Recent research of a cohort of HIV exposed sero-negative (HESN) female commercial sex workers in Nairobi has revealed an Immune Quiescent phenotype; characterized by reduced T cell activation and higher regulatory T cells (Tregs) in peripheral blood. HESN women also express lower levels of interferon regulatory factor-1 (IRF-1), a critical regulator known to negatively impact Treg development in mice. In this study, we analyzed the functional capacity of Tregs by an in vitro depletion assay and measured functionality by flow cytometry. Data showed Tregs suppressed CD4+ and CD8+ proliferation responses. We characterized the link between Tregs and IRF-1 in HESN and observed an inverse correlation between IRF-1 expression and Treg proportions. We also established reduced expression of IRF-1 in Tregs of healthy donors by flow cytometry. In a separate study, flow cytometric analysis of high-risk sex-workers revealed that CTLA-4 expression in memory CD4+cells, not Treg frequency, was associated with HIV seroconversion. / February 2017

The study of regulatory T cells (Tregs) is a relatively new field. Within the past few decades, research on Tregs has greatly deepened scientists’ understanding of the link between the immune system and cancer. The study of melanoma is one such cancer that has benefited greatly from this area of study. Tregs are a subset of CD4+ T cells (TCs) that are either generated in the thymus or in the periphery. The main role of Tregs in normal immune physiology is to suppress immune cells. This is an essential component in the prevention of autoimmunity. In melanoma, however, Tregs prevent components of the immune system from mounting a robust response to cancerous lesions and tumors. Tregs have been observed to infiltrate melanoma tumors due to chemokines and other soluble signaling molecules such as CCL1 and CCL22. Once Tregs accumulate inside melanoma tumors, they generate an immunosuppressive microenvironment in a contact-dependent and contact-independent manner. IL-10 secretion and use of the CTLA-4 pathway were observed to be the most well characterized modes of suppression but other mechanisms are still being discovered. Clinicians can take advantage of new therapeutics to modulate the activity of Tregs. Exogenous administration of antibodies that bind to CTLA-4, PD-1, CCR4 and other receptors and molecules can prevent Treg development and action. Preventing Tregs from carrying out their suppressive function may allow other elements of the immune system, such as CD8+ TCs, to target and destroy melanoma cells. Clinicians can also measure the relative abundance of Tregs or use the ratio of effector TCs (Teff) and Tregs to predict patient outcomes and survival. More research is needed to determine that precise mechanisms of Treg infiltration and accumulation within the tumor and the mode of Treg suppression. This paper finds that there is no standard Treg identification marker. This can lead to aberrant results and failures such as the inability to distinguish Tregs from melanoma cells that also express Treg-like markers or a failure to identify other Treg subtypes. Lack of consensus also extends to the prognostic value of Tregs due, in part, to small sample sizes and the inability to accurately identify Tregs in vivo. Future research must focus on Treg identification, action, and the elucidation of therapeutic mechanisms. These future studies will ensure that clinicians have the correct information to choose the proper melanoma treatment that will target the specific Treg populations found within patients’ melanoma tumors.

