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Exam 12: The Mucosal Immune System

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In a second experiment, the isolated splenic and lamina propria macrophages were treated with LPS and then incubated with the protein antigen, chicken ovalbumin. CD4 T cells specific for a peptide of ovalbumin (OVA) bound to MHC class II were then incubated with each population of macrophages, and three days later, the culture supernatants were analyzed for IL-2 and IFN- $\gamma$ production. Which of the graphs in Figure most likely represents the results of this experiment?

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Oral inoculation with rotavirus, an intestinal pathogen, induces adaptive immune responses that are initiated in gut-associated lymphoid tissue, such as mesenteric lymph nodes and Peyer's patches. The rotavirus-specific effector T cells that are generated in this response are never found in the circulation, but home directly from the lymphoid tissue to the epithelium without ever leaving the intestinal environment.

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NOD proteins are intracellular sensors of infections that recognize cell wall or peptidoglycan components of many bacteria. The importance of these sensors in maintaining homeostasis in the intestine is indicated by the consequences for individuals lacking one of these sensors, NOD2. Specifically, NOD2 mutations have been found in a large percentage of individuals with a form of inflammatory bowel disease known as Crohn's disease. How might the absence of NOD2 lead to loss of barrier integrity and thus to increased inflammation in the gastrointestinal epithelium?

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 a $\alpha$ 4: $\beta$ 7 integrin or for E/P-selectin ligand expression and analyzed by flow cytometry. The results are shown in the upper graph in Figure. 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. 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?

The TACI receptor on B cells, which binds to BAFF and APRIL, is important in IgA antibody secretion by B cells in the gastrointestinal lamina propria.

Infection of mice with the bacterial pathogen, Citrobacter rodentium, elicits a protective immune response in the gastrointestinal tract. This protective response is characterized by two sequential waves of IL-22 production, both induced by IL-23. The early wave of IL-22 production (<day 8 post-infection) is likely produced by:

IPEX syndrome is a genetic disease due to mutations that disrupt the function of an important transcription factor expressed in subset of CD4 T cells. Individuals with this disease generally begin showing symptoms shortly after birth. This disease is often fatal. One of the most prominent symptoms of IPEX is severe gastrointestinal inflammation accompanied by severe diarrhea. This transcription factor is most likely:

Oral tolerance to food antigens and immune tolerance to gut microbiota share the property that foreign antigens encountered in the gastrointestinal (GI) tract-food and commensal microbes, respectively-do not elicit immune effector responses. Yet, these processes differ in that commensal microbes will still elicit protective adaptive immune responses if they cross the GI epithelium and enter the body.

Reovirus is an enteric virus that infects mice by adhering to intestinal M cells and then using M cell transport to enter Peyer's patches. Mice that were orally inoculated with reovirus cleared the primary infection, and upon secondary challenge 21 days later, had no detectable virus in their Peyer's patches. In contrast, naive controls that did not receive the primary inoculation had >10³ PFU of virus/mg of tissue following oral challenge with virus. The protective response induced by the primary oral inoculation would also be eliminated in:

Macrophages were isolated from the spleen or the intestinal lamina propria, and stimulated in vitro with the indicated TLR ligands. Twenty-four hours later, the culture supernatants were tested for the amounts of IL-10, the IL-12p40 subunit (shared between IL-12 and IL-23) , and for IL-12, and the results are shown in Figure . These data indicate that the lamina propria macrophages:

Analysis of human milk from lactating mothers shows that it contains IgA antibodies against infections that were recent (<3 weeks earlier) and those from the distant past (>1 year) . These antibodies are directed against a host of organisms, including viruses, such as enteroviruses, herpes simplex viruses, respiratory syncytial virus, rubella, reovirus, and rotavirus. In addition, IgA antibodies against many bacteria are found in human milk, including those reactive to E. coli, Shigella, Salmonella, Campylobacter, Vibrio cholerae, H. influenzae, S. pneumoniae, Clostridium difficile, C. botulinum, and Klebsiella pneumoniae. IgA antibodies to the parasite Giardia and the fungus, Candida albicans, are also seen in human milk. Since most of these infections were localized in the gastrointestinal tract of the mother, these IgA antibodies ended up in breast milk by:

Wild-type mice are adoptively transferred with naive CD4 T cells isolated from OT-II T-cell receptor transgenic mice. These OT-II T cells are specific for a peptide of the chicken ovalbumin protein (OVA) bound to MHC class II. After transfer, the OT-II cells rapidly disperse to all of the secondary lymphoid organs in the recipient mice. Half of the recipient mice are then given OVA protein dissolved in their drinking water, and the other half of the mice receive normal drinking water for 5 days. On the following day (day 6), lymphocytes from the spleen, the peripheral lymph nodes (LN), mesenteric lymph nodes (MLN), Peyer's patches (PP) and lamina propria (LP) are isolated and examined for the proportions of CD4 T cells in each organ expressing FoxP3. The results are shown in Figure. a) What is the conclusion from this experiment? To investigate this further, dendritic cells are isolated from the spleen (spl-DC) or from the lamina propria of the small intestine (LP-DC). Each subset of dendritic cells is pulsed with the OVA protein, and then incubated with naive OT-II CD4 T cells for 5 days in the presence or absence of TGF- $\beta$ . The CD4 T cells are then examined for FoxP3 expression; the results are shown in Figure. b) Why is TGF- $\beta$ added in this experiment? c) What is the major difference between the splenic dendritic cells and those isolated from the lamina propria? The CD4 T cells from the experiment above, in which the T cells were cultured for 5 days with OVA-pulsed splenic or lamina propria dendritic cells in the presence or absence of TGF- $\beta$ , were also assessed for their surface expression of the integrin $\alpha$ 4 $\beta$ 7. d) Of the four groups of T cells shown in the graph, which ones would be expected to show high levels of $\alpha$ 4 $\beta$ 7?

Vibrio cholerae causes an acute diarrheal illness that can be fatal if not treated. Several vaccines have been developed in an effort to prevent this disease. The oral cholera vaccine is a mixture of killed Vibrio cholerae bacteria plus additional inactivated cholera toxin protein. Efficacy studies of this vaccine indicate that it prevents 50-60% of the cases of cholera infection observed in non-vaccinated individuals. In contrast, injectable vaccines made from killed bacteria or purified bacterial subunits are substantially less effective at preventing infections. This is likely due to the fact that:

Mice deficient in the enzyme MMP7, that cleaves prepro- $\alpha$ -defensin to the active $\alpha$ -defensin, show an increased susceptibility to the enteric pathogen, Salmonella typhimurium. The cell type in the gastrointestinal (GI) epithelium most likely to express the highest levels of MMP7 is:

Mice lacking the poly Ig receptor (pIgR) have an immunodeficiency disease characterized by increased susceptibility to mucosal infections and an increase in the penetration of commensal microbes into the body's tissues. Yet, a genetic deficiency in the production of IgA antibodies is the most common form of human immunodeficiency, and is generally a mild disease and often even asymptomatic. This dichotomy can be explained by:

Mice lacking IL-15 or the IL-15R $\alpha$ chain have substantially reduced numbers of type b IELs, including both $\alpha$ : $\beta$ and $\gamma$ : $\delta$ T-cell receptor-positive subsets. Normal numbers of type b IELs can be restored in il15^-/- mice by cell-type specific expression of IL-15 in:

Secretory IgA produced in the epithelium of mucosal surfaces has several functions in protective immunity. Among these are neutralization of pathogens or toxins in the gastrointestinal tract lumen or in epithelial cell endosomes, neutralization of pathogens or toxins that cross the epithelial barrier, and transport of pathogens or toxins across M cells for delivery to lamina propria dendritic cells. All of these functions share the common feature that they:

IL-10-deficient mice develop spontaneous colitis, a disease due to chronic inflammation in the gastrointestinal (GI) tract. However, when these mice are housed in germ-free conditions, in which they lack all commensal microbes in their GI tract, no colitis was observed. In addition, one would expect germ-free IL-10-deficient mice to also show:

A healthy intestinal mucosa is one in which induced adaptive immune responses to pathogenic infections are balanced by the lack of responses to innocuous food antigens and commensal microbes. This balance is maintained by an array of different subsets of effector T cells and regulatory T cells that reside in the intestinal epithelium and lamina propria. Although these different T cell subsets have diverse patterns of cytokine production and other effector functions, they share:

Patients receiving hematopoietic stem cell transplants often suffer from graft-versus-host disease (GVHD) , involving immune-mediated damage to the gastrointestinal (GI) tract. The symptoms of GVHD include nausea, vomiting, diarrhea, and abdominal cramping due to epithelial cell apoptosis and inflammatory leukocyte infiltration into the GI epithelium. One proposed treatment has been tested in mouse models of GVHD for potential therapeutic benefit. This treatment is: