1. Affinity maturation: process by which the affinity of Abs produced in response to a protein antigen increases with prolonged or repeated exposure to that antigen a. Increase in affinity caused by point mutations in the V regions of the gene encoding the antibodies produced 2. Affinity maturation occurs in the germinal centers of lymphoid follicles and is the result of somatic hypermutation of Ig genes in dividing B cells, followed by the selection of high-affinity B cells by antigen a. AID converts C to U, which are converted to Ts during DNA replication or are removed and repaired by error prone mechanism that lead to mutations b. Follicular dendritic cells express receptors for the Fc portions of antibodies and for complement proteins, and B cells that recognize the antigen are activated to survive c. B cells also bind the antigen, process it and present it to helper T cells in germinal enters, which provide critical survival signals d. B cells selected to survive must be able to bind antigen at low concentrations, have high affinity e. Cells enter circulation and tend to migrate to bone marrow, where they mature in plasma cells f. Fraction of activated B cells become memory cells i. Do not secrete antibodies but can responds rapidly if antigen is reintroduced Antibody responses to T-Independent Antigens A. Polysaccharides lipids and other nonprotein antigens 1. Can crosslink many antigen receptors on a specific B cell which may activate B cells enough to stimulate their proliferation and differentiation Regulation of Humoral Immune Response: Antibody Feedback A. Antibody feedback 1. Most B cells die by a process of programmed cell death contribute to decline of humoral immune response 2. Antibody feedback antibody bound to antigen inhibits further Ab production

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c. Summary: I. II.

Secreted antibody forms complex with antigen Complexes interact with B cells specific for the antigen, with the membrane Ig antigen receptors recognizing epitopes of the antigen and a certain type of Fc receptor (FcRIIBI) recognizing bound Ab Fc receptors block activating signals from antigen receptor, terminating B cell activation

Humoral responses is mediated by antibodies that bind to extracellular microbes and their toxins, which are neutralized or targeted for destruction by phagocytes and the complement system Humoral responses to nonprotein antigens are initiated by recognition of the antigens by specific Ig receptors

Review Questions 1. What are the signals that induce B cell responses to protein antigens and polysaccharide antigens? 2. What are some of the differences between primary and secondary antibody responses to a protein antigen? 3. How do helper T cells specific for an antigen interact with B-lymphocytes specific for the same antigen? Where in a lymph node do these interactions mainly occur? 4. What are the mechanisms by which helper T cells stimulate B cell proliferation and differentiation? What are the similarities between these mechanisms and the mechanisms of T cellmediated macrophage activation? 5. What are the signals that induce heavy-chain isotype switching, and what is the importance of this phenomenon for host defense against different microbes? 6. What is affinity maturation? How is it induced, and how are high-affinity B cells selected to survive? 7. What are the characteristics of antibody responses to polysaccharides and lipids? What types of bacteria stimulate mostly these types of antibody responses?