1 Name. 1. (3 pts) What is apoptosis and how does it differ from necrosis? Which is more likely to trigger inflammation?

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1 1 Name MCB 150 Midterm Eam #1 (100 points total) Please write your full name on each page of the eam!! The eam consists of 17 questions (6 pages). Each has a different point count as indicated. Please limit your answer to the space provided. 1. (3 pts) What is apoptosis and how does it differ from necrosis? Which is more likely to trigger inflammation? Apopsis:programmed cell death, tidy, material degraded by phagocytes/ necrosis: death from outside, messy, material released into etracelluar area promotes inflammation 2. (4 pts) What is a cytokine? Why do some viruses encode decoy cytokine receptors? Small secreted proteins mediates intercellular communication. Viruses use decoy receptor to block immune responses. 3. (5 pts) From a mouse s perspective, having a mutation in the TLR4 gene can be either a detriment or a benefit. Eplain. Detriment: susceptibility to bacterial infection Benefit: resistant to shock 4. (3 pts) What is the difference between a lymphoid organ and a secondary lymphoid organ? What is the main lymphoid organ in adults for B cells. Primary: where B cells and T cell develop/secondary where they respond to antigen /bone marrow is for B cells 5. (5 pts) How many antigen binding sites are found per molecule of: IgA 4 IgG 2 10 For an IgA and an IgG with the identical antigen binding site, which would bind more tightly to a multivalent ligand? Full credit given for all answers because of confusion during eam. For an IgA and an IgG with the identical antigen binding site, which would bind more tightly to a monovalent ligand? Full credit given for all answers because of confusion during eam.

2 2 Name 6. (7 pts) For each of the 5 major antibody isotypes indicate whether the following statements apply. IgG IgA IgE IgD Transported across epithelium into secretions () Eists in a membrane form only Triggers Mast cell degranulation () The earliest Ig secreted after infection Transported across the placenta Present at lowest concentration in blood Good at fiing complement () 0.5 pt for each missing or etra check / () means OK if they did or did not check it 7. (4 pts) For each of the following conditions indicate whether you would epect a precipitate to form? a.polyclonal antibody mied with a multivalent antigen b. Monoclonal antibody mied with a multivalent antigen c. Monoclonal antibody mied with a monovalent antigen d. Monoclonal antibody mied with a multivalent antigen in the presence of a large ecess of the same antigen in a monovalent form. Answer: a, b (not c, d) Full credit given for all answers because of confusion during eam. 8. (4 pts) You have isolated a viral protein (protein D) from a deadly virus and are testing this protein for its possible use as a vaccine against the virus. You first inject rabbits intravenously with purified protein D, then test for the effectiveness of your vaccination by eposing the treated rabbits to the deadly virus. You find that all the treated rabbits die. Provide two possible eplanations as to why your vaccine did not work and suggest two ways that you could improve it. Answer: Protein D may not be immunogenic enough to mount an immune response and thus you need to add an adjuvant (such as Freund s) with the vaccine strain to trigger the immune system. Additionally, subcutaneous administration is better then intravenous injection. (Also possible that you produced antibodies, but they were not protective) Other common answers I accepted: Protein D is similar to a rabbit protein so cells recognizing it had been deleted. Not enough time between the vaccine & eposure to the virus for an adaptive immune response to take place.

3 3 Name 9. (5 pts) Draw lines to match each receptor with its ligand: Receptors ligand TLR9 Constant region of Ig heavy chain TCR Ig TLR4 Fc receptor lipopolysacharide (LPS) bacterial DNA (CpG) peptide antigen-mhc soluble antigen 10. (4 pts) To the right is a diagram of an antibody molecule. Indicate on the diagram the location of: Antigen binding site Hinge region V H Ig domain Constant domain of light chain 11. (6 pts) You have three monoclonal antibodies directed against different epitopes of the same soluble, secreted protein antigen. Monoclonal A recognizes a conformational, surface epitope, and Monoclonal B recognizes a linear epitope that is only eposed when the protein is denatured and Monoclonal C recognizes a C-terminal linear, surface accessible epitope. For each technique listed below indicate which antibody/antibodies would be useful for detecting the antigen and briefly eplain why. Western blot B, C: linear epitopes maintained upon denaturation Flow cytometry None: antigen is secreted: wont be on cell surface. Sandwich ELISA A, C recognize epitopes eposed in soluble protein.

