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Activation of B cells and Production of Antibodies Learning Objectives of lecture: Describe the key changes that occur in the B cell upon binding antigen.

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Presentation on theme: "Activation of B cells and Production of Antibodies Learning Objectives of lecture: Describe the key changes that occur in the B cell upon binding antigen."— Presentation transcript:

1 Activation of B cells and Production of Antibodies Learning Objectives of lecture: Describe the key changes that occur in the B cell upon binding antigen Understand the 2 classes of antigen, T-independent & T-dependent Explain what a B cell must do in order to receive T cell help Describe the major components of T cell help and appreciate the defects that can cause humoral immunodeficiency Explain how a conjugate vaccine works and how you can make an antibody response to a hapten Explain how isotype switching occurs Describe the properties of plasma cells Describe the process of antibody affinity maturation in germinal centers, recognizing the 2 key processes involved and the cellular outputs Understand the main Ig isotypes made during memory responses and why these responses are faster and of greater magnitude Jason Cyster, PhD

2 Hemagglutinin of influenza H1N1 Fab of broadly neutralizing antibody Activation of B cells and Production of Antibodies How is it that we can make an antibody against any foreign surface?

3 The life history of B lymphocytes Newly produced B cells leave bone marrow and enter circulation Migrate through secondary lymphoid organs and survey for antigens Antigen-recognition; Interaction with helper T cells; clonal expansion; (isotype switching) Low-affinity Plasma cells -> Antibody Germinal Center formation: somatic mutation and affinity maturation High-affinity Plasma cells (->Antibody) and memory B cells (continual) (a few days)(1-2 weeks) (weeks)

4 (BCR) -> Changes in gene expression include upregulation of B7

5 T cell clonal expansion; differentiation 'activation' signal but no clonal expansion present Ag T-independent (TI) T-cell dependent (TD) Types of B cell Antigens: T-independent and T-dependent clonal expansion; differentiation BCR Ag T-independent antigens are multivalent (e.g. bacterial polysaccharides or repeating determinants on the surface of viruses) –fast (within 1-2 days) and predominantly IgM –weak in infants and young children T-dependent antigens must contain a protein component (true of most antigens) so that T cell help can be received –slower (initiate over several days), involve all Ig isotypes (IgM, IgG, IgA, IgE) –can lead to antibody affinity maturation and memory

6 (T zone) Antigen (red) Specific B cell (green)

7 Antigen presentation by B lymphocytes to helper T cells B cells present antigen they are specific for 100,000 times more efficiently than a non-specific antigen

8 Mechanisms of helper T cell-mediated activation of B lymphocytes

9 Role of CD40 in B cell activation TCR triggering up- regulates CD40L on T cell CD40 signaling promotes B cell activation, isotype switching CD40 also important in DC, Macrophage function CD40L-deficiency = 'hyper-IgM syndrome’ (X-linked) increased expression of cell cycle molecules, survival molecules, promotes isotype switching ICOS – ICOSL also required for Germinal Center responses ICOS deficiency is a cause of Common Variable Immunodeficiency (CVID)

10 Linked help and the Conjugate Vaccine concept Many bacteria are heavily coated with surface polysaccharides Vaccines against these bacteria aim to induce antibodies specific for the polysaccharide e.g. Haemophilus influenzae Type b vaccine Pneumococcal vaccine Meningococcal vaccine But infants and young children mount poor T-independent antibody responses Conjugate vaccines link the polysaccharide to an immunogenic protein carrier so that a T-dependent antibody response can be induced

11 Mounting a T-dependent antibody response to a polysaccharide in a conjugate vaccine foreign protein sugar (polysaccharide) Polysaccharide Specific B cell T Foreign protein specific T cell CD40L Cytokines BCR MHC II Ab endosome foreign peptide

12 Haptens and hypersensitivity reactions Small organic molecules do not provoke antibodies by themselves Antibodies can be raised against them if attached to a protein carrier Termed haptens (from the Greek haptein, to fasten) Some drugs (e.g. Penicillin) can act as haptens and induce antibody-mediated allergic reactions

13 Making an antibody response to a hapten proteinhapten Hapten Specific B cell + 1. Hapten covalently attaches to self-protein 2. Hapten specific B cell binds haptenated-protein 3. Complex is internalized and degraded to haptenated peptides 4. Haptenated peptides are presented to T cells 5. B cell receives help and secretes hapten specific antibody CD40L Cytokines T Haptenated- peptide Specific T cell haptenated self-peptide Ab

