Humoral Immunity Week 8 PDF

HU MO R A L IMM UN ITY ANN DONACHEY [email protected] 1-613-868-2284  SIGN IN FOR EACH SESSION YES, EVEN IF YOU’RE WATCHING THE RECORDING OR JUST DOING THE PRACTICE PROBS  B CELL RECAP PRECURSOR: LYMPHOID PROGENITOR DEVELOP IN THREE STAGES: maturation:...

HU MO R A L IMM UN ITY ANN DONACHEY [email protected] 1-613-868-2284  SIGN IN FOR EACH SESSION YES, EVEN IF YOU’RE WATCHING THE RECORDING OR JUST DOING THE PRACTICE PROBS  B CELL RECAP PRECURSOR: LYMPHOID PROGENITOR DEVELOP IN THREE STAGES: maturation: maturation of immunocompetent B lymphocytes in bone marrow activation: contact with antigen differentiation: plasma cells (antibody production) & memory B cells SITES OF DEVELOPMENT: BONE MARROW, BURSA OF FABRICUS (BIRDS) & PEYERS PATCHES (RABBITS, PIGS & OTHER) site of induction: external antigens activate in lymph node, blood borne antigens activate in spleen PLASMA CELLS: SECRETE IMMUNOGLOBULINS SPECIFIC TO ANTIGENS B CELL RECEPTORS RECALL: 200,000- 500,000 identical BCR per B cell have two heavy chains and two lights chains soluble form= antibody All BCRs have their besties that help transduce the signal: Ig-a and Ig-b (CD79a & CD79b) glycoprotein heterodimers longer tails called ITAMs (immunoreceptor TYROSINE based activation motif) that extend into the cell to signal B CELL T-independent antigens are ACTIVATION usually weak stimulators =polysaccharides, nucleic acids and lipids driven by presence of antigen multivalent antigen (repeated epitopes) repeat determinants naive B cells that do not engage with antigen die within a few weeks T-independant antigens can act directly (T-independent B cell activation) no helper T cell involvement weak immune response short lived plasma cells no memory cells and only low-affinity IgM T-independant antigens (aka thymus independent (TI) or thymus dependant (TD)) mitogenic antigens (aka TI-1): activate polyclonal B cell response and can act on BCR or TLR cross-linking antigens (aka TI-2): connect to multiple B cell receptors the B cells that act independantly are called Marginal Zone B cells (secondary lymphoid organs) or B-1 cells (mucosal tissues & peritoneal cavity)  T-cell dependent B CELL antigens are ACTIVATION usually strong= Has helper T cell support occurs in secondary lymphoid organs proteins 1 activated B cell can activate up to 5000 antibody secreting cells each antibody secreting cell can produce 2000 antibodies per second isotype switching: changes from crappy IgM to must more durable, high affinity IgG, IgA or IgE affinity maturation: B cells that produce high affinity antibodies will dominate the response (only the best players get to stay on the field) long-lived plasma cells memory B cells Mediated by Follicular B cells (B-2 cells) in lymphoid organs HUMORAL RESPONSE Primary response: naive (unstimulated) B cell initial contact with antigen Has a characteristic lag time caused by: clonal selection: select B cell with highest specificity to antigen clonal expansion: make a little army of the selected B cell differentiation: development of plasma and memory cells antibody production: mostly IgM and IgG for primary reaction Characterized by low antibody level and IgM dominance- this is why we booster vaccines! Response: antibody production by plasma cells and memory cells duration of lag time depends on: antigen, the species and their immunization status, the route of admin and the presence of an adjuvant HUMORAL RESPONSE Secondary response: memory B cells stimulated by subsequent contact with antigen dependent on the existence of memory B and helper T cells rapid stronger last longer secretes higher affinity antibodies instant response characterized by higher levels of IgG than IgM remember IgM is our starter molecule, but its bulky, so we swap it out with the stealthier IgG or higher affinity IgA or IgE Response: waaaaay better  CO-STIMULATION OF B CELLS RECALL: B cells are only acting on mature helper T cells in a second exposure; the first exposure to a new antigen, a DC cell will create the helper T cell from a naive T cell! B cells interacting with helper T cells requires TWO FACTOR SIGNAL to activate Signal 1: helper TCR and MHC class 2 on the B cell Signal 2: CD40-CD40L induces B7 and CD28 (co-stimulating) interaction cytokines are released  HUMORAL IMMUNITY 1. Antigen arrives in the secondary lymphoid organ 2. immune response initiated by recognition of antigens by follicular B cells B cells act as antigen presenting cells BCR binds antigen, Ig-a/Ig-b transduce signal to activate B cell BCR internalizes antigen and processes it on an MHC-2 for helper T cell recruitment 3. TWO THINGS HAPPEN SIDE BY SIDE BUT NOT YET INTERACTING: a. A dendritic