The vertebrate immune system contains a diverse inventory of genetic, epigenetic, molecular and cellular mechanisms dedicated to distinguishing and to determining appropriate responsiveness to "self" and "non-self." Autoimmune diseases such as type 1 diabetes (T1D), caused by immune destruction of insulin-producing beta cells in the pancreas, are the result of breakdowns in these mechanisms. Recent T1D research efforts have uncovered the opposing functions of pancreatic Foxp3+ regulatory T \((T_{reg})\) cells and natural killer (NK) cells as critical determinants of tolerance versus autoimmunity. Here, we examine the extrinsic adaptive regulation of NK cells by \(T_{reg}\) cells, and profile the tissue-specific heterogeneity of NK cells for intrinsic mediators of NK cell tolerance. Depletion of \(T_{reg}\) cells in the BDC2.5/NOD model resulted in destruction of pancreatic islets and a high penetrance of T1D. Prior to the activation of T cells, there was a rapid and localized activation of pancreatic NK cells, including their proliferation and production of diabetogenic \(IFN-\gamma\). How \(T_{reg}\) cells exerted their dominant tolerance on NK cells in this setting was unclear. We explored the molecular mechanisms underlying this NK/\(T_{reg}\) cell axis, following leads from a kinetic exploration of gene-expression changes early after punctual perturbation of \(T_{reg}\) cells. Our data supported a scenario in which \(T_{reg}\) cells controlled NK cell functions by limiting the bioavailability of T cell-derived IL-2 in the islets, representing a novel intertwining of innate and adaptive immunity. Cell intrinsic regulatory mechanisms, such as the expression of the Ly49 receptor family during NK cell education, tune NK cells to be functionally self-tolerant. However, the transcriptional repertoire of Ly49 receptors had never been comprehensively explored. We performed RNAseq-based profiling of the Ly49 receptors, and uncovered a subtle difference in the expression of Ly49E and Ly49H on pancreatic NK cells compared to spleen. We also expanded the phenotypic profiling of pancreatic NK cells using high-dimensional mass cytometry and uncovered greater diversity than had previously been described. Taken together, these studies highlighted a new degree of heterogeneity in pancreatic NK cells and uncovered a novel regulatory mechanism, originating from the adaptive immune system, responsible for maintaining NK cell tolerance.

At least 1.4 million of Americans suffer from Inflammatory Bowel Diseases (IBD). IBD (Crohn’s disease and ulcerative colitis) is a spontaneously relapsing, immunologically mediated disorder of the gastrointestinal tract. Complete medical cure remains a challenge and the probability of relapse is over 70%. Curcumin has been shown to have a protective role in mouse models of inflammatory bowel diseases (IBD) and to reduce the relapse rate in human ulcerative colitis (UC), thus making it a potentially viable supportive treatment option. The objective of this research project was to provide a preclinical evaluation of curcumin’s efficacy in relevant models of human IBD, and to investigate the molecular mechanisms of its protective mechanism of action. (1) We investigated the effect of dietary curcumin in trinitrobenzene sulfonic acid (TNBS)-induced colitis in SJL/J mice (Th-1/Th-17 response) and in BALB/c mice (Th-1/Th-2 response). We demonstrated that the efficacy of dietary curcumin varies in the two strains. Although the exact mechanism underlying these differences remains unclear, our observations suggest that the therapeutic value of dietary curcumin may vary depending on the nature of immune dysregulation. (2) We further confirmed those findings and we investigated the effects of curcumin on the development of colitis, immune activation, and in vivo NF-κB activity in germ-free IL-10^(–/–) colonized with specific pathogen-free microflora. In this model resembling CD, we demonstrated that IL-10 and curcumin act synergistically to downregulate inflammation. (3) Neutrophil aberrant accumulation at the intestinal mucosa is a characteristic hallmark of inflammatory conditions such as ulcerative colitis. Neutrophil transepithelial migration leads to an impaired epithelial barrier function, perpetuation of inflammation and tissue destruction. Therefore, we investigated the effect of curcumin on neutrophil polarization and motility. Our results indicated that curcumin interferes with colonic inflammation partly through chemokine expression inhibition and neutrophil chemotaxis and chemokinesis inhibition. We also demonstrated that curcumin significantly reduced epithelial tissue injury generated by neutrophil transepithelial migration and protease release. Those findings significantly add to our understanding of the mechanism by which curcumin affects the innate and adaptive immune response in IBD and may help develop innovative therapeutic strategy for IBD.

