HE303 B Cells PDF

The adaptive immune response B cells – receptors, activation, effector functions BCRs Surface immunoglobulin (mIgM) Complexed with Ig⍺ and Igβ chains These have ITAMs for intracellular signaling Recognize three-dimensional (tertiary) structure of antigen ...

The adaptive immune response B cells – receptors, activation, effector functions BCRs Surface immunoglobulin (mIgM) Complexed with Ig⍺ and Igβ chains These have ITAMs for intracellular signaling Recognize three-dimensional (tertiary) structure of antigen What do lymphocyte receptors bind to? BCRs bind to the three dimensional (or tertiary) structure of an antigen (ex. part of a pathogen) It can be a protein, carbohydrate, lipid, nucleic acid TCRs do not bind to pathogens directly They bind to short fragments of peptide (primary structure) presented in a peptide receptor called MHC (major histocompatibility complex) Class I MHC are expressed on every cell in the body and present intracellular peptides to TCRs Class II MHC are expressed on specialized antigen-presenting cells (APCs; macrophages, dendritic cells) and generally present extracellular peptides to TCRs  * Somatic recombination and diversity There are many combinations of V(D) and J which can lead to a high level of diversity Recombination of lymphocyte receptor DNA occurs early in lymphocyte development and mature lymphocytes express rearranged receptors on their surface This process = lymphocyte diversity Lymphocyte receptor rearrangement is a random process which results in each person generating a very large and unique TCR and BCR repertoire B lymphocyte diversity takes place in the bone marrow T lymphocyte diversity takes place in the thymus BCR with V domain (VH and VL) that bind the antigen Both V domains are made by somatic recombination BCR rearrangement BCRs are made up of heavy and light chains Heavy chain genes are found on chromosome 14 and contain multiple V, D, J and C exons There are two kinds of light chain genes Kappa (κ) genes are found on chromosome 2 Lambda (ƛ) genes are found on chromosome 22 Light genes only contain V, D and C exons (NOT D) Each rearranged BCR expresses either ƛ or κ light chains (not both) BCR genes are commonly referred to as immunoglobulin (Ig) genes BCRs are antibodies stuck to the B cell surface Somatic recombination  Number of functional gene segments in human immunoglobulin genes This will vary in different people, due to genetic polymorphism But….this isn’t the only place for BCRs to generate diversity! B cell recombination – Bone marrow HSC produces B cell Pro—B cell committed to B cell lineage Starts recombining Ig genes Once IgM is on the surface the immature B cell leaves the bone marrow to complete maturation in the spleen Positive and negative selection happens in the bone marrow and spleen: Positive selection: does the BCR bind ligands? if yes, then allowed to survive Negative selection: Does the BCR bind self-ligands? If yes, then the cell tries to fix the receptor, if it can’t be fixed - deleted Positive selection BCR somatic hypermutation (SHM) More diversity! During an immune response, the BCR genes in activated (mature) B cells can undergo mutation to generate even more diversity Generation of high affinity antibodies to the pathogen Point mutations are introduced at a high frequency in dividing (activated) B cells – 103/cell division Mediated by enzymes called AID and UNG This further diversifies the variable regions of activated B cells Affinity maturation Somatic hypermutation leads to affinity maturation The ability of B cells to make very high-affinity secreted antibodies which can bind more tightly to the antigen Increase the quality of the specific antibody effector functions Both SHM and affinity maturation happen in germinal centres of the lymph nodes Germinal centres form in the lymph nodes approx. 6 days after primary immunization Proliferating B cell and helper T cells (follicular helper T cells, Tfh) Germinal centres Germinal centres (GCs) form in lymph nodes 6 days after primary immunization and are the focus of proliferating B cells and specialized help T cells (T follicular helper cells, Tfh) There are two areas Dark zone: B cell clonal expansion and somatic hypermutation Light zone: Selection for highest affinity BCRs The light zone also has follicular dendritic cells that: Retain/trap antigen long term to support B cell affinity maturation Maintain the GC structure  Dividing B cells containing these point mutations are competing for binding antigen on FDCs in the light zone of the germinal centre Favorable mutations allow the B cell to bind strongly to the antigen, these cells will get signals to remain activated Disadvantageous mutations mean the B cell can’t bind the antigen and won’t get any more activating signals Primary vs secondary response Somatic recombination and affinity maturation results in increased sequence diversity (increasing our BCR repertoire) Respond to a very wide range of pathogens we are exposed to throughout our lifetime Generate antibodies with increasing affinity as our immune response develops Memory cells will have the highest affinity BCRs for the pathogen Why our secondary immune response is better than our primary immune response -> the receptors recognize the pathogen better Activation of the adaptive immune system Now that our B cells have receptors that are capable of binding antigen but not ‘self’ they are ready to detect invading pathogens Main cell types of the adaptive immune response Conventional B cells: have diverse BCRs (referred to as B2 cells) which rely on T cell help for activation Plasma cells: activated B cells that produce antibodies (the Ig molecule released has exactly the same specificity as the membrane bound BCR) Unconventional B cells: less diverse BCRs (referred to as B1 cells) which are less dependent on T cell help for their activation Helper T cells: express the accessory molecule CD4 (which binds to an invariant portion of MHC class II) Cytotoxic (killer) T cells: express the accessory molecule CD8 (which can bind to the alpha 3 domain of MHC class I) TRegs: Regulatory T cells that suppress the activity of the above two populations and control immune responses Lymphocytes that have never seen an antigen before are called ‘naïve’ lymphocytes B cell activation The process has some similarities to T cell activation They need three signals Most of the time B cells get these signals from T H cells Sometimes they get the signals from the innate immune response  T cell dependent B cell activation 1. BCR binds antigen Directly, not through MHC 2. CD40 on B cell binds co- stimulatory molecules on TH cell (CD40L) 3. TH cell releases cytokines to enable B cell differentiation, proliferation and survival of the B cell T cell independent activation of B cells These are with antigens that carry a lot of antigens in one spot (multivalent) In this case: 1. Multiple BCRs bind the same pathogen to create receptor clustering Stronger BCR signaling 2. PRRs also bind antigen Stimulate a second set of signaling cascades within the cell For simplicity, we are going to focus on the T cell dependent response How does the antigen get to the B cell? Antigen enters the node via the afferent lymphatic vessel Along with cytokines and chemokines Antigen can be bound to BCRs that match either: When soluble When bound to local macrophages or follicular dendritic cells (FDCs) B cell activation Once the B cell finds an antigen that matches its receptor it will: Take up antigen, process it, and present it in its MHC class II in order to find its matching TFH cell and ‘talk’ to it Move to the border between the T cell and B cell zones in the lymph node and search of a matching TFH cell  BCR activation B cells have syk (=Zap70 in T cells) Recruited to P-ITAMs Syk recruitment leads to activation of phospholipase C-ɣ (PLC-ɣ) Causes to production of secondary messengers IP3 and DAG This leads to activation of the transcription factors: CREB, Jun, Elk-1, Egr-1 and NFAT The B cell moves towards activation Still needs signals from innate immune response or TH cells  BCR activation Ig⍺ an Igβ associate with the BCR and have ITAMs Binding of matching antigen to the BCR will cause tyrosine kinases to be activated (Btk, Fyn and Lyn) This starts with BCR clustering Phosphorylates the ITAMs on Ig⍺ an Igβ The B cell co-receptor, CD19, CD21 forms a complex with the BCR and contributes to downstream signaling Complement coated pathogens will bind CD21 (AKA CR2) and be localized to the BCR complex B cell activation Soluble antigen that is opsonized by complement enters the lymph node via the afferent lymphatics Here it binds the complement receptors on the subcapsular sinus macrophages (SCSMs) which deliver the antigen into the node The antigen coats the follicular dendritic cells (FDCs) Naïve B cells sample