B Cell Development PDF

Lymphocyte Development ▪ B cells, T cells, and NK cells Derivation: Hematopoietic stem cells → Multipotent progenitor cells → Common lymphoid progenitor cells → Lymphocytes This whole process is orchestrated by stromal cells and cytokines Humoral Immunit...

Lymphocyte Development ▪ B cells, T cells, and NK cells Derivation: Hematopoietic stem cells → Multipotent progenitor cells → Common lymphoid progenitor cells → Lymphocytes This whole process is orchestrated by stromal cells and cytokines Humoral Immunity ▪ Adaptive humoral Effector functions of B cells Mostly against extracellular microbes and their products Effector B cells are antibody secreting plasma cells Remember Ig lecture for effector functions of the antibodies B Cell Subsets ▪ Two classes of B cells B-1 B cells Primarily mature in the fetal liver and can self-renew Follicular (B-2) B cells Mature in the bone marrow – What we talk about here HSC = hematopoietic stem cell B Cell Receptor and Antibody Structure ▪ Mature, naïve B cells express BCR on their surface Composed of four peptide chains Joined by disulfide bonds 2 identical heavy chains 2 identical light chains – Each with variable and constant regions Transmembrane region anchors BCR to cell surface B Cell Receptor and Antibody Structure Transmembrane region anchors BCR to cell surface Interacts with CD79 (Igα + Igβ) for signaling The BCR complex (BCR + Igα + Igβ) is expressed by all B cells. It is important in signal transduction during antigen-induced B cell activation Note: Igα and Igβ are analogous to CD3 + ζ chains on the T cell B cell Development ▪ Development occurs in bone marrow Stromal Cells in bone marrow direct the development The BCR is developed during this process Random somatic recombination of V, J (light) and V, D, J (heavy) gene segments B cell Development ▪ Follows a fixed path of genetic rearrangement of Ig genes Starts with rearrangement of heavy chain http://www2.talkdesign.org/cs/evolving_immunity B cell Development ▪ Early Pro-B Cell starts with D-J rearrangement RAG1 and RAG2 B cell Development ▪ Late Pro-B Cell continues with V-DJ rearrangement RAG1 and RAG2 B cell Development ▪ Late Pro-B Cell continues with V-DJ rearrangement B cell Development ▪ Large Pre-B Cell Critical Step – block this, block further B cell development Occurs in ER Checks for functional heavy chain Checks compatibility with surrogate light chain – Note similarity to T cells and pTα Expression of Pre-BCR Survival and proliferation of the line Light chain recombination and inhibition of surrogate light chain synthesis What do you predict would happen to a patient with genetic deficiency in VpreB? Allelic Exclusion ▪ Ensure B Cell expresses only receptor type Why don’t we want B cells making more than one type of BCR? B Cells expressing multiple versions of BCR would be detrimental to immune response – Prevent high avidity binding or cross-linking of multiple BCRs bound to repeated antigens – Hamper T-independent B cell response Clinical Application: XLA ▪ X-linked agammaglobulinemia Identified in horses and humans Mutation in BTK – a kinase important in signal transduction from pre-B cell receptor If a functional pre-B cell receptor is made, signaling occurs for proliferation and differentiation of pre-B cells Btk mutation cannot transmit the signal, apoptosis of pre-B cell Disease is characterized by lack of B cells and lack of serum antibodies B cell Development ▪ Large Pre-B Cell Critical Step – block this, block further B cell development Occurs in ER Checks for functional heavy chain Checks compatibility with surrogate light chain – Note similarity to T cells and pTα Expression of Pre-BCR Survival and proliferation of the line Light chain recombination and inhibition of surrogate light chain synthesis B cell Development ▪ Small Pre-B Cell RAG1 and RAG2 B cell Development ▪ Multiple rearrangements possible To avoid cell loss RAG1 and RAG2 RAG1 and RAG2 RAG1 and RAG2 B cell Development ▪ Multiple rearrangements possible Kappa () or Lambda () Ratio of : varies by species – Humans 60:40 – Pigs 52:48 – Dogs 90:10 Less than half have productive rearrangements Use of  is done simply to increase odds of a successful rearrangement B cell Diversity ▪ Combinatorial Diversity Which V, D, and J segments are used in development of the BCR allele? How do the heavy chain and light chains interact? ▪ Junctional Diversity During splicing, which specific nucleotides are added or deleted? B cell Development ▪ Checkpoints for possible trouble Immature B Cells ▪ Makes a functional surface IgM receptor only ▪ Enters negative selection process Immature B cell ▪ Rearrangement ceases ▪ Express a functional surface IgM ▪ Goes through negative selection No strong reaction to self molecules → B cells mature Move out of bone marrow Reaction to self molecules leads to one of four outcomes Clonal deletion – removal of cells of a specific antigen specificity Receptor editing – further genetic rearrangement to replace BCR with one that does not self-react Anergy – permanent state of unresponsiveness, eventually leading to death Immunological ignorance – cells have affinity for self antigens, but do not respond Immature B Cells ▪ Negative selection ~75% of immature B cells have some affinity for self These are sent for receptor editing ▪ Receptor Editing Chance to save auto-reactive B cell VJ rearrangements on the light chain can occur Light chain editing only – Kappa followed by lambda Heavy chain remains the same Why? Immature B Cells ▪ Receptor Editing Heavy chain remains the same Why? – Structure of the heavy chain genetic region » The diversity segment in between variable and joining segments prevents further editing Immature B Cells ▪ Receptor Editing Immature B cell makes new light chain and new IgM Again undergoes negative selection process Light chain can continue to rearrange if still self- reactive Eventually killed via apoptosis Non-reactive cells enter blood Mature B Cells ▪ After selection, alternative RNA splicing of the primary RNA transcripts occurs in B-2 cells, producing two different mRNAs: VDJ region combined with the mu (μ) constant region VDJ region combined with the delta (δ) constant region ▪ Alternative RNA splicing allows B-2 B cells to synthesize both IgM & IgD Mature B-2 B cells expressing both mIgM & mIgD are released from the bone marrow Also express CD21 (CR2) B Cell Maturation ▪ Production of B cells 2.5 billion B cells PER DAY enter the B cell development protocol 30 billion B cell progeny leave the bone marrow Competition between B cells in the lymphoid tissue is intense – Most immature B cells fail to even enter the follicle and die – Mature B cells have a 100-day half-life B Cell Development Summary ▪ Generation of mature B cells occurs in stages that are defined by certain proteins that are expressed on the cell surface. Summary of major events: Progenitor B cell (Pro-B cell) Initial expression of CD19 & CD20 Initiation of somatic recombination of the heavy chain Precursor B cell (Pre-B cell) Successful recombination and expression of the heavy chain as a Pre-BCR Initiation of somatic recombination of the light chain Immature B cell Successful recombination and expression of a membrane-bound BCR complex (BCR is IgM) Negative selection; if necessary, light chain editing occurs Mature, naïve B cell B-1 B cells: expression of mIgM plus CD5 B-2 (follicular) B cells: expression of both mIgM & mIgD plus CD21 B Cell Development Summary Note: This summary is general for both cells; specific to B-2 B cells for the last step B Cell Activation ▪ B cell maturation is antigen-independent ▪ Antigen dependent phases: B cell activation T-independent antigens T-dependent antigens B cell differentiation B-2 B cells: effector B cells and memory (plasma) cells B-1 B cells: effector B cells B Cell Activation ▪ B cell recognition of antigens BCR binds free, unprocessed antigens MHC B Cell Activation ▪ B cell recognition of antigens https://www.nature.com/articles/nrmicro3415 T-dependent (TD) antigens Protein antigens that lead to B cell activation with helper T-dependent T cells T-independent (TI) antigens Non-protein antigens that T-independent lead to B cell activation without helper T cells B Cell Activation ▪ B-2 (follicular) vs. B-1 B cells B-2 B cells are in the follicles (hence the name) and respond mostly to T-dependent (TD) antigens B-1 B cells located in the mucosal tissues and pleural/peritoneal cavities and respond mostly to T- independent (TI) antigens Non-protein antigens that lead to B cell activation without helper T cells B Cell Activation ▪ Follicular B cell migration Circulate between blood and lymphoid tissues to find antigen As these B cells enter, they reside in & circulate through the follicles within the secondary lymphoid tissues Follicular B cells are attracted to the follicles by B cell specific chemokines produced by follicular dendritic cells (FDCs) If unactivated, B cells will exit following the S1P gradient – As learned for T cells B Cell Activation ▪ Follicular B cell migration Antigens enter the secondary lymphoid organs from the lymph/blood, by crossing epithelial barriers or by being carried in by dendritic cells to interact with receptors on the follicular B cells B Cell Activation ▪ Activation of B cell subsets occurs differently: B-2 (follicular) B cells respond primarily to TD (protein) antigens and result in: Their ability to process and present antigens to TH cells to activate and/or stimulate TH cells to provide signals to the B cell Differentiation into subpopulations of plasma cells, some are short-lived while others are long-lived Some B cells may undergo isotype switching