Proefschrift--Universiteit Leiden, 2007. / Description based on print version record. Includes bibliographical references.

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.

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Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Jodi L. McGill / The causative agent of tuberculosis (TB) in cattle is Mycobacterium bovis (M. bovis). γδ T cells are a unique subset of nonconventional T cells that play major roles in both the innate and adaptive arms of the immune system. Bovine γδ T cells have the capacity for multiple immune functions during infection with M. bovis. However, the alternative functions of γδ T cells as well as the responses of γδ T cells in vivo at the site of infection remain unclear. To identify novel functions for γδ T cells in response to M. bovis infections, RNA sequencing and transcriptomics analysis was completed on peripheral blood γδ T cells isolated from virulent M. bovis-infected cattle. Differentially expressed genes were confirmed with real-time PCR. In an attempt to model in vivo cell-to-cell interactions at the site of infection, γδ T cells were also isolated from naïve and M. bovis-infected calves and co-cultured with autologous, BCG-infected, monocyte-derived macrophages. γδ T cell chemokine and cytokine expression was analyzed via ELISA and real-time PCR. The characteristic lesions of bovine tuberculosis are well-organized pulmonary granulomas. To determine the relevance of the RNA-sequencing and in vitro co-culture results to in vivo infection, tissue samples from granulomatous lesions in the lungs and mediastinal lymph nodes of virulent M. bovis-infected cattle were collected 3 months after infection. mRNA transcripts for γδ T cells expression of-- IFN-γ, IL-17, IL-10, IL-22, and CCL2 were microscopically evaluated within the granulomas using an in situ hybridization system, RNAScope (Advanced Cell Diagnostics Inc.). Co-culture experiments and transcriptomics analysis revealed increased expression of chemokines and various cytokines by γδ T cells responding to M. bovis infection. The novel in situ hybridization assay revealed that cytokine expression by γδ T cells varied within the lesions, with significant levels of CCL2 and IFN-γ, and low expression of IL-10, IL-22, and IL-17 in situ at this time-point after infection. Co-culture experiments also revealed that γδ T cells from virulent M. bovis-infected cattle have the capacity to directly impact the viability of M. bovis in vitro. Our results suggest that γδ T cells accumulate within the granulomas, and influence host immunity to M. bovis by secretion of cytokines and chemokines, and direct cytotoxicity, in response to infected macrophages.

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The innate immune response mediates immediate control of the pathogen and is followed by the acquired immune response which is slower but ensures comprehensive elimination of the pathogen. Dendritic cells are unique innate immune cells that can phagocytose the pathogen and generate pathogen-associated antigenic peptides for presentation to T cells in order to initiate the acquired immune response. Dendritic cells also express cytokines which facilitate pathogen control and development of acquired immune responses, thus acting as a bridge between innate and acquired immune responses. CD8+ T cells are important cells of the adaptive immune system that play a key role in mediating clearance and protection against intracellular pathogens. Upon engagement by antigen-presenting cells, CD8+ T cells undergo massive expansion followed by a swift, extensive contraction to restore homeostasis. The mechanisms behind the expansion and contraction of CD8+ T cells are yet to be completely elucidated. FoxO3a is a transcription factor that is involved in the regulation of various vital cellular processes ranging from cell proliferation and cell metabolism to stress resistance and cell death. I have, therefore, investigated the role of FoxO3a signaling in the activation of dendritic cells and CD8+ T cells. My initial experiments indicated that FoxO3a regulates the homeostasis of various immune cells including CD8+ T cells and dendritic cells. CD8+ T cells lacking FoxO3a displayed enhanced proliferation, as evaluated by cell imaging, CFSE dilution and Ki67 staining, upon polyclonal stimulation in vitro. The modulation of cell proliferation by FoxO3a seemed to be p27kip-independent, as evaluated by western blotting. At later stages of stimulation, FoxO3a-deficient CD8+ T cells underwent reduced cell death, as assessed by cell counting and 7-AAD staining, and this seemed to be independent of Bim, Caspase 8 or Caspase 3 activation. In addition, FoxO3a regulated cytokine expression by CD8+ T cells while displaying similar NFκB activation in comparison to WT CD8+ T cells. Similar results were observed in dendritic cells upon LPS stimulation in vitro, wherein cytokine expression was higher in the FoxO3a-deficient dendritic cells and they also displayed enhanced antigen presentation to CD8+ T cells, as evaluated by CFSE dilution. Taken together, these results indicate that FoxO3a acts as a negative regulator of CD8+ T cell and dendritic cell activation.

