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Activation of B cells and Production of Antibodies

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1 Activation of B cells and Production of Antibodies
Jason Cyster, PhD 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

2 Activation of B cells and Production of Antibodies
Fab of broadly neutralizing antibody Hemagglutinin of influenza H1N1 How is it that we can make an antibody against any foreign surface? Human mAb CH56 from a vaccinated individual: neutralizes infectivity of 30 out of 36 H1N1 strains tested. Whittle…Harrison PNAS Aug 2011

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) (continual) (continual) (a few days) (1-2 weeks) Development, VDJ recombination and generation of a diverse B cell repertoire has been discussed, as has recirculation and migration of B cells through lymphoid organs. Here we are going to pick up on the events from antigen-recognition. Low-affinity Plasma cells -> Antibody Germinal Center formation: somatic mutation and affinity maturation High-affinity Plasma cells (->Antibody) and memory B cells (weeks)

4 (BCR) -> Changes in gene expression include upregulation of B7
Syk is very similar to the T cell kinase, ZAP70 -> Changes in gene expression include upregulation of B7

5 Types of B cell Antigens: T-independent and T-dependent
T-independent (TI) T-cell dependent (TD) Ag Ag Ag present T cell Ag BCR clonal expansion; differentiation 'activation' signal clonal expansion; differentiation but no clonal expansion 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 T-dependent antigens - require a protein component, but B cell may recognize a non-protein part of the complex (e.g. a surface sugar or lipid of a virus)

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 BCR crosslinking by antigen leads to internalization of the antigen-BCR complex, degradation of the antigen, association of peptides from the degraded antigen with MHC class II molecules and then presentation of these MHC-peptide complexes on the surface of the B cells for recognition by the TCR on CD4 helper T cells

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) NB: Five types of hyper IgM syndrome have been characterized: Hyper-IgM syndrome type 1, characterized by mutations of the CD40LG gene. In this type, T cells cannot tell B cells to switch classes. Hyper-IgM syndrome type 2, characterized by mutations of the AICDA gene. In this type, B cells cannot recombine genetic material to change heavy chain production, which is a required step in switching classes. Hyper-IgM syndrome type 3, characterized by mutations of the CD40 gene. In this type, B cells cannot receive the signal from T cells to switch classes. Hyper-IgM syndrome type 4, which is a defect in class switch recombination downstream of the AICDA gene that does not impair Somatic Hyper Mutation.[1] Hyper-IgM syndrome type 5, characterized by mutations of the UNG gene. 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 Explain these concepts while showing the figure

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

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 Urushiol in poison oak and the nickel in earrings that cause contact dermatitis are both haptens involved in T cell-mediated DTH reactions

13 Making an antibody response to a hapten
Hapten covalently attaches to self-protein Hapten specific B cell binds haptenated-protein Complex is internalized and degraded to haptenated peptides Haptenated peptides are presented to T cells B cell receives help and secretes hapten specific antibody hapten protein Ab + CD40L Cytokines T Haptenated-peptide Specific T cell haptenated self-peptide Penicillin can induce both IgE and immediate type HS and IgG, leading to immune complex HS Why do some people respond to Penicillin if there are no costimulatory inputs? Perhaps there are i.e. penicillin is given to people with bacterial infection Note that allergies of this type have a genetic basis and tend to run in families Hapten Specific B cell

14 After appropriate activation the B cell differentiates into an antibody secreting cell (or Plasma Cell) B cell Plasma Cell membrane Ig secretory Ig Electron microscopy Images from Molecular Biology of the Cell Alberts et al., 3rd Edition., 1994 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
B cell Plasma Cell membrane Ig (BCR) secretory Ig (Ab) V C tm cy V C Maybe delete this slide H H H H polyA polyA B cells express Ig Heavy chain transcripts that include transmembrane and cytoplasmic domains Plasma cells express Ig Heavy chain transcripts that stop after the CH domains, thereby encoding the same antibody but in a secreted form

16 B cell antibody response -> clonal replication enters into a
higher order upon plasma cell differentation (Note: the exact numbers are not important) 3-4 days 12 divisions 1 day differentiation 1 day 104 Ab/cell/sec naive B cell activated B cells plasma cells antibodies 4,096 >1012 1 212 = 4,096 The exact numbers are not important, just the overall concept that by becoming antibody secreting cells, B cells are able achieve huge clonal expansion of their antigen-specific effector molecule (their antibody); of course, early innate protection mechanisms help keep bacterial numbers in check while adaptive immune response comes into play bacteria - possibly dividing every ~60 min 5 days = 2120 divisions