14 membrane Ig secretory Ig B cell Plasma Cell After appropriate activation the B cell differentiates into an antibody secreting cell (or Plasma Cell) After their generation in secondary lymphoid organs, many Plasma Cells home to the bone marrow or mucosal surfaces (or lactating mammary gland) where they live for many months, continually secreting antibody

15 Production of membrane vs secreted Ig membrane Ig (BCR) C H tm cy polyA secretory Ig (Ab) B cell Plasma Cell V H - B cells express Ig Heavy chain transcripts that include transmembrane and cytoplasmic domains - Plasma cells express Ig Heavy chain transcripts that stop after the C H domains, thereby encoding the same antibody but in a secreted form C H V H

16 naive B cell activated B cells 3-4 days 12 divisions plasma cells 1 day differentiation 1 day 10 4 Ab/cell/sec antibodies 12 12 = 4,096 4,096>10 12 B cell antibody response -> clonal replication enters into a higher order upon plasma cell differentation bacteria - possibly dividing every ~60 min 5 days = 2 120 divisions (Note: the exact numbers are not important)

17 Ig Heavy chain class (isotype) switching VDJ    55 kb (cytokines, CD40L) T cell help antigen IgM+ naive B cell IgG+ memory cell IgG secreting plasma cell constant variable Why make the different Ig isotypes? -> We will discuss antibody effector mechanisms in the next lecture

18 Affinity Maturation Affinity maturation occurs in germinal centers and is the result of (1) somatic hypermutation of Ig-genes in dividing B cells followed by (2) selection of B cells for their ability to bind more strongly (with higher affinity) to the inducing antigen The high affinity B cells emerging in germinal centers give rise to long-lived plasma cells and memory B cells

19 VHVH VLVL CLCL C H1 CDR 1 23 C H2 C H3 CDR 12 3 Ag Mutations are targeted to antigen binding region of antibody CDR = complementarity determining region, also known as the hypervariable region (part of V domain that binds the antigen) Ag before Affinity maturation improves the ‘fit’ of the antibody for the inducing antigen after Ag - increasing the binding affinity

20 Affinity maturation and antibody responses

21 AID dependent mutator complex DNA replication error ATG... GGC TAT GCT CAC CGT... V C H1...GGC, CCT... Met... Gly Tyr Ala His Arg......Gly, Pro... AID = Activation Induced Deaminase (-> deaminates Cytosine on Uracil -> repair proteins then come in and this leads to error prone repair) Somatic mutation of Ig V region in GC B cell -> mutations are actively induced in the V-regions of the antibody heavy and light chain genes

22 ATG... GGC TAT GTT CAC CGT... Met... Gly Tyr Val His Arg... T Val...GGC, CCT......Gly, Pro... V C H1 Somatic mutation of Ig V region in GC B cell -> now encodes antibody molecule with slightly altered antigen binding site -> sometimes, by chance, this site will have an improved ability to bind the inducing antigen (i.e. a higher affinity)

23 mantle zone GC light zone (bright green staining, FDCs) GC dark zone T zone (red - cell cycle marker-high cells that are rapidly dividing GC B cells) (naive B cells) Germinal Center in Human Tonsil

24 Germinal Center Dynamics B B B B B B B B B B B B MØ B T F F B B B B B T T T B B 2. Dark Zone -> GC B cells (blasts) undergo proliferation and somatic mutation of Ig V genes 3. Light Zone -> GC B cells compete to bind antigen displayed on Follicular Dendritic Cells (F) and to receive T cell help -> selection occurs for cells with higher affinity BCR -> cells that fail to bind antigen die and are engulfed by macrophages ( MØ) 1.Seeding GC seeded by low affinity B cells that bound antigen and received T cell help 4. Differentiation & Exit -> high affinity (selected) B cells differentiate into long-lived plasma cells and memory B cells that exit the GC MB PC antigen Response takes 1-2 weeks

25 Memory B cells Generated in germinal centers –therefore we only have strong humoral memory to T-dependent antigens Small, recirculating cells Often isotype switched (e.g. IgG + or IgA + ) Typically have higher affinity for the inducing Ag Longer lived than naïve B cells –Persistence of memory B cells after an immune response ensures that we have increased numbers of B cells specific for the antigen and ready to respond on re-encounter

26 Features of primary and secondary antibody responses

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