Surgical resection is the leading treatment of most solid tumours, however surgical stress creates an immunosuppressive environment that promotes metastases. A global decrease in T cell numbers and function post-surgery has been documented. However, the effect on tumour associated antigen (TAA)-specific T cells remains unclear. The objective is therefore to evaluate the impact of surgical stress on TAA-specific adaptive T cell immunity. Melanoma tumour-bearing C57BL/6 mice were vaccinated using AdhDCT, an adenovirus expressing dopochrome totaumerase (DCT), a melanoma TAA, and underwent abdominal nephrectomies to induce surgical stress. Surgical stress decreased the number of splenic cytotoxic T cells (CTLs) and their capacity to produce immunostimulatory cytokines (IFNγ and TNFα), as determined by flow cytometry. A perioperative accumulation in CTL-suppressive MDSCs was observed and demonstrated a direct suppression of CTL IFNγ and TNFα production and secretion. Understanding the mechanisms of perioperative T cell dysfunction will facilitate the development of targeted immunotherapies.

Interleukin (IL)-7 is an essential non-redundant cytokine and throughout the life-span of a T cell signaling via the IL-7 receptor influences cell survival, proliferation and function. It is therefore no surprise that expression of the IL-7 receptor alpha-chain (CD127) is tightly regulated. In this study I establish IL-7 down regulates CD127 gene transcription and surface protein expression in primary human CD8 T cells through two mechanisms. Upon binding IL-7, surface CD127 is rapidly internalized and phosphorylated at the critical tyrosine residue Y449. Concurrent activation of the JAK/STAT5 pathway stimulates expression of CIS, a member of the SOCS family of proteins. CIS protein already expressed at basal levels and induced by IL-7 bind directly to CD127 as demonstrated by Coimmunoprecipitation assays and colocalize with both CD127 and the early endosomal marker EEA1. Subsequent proteasomal degradation of CD127 and CIS is dependent on an E3 ligase. Through siRNA-mediated knockdowns I confirm CIS plays a predominant role in the IL-7 mediated degradation of CD127. The mechanism by which IL-7 suppresses CD127 transcripts in primary human CD8 T cells was also examined. Through qPCR and nuclear run-on assays I illustrate that IL-7 suppresses CD127 gene transcription in a time- and dose-dependent manner. The IL-7 mediated suppression of CD127 transcripts is dependent on JAK/STAT5 signaling. Notably, cycloheximide blocked IL-7’s ability to down-regulate CD127 transcripts suggesting IL-7 stimulates the de novo synthesis of a transcriptional repressor of the CD127 gene. Through PCR arrays, qPCR and Western blot analysis the IL-7 inducible transcription factor c-Myb was identified as a candidate repressor. The region within the CD127 gene promoter required for IL-7 mediated transcriptional suppression was identified through progressive truncations using firefly luciferase as a reporter gene and is located from -1760 to -2406 bp upstream of the TATA box and contains three putative c-Myb binding sites. Using siRNA-mediated knockdown and transient over-expression, I illustrate c-Myb suppresses CD127 gene transcription in primary human CD8 T cells. A thorough understanding of the mechanisms by which IL-7 regulates CD127 expression is imperative and may reveal novel insights into the contribution of abnormal IL-7 signaling to diseases affecting immune function.