cell activates a naive T cell with the antigen in the extrafollicular space b. Our follicular B cell from step 2 migrates out of the follicle 4. B cells present antigen in MHC-2 to the recently activated CD4 T cell at the T cell zone a. signal 1: MHC-2 (on B cell) connects to TCR + CD3 (on recently activated helper T) b. signal 2: CD40/CD40L + CD28/B7 + cytokines 5. Helper T cell induces initial B cell proliferation and differentiation= extra-follicular B cells w/ early antibody response a. short lived plasma cells to pump out the first few antibodies so this infection isn’t raging while we get the rest of our reaction sorted 6. Activated T and B cell migrate into the follicle, forming a GERMINAL CENTER where the reaction gets specialized a. affinity maturation b. isotype switching c. memory cell formation d. long lived plasma cell formation  GERMINAL CENTERS Germinal centers: within the follicle of secondary lymphoid tissues, composed of a basal dark zone and light zone. Dark zone: contains proliferating B cells Light zone: contains follicular dendritic cells and follicular helper T cells to cause somatic mutation, affinity maturaton, and isotype switching High affinity antibody secreting cells and memory cells will exit the germinal center.  GERMINAL CENTERS 1. iniation of germinal centre by T cell and B cell entry from follicle 2. proliferation of B cells 3. somatic mutations in Ig genes and isotype switching 4. interaction with follicular DC and follicular helper T cells to selection the highest affinity B cells (affinity maturation) 5. selection 6. differentiation into long-lived plasma cells and memory B cell formation ISOTYPE SWITCHING CHANGING IMMUNOGLOBULIN (IG) MOLECULE IN T-DEPENDENT RESPONSES, B CELLS UNDERGO HEAVY CHAIN SWITCHING drecall: default BCR is IgM can switch to IgG, IgA and IgE recall: IgD is just a maturity marker Ig class switching is a somatic hypermutation= causes a shift in the VDJ of the heavy chain via AID (activation-induced deaminase) this is regulated by cytokines produced by the helper T cells that are activated by microbes if no T cell = no cytokines= no isotype switching= only IgM T cell activation releases cytokines: IFN-y: IgG opsonization, phagocytosis, complement activation, neonatal immunity IL-4: IgE and IgG immunity against helminths and mast cell degranulation Mucosal tissues and cytokines release TFG-b, APRIL, BAFF and other cytokines: IgA mucosal immunity AFFINITY MATURATION SOMATIC MUTATIONS (POINT MUTATION, DELETION AND INSERTION) OF IG GENES AND SELECTION OF HIGH AFFINITY B CELLS Creates high affinity antibodies for each specific antigen better binds, better neutralizers, better eliminators done in the variable region CLONAL SELECTION AND DELETION B CELLS BIND ANTIGENS IN GERMINAL CENTERS ARE SELECTED TO SURVIVE BECAUSE OF THEIR HIGH AFFINITY best able to bind with antigen on follicular dendritic and T cells B CELLS WITH LOW AFFINITY TO FOREIGN ANTIGENS ARE ELIMINATED THROUGH CLONAL DELETION only cells with high affinity receptors selected to survive if they don’t interact with the pathogen, they die PLASMA CELL DIFFERENTIATION Plasma cells are permanently differentiated B cells plasma cells are larger than B cells Committed to antibody production membrane associated BCR becomes secreted antibody they lose their BCR! Two types: SHORT LIVED: result of T-independent response can be seen in early T-dependent responses, during extra-follcicular response found in secondary lymphoid organs and other non-lymphoid tissues LONG LIVED: generated by T-dependent responses in germinal centers produce massive amounts of antibodies continue to secrete antibodies for decades after antigen is removed these plasma cells send antibodies into the periphery, but they themselves do not recirculate MEMORY B CELLS generated during germinal center reaction so mostly during T-denpendent responses due to location, but can still occur in T-independent make rapid response to subsequent antigen encounters survive for long periods without continued antigen stimulation because of anti-apoptotic BCL-2 some memory B cells remain in the lymphoid organ, others recirculate between the blood and lymphoid organs vaccines must induce affinity maturation and memory B cell formation ANTIBODIES  ANTIBODY FUNCTIONS neutralization opsonization (labels for immune cells with Fc receptors) ADCC (labels for NK and T cells with Fc receptors) complement activation (RECALL: only the classical complement pathway!) Found in serum +/- milk, tears, saliva, mucous and bile  ANTIBODY STRUCTURE Antibodies are heterodimers made from 4 polypeptide chains: 2 heavy chains Variable region: first 110 amino acids 5 isotypes (alpha, gamma, delta, epsilon