The diagnosis of infectious diseases is based upon the isolation and identification of the infecting organism. At times it is difficult or even impossible to isolate the causative organism and one has to rely upon serological methods for diagnosis. Infections by Mycobacterium tuberculosis present a special case. [...]

<p>Proper regulation of the immune system with regards to development, reaction specificity, and duration is necessary to prevent immune reactions directed against the host that can have a number of potentially lethal consequences. This regulation is controlled in part through a wide variety of transcription factors that function to ensure the proper development, function, and regulation of the immune system. </p><p>E-proteins are widely expressed and have a multitude of functions in the immune system including proper lymphocyte development and function. E-proteins can be regulated by class V HLH factors, including the inhibitor of differentiation (Id). genes 1-4. Id proteins are expressed throughout the hematopoietic system and have crucial roles in cell fate decisions, differentiation and proliferation in a multitude of tissues and cell types.</p><p>Id3 plays a variety of important roles in the immune system including T cell homeostasis, activation, and effector function. The importance of Id3 has been demonstrated using a number of mouse models, and mice that lack Id3 develop an autoimmune condition similar to that of Primary Sjögren's syndrome (PSS). The goal of this dissertation is to further characterize disease initiation and progression in Id3-/- mice, with a focus on the specific targeting of exocrine glands and associated lymphoid tissues by the immune system. </p><p>T cells play a crucial role in the development of disease symptoms in Id3-/- mice, though much remains unknown about the relative contribution of the two major subsets of T cells, &#945;&#946; and &#947;&#948; T cells to disease severity. The importance of both &#945;&#946; and &#947;&#948; T cells is demonstrated in part by the use of two newly generated models, Id3/&#946;-/- and Id3/&#948;-/- mice, which lack &#945;&#946; or &#947;&#948; T cells, respectively. These mice have allowed for a better understanding of the relative contribution of T cells in disease initiation and progression in Id3-/- mice.</p><p>Analysis of serum cytokine levels show that Id3-/- mice develop elevated levels of IL-13 at an early age, and this cytokine is associated with impaired saliva function. Significant populations of IL-13+ T cells have been identified in the peri-glandular lymph nodes of Id3-/- mice, though it appears that there are significant IL-13 producing cells not of the T cell lineage. Reduction of serum IL-13 levels via anti-IL-13 antibody treatment resulted in improved saliva production in response to cholinergic stimulation and reduced the number of mast cells detected in the mandibular and lachrymal glands. The importance of IL-13 in the initiation and progression of exocrinopathy in Id3-/- is being further investigated using a recently acquired IL-13 reporter mouse model. Additionally, IL-13R&#945;1+ cells have been identified in and around the gland tissues of Id3-/- mice, and while the origin and function of these cells remains unknown, these cells have a potential to serve as a biomarker for disease progression and severity.</p><p>Lastly, analysis of Id3-/- mice reveals an increased presence of mast cells in the peri-glandular lymph nodes and gland tissues as compared to wild type controls. These mast cells are localized in areas of significant tissue remodeling, serve as a potential source of IL-13 and are associated with impaired saliva production. These findings suggest an important role of mast cells in disease development in Id3-/- mice. Together, these studies have revealed a number of findings that will likely contribute to our understanding of the initiation and progression of exocrinopathy in Id3-/- mice.</p> / Dissertation