the FDCs or soluble antigen for antigen matching their BCR How does the TFH CD4+ T cell find the B cell? Antigen enters the node (both on its own and presented in MHC class II on the APC) Naïve CD4+ T cells sample the APC, if there is a match the CD4+ T cell activates, proliferates and differentiations into TFH CD4+ cells (if the correct polarizing cytokines, IL-16 and IL-21, are present)  At the same time a B cell with a matching BCR binds the antigen and start to activate, they put the Ag into the MHC class II The activated TFH cells move from the T cell zone to the border of the B cell zone The activated B cells move to the border of the T cell zone The TFH cell and the B cell interact  The TFH TCR binds to the Ag in MHC class II on the B cell The matching TFH and B cell make an immune synapse The TFH cell produces CD40L that binds CD40 on the B cell (signal 2) The TFH cell produces cytokines to encourage B cell activation, proliferation and differentiation into plasma cells (IL-4 and IL-21) TFH cell and B cell activation In this case the B cell is the APC Presenting extracellular Ag in MHC class II A TFH cell with a matching TCR will provide cytokines such as IL-21 and IL-4 Induce B cell proliferation and differentiation into antibody producing plasma cells What happens to an activated B cell? A B cell that receives T-cell help can do one of two things: It can differentiate into a plasma cell immediately, leave the node and make a low affinity IgM antibody It can join other activated B cells to form a germinal centre in the node Here the B cells undergo affinity maturation and class switching  Reminder: activated B cells undergo somatic hypermutation (SHM) via point mutations to produce variation in the V domain of their BCRs (this happens in the dark zone of a follicle) B cells that produce higher affinity BCRs out compete low affinity BCRs for binding to the FDCs → binding FDCs = survival signals, so only the highest affinity BCRs survive  These survival signals (affinity selection or affinity maturation) occur in the light zone Also in the light zone is class switching Class switching The first plasma cells activated will make IgM antibodies But there are a number of different antibody classes possible: IgM, IgG, IgA, IgD, IgE They all mediate different effector responses Therefore, depending on the type of infection the B cell will need to switch the type of antibody it produces Class switching AID (activation-induced cytidine deaminase) mediates class switching Class switching involves genomic deletions AID deaminates cytidine to uridine in the switch regions (S) in the genome on both strands These uridines are converted into double-stranded breaks The breaks in the DNA come together to put a new constant domain downstream of the V domain This produces a different class BCR and secreted antibody The DNA in between gets excised from the genome Cytokines sensed by the B cell dictate which S region binds AID Class switching  Migration B cells with high affinity, class switched BCRs are allowed to differentiate into plasma cells to produce antibodies Plasma cells move out of lymph node to the bone marrow or gut and lung mucosal tissues Antibodies enter blood stream or lumen of mucosal surfaces (IgA) Memory B cells Memory B cells Three types of memory B cells 1. Early subset of memory B cells 2. Later subsets of memory B cells 3. Long lived plasma cells Early subset of memory B cells These are the B cells that are first activated during an infection They produce IgM BCRs and are low affinity for the pathogen Later subsets of memory B cells These memory B cells are produced after somatic hypermutation and class switching BCRs are not IgM and their V domains are higher affinity for the pathogen These memory cells can remain in or near the germinal centre or migrate to sites of antigen drainage (marginal zone of spleen) and the mucosal epithelium of the gut, lungs and tonsils Areas where antigen is likely to be sensed first during re- infection Long lived plasma cells Not a memory B cell per se, as they are differentiated plasma cells Long-lived plasma cells (LLPCs) are a subset of plasma cells Reside in bone marrow or near barriers (gut, lung, mucosa) Can produce antibodies for a long period of time after antigen stimulation without the need for further antigen re-exposure Ex. Recipients of the smallpox vaccine can have LLPCs 75 years after vaccination! Thus LLPCs