Some B cells will undergo affinity maturation Development of memory B cells B-1 B cells respond primarily to TI (non-protein) antigens and result in: Differentiation into plasma cells that are generally short-lived Essentially no class-switching; usually just IgM is produced Essentially no memory B cell production B Cell Activation ▪ B-2 (follicular) B cell activation and differentiation When a follicular B cell recognizes a protein antigen, it becomes activated, resulting in proliferation and differentiation into effector B cells (plasma cells) and memory cells. Follicular B cells also can undergo isotype (class) switching, affinity maturation, and development of memory cells when helper T cells are involved B Cell Activation ▪ B-2 (follicular) B cell activation Signal 1: Follicular B cells are initially activated upon antigen binding to the BCR For signal transduction, aggregation of receptors is required to induce proliferation 1. Antigen binding and cross-linking two or more BCRs B Cell Activation ▪ B-2 (follicular) B cell activation * Signal 1: Follicular B cells are initially activated upon antigen binding to the BCR For signal transduction, aggregation of receptors is required to induce proliferation 1. Antigen binding and cross-linking two or more BCRs 2. Antigen cross-linking a BCR with the CR19/CD21 coreceptor *C3d is a breakdown product of C3b B Cell Activation ▪ B-2 (follicular) B cell activation Follicular B cells activated by 1st signal migrate to the outer edge of the follicle into the T cell zone B cell can interact with an activated Th cell (most likely) or the B cell can activate antigen-specific Th cells if unactivated previously (less likely) – B cells receive their second signal from Th cells B Cell Activation ▪ Reminder: B cells are professional APCs Follicular B cells present antigens to activate Th cells if they aren’t already activated by a dendritic cell CD40L * Your book labels CD40L as CD154 B Cell Activation ▪ Linked Recognition Activated by helper T cells that respond to same antigen CD40L That does not mean they bind the same sequence Peptide TCR binds is different from BCR epitope, but belongs to the same antigen BCR internalizes antigen, processes, and then displays the MHC their surface – Which class of MHC? Ensures self tolerance B cell by itself cannot instigate entire immune response T cell to the same antigen must also be present Linked Recognition ▪ Used in Vaccine development: ex: Haemophilus influenzae Adults make strong TI response to polysaccharide in capsule Infants do not make proper TI response to polysaccharides Polysaccharide is linked to tetanus toxoid B cells binding the polysaccharide are then activated by T cells that bind peptides in the tetanus toxoid B Cell Activation ▪ B-2 (follicular) B cell activation Second signal for follicular B cell activation comes from both the interaction of the B cell with the Th cell and the Th cell cytokines: CD40 - CD40L Th cytokines – IL-4 induces proliferation B Cell Differentiation ▪ After activation and proliferation A subset of the follicular B cells differentiate rapidly into antibody- producing plasma cells Located outside of follicle = extrafollicular focus Short-lived plasma cells producing low affinity IgM B Cell Differentiation ▪ After activation and proliferation Undifferentiated B cells migrate back into the follicle. Extrafollicular helper T cells also migrate into the follicle and differentiate into follicular helper T cells (Tfh) Site of the germinal center reaction B Cell Differentiation ▪ Germinal Center Reaction Follicular B cells interact with FDCs and Tfh cells Isotype (class) switching – Switch from IgM to another isotype Affinity maturation (somatic hypermutation) – Mutations to increase binding affinity of antibodies to the target – Both of these require Tfh assistance After these processes, the follicular B cells will differentiate into effector and memory cells B Cell Differentiation ▪ Class Switching Directed by Tfh interaction via CD40-CD40L and cytokines – Know the circled cytokines/antibody induction B Cell Differentiation ▪ Class Switching Recall: During B cell development, V-D-J recombination of the DNA encoding the heavy chain variable domain (V) occurred Downstream of the recombined V region are the constant gene segments encoding for mu, delta, gamma, epsilon, and alpha heavy chain Production of other isotypes (other than IgD) requires recombining the V region with a different constant region – Enzyme responsible: AID (Activation-induced cytidine deaminase) Clinical Application: Hyper IgM syndrome ▪ AID functions by intentionally introducing mutations that must be repaired These mutations lead to different amino acids in the antibody (somatic hypermutation) and potentially chromosome breaks (class switching) ▪ AID deficiency Produces normal or high