and mu) which on N terminals of heavy and light chain determine the class of Ig highly variable for each antibody subtypes of an immunoglobulins have differences in forms our antigen binding site slight amino acid sequences (IgG1, 2, 3 etc) Constant region: constant and unchanging amino acid sequence 2 light chains: in both heavy and light chains 2 subtypes: lambda and kappa which are identical functionally connected by disulfide bonds and non-covalently hydrogen and Fab: fragment antigen binding hydrophobic bonds antigen binding two identical fragments, which is why Heavy chains: Light chains: we have two binding sites 400-500 amino acids 220 amino acids Fc: fragment crystalizable 4-5 domains depending on the Ig 2 domains crystallizes at low temperatures 1 variable domain 1 variable 3-4 constant domains 1 constant TALLY: 2 light chains 2 heavy chains 2 variable regions= TWO BINDING SITES FOR ANTIGENS one constant region  ANTIBODY STRUCTURE HOW WE FIGURED ALL THAT CRAP OUT: Reduced and acidified an Ig into light and heavy chains Digested Ig with enzyme Papain into two Fab regions (TWO separate ANTIGEN BINDING SITES) and a Fc region cleaved above the disulfide bond Digested Ig with Pepsin into an Fab complex (both biding sites reman associated) and one partial Fc region cleaved below the disulfide bone  IMMUNOGLOBULIN CLASSES Each Ig class has a unique amino acid sequence for the heavy and light chain in the constant region IgG structure: gamma heavy chains either kappa or lambda 4 domains 180kDa weight highly mobile due to hinge region and can therefore enter tissues MOST ABUNDANT IN SERUM functions: neutralization opsonization classical complement pathway activation ADCC (by NK cell) neonatal immunity- only antibody transferred via colostrum  IMMUNOGLOBULIN CLASSES Each Ig class has a unique amino acid sequence for the heavy and light chain in the constant region IgM structure: 5 domains 900 kDa free IgM is 5-6 subunits (usually pentamer) ninja star total of 10 binding sites but can only bind 5 large antigens the monomer is found on the surface of B cells (BCR) functions: IgM first immunoglobulin synthesized by newborns first one made during humoral response second highest concentration in serum but RARELY enters tissues (too heckin big) MOST EFFECTIVE in activating complement (even more than IgG)  IMMUNOGLOBULIN CLASSES IgA structure: alpha heavy chain 4 domains usually a 360kDa dimer functions: dominating Ig in body secretions like milk, tears and mucous of resp, Gi and repro tracts secretory IgA- protects from pathogens that may enter at vulnerable mucosal portals IgA dimers in secretions are called secretory IgA and is found in submucosal membrane process: translocation through epithelial cells in mucous membranes IgA dimer binds to the poly-Ig receptor (plgR) on basolateral epithelial cell surface this transports IgA via endocytosis with the poly-Ig receptor onto the apical surface the poly-Ig receptor is cleaved enzymatically and becomes a part of the dimer the poly-Ig secretory component protects IgA hinge from proteolytic enzyme digestion in the mucosa antigen-antibody complexes formed at surface removed by cilia of resp and peristalsis of GI IMMUNOGLOBULIN CLASSES  IMMUNOGLOBULIN CLASSES IgE IgD structure: structure: epsilon heavy chain delta heavy chain 4 domains 5 domains 170 kDa weight 190kDa weight not found in rabbits or cats shortest half life (2-3 days) function: function: NONE IgD is found on the surface of B cells small amounts in serum no biological effector function in animals allergic reactions (hypersensitivities) in humans it can bind to basophils binds to Fc receptors on basophils and mast cells parasite immunity  IMMUNOGLOBULIN SUMMARY 5 domain antibodies: M&E 4 domain antibodies: D, A, G Epithelium: IgE (skin gets itchy with allergies) Dermis: IgG (where we are guarding from pathogens) Mucosal surface: IgA (only one that can secrete) Blood: IgM and IgG  ANTIGENIC DETERMINANTS ON IG Ig are immunogens= immunogenic antigens= have immunogenicity Recall immunogenicity: immunogens capable of inducing an immune response when administered to other species, producing anti-Ig antibodies. There are three categories of antigenic Ig: Isotypic: determine the isotypes of the immunoglobulin (within the population) constant regions of heavy and light chains & subclasses each species have different isotypes, so antibodies from one species will cause an immune reaction in another Ex: cats cannot donate blood to dogs Allotypic: determine the differences within a given species (within the species) each species has the same isotype, but alleles (amino acids) can differ within these isotypes Ex: O-, A+, B+ or AB...blood types