In multiple sclerosis (MS), pathogenic T effector cells (Teff) are believed to orchestrate immune-mediated destruction of the central nervous system (CNS) myelin sheath. In experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, CNS infiltration by regulatory T cells (Treg), producing the anti-inflammatory cytokine IL-10, promotes the resolution of disease. Currently, little is understood about how Treg function within the inflamed CNS and on which cells they exert their suppressive function. There is a debate as to whether Treg in MS patients are capable of infiltrating the CNS and if they do, it is unclear whether they are functional. Understanding Treg function in EAE and MS could open up new possibilities for treatment, as Treg could be modulated for immunosuppressive therapy. A key step in the development of EAE (and presumably MS) is the ability of Teff cells to cross the blood brain barrier (BBB) and enter the CNS parenchyma. The hypothesis of this work was that Treg facilitate resolution of the inflamed CNS by preventing entry of the pathogenic T cells into the CNS parenchyma, thus preventing further damage. As such, it is important to understand with which immune cells and CNS resident cells Treg communicate to achieve resolution of disease. The presence of Treg in MS lesions was investigated with double immunohistochemistry (IHC) in frozen post-mortem MS brain tissue. CD4+Foxp3+ Treg were present in a subset of patients and their presence was associated with perivascular retention of CD4+Foxp3- and CD8+Foxp3- T cells. Foxp3+ cells in MS lesions predominantly expressed IL-10, indicating regulatory activity, although low-level production of IL-17, TNF-α, IFN-γ and GM-CSF was observed as well. Generally, analysis of total cytokine expression identified distinct patterns of cytokine production between lesions. Nonetheless, these could not be used to discriminate individual patients. These studies were repeated in C57BL/6 mice in which the Treg population was depleted before onset of EAE to mimic lesions with and without Treg presence, as found in MS patients. An immunofluorescent technique to study up to 5 fluorochromes simultaneously was developed to study antigen presenting cell (APC), Teff and Treg location, spatial relationship and function (as measured by cytokine expression) in the CNS of EAE mice at different stages of disease. Using this technique it was found that CD4+Foxp3- Teff and CD4+Foxp3+ Treg were located within 50-100μm of CD11c+ APC in the CNS of EAE affected mice. CNS Teff and Treg predominantly produced IFN-γ or IL-10, although low levels of IL-17 were detected in Teff and Treg as well. IL-17+ Treg were close to IL-17+ Teff, IFN-γ+ Treg were close to IFN-γ+ Teff, but IL-10+ Treg were not in close proximity to IL-10+ T cells in the CNS during EAE. In conclusion, there is evidence for functional Treg in EAE and MS lesions, supporting the concept of enhancing Treg activity as a clinical intervention. Treg seem to be capable of retaining pathogenic T cells at the blood brain barrier in MS lesions. In addition, studies of cytokine expression in MS lesions indicated that there is no sound basis for patient stratification based on peripheral blood cytokine profile. This thesis advances our understanding of Treg location, function and spatial relationship with other immune cells within the inflamed CNS.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Histopathologic examination of the tumor microenvironment demonstrates the presence of a vast repertoire of infiltrating lymphocytes and antigen presenting cells (APC’s). Recent studies establish a strong correlation between the tumor microenvironment cell composition and prognostic value in terms of cell type, location and ratio, referred to as a tumor’s immunoscore. More specifically, the relationship between T regulatory (Treg) cell to T effector (Teff) cell percentage predominates as a mechanism of tumor immune evasion. Further investigation of the factors influencing the development of Treg and Teff cells is therefore warranted. Gammainterferon‐inducible lysosomal thiol reductase (GILT) acts to influence antigenic processing and presentation by MHC class II cells, ultimately impacting lymphocyte development. Evaluation of the role of GILT expression in MHC class II+ APC’s with respect to Treg and Teff cell development in primary melanoma lesions, to our knowledge, has not been reported. Therefore our investigation focuses on elucidating a plausible relationship between GILT presence and Treg to Teff cell ratio. The aim of our study is to examine a possible association between GILT expression in APC’s and Treg:Teff cell ratio. We hypothesized GILT expression in melanoma cells would result in a decreased Treg to