17 Ig Heavy chain class (isotype) switching
variable constant m g VDJ e a 55 kb T cell help (cytokines, CD40L) IgG+ antigen memory cell IgM+ naive Tony may have gone over this already B cell IgG secreting Why make the different Ig isotypes? -> We will discuss antibody effector mechanisms in the next lecture plasma cell

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 Mutations are targeted to antigen binding region of antibody
Ag before Affinity maturation improves the ‘fit’ of the antibody for the inducing antigen Ag after Ag - increasing the binding affinity CDR 1 CDR 1 2 3 2 3 VL VH CH1 CL CH2 CDR = complementarity determining region, also known as the hypervariable region (part of V domain that binds the antigen) CH3

20 Affinity maturation and antibody responses

21 V CH1 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 V CH1 Met ... Gly Tyr Ala His Arg ... ...Gly, Pro... ...GGC, CCT... ATG ... GGC TAT GCT CAC CGT ... DNA replication AID dependent mutator complex error Mutations are ACTIVELY induced SELECTIVELY in the V region (made up by the V-D-J elements in the heavy chain and the V-J elements in the light chain) Occasional induction of mutations at other sites (in oncogenes) explains why several types of lymphoma emerge in germinal centers NB: AID (activation induced deaminase) deaminates Cytosine to Uracil; a repair protein comes in and this then leads to error prone repair Hyper-IgM syndrome type 4, which is a defect in class switch recombination downstream of the AICDA gene that does not impair Somatic Hyper Mutation.[1] AID = Activation Induced Deaminase (-> deaminates Cytosine on Uracil -> repair proteins then come in and this leads to error prone repair)

22 V CH1 Somatic mutation of Ig V region in GC B cell
ATG ... GGC TAT GTT CAC CGT ... Met ... Gly Tyr Val His Arg ... T Val ...GGC, CCT... ...Gly, Pro... CH1 -> 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) Cytidine deamination to uracil explains some of the known biases of somatic hypermutation such as transition-type mutations from C to T or G to A. More difficult to explain is how deamination of C could give rise to mutations at A-T base pairs, which are also common in SHM. It is possible that when repair mechanisms are triggered by a U-G mismatch, nicks in DNA are created and there is more extensive and error-prone repair through DNA replication.

23 Germinal Center in Human Tonsil
mantle zone (naive B cells) GC light zone (bright green staining, FDCs) GC dark zone (red - cell cycle marker-high cells that are rapidly dividing GC B cells) from Liu et al., Immunology Today 13, (1992) Consider whether to mention ICOS-ICOSL; follicular helper T cells T zone

24 Germinal Center Dynamics
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) antigen 4. Differentiation & Exit -> high affinity (selected) B cells differentiate into long-lived plasma cells and memory B cells that exit the GC B T B F F B T T PC B B B B B MB B B B B B Follicular Dendritic Cells (FDCs) have FcR and complement receptors and display antigen for long periods. B cells compete to bind this antigen. GC gives rise to plasma cells and memory B cells. Memory B cells are small B cells that look much like a naïve B cell, but they are often isotype switched so express surface IgG (or IgA or IgE) instead of IgM/D, and they often have higher affinity for the inducing antigen. They live for a long time and are able to recirculate between lymphoid organs looking for antigen. B B B Response takes 1-2 weeks B 2. Dark Zone -> GC B cells (blasts) undergo proliferation and somatic mutation of Ig V genes B B B T Seeding GC seeded by low affinity B cells that bound antigen and received T cell help

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
(may come to this summary slide in the Effector lecture). In the first week, before the GC response, IgM and some IgG are produced but mostly low affinity. By day 10 the GC response has gotten underway and you start to make high affinity antibody. Antibody secreting cells are the same thing as Plasma cells - they are present in secondary lymphoid tissues, bone marrow and in the mucosal epithelia. Memory B cells arising from GC are higher affinity. On secondary exposure you are able to make a more rapid response because you have more B cells present that are specific for the antigen (and they have other differences that make them fast responders). Plasma cells emerging from GC home preferentially to the bone marrow and live for many months or even years, contributing to long term antibody production.

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