may last for life Summary The effector responses of B cells Adaptive and innate immune responses linked You’ll notice as we go through the adaptive immune response’s effector mechanisms that the adaptive immune response often reuses innate effector mechanisms But adapts them so they can be activated in a more specific manner Antibodies as effector molecules Some antibody reminders first Antibodies are released by activated B lymphocytes called plasma cells The antibodies secreted have identical specificity to the membrane bound BCR Antibody structure and functional domains Structure Two identical light chains Two identical heavy chains Functional domains Two identical antigen binding domains VH+VL x 2 VH+VL = specific recognition of antigen =Fab One Fc domain Area of antibody that binds receptors and mediates immune effector mechanisms Antibody classes Antibody class is determined by the C domains IgG Subclasses: IgG1, IgG2, IgG3, IgG4 IgD IgE IgA Subclasses: IgA1, IgA2 Forms a dimer in mucosal secretions (but not plasma) IgM Forms a pentamer or hexamer in plasma Antibody multimers increase the number of antigen binding sites Antibody distribution Antibody classes are distributed differently throughout the body IgG is most abundant in serum, tissues and can transfer across the placenta IgM is found is serum but is too big to easily enter tissue Secretory IgA is mostly found at mucosal surfaces May be the most prevalent antibody in the body IgE is associated with mast cells Mast cells are found close to our barriers ex. epithelium of skin and mucosa IgD is the least understood Monomer, found in airway mucosa and on the surface Fc receptors of basophils Classes and effector responses Different antibody classes and subclasses can activate different effector responses It all has to do with the Fc region of antibody Fc receptors (FcR) on immune effector cells recognize different Fc types A phagocyte, NK cell, or mast cell expressing a specific FcR will recognize a specific Fc type and activate to initiate a specific effector response FcR Nomenclature (Table 12-2) Fc-Greek letter – R – subtype FcɣR1 binds IgG Expressed on: dendritic cells, monocytes, macrophages, granulocytes, B cells Triggers: phagocytosis, cell activation, respiratory burst, ADCC Fc𝜀R1 binds IgE Expressed on: eosinophils, basophils, mast cells Triggers: degranulation of granulocytes Fc⍺R binds IgA Expressed on: monocytes, macrophages, granulocytes, dendritic cells Triggers: phagocytosis, cell activation, ADCC Fc⍺/µR binds IgM (high affinity), IgA (moderate affinity) Expressed on: B cells, macrophages, FDCs, kidney mesangial cells Triggers: endocytosis, phagocytosis PolyIgR binds IgA and IgM Expressed on multiple types of epithelial cells Triggers: transport of IgA from blood to the lumen of the gut, respiratory and reproductive tracts and secretory glands Immunobiology: The Immune System in Health and Disease. 5th edition. Janeway CA Jr, Travers P, Walport M, et al. New York: Garland Science; 2001. IgA transcytosis Plasma cells in tissue make dimeric IgA Dimeric IgA binds polyIgR on the basal side of the epithelium Dimeric IgA is endocytosed, transported through the epithelial cell and and released by exocytosis from the apical surface of the cell Now the dimeric IgA is in the lumen of the gut, lung etc. IgA accumulates in bodily secretions Figure 12-5(e) A pathogen activates a polyclonal antibody response A single antigen will have multiple epitopes Epitope = a spot on a pathogen recognized by an antibody (or BCR or TCR) Therefore, multiple different B cells will be activated in an infection And multiple different antibodies, specific for their epitope will be produced Monoclonal antibodies are produced when a plasma cell is isolated, merged with an immortal cell to produce a hydridoma that produces a singly antibody Antibody effector mechanisms 1. Antibody neutralization of pathogens and toxins Blocking virus entry into host cells IgA plays a very important role in blocking entrance of mucosal pathogens 2. Antibody mediated agglutination of pathogens 3. Antibody-mediated opsonization and phagocytosis 4. Antibody activated classical pathway of complement 5. Antibody dependent cellular cytotoxicity (ADCC) 6. Antibody-mediated granulocyte activation 1. Antibody neutralization - pathogen IgG and IgA Viruses and some bacteria need to bind to a cell or tissue to enter it Ex. SARS-CoV-2 binds ACE2 on the surface of the cell to enter A neutralizing antibody binds to the pathogen and blocks its ability to bind the surface receptor needed for entry PROBLEM: viruses can mutate their surface proteins to evade antibody binding A particular problem with HIV https://www.nature.com/articles/s41577-020-0321-6 Other neutralization options Antibodies can immobilize or ‘paralyze’ pathogens Antibodies against Pseudomonas aeruginosa flagella inhibit the bacteria’s motility Antibodies against HIV-1 may slow down random movement of HIV-1 in the vaginal mucosa, reducing contact time between virus and host epithelium Antibodies can destabilize pathogens, making them non- infectious The monoclonal antibody 4C9 binds foot-and-mouth-disease virus, disrupting the virion capsids 1. Antibody neutralization - toxins Dendritic cells can also detect toxins and induce a B cell response against them Neutralizing antibodies bind toxins and blocks the toxin’s ability to damage the cell The tetanus vaccine contains inactive versions of the tetanus toxin produced by Clostridium tetani bacteria 2. Antibody agglutination Can involve any antibody class Antibodies have 2 (ex. IgG), 4 (ex. dimeric IgA) or 10 (ex. pentameric IgM) antigen binding sites This gives antibodies the potential to bind multiple pathogens at a time  Agglutinated pathogens can’t infect A pathogen can’t infect a cell or tissue if it is stuck in a clump They can also be easier to clear Intestinal bacteria that are agglutinated cannot attach to the intestinal wall, they get caught in mucous and are cleared from the gut by peristalsis Non-immunized mice Immunized mice Blue: nasal cells Red: bacteria Green: tissue Streptococcus pneumoniae agglutination in nasal cavity of immunized mice 3. Antibody-mediated phagocytosis Dominant antibody in serum is IgG Phagocytes express FcR for IgG (FcɣRs) and monomeric IgA (Fc⍺R1) Interaction of a specific antibody Fab with a pathogen surface causes a conformational change in the Fc domain so it can now bind its FcR The Fc portion of soluble antibodies do not bind FcRs Antibody binding to the surface of pathogen (opsonization) increases the efficacy of internalization of pathogens and digestion of the pathogens within phagolysosomes NOTE: phagocytes can take up pathogens using their innate immune phagocytic receptors too!  Fc FcɣRs or Fc⍺R1 4. Complement fixation Some antibody classes/subclasses will activate the complement cascade once bound to a pathogen Classical pathway Good example of an adaptive effector immune response using innate immune effector mechanisms IgM and IgG activate the classical pathway (IgM>IgG) IgA and IgE do not When antibodies bind to a pathogen they undergo a conformational change The Fc portion of the antibodies can bind the C1 complex C1q + 2 serine proteases (C1r and C1s)  Same outcomes as other pathways: 1. Opsonization 2. Lysis 3. Inflammation 5. Antibody-dependent cell-mediated cytotoxicity NK cells express FcR for IgG (FcɣRIII; CD16) Virus-infected cells express viral proteins on their surface Antibodies can bind these virus proteins The antibodies then coat the cell NK cell’s FcɣRIII binds to the Fc region of the antibodies on the surface of the cell The FcɣRIIIs cross-link which activates the NK cell NK cell forms a synapse and releases perforin and granzymes to kill the infected cell ADCC can also be used to kill tumor cells  6. Antibody-mediated granulocyte activation Intestinal helminths are multi-cellular eukaryotic pathogens They induce a strong Th2 immune response and the activation of IgE-secreting plasma cells Mast cells and basophils express Fc𝜀R1 which binds IgE with very high affinity As soon as IgE enters the blood it is bound to mast cells (in tissue) and basophils (in blood) Cross-linking of surface bound IgE to multivalent antigens on the helminth will lead to: Degranulation and release of pro-inflammatory granule contents (ex. histamine, lysosomal enzymes, proteases, pre-formed cytokines and chemokines, growth factors) Synthesis and release of lipid mediators of inflammation (ex. prostaglandins, leukotriene) Synthesis and release of cytokines (ex. TNF-alpha, type I and II IFNs, IL-12, IL-8)  Mast cells can also express FcɣRs and degranulate in response to cross-linked IgG1 Eosinophils (blood borne) express FcɣRs, Fc⍺Rs and low levels of Fc𝜀Rs, so they can be activated by IgG, IgA, and IgE This type of immune response is referred to as ‘type 2’ immunity