Activation of B cells and Production of Antibodies

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?

3. The life history of B lymphocytes (continual) 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) (a few days) (1-2 weeks) Germinal Center formation: somatic mutation and affinity maturation High-affinity Plasma cells (->Antibody) and memory B cells Low-affinity Plasma cells -> Antibody (weeks)

4. (BCR) -> 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 T cell present 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 BCR clonal expansion; differentiation clonal expansion; differentiation 'activation' signal but no clonal expansion

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

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 self-peptide Haptenated-peptide Specific T cell Hapten Specific B cell

14. After appropriate activation the B cell differentiates into an antibody secreting cell (or Plasma Cell) Plasma Cell B cell membrane Ig secretory Ig 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 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 bacteria - possibly dividing every ~60 min 5 days = 2120 divisions

17. Ig Heavy chain class (isotype) switching variable constant m g e a VDJ 55 kb T cell help (cytokines, CD40L) IgG+ antigen memory cell IgM+ naive 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. Affinity maturation improves the ‘fit’ of the antibody for the inducing antigen CDR CDR 1 2 1 3 2 3 Ag Ag before after - increasing the binding affinity Mutations are targeted to antigen binding region of antibody Ag 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. DNA replication error 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 ... AID dependent mutator complex AID = Activation Induced Deaminase (-> deaminates Cytosine on Uracil -> repair proteins then come in and this leads to error prone repair)

22. Met ... Gly Tyr Val His Arg ... Val ...Gly, Pro... ...GGC, CCT... ATG ... GGC TAT GTT CAC CGT ... T Somatic mutation of Ig V region in GC B cell V 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)

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) 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) 4. Differentiation & Exit -> high affinity (selected) B cells differentiate into long-lived plasma cells and memory B cells that exit the GC antigen B T B F B F T PC T B B B B B MØ MB MØ B B B B B B B B Response takes 1-2 weeks B B 2. Dark Zone -> GC B cells (blasts) undergo proliferation and somatic mutation of Ig V genes B T B • 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