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[answered] 1) Purified CD4 T cells were stimulated in vitro with anti-


Can someone please help me with the questions in the attached file? ?There are six Immunology homework questions that I am struggling to answer.

Thank you for the help in advanced?

1) Purified CD4 T cells were stimulated in vitro with anti-CD3 plus anti-CD28 antibodies to activate


them, and cultured for two days in the presence or absence of all-trans retinoic acid (RA). The T


cells were then stained with antibodies to either a4:7 integrin or for E/P-selectin ligand


expression and analyzed by flow cytometry. The results are shown in the upper graph in Figure


Q1B. In a second assay, the cells were tested for their ability to migrate in response to a panel


of chemokines, and the percentages of migrated cells were measured, as shown in the lower


graph. Note that these chemokines are ligands for the receptors as indicated in Figure Q1.A. In


the absence of infection or inflammation, where are naive T cells encountering RA, and based


on these data, what effect will this have on their subsequent behavior? Figure Q1.A Figure Q1.B 2) Mice and humans with inactivating mutations in the gene encoding activation-induced cytidine


deaminase (AID) have an immunodeficiency disease known as ?hyper IgM type 2?. Since AID is


the enzyme that catalyzes the conversion of cytosines in the DNA to uracils, thereby initiating


the process of somatic hypermutation, why do individuals with this deficiency only produce IgM




3) Irradiation of mice with a dose of 600 rad of total body irradiation eliminates 95% of total


lymphocytes from spleen and lymph nodes and also eliminates all antigen-specific memory B


cells. Nonetheless, when Influenza A-infected mice are subjected to this irradiation at 60-days


post-infection, and then reconstituted with bone marrow cells from a naive mouse (this


replenishes all of the lymphocyte populations), the levels of circulating anti-Influenza A IgG


antibodies show nearly no decline when mice are monitored for the following year. What is the


explanation for this finding? Page 1 of 5 4) In addition to producing distinct innate responses locally at the site of infection, the different


cytokines produced during type I, type 2, or type 3 immune responses also induce distinct


adaptive immune responses that are tailored to the eradication of the three different classes of


pathogens. One example is the production of different classes of antibodies during type I, type


2, or type 3 responses. Which step during the induction of the adaptive immune response is the


key to generating and coordinating the three different immune modules?


5) MPSV-4 is a tetravalent polysaccharide vaccine designed to elicit antibodies to four different


serotypes of Neisseria meningitidis, a bacterial infection that causes meningitis and sepsis in


susceptible individuals. However, the protection induced by this vaccine is short-lived, and


normally wanes to undetectable levels by 2?3 years post-vaccination. In addition, MPSV-4 does


not induce mucosal immunity to Neisseria meningitidis; instead, it only protects against bacteria


that access the bloodstream. Two newly developed vaccine candidates (A and B) are tested in


mice for their ability to elicit high concentrations of anti-meningococcal antibodies that would


provide mucosal as well as bloodstream protection. Also, the ideal candidate vaccine should


also provide long-lasting immunity to the infection. Figure Q5.A is a diagram of the results from


the primary immunization with both candidate vaccines: Figure Q5.A


a) what is the predominant antibody isotype elicited by the primary immunization with these


candidate vaccines? In which part of the body is that antibody primarily found? Figure Q5.B


shows the responses to a primary, followed by a secondary immunization to each of the two


candidate vaccines. Figure Q5.B


b) Which candidate vaccine elicits the preferred response? What are the three aspects of the


preferred response that make it the candidate vaccine of choice? Page 2 of 5 The vaccine developers refuse to divulge the components of the candidate vaccines A and B.


c) What is the likely composition of each vaccine and what evidence from the information above


are used to lead to your conclusions?


d) To confirm the choice of the preferred candidate vaccine, what type of additional information


from the vaccine trials in mice shown above would support this conclusion? Name two


additional features of the secondary antibody response to each candidate vaccine that could be


assessed, and what results would be expected for each of the candidate vaccines.


e) To assess whether either candidate vaccine might provide mucosal immunity in addition to


immunity in the bloodstream, what feature of the response to each vaccine should be




6) Hyper-IgE syndrome, also known as Job?s syndrome, is an immunodeficiency disease


resulting from the lack of function of a single gene (gene ?X?). Patients with this disease are


highly susceptible to infections with extracellular bacteria and fungi, most frequently including


Staphylococcus aureus infections and Candida albicans infections in the skin. Analysis of the


various immune cell compartments indicates that these patients have normal numbers of each


cell lineage (i.e., CD4 and CD8 T cells, B cells, monocytes, dendritic cells, NK cells,


granulocytes, etc.), and normal levels of IgG, IgA, and IgM antibodies, but higher than normal


levels of IgE.


a) Given this information, name a likely component of the immune response that could be


impaired in these patients.


To investigate the immune mechanism impaired in these patients, a mouse model of this gene


deficiency was generated. Conditional knockout mouse lines were generated in which gene X


was knocked out in either the T cells, the B cells, or the myeloid cells of the mouse. For each


conditional knockout line, mice were challenged with Candida albicans, and the ability to clear


the infection was assessed. In mice, infection of the oral cavity with Candida albicans has been


shown to be a valid model for mucosal Candida albicans infections in humans. After infection,


the response was assessed by measuring fungal burden (CFU/g tissue) on the tongue. The


resulting data are shown in Figure Q6.A. Figure Q6.A Page 3 of 5 b) Based on these data, what is the most likely immune function impaired in the Gene Xdeficient




Histological examination of tongue sections from Candida albicans infected mice were


examined, and the numbers of infiltrating leukocytes (white blood cells) were quantified in each


microscopic field of each section, and the results are shown in Figure Q6.B. Figure Q6.B


c) Do these data support or refute your hypothesis stated in response to question (b)? Why or


why not?


To examine the details of T cell responses when Gene X is absent from the T cells, a series of


in vitro experiments were performed. CD4 T cells were isolated from wild type mice and from T


cell-deleted Gene X knockout mice, and were stimulated in vitro with a combination of anti-CD3


and anti-CD28 antibodies to activate the T cells. In addition, each culture was supplemented


with one of the following cytokine conditions: (1) IFN- plus IL-12; (2) IL-4; or (3) IL-6, TGF-,


IL-1 plus IL-23. After four days, the cells were examined for the expression of transcription


factors by RT-PCR, as shown in Figure Q6.C. Note that Gene X does not encode T-bet,


GATA3, or RORt. Instead, these data indicated impaired responses of Gene X-deficient T cells


to the cytokines used in these in vitro culture experiments. Figure Q6.C


d) Based on these data, name three candidate genes that could be Gene X. Page 4 of 5 Page 5 of 5


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