concentrations of IgM with low or absent IgG, IgA, and IgE Patients have recurrent bacterial respiratory and GI infections B Cell Differentiation ▪ Affinity maturation Leads to increased binding affinity of Ig for its antigen Does not change Ig target Occurs via AID-induced somatic hypermutation within the gene segments encoding the variable domains of heavy and light chains Most likely to occur in hypervariable regions (aka complementarity determining regions) Each exposure to an antigen leads to a new round of affinity maturation Vaccine boosters re-up memory cells and also induce somatic hypermutation B Cell Differentiation ▪ After affinity maturation Mutations occur in proliferating B cells in the follicle Results in the generation of numerous follicular B cells, each having different mutations in the variable domains of the heavy and light chains – Altered B cells may now express a membrane-bound antibody (BCR) with a higher or lower binding affinity for the original antigen – After affinity maturation, the BCR expressed on a follicular B cell needs to be “tested” to it is still able to bind to the original antigen and do so with higher affinity » Undergo a second round of selection, mediated by follicular dendritic cells (FDCs) B Cell Differentiation ▪ After affinity maturation After isotype switching and somatic hypermutation, follicular B cells interact with follicular dendritic cells (FDCs) displaying the antigen using PRRs Follicular B cells expressing the highest affinity BCRs competitively bind to the antigen displayed by FDCs High affinity binders survive Lower affinity (or no affinity) binders undergo apoptosis B Cell Differentiation ▪ Exit from the lymphoid tissue High affinity follicular B cells differentiate into memory and plasma cells Some long-lived plasma cells migrate to the bone marrow and produce antibodies for months to years after the antigen is no longer present Memory follicular B cells enter the blood and circulate B Cell Differentiation ▪ Plasma Cells Essentially wholly committed to making antibodies Up to 20% of protein made by cells are antibodies No longer express MHC class II or co-stimulatory molecules – cannot present antigens Some plasma cells have short lives… others longer lived, provide for continued production B Cell Differentiation ▪ Memory B cells Memory cells circulate for months to years, possibly a lifetime Ready to respond rapidly if antigen is re-encountered Activation of memory B cells = secondary immune response Express a membrane-bound BCR that will be of the same isotype generated after class-switching with a higher affinity Review of Primary and Secondary Responses ▪ Primary immune response: Activation of naïve B cells Synthesize IgM first (short-lived plasma cells), affinity mature and class-switch to synthesize IgG, IgE, or IgA (long-lived plasma cells) Memory B cells are produced ▪ Secondary immune response: Faster activation of memory cells Synthesize IgG (or others); no IgM made Affinity maturation occurs creating higher affinity antibodies More memory B cells are produced B Cell Activation Overview ▪ Activation of B cell subsets occurs differently: B-2 (follicular) B cells respond primarily to TD (protein) antigens and result in: Their ability to process and present antigens to TH cells in order to activate and/or stimulate TH cells to provide signals to the B cell Differentiation into subpopulations of plasma cells, some are short-lived while others are long-lived Some B cells may undergo isotype switching Some B cells will undergo affinity maturation Development of memory B cells B-1 B cells respond primarily to TI (non-protein) antigens and result in: Differentiation into plasma cells that are generally short-lived Essentially no class-switching; usually just IgM is produced Essentially no memory B cell production Now, let’s examine the differences with B-1 B cells B-1 B Cell Activation ▪ B-1 B cells are important for antibody responses against T-independent (TI) antigens TI antigens are non-protein antigens such as polysaccharides, nucleic acids, and glycolipids and are often multivalent antigens (repeat epitopes) B-1 B Cell Activation ▪ The development of B-1 B cells is not fully understood. Mature B-1 B cells are produced from the HSC primarily during fetal development Mature B-1 B cells migrate to peritoneal and pleural cavities and to mucosal areas Mature B-1 B cells have the capacity to self-renew in these locations ▪ B-1 B cells serve as a front line of defense against infections at these locations. B-1 B Cell Activation ▪ A multivalent antigen binding and cross-linking of multiple BCRs acts as both the first and second activation signals Sufficient receptor aggregation initiates B cell activation No T cell involvement Generates short-lived, IgM producing plasma cells No memory cells Rapid antibody response Summary