L#5 : Antibodies, Antigens, B Cell Development.
L#5 : Antibodies, Antigens, B Cell Development. 0530
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This 4 page Class Notes was uploaded by Denise Croote on Saturday January 23, 2016. The Class Notes belongs to 0530 at Brown University taught by Dr. Richard Bungiro in Fall 2013. Since its upload, it has received 28 views. For similar materials see Principles of Immunology in Biology at Brown University.
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Date Created: 01/23/16
Lecture Five: Ab-Ag Interactions Antigens interact with the adaptive immune system via ARMS and immunogens are antigens capable of inducing a specific antibody or cell mediated response. Antigens are judged on how well they bind, whether they cause an immune response or not, whether they cause and allergic reaction or not, and whether they are “tolerant” (i.e. whether they induce a state of nonresponsiveness) Immunogens can be polypeptides, proteins, lipoproteins, polysaccharides, nucleoproteins, and hapten protein complexes. They contain epitopes and can be recognized in the 3D by B cells or processed and presented as linear peptide fragments for a T cell response Immunogenicity is influenced by foreignness, molecular size, chemical complexity (need to be heterogeneous to produce shapes that will be recognized as non-self) and degradability (need to be digested by enzymes within APCs and presented via the MHC. Insoluble antigens are more likely to be engulfed and therefore processed.) Immunogenicity is also influenced by the route of exposure (mucosal vs. subcutaneous ex.)), the dosage, the host MHC cells, and the presence of adjuvant Adjuvants are substances that enhance the immune response, they could be cytokines, Ag persistence, and dendritic cell activation B cell epitopes are generally natural 3D structures of proteins that are non-sequential, they may be comprised of linear sequences but their shape is most important o Hence if two antigens have the same primary sequence and one it a closed loop and the other a semi-circle, than the B cell may only recognize ones shape. o If two things are close enough in shape then they could compete for ARMS o Antigen are usually water loving and soluble Haptens: Haptens are small organic molecules that can bind to ARMS but cannot induce an immune response on their own o they can bind to antibodies but not T cells o they can be coupled with a carrier protein that incites a T cell response o Penicillin can form a hapten complex and cause individuals to be hyper sensitive o Antibodies can form to the hapten protein complex or to just the protein complex but not just the hapten T cell epitopes are comprised of processed antigens. o The antigens could either be natural or denatured (because only the amino acid sequence matters) o T cell epitopes are not usually accessible from the exterior of the antigen and need to be broken down to be recovered o A complex consisting of TCR, PEPTIDE, and MHC forms o The complex is made up of a epitope – the complex where the peptide binds to the TCR and an agregtope the complex where the peptide binds to the MHC on the antigen presenting cell o Antigens are not soluble Ab-Ag Interactions: they bind reversibly and noncovalently, the individual attractions are pretty weak but gain strength in that there are a lot of them, they occur over a short distance, and they interact with a “tight” fit o They interact via noncovalent bonds such as van der Wallas forced, Hydrogen bonds, and ionic bonds. However, their binding can lead to phosphorylation and that phosphorylation could create a more permanent covalent bond o Affinity describes the strength of an individual interaction o Avidity measures the strength of multiple interactions between multivalent antibodies and antigens o IgG/D/E have 2 binding sites, IgA has four binding sites, and IgM has 10 binding sites o The affinity for IgG binding sites can be larger than the affinity for IgM sites but M will still be stronger because there are MORE interactions. Not all IgM sites may be bound by antibodies at once because that would create significant steric hinderance, but it is likely that most will be bound at one given point. o If the antigens of two separate pathogens are close in structure one type of antibody may react to both of them (cross reactivity) This was good in the case of the vaccinia cowpox and variola small pox viruses but not good for transfusions How does the shape of antibodies promote binding? o They are Y shaped and in the middle of their Y is a hinge, this hinge can change the distance between the binding sites on the tips of the Y, allowing the antibody to bind with antigenic determinants arranged at different distances on the antigen. o The variable regions of the heavy and light chains can rearrange to form a variety of combinations and this allows for the creation of a diverse array of antigen binding sites. o The constant regions on antibodies can form special bridges with phagocytic cells to insure their destruction when an antigen binds and also with complement proteins to elevate the reaction. Blood Types: o If you are B type blood you have B antigens so you will NOT have B antibodies because then you would self react o If you are A blood you would have A antigens so you will not have A antibodies because you would self react o If you are AB blood you have both antigens so you have no antibodies o If you are O blood you have no antigens so you have BOTH antibodies o Therefore if you are transferring serum, the donor’s antibodies may react with the antigens on the host’s RBCs. o If you are transferring RBCs then the antigens on these RBCs may react with the recipient’s antibodies. Precipitation: this occurs when Ab-Ag bind and form complexes that are insoluble. They form complexes when they are both multivalent and can attach to several partners. They require an equivalence zone – which is the optimal ratio. If you have too much Ab it won’t have anything to bind too, if you have a setup where everyone gets their own partner there will be no incentive to bind to others. Agglutination Reactions: o Hemagglutination – antibodies cross link red blood cells by binding their surface antigens. This forms a clump that is big and bulky and does not sit densely in a test tube o You can attach antigens to the surface of RBC and then bind them together with an antibody like IgM ELISA: Indirect o 1.) add the antigen of interest to the wells and it will bind to the sides of the well o 2.) wash out the unboard proteins o 3.) Block the plate with milk or BSA so that when you