between individuals can differ, even though they all still count as blood Idiotypic: determined the difference between specificities of antibodies (within the individual) antigenic determinants found in variable regions of heavy and light chains Ex: IgG1 antibodies against streptococcus are not going to be the same against IgG1 antibodies against rhodococcus, even though they are both IgG1, because there are epitope differences in that vairable antigen binding site  EFFECTOR FUNCTIONS bind antigens DO NOT KILL PATHOGENS OR DIRECTLY REMOVE THEM induce effector activity of other components of immunoty which will kill/remove the FC region is what interacts with other cells What they do: neutralization (phagocytosis) opsonization (phagocytosis): covers the antigen and facilitates phagocytosis by macrophages and neutrophils, they have Fc receptors on their surfaces which bind to Fc fragments on antibodies agglutination (phagocytosis) precipitation (phagocytosis) activation of complement and MAC (cell lysis) antibody dependent cellular cytotoxicity ADCC antibody binds antigens on surface NK cells recognition Fc regions using their CD16 (FcReceptor3) cross-linking of Fc receptors to NK cause apoptosis  FC RECEPTOR FC receptors are found on the surface of many cells They are responsible for many biological functions of the antibodies: transcytosis mediation of phagocytosis antibody dependent cell cytotoxicity ADCC Antibodies induce signals to activate or inhibit the cells with Fc receptors Types: Poly-Ig receptors: transcytosis of IgA and IgM through epithelial cells FcRn: neonatal FC receptor transports IgG through placenta and regulates level of IgG in serum FcaR binds IgA FceR binds IgE FcyR found in many forms but they bind IgG and its subclasses  MONOCLONAL ANTIBODIES POLYCLONAL: Most antigens have many epitopes which induce proliferation and differentiation of many different clones of B cells that produce different antibodies serum in these circumstances i heterogenous contains mixture of antibodies with specificities for different antigens suited to various effector functions inside the host not suitable for diagnostic or therapeutic procedures because we cannot purify them MONOCLONAL: antibodies produced by single clone and are specific for a single antigenic epitope three Nobel prize winners fused normal plasma cells and myeloma cells creating hybrid cells they are immortal and capable of continuously produced antibodies of single specificity useful for diagnostics of infectious diseases and cancer 80 approved monoclonal antibodies for human health therapy, called “-umab” canine monoclonal antibodies for puritis= CYTOPOINT neutralizes IL-31 which causes itch LABEL ME LABEL ME 1. heavy chain 2. light chain 3. Fab 4. Fc  Which of the following is correct? o a) development of B cells is differentiation, maturation, activation b) B lymphocytes develop in the bone marrow c) Plasma cells secrete antigens d) cytotoxic T cells help stimulate B cells to produce high affinity antibodies  Which of the following is correct? a) development of B cells is differentiation, maturation, activation b) B lymphocytes develop in the bone marrow c) Plasma cells secrete antigens d) cytotoxic T cells help stimulate B cells to produce high affinity antibodies Which of the following is not necessary for t-dependent B cell activation? a) Iga-Igb b) TLR or CR2 c)CD40-CD154 d) polyIg Which of the following is not necessary for t-dependent B cell activation? a) Iga-Igb b) TLR or CR2 c)CD40-CD154 d) polyIg Which of the following will not be a major occurance when T-dependent antigens prompt B cells activation: a) antibody secretion b) isotype switching c) affinity maturation d) memory cell formation e) short lived plasma cells  Which of the following will not occur when T-dependent antigens prompt B cells activation: a) antibody secretion b) isotype switching c) affinity maturation d) memory cell formation e) short lived plasma cells although they do occur in small numbers during the focus Tcell-Bcell interaction Which of the following supports transduction of activating signals? o a) ITAM b) BCRs c) IgD c) TI-2 Which of the following supports transduction of activating signals? a) ITAM b) BCRs c) IgD c) TI-2  True or False the primary B cell immune response is characterized by a dominating IgG reaction.  True or False the primary B cell immune response is characterized by a dominating IgG reaction. IgG will dominate the secondary response  true or false one activated B cell can cause the differentiation of 2000 plasma cells, each of which can produce 500 antibodies per second.  true or false one activated B cell can cause the differentiation of 2000 plasma cells, each of which can produce 500 antibodies per second. 