The ability of cells within the adaptive immune system to develop into specialized subsets allow for a robust and tailored immune response in the advent of an infection or injury. Here, CD4+ T-cells are a crucial component within this system, with subsets such as TH1, TH2, TH17, TFH and TREG cells playing vital roles in propagating cell-mediated immunity. For example, TH1 cells are essential in combating intracellular pathogens such as viruses, while TFH cells communicate with B-cells to optimize antibody responses against an invading pathogen. The development (and functionality) of these subsets is ultimately dictated by the appropriate integration of extracellular cues such as cytokines with cell intrinsic transcription factors, thereby promoting the necessary gene profile. Moreover, the observation that T-helper cells could exhibit a flexible nature (i.e having shared gene profiles and effector functions) not only demonstrate the efficiency of our immune system but also how such flexibility could have unintended consequences during adverse events such as autoimmunity. An important mediator of such flexibility is cytokines. However, the complete network of factors that come together to co-ordinate cytokine mediated plasticity remain unknown. Thus, the work in this dissertation hope to delineate the factors that collaborate to regulate cytokine induced T-helper cell flexibility. As such, we see that in the presence of IL-2, the Ikaros Zinc Finger (IkZF) transcription factor Eos is upregulated in TH1 cells, with this factor playing a significant role in promoting regulatory and effector functions of TH1 cells. Moreover, we show that Eos forms a novel protein complex with STAT5 and promotes STAT5 activity in TH1 cells. However, depleting IL-2 from the micro-environment leads to the upregulation of two other members within the IkZF family, Ikaros and Aiolos. Aiolos in turn collaborate with STAT3, induces Bcl-6 expression within these cells, thus promoting these cells to exhibit characteristic features of TFH cells. The work in this dissertation hopes to advance our understanding of the regulatory mechanisms involved in cytokine mediated T-cell flexibility thereby hoping to open new avenues for the development of novel therapeutic strategies in the event of autoimmunity. / Ph. D. / T-helper (TH) cells are an important component of the immune system, as these cells aid in the fight against pathogens by secreting factors that either accentuate the inflammatory response during infection or attenuate immune responses post infection. Such effects are made possible because T-helper cells can differentiate into a variety of subsets, with each subset being an important mediator in maintaining immune homeostasis. For example, the T-helper cell subset called TH1 plays a vital role in the fight against intracellular pathogens such as viruses and certain parasites, while T-follicular helper (TFH) cells aid in the production of antibodies specific to the invading pathogen. The development of such subsets occur when cell extrinsic signals, called cytokines, lead to the activation or induction of cell intrinsic proteins called transcription factors. Interestingly, research over the years have shown that T-helper cells are highly adaptable in nature, with one subset having the ability to attain certain characteristic features of other subsets. This malleable nature of T-helper cells relies on several factors, with cytokines within the micro-environment being an important one. Although this form of flexibility is efficient and beneficial at times, it can also be detrimental, as such flexibility is known to promote certain autoimmune diseases such as multiple sclerosis, rheumatoid arthritis and type 1 diabetes. Such detrimental effects are thought to be due to cytokines within the environment. Therefore understanding how cytokines influence the flexible nature of T-helper cells is important; as controlling such flexibility (either by regulating cytokines or the transcription factors activated as a consequence) could prevent the propagation of undesired T-helper cell functions. As such, the work in this dissertation hopes to uncover how one such cytokine, termed Interleukin-2 (IL-2) mediates the flexibility between TH1 and TFH cells. The work highlighted in this dissertation broadens our understanding of how cytokines influence T-helper cell development and flexibility, and consequently allows the design of novel therapeutic strategies to combat autoimmune diseases.

Dietary n-3 polyunsaturated fatty acids (PUFA) have been shown to potently attenuate T cell-mediated inflammation, in part, by suppressing T cell activation and proliferation. Apoptosis is an important mechanism for preventing chronic inflammation by maintaining T cell homeostasis through the contraction of populations of activated T cells. We hypothesized that dietary n-3 PUFA would promote T cell apoptosis, thus, providing an additional mechanism to explain the anti-inflammatory effects. We specifically examined activation-induced cell death (AICD) since it is the form of apoptosis associated with peripheral T cell deletion involved in immunological tolerance and T cell homeostasis. Female C57BL/6 mice were fed diets containing either n-6 PUFA (control) or n-3 PUFA for 14 d. Splenic T cells were stimulated with CD3/CD28, CD3/PMA, or PMA/Ionomycin for 48 h followed by reactivation with the same stimuli for 5 h. Apoptosis was measured using Annexin V/propidium iodide and flow cytometry. Cytokine analyses revealed that n-3 PUFA enhanced AICD only in T cells expressing a Th1-like cytokine profile (high IFN, low IL-4) compared to mice fed the n-6 PUFA control diet. Dietary n-3 PUFA significantly altered the fatty acid composition of phosphatidylcholine and phosphatidylethanolamine in T cell membranes. To examine the apparently selective effect of dietary n-3 PUFA on AICD in Th1 cells, CD4+ T cells were polarized in vitro to a Th1 phenotype by culture with IL-4, IL-2, and IL-12 for 2 d, followed by culture with IL-2 and IL-12 for 3 d in the presence of diet-matched homologous mouse serum (MS) to prevent loss of cell membrane fatty acids. Following polarization and reactivation, we observed that n-3 PUFA enhanced Th1 polarization and AICD only in cells cultured in the presence of MS, but not in fetal bovine serum. The n-3 PUFA enhancement of Th1 polarization and AICD was associated with the maintenance of diet-induced changes in EPA (20:5n-3) and DHA (22:6n-3) in plasma T cell membrane lipid rafts. Overall, these results suggest that dietary n-3 PUFA enhance both the polarization and deletion of pro-inflammatory Th1 cells, possibly as a result of alterations in lipid raft fatty acid composition.