4 4 Name 12a. (4 pts) Above is a simplified map of the human germ-line light chain kappa locus. Indicate the positions of the following structures (if applicable): V segment D segment J segment C eon RSS 12b. (3 pts) Draw a diagram of the same locus after V(D)J rearrangement. Indicate the locations of the CDR1, CDR2 and CDR3 coding regions on the rearranged locus. 13. (5 pts) Eplain why an individual B cell can simultaneously produce and IgD, whereas a B cell epressing other isotypes (IgG, IgA, IgE) cannot epress. Alt mrna processing for /IgD CSR for other deletes locus. 14. (8 pts) You have identified a novel gene X in the mouse that is predicted to encode a member of the TLR family based on protein homology. You also have a potential ligand, y that is a component of the yeast cell wall that is know to trigger innate immunity but for which a corresponding TLR has not yet been identified. Describe how you would determine whether X induces innate immune responses to y. (You should mention the various methods you would use, and any essential controls, but you don t need to describe each method in great detail.) many possible answers including: Knock out gene X in a mouse using homologous recombination (X-/-). Epose X-/- and wild-type (wt, X) mouse to ligand y. Etract serum and test for production of cytokines using an ELISA assay. Isolate macrophages and dendritic cells and run flow cytometry to look for up-regulation of surface markers. If ligand y stimulates the novel TLR X, then you should see cytokine production and surface marker upregulation in the wt stimulated mouse but not in the X-/- mouse. As a positive control you can use other known TLR stimulants such as LPS which should induce cytokine production and upregulation of surface markers in both wt and X-/- mice.

5 5 Name 15. (12 pts) You discover a novel carbohydrate antigen (X) that is present on the red blood cells of a certain inbred mouse strain a, but not on the cells of another mouse strain b. Would you epect to find antibodies reactive to X in an a strain mouse? In a b strain mouse? Briefly eplain your answer. Not in a due to self tolerance Could be in b, if cross-reactive to a pathogen Briefly describe a procedure you might use to quantitate the presence of anti-x antibodies in mouse serum. Agglutination of RBC from B mouse Could use ELISA if have soluble form of X available In order to study B cell tolerance, you generate a transgenic mouse epressing HC and LC rearranged BCR transgenes that encodes an antibody reactive to X. Using a monoclonal antibody that is specific to the particular HC and LC combination of your transgene to detect the presence of the transgene encoded BCR, and an antibody that recognizes part of the Fc portion of, you perform flow cytometry of the spleen cells of transgenic mice and obtain the following results: Non transgenic Transgenic on a background Transgenic on b background Based on your flow cytometric analysis, what mechanism of B cell tolerance do you think is operating in this system? Briefly eplain how you know referring specifically to the cell populations in the above plots. Clonal deletion. + + cell are present in b but not a mice. In a perfect answer, would say that + B cells present in non tg, don t have specific BCR. In b background specific BCR epressing B cells present(upper rt quadrant), all B cells deleted in a background (no + or specific BCR+ cells present.) Draw a plot you might have seen if anergy were the mechanism of tolerance in this system. Transgenic on a background

6 6 Name 16. (8 pts) For each of the following phrases, say whether they are likely to apply to an initial () B cell response to a protein antigen, or to a B cell response generated after repeated immunizations with the protein antigen (secondary response), or to both. Requires T cell help. both Frequency of antigen specific B cells < 1/10000 Frequency of antigen specific B cells > 1/1000 secondary K d of antibodies < 10-6 M secondary K d of antibodies > 10-6 M High rate of somatic hypermutation secondary Very little somatic hypermutation Primarily produced 17. (10 pts) What is (are) each of the following and how are they important. (Two sentence limit for each): Allelic eclusion Mechanism that ensures that B cells only epress one of their HC and LC genes Hybridoma Hybrid between a myeloma cell and a plasma B cell: used to produce monoclonal antibody CD40 Receptor on B cell that binds to CD40L on helper T cell during T-B collaboration follicular dendritic cell cell that presents antigen to B cells in germinal center. Responsible for affinity maturation. λ5 component of surrogate light chain/prebcr that promotes pro > pre B cell transition and allelic eclusion.