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


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

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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

 

antibodies?

 

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

 

examined?

 

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

 

patients?

 

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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