put the antibody in it can’t bind to any open spaces on the walls o 4.) Add primary antibodies and if the patent is infected they will have antibodies for the antigen and will bind to the sides of the wall. If they are uninfected their antibodies will just float in the serum because none will be specific for the antigen o 5.) put in secondary antibodies to bind to the first antibodies o 6.) wash out any extra antibody o 7.) put in a substrate that will be attacked by an enzyme on the secondary antibody o 8.) measure a color change as the substrate and enzyme react Can quantify Ab-Ag reactions with serial dilutions. After the first immunization you have some antibodies, and it may take you two dilutions to get to the background color. After a second immunization it may take you 5 dilutions to get to the background color. This indicates that there were more antibodies present after the second immunization (which makes sense) For the Indirect ELISA you are not changing the amount of antigens on the walls, you are just measuring the difference in antibody concentration over time or after immunizations ELISA: Sandwich – this is the same except you add antibody first and that sticks to the well and then you add the unknown antigen in differing amounts depending on the time samples were take ect., and then you add a detection antibody with the substrate enzyme interactions feature Western Immunoblotting: is very similar in that you are separating proteins in an electrophoresis gel, applying them to a membrane, and the putting 1 and 2 antibodies on them cause a response and emit light (immunofluorescence) Can use fluorochromes to analyze the cell surface for different surface markers. o For example a certain fluorochromes could be color specific for CD4 markers on T cells and others for CD8 markers on T cells. Based on the color emitted, you can tell which is more prevalent Flow Cytometry: This is a powerhouse that can analyze 10,000 cells in a few minutes. It can analyze the intracellular components, like the cell size and DNA content. It can even sort into different compartments based on what is present on the cell o Good for …..Analyzing cell development (which stage), immune response (ratio of types of cells that are present), enriching populations with only the cell types you want, quantitatively determining the distribution of antigens and level of expression. Lecture Six: B Cell Development Generation: of B cells in the primary lymphoid organs through gene rearrangement Activation: of B cells by foreign antigens, typically in the secondary lymphoid organs (with the help of the T cells Differentiation: of the B cells into plasma cells and memory cells (associated with affinity maturation – which is the increased affinity of an antibody for an antigen throughout the course of infection or after subsequent exposures.) (also associated with class switching – which is the genetic recombination of antibody heavy chains) Most Ab development occurs in the bone marrow and finishes for IgM and IgD in the spleen. B cells have variable, diverse, and joining gene segments (VDJ) rearranging these gives the different combinations for immunoglobulins Variable, diverse, and joining segments are present in Heavy chain rearrangements but diverse is lacking from Light chain rearrangement o Steps: (heavy chain) 1.) D and J segments of heavy chain recombine, in both heavy chains, if one of the recombination’s is successful then the VDJ proceeds 2.) V gene segment joins this powwow 3.) Constant region is added 4.) Translation of the heavy gene protein 5.) H chain associated with the signaling chains (lg alpha and lg beta) and the surrogate light chains (V preB and lambda 5) together making the pre-B cell receptor 6.) If this is functional and the heavy chain and surrogate light chain have efficiently paired other H alleles are silenced (allelic exclusion). You silence the lg rearrangements by downregulating RAG, the gene known to regulate lg rearrangement 7.) becomes responsive to IL growth factor and makes clones of itself because it has found a successful H chain. Makes about 256 baby cells and these are free to go after a variety of light chain cells. o Steps: (light chain) 1.) since no D is present V and J recombine 2.) Constant Region is added 3.) Translation of the light gene protein occurs Pre -ProB DJ heavy chain rearrangement occurs and VDJ heavy chain recombination starts ProB --> PreB complete the VDJ heavy chain recombination start the VJ light chain rearrangement Immature B Cells Negative Selection of self attacking cellsdelete and edit receptors Stromal cells like osteoblasts, IL-7 growth factors, and endothelial cells help B cell development, they can release stem cell factors and have certain receptors designed to bind stem and progenitor cells (called c- kit receptors). The stem cells have special markers (called sca-1) on the progenitor and stem cells that flag this binding Developing B cells are marked by the presence of B220 (an early lineage marker) Removing Self Reactive B Cells: o Clonal Deletion: apoptosis of self reactive B cells in the bone marrow o Anergy: functional inactivation that will occur in the periphery, this is not considered death o Receptor Editing: revision of the BCR via secondary recombination, typically occurs in light chains o Only 10% of B cells make it out of the bone marrow, the rest are killed because of the potential to self react Nemazee and Burki Experiment: o They made a gene that would express the immunoglobulin Kk (and called it anti Kk). Its H and L chains were already rearranged so once inserted it overrode all of the other gene rearrangements o The K allele is a part of MHC Class I so it is found on most cells. o Mice were either homozygous in expressing only the Kd allele on the MHC or heterozygous and expressing both the Kd and Kk alleles o Found that no antiKk was expressed on cells in the d/k mice because these mice have Kk so antiKk was marked as a self attacking antibody and deleted o Found antiKk in the Kd allele mice because it was not marked as self because these mice don’t have Kk o Found some Kd and Kk cells with antiKk and this was because of light chain editing. Light chain eiditing changed the light chain so that it wouldn’t self react with the Kk on the cell o Deleting the H chain leads to a rapid decay of antibodies o OVERALL pre-BCRs select successful H chain which allow for continued development with light chains. B cells obtain a tolerance to self and need membrane bound immunoglobulins if they hope to survive in the periphery.
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