The healthy immune system is a delicate and precisely orchestrated balance between activation and suppression. It is well established that regulatory T cells (Tregs) have substantial immunosuppressive properties and play a pivotal role in maintaining this balance. Many autoimmune states have been characterized by disproportionately high numbers of T effector cells, and comparatively low numbers of regulatory T cells (Hori et al., 2003; Sakaguchi et al., 1995; Choileain et al., 2006). Furthermore, mouse models in which regulatory T cells are removed or rendered ineffective show rapid development of autoimmunity. It is therefore hypothesized that regulatory T cells are essential to the acquisition and maintenance of self-tolerance. Type 1 diabetes is an increasingly common autoimmune condition, with 30,000 new diagnoses each year (JDRF Fact Sheet). Pancreatic islet transplantation holds great promise as a potential cure for this difficult disease; however human trials have had limited success. Attempts to promote self-tolerance or maintain a physical barrier to the transplanted islets have largely failed (Groot et al., 2004). Because of this, insulin dependence normally resumes fiver years post-operation. The deleterious effects of long-term immunosuppression to promote extended islet survival are considered too great to justify this treatment. Because of their important role in promoting self-tolerance, many immunologists believe regulatory T cells are the key to developing tolerance of islet allograft tissue. Rapamycin and anti-CD154 are immunoregulatory treatments that specifically inhibit the activation of T effector cells and promote the growth of regulatory T cell populations. As regulatory T cell numbers increase, self-tolerance is established and the need for immunosuppressant drugs is eliminated. Unfortunately, treatments such as anti-CD154 and rapamycin have had limited success due to the ability of toll-like receptor (TLR) pathways to bypass such activation blockades. TLR stimulation results in a potent and direct activation that acts to bolster the immune response. This TLR activation results in the release of inflammatory cytokines, which render regulatory T cells unstable. Regulatory T cells have been shown to adopt effector phenotypes in such environments and may have pathogenic potential. This study aims to elucidate aspects of Treg plasticity that result from TLR activation. In vitro models were used to demonstrate how TLR agonists change Treg phenotypic expression. Our findings indicate that the presence of lipopolysaccharides (LPS) has a relatively significant effect on regulatory T cell phenotypes. Specifically, our findings indicate that LPS causes increased GATA3 expression in Tregs, promoting differentiation to a TH2 phenotype (p= 0.0543). Regulatory T cells were also examined for the expression of RORγt and Tbet transcription factors. Neither transcription factor was significantly expressed, indicating the absence of TH17 and TH1 phenotypes, respectively. It is also worth noting that stability of the foxp3 transcript appeared to be greater in cells treated with LPS, than in those without (p= 0.0009). In addition, this study utilized an in vivo model for tracking regulatory T cell changes after pancreatic islet transplantation. Diabetic reporter mice received pancreatic islet transplants, as well as TLR agonist to induce allograft rejection. Mice were treated with rapamycin, anti-CD154 and TLR agonist. After 12 days, regulatory and ex-regulatory T cells were harvested from the transplanted area and analyzed. This experiment is still in progress and results have yet to be determined. This study establishes proof of concept of an effective system for the study of regulatory T cell plasticity. Additional investigation must be done in order to more thoroughly understand these important cells. This study is not complete, but our progress thus far is a strong foundation for further experimentation.

Superantigens are exotoxins produced by Staphylococcus aureus and induce extensive T cell proliferation and proinflammatory cytokines, leading to toxic shock syndrome at high concentrations. However, the role of superantigens produced at relatively low concentrations during asymptomatic colonization or chronic infection has not been well established. In this dissertation, we demonstrated that stimulation of human PBMCs with staphylococcal enterotoxin C1 (SEC1) at the dose inducing a half maximal T cell proliferation (suboptimal stimulation) induced immunosuppressive CD4+CD25+FOXP3+ and CD8+CD25+FOXP3+ T cells. The suppression of these cells was mainly mediated by the galectin-1. We found that suboptimal stimulation with SEC1 induced differential activation of PI3K-mTOR-Akt pathway, leading to expression of FOXP3 isoforms preferably localized to the nucleus and induction of PTEN that contributes to maintain stability and suppressive activity of regulatory T cells. Taken together, these results demonstrate the important role of superantigen produced at low concentration during asymptomatic colonization that induce immunosuppressive CD4+ and CD8+ regulatory T cells to promote survival, propagation, and colonization for S. aureus in the host.

Regulatory T cells (TRegs) are stable markers of immune functioning, acting to suppress inflammation. TRegs are important during implantation and early pregnancy where they suppress immune-mediated rejection of the embryo. Given the role of TRegs in the maintenance of pregnancy, their depletion can be associated with obstetric complications. Through the completion of two studies, this thesis seeks to identify the role of TRegs in two forms of perinatal pathology: depression and arterial thickening. The first study examines whether decreased TReg levels during pregnancy are associated with an increase in depressive symptoms, and if this relationship is mediated by maternal stress. We predicted that the TReg-depression relationship would be unique to pregnancy, and not occur in the postpartum. In the second study we assessed if decreased TRegs were inversely correlated with carotid arterial thickness. TReg samples were obtained from women between 24 and 32 weeks gestation (N=16), and at 12 weeks postpartum (N=19). Depression was assessed using the Edinburgh Perinatal Depression Scale (EPDS) and the Mongomery-Asberg Depression Rating Scale (MADRS) , and stress with the Perceived Stress Scale (PSS). TRegs were measured using flow cytometry. In the first study, we showed that lower TRegs were associated with increased levels of depression in pregnancy, and that this association was mediated by perceived stress. In the postpartum period, TRegs were not associated with changes in mood. In the second study, we found no relationship between TRegs and carotid arterial thickness. Our results suggest that TReg changes in pregnancy may be associated with maternal mood in pregnancy, but not in the postpartum period. Despite the fact that we failed to find a correlation between TRegs and carotid arterial thickness during pregnancy, our limited sample size leads us to recommend that the presence of an inverse correlation between these two markers not be ruled out, but suggest that these links be further examined using a larger sample and more precise imaging. Together, these two studies may provide very early insights into the role of TRegs in perinatal mood disorders and cardiovascular health and highlight the need for further research. / Thesis / Master of Science (MSc)

No description available.

The germinal centre (GC) response is critical for generating highly effective humoral immune responses and immunological memory that forms the basis of successful immunisation. Control of the output of the GC response requires Follicular regulatory T (Tfr) cells, a subset of Foxp3+ Treg cells located within germinal centres. Tfr cells were first characterised in detail in 2011 and because of this relatively little is known about the exact role of Tfr cells within the GC, and the mechanism/s through which they exert their suppressive function. At the outset of this work, the major barrier to understanding Tfr cell biology was the lack of appropriate tools to study Tfr cells specifically, without affecting Tfh cells or other Treg cell subsets. This thesis set out to develop a strain of mice that specifically lacks Tfr cells. A unique feature of Tfr cells is their CXCR5-dependent localisation within the GC. Therefore, genetic strategies that exclude Treg cells from entering the GC are a rational approach to generating a mouse model that lacks Tfr cells. To this end, I generated a strain of mice that lacks CXCR5 on Foxp3+ Treg cells. These animals show a ~50% reduction in GC localised Tfr cells, and a GC response that is comparable to control animals. These data indicated that redundant mechanisms are involved in Treg cell homing to the GC. I identified CXCR4 as a chemokine receptor that is also highly expressed on Tfr cells, and hypothesised that it may also be involved in Tfr cell localisation to the GC. Surprisingly, simultaneous deletion of both CXCR4 and CXCR5 in Treg cells resulted in a less marked reduction in Tfr cells compared to deletion of CXCR5 alone, suggesting that CXCR4 might be involved in negative regulation of Treg homing to the GC. These data identify both CXCR4 and CXCR5 as key regulators of Tfr cell biology. Bcl6 drives Tfr cell differentiation, but how this transcriptional repressor facilitates commitment to the Tfr cell subset is unknown. I hypothesised that Bcl6 drives Tfr cell differentiation by repressing Tbx21, the transcriptional regulator involved in the differentiation of Th1-like Treg cells. I tested this hypothesis in Bcl6fl/fl CD4cre/+ animals and unexpectedly found that loss of Bcl6 regulates Treg cell differentiation in the absence of immunisation or infection. I have demonstrated that thymic loss of Bcl6 results in an increase in activated effector Treg cells, which occurs very early in life. These data point to a novel role for Bcl6 in preventing early thymic Treg activation, indicating that Bcl6 has a global role in Treg development and differentiation that is not simply limited to Tfr cells.

Background: Nociceptive and neuropathic pain occurs as part of the disease process after traumatic brain injury (TBI) in humans. Central and peripheral inflammation, a major secondary injury process initiated by the traumatic brain injury event, has been implicated in the potentiation of peripheral nociceptive pain. We hypothesized that the inflammatory response to diffuse traumatic brain injury potentiates persistent pain through prolonged immune dysregulation. Results: To test this, adult, male C57BL/6 mice were subjected to midline fluid percussion brain injury or to sham procedure. One cohort of mice was analyzed for inflammation-related cytokine levels in cortical biopsies and serum along an acute time course. In a second cohort, peripheral inflammation was induced seven days after surgery/injury with an intraplantar injection of carrageenan. This was followed by measurement of mechanical hyperalgesia, glial fibrillary acidic protein and Iba1 immunohistochemical analysis of neuroinflammation in the brain, and flow cytometric analysis of T-cell differentiation in mucosal lymph. Traumatic brain injury increased interleukin-6 and chemokine ligand 1 levels in the cortex and serum that peaked within 1-9 h and then resolved. Intraplantar carrageenan produced mechanical hyperalgesia that was potentiated by traumatic brain injury. Further, mucosal T cells from brain-injured mice showed a distinct deficiency in the ability to differentiate into inflammation-suppressing regulatory T cells (Tregs). Conclusions: We conclude that traumatic brain injury increased the inflammatory pain associated with cutaneous inflammation by contributing to systemic immune dysregulation. Regulatory T cells are immune suppressors and failure of T cells to differentiate into regulatory T cells leads to unregulated cytokine production which may contribute to the potentiation of peripheral pain through the excitation of peripheral sensory neurons. In addition, regulatory T cells are identified as a potential target for therapeutic rebalancing of peripheral immune homeostasis to improve functional outcome and decrease the incidence of peripheral inflammatory pain following traumatic brain injury.

The development and function of T helper (Th) cells and regulatory T cells (Tregs) are plastic processes that are regulated by cytokines. In our project we first analyzed the effect of different cytokines on the development of induced (i) Tregs. It has been demonstrated that iTregs arise from CD4+ CD25- T cells upon stimulation with alloantigen in the presence of transforming growth factor β (TGF-β). The development of these Tregs and their proliferation were inhibited by interleukin (IL)-4 and IL-12. The aquired results also demonstrated distinct responses of naturally occuring (n) Tregs and iTregs to the regulatory action of IL-4 and an opposite role of IL-4 in maintenance of nTregs and iTregs phenotype. An important role in the induction of T cell subsets may play also mesenchymal stem cells (MSCs) which can, under specific conditions, produce TGF-β and IL-6. Depending on the current production of TGF-β or IL-6, MSCs can qualitatively regulate the ration between Tregs and Th17 cells. Anti-inflammatory Tregs and pro-inflammatory Th17 cells are induced upon stimulation in the presence of TGF-β and TGF-β and IL-6, respectively. In addition to our previous work we studied the role of IL-12 in the development of Tregs and Th17 cells. It was shown that Treg and also Th17 cell differentiation was...

Regulatory T cells (Treg) are critical to the maintenance of immune tolerance, partly by controlling the unwanted activation of effector T cells (Teff) and thereby enhancing the resolution of autoimmune and allergic inflammation. Recent data suggest that Treg can specialize to better control different types of inflammation by using transcriptional machinery which controls differentiation and function of Teff. This thesis addresses questions related to the efficacious use of Treg, notably their ability to adopt distinct phenotypic profiles under different inflammatory contexts and their need to recognize antigen in the inflamed organ. Two differentially mediated mouse disease models were used in this project, namely Th1/Th17-mediated experimental autoimmune encephalomyelitis (EAE) as a model of multiple sclerosis and Th2-mediated allergic airways inflammation (AAI) as a model of asthma. A new model of rMOG-induced AAI was developed to specifically answer the questions on the importance of cell phenotype versus antigen-reactivity for the effective Treg-mediated suppression. It was demonstrated that Treg from the inflamed CNS in EAE had an upregulated expression of Th1 master regulator T-bet and Th1-associated chemokine receptor CXCR3, whereas Treg derived from the inflamed lung in AAI had an increased expression of Th2 master regulator GATA-3, lacked expression of T-bet and displayed decreased levels of CXCR3. This specialized and activated phenotype was restricted to tissue-derived Treg. The importance of appropriate Treg phenotype for effective suppression was suggested by the observed inability of CNS-derived Treg to inhibit AAI. A different Treg subset, TGF-β-induced Treg (iTreg), was shown to express high levels of T-bet and CXCR3, but not GATA-3 upon induction in vitro. iTreg effectively suppressed both Th1 and Th2 types of inflammation and the antigenreactivity was key to this. This thesis demonstrates that Treg are capable of acquiring a distinct phenotype corresponding with a CD4+ T cell response driving inflammatory disease and identifies antigen-reactivity as key to the efficacious suppression of inflammation. It also highlights substantial phenotypic differences between iTreg and naturally-occurring Treg which could be associated with different modes of suppression.