1 B cell can cause 5000 plasma cells to differentiate. Each plasma cell can make 2000 antibodies per second You are part of a cool, new study to examine B cell differentiation in recently vaccinated patients. Your role is to examine the lymphoid organs during B cell differentiation. Where would you expect to see B cell proliferation? a) dark zone of liver b) light zone of spleen od) light zone of lymph node c) dark zone of lymph node You are part of a cool, new study to examine B cell differentiation in recently vaccinated patients. Your role is to examine the lymphoid organs during B cell differentiation. Where would you expect to see B cell proliferation? a) dark zone of liver b) light zone of spleen c) dark zone of lymph node d) light zone of lymph node  You’re still working away, but now you’re looking for signs of somatic mutation. Where would you expect to see B cell’s in this stage? (select all that apply) a) dark zone of liver b) light zone of spleen c)8 dark zone of lymph node d) light zone of lymph node  You’re still working away, but now you’re looking for signs of somatic mutation. Where would you expect to see B cell’s in this stage? a) dark zone of liver b) light zone of spleen c) dark zone of lymph node d) light zone of lymph node  no True or False Ig class switching requires the recombination of the amino acids in the light chain  True or False Ig class switching requires the recombination of the amino acids in the light chain this would need to say heavy for this to be correct  Your patient has a recurrent bacterial infection. Which of the following antibodies would be found in highest number in their serum? a) IgA b) IgE x̅ c) IgD d) IgG  Your patient has a recurrent bacterial infection. Which of the following antibodies would be found in highest number in their serum? a) IgA b) IgE c) IgD d) IgG  A virus has entered the lungs of a herd of livestock. Before it has the chance to cross the mucosa, which of the following immunoglobulins will act, and how will that pathogen ultimately be removed? a) IgA- complex formation for mechanical removal b) IgE- mast cell degranulation 0 c) IgA- complement attack complex as part of the innate response d) IgG- opsonization in the mucosa  A virus has entered the lungs of a herd of livestock. Before it has the chance to cross the mucosa, which of the following immunoglobulins will act, and how will that pathogen ultimately be removed? a) IgA- complex formation for mechanical removal b) IgE- mast cell degranulation c) IgA- complement attack complex as part of the innate response d) IgG- opsonization in the mucosa  Which of the following is true in regards to IgM? a) first Ig synthesized in newborns b) most effective complement activator c) structurally, its five subunits joined by joining chain d) all of the above O  Which of the following is true in regards to IgM? a) first Ig synthesized in newborns b) most effective complement activator c) structurally, its five subunits joined by joining chain d) all of the above  Fill in the word The reason we cannot use feline blood for canine transfusions is due to ( ), whereas the reason blood within a species is not universal is because of ( ), however the unique, individual differences between antibodies would be an example of ( ). isotypic allotypic idiotypic 7  Fill in the word The reason we cannot use feline blood for canine transfusions is due to isotypic, whereas the reason blood within a species is not universal is because of allotypic, however the unique, individual differences between antibodies would be an example of idiotypic.  Put these in chronological order: B cell and T cell migrate into follicle to create germinal centre T cell gets activated in periphery by DC cell B cell gets activated in follicle 3 Formation of focal, short lived plasma cells that secrete IgM Somatic mutation, isotype switching and memory formation Activated B and T cell meet at the edge of the follicle  Put these in chronological order: please note this is not all inclusive! I intentionally left out steps- go find them and build this pathway to completion. T cell gets activated in periphery by DC cell B cell gets activated in follicle (these are happening side by side after antigen exposure) Activated B and T cell meet at the edge of the follicle (where B cell presents antigen to helper T cell) Formation of focal, short lived plasma cells that secrete IgM (temporary solution while we get the full reaction underway) B cell and T cell migrate into follicle to create germinal centre somatic mutation, isotype switching and memory formation (happen in the light zone of the germinal center) THANK YOU! GOODLUCK