<p>The chromatin architecture of antigen receptor loci has been hypothesized to facilitate the assembly of variable (V), diversity (D), and joining (J) gene segments during lymphocyte development. The 1.6 megabase Tcra/Tcrd locus is unique since it undergoes highly divergent Tcrd and Tcra recombination programs in CD4&ndash;CD8&ndash; double negative (DN) thymocytes and CD4+CD8+ double positive (DP) thymocytes, respectively. In this dissertation, we asked whether these divergent recombination programs are supported by distinct conformational states of the Tcra/Tcrd locus by using three-dimensional fluorescence in situ hybridization (3D-FISH) and chromosome conformation capture (3C). </p><p>Using 3D-FISH, we found the 3' portion of the locus is contracted in both DN and DP thymocytes as compared to B cells. Remarkably, the 5' portion of the locus is contracted in DN thymocytes, but is decontracted in DP thymocytes. We propose that the fully contracted conformation in DN thymocytes allows Tcrd rearrangements involving V&alpha; gene segments distributed over one megabase, whereas the unique 3'-contracted, 5'-decontracted conformation in DP thymocytes biases initial Tcra rearrangements to the most 3' of the available V&alpha; gene segments. This would maintain a large pool of distal V&alpha; gene segments for subsequent rounds of recombination.</p><p>To study the conformational changes at the molecular level, we used 3C to detect interactions between different sites spanning 400kb in the contracted 3' portion of the locus. The Tcra enhancer (E&alpha;) is known to activate V&alpha; and J&alpha; segment promoters and to stimulate V&alpha;-to-J&alpha; recombination in DP thymocytes. We detected various pair-wise interactions between elements essential for initial Tcra recombination, including proximal V&alpha; segments, TEA promoter, 5' J&alpha; array and E&alpha;. Notably, these interactions occur specifically in DP thymocytes and all are E&alpha;-dependent. We proposed that in addition to regulating transcriptional activity, E&#945; promotes synapsis of RSSs by tethering proximal V&alpha; and 5'J&alpha; segments together to facilitate initial Tcra recombination. </p><p>We also asked whether a known chromatin organizer, CTCF, regulates the formation of the DP stage-specific, E&alpha;-dependent chromatin hub. Using ChIP-seq, we identified CTCF binding sites at E&alpha;, TEA promoter, and many V&alpha; promoters in DN and DP thymocytes. Loss of CTCF in DP thymocytes resulted in impaired primary V&#945;-to-J&#945; recombination, reduced V&alpha; and TEA germline transcription, and reduced interactions between E&alpha; and Tcra genes. Strikingly, we also observed aberrantly increased Tcrd gene transcription and interactions between E&alpha; and Tcrd gene segments in CTCF-deficient DP thymocytes. Our data suggest that CTCF helps E&alpha; to organize a DP stage-specific chromatin hub that sets the stage for synapsis and recombination of proximal V&alpha; and 5' J&alpha; segments in DP thymocytes.</p> / Dissertation

Thesis (Ph. D.)--University of Toronto, 2005. / Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin--Madison, 1963. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 61-64).

Thesis (Ph. D.)--Universite de Sherbrooke, 2004. / Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin--Madison, 1971. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Activating antibodies against the T-cell costimulator CD28 fall into two categories: those that require an additional stimulus, such as an anti-TCR antibody, to fully active a T cell are termed "conventional antibodies" (CAs), and those that induce such strong signalling that T cells become activated in the absence of TCR ligation are referred to as "superagonists" (SAs). It was proposed that SAs are particularly potent because they sterically exclude large phosphatases such as CD45 more efficiently from signalling receptors versus CAs. To test this, we stimulated T-cell lines with an anti-CD28 SA or CA immobilised on glass surfaces. Although no TCR ligand is present, we confirmed that the expression of the TCR is nevertheless required for SA-induced signalling on glass. We showed that the SA is only active on glass when it is immobilised via its Fc, and that signalling is sensitive to the dimensions of the antibody/CD28 complex relative to those of CD45. We then visualised the organisation of these proteins by imaging. When treated with the SA, T cells form contacts with the glass slide that exclude CD45 ~ 2-fold more efficiently versus CA treatment, and both antibody-bound CD28 and CD45 slow down their diffusion to a greater extent under SA versus CA treatment. Both the exclusion of CD45 and the restriction of CD28 and CD45 diffusion appear to be steric effects, caused by the SA bridging a narrow gap between cell and glass surface. We hypothesize that SAs induce particularly potent signalling by protecting CD28 from dephosphorylation by CD45 via (1) spatially segregating the two proteins, and (2) reducing their encounter rate by slowing them down, while kinases continue to diffuse and have access to CD28. In summary, our results provide evidence that antibody superagonism is driven by size-dependent local perturbations in kinase and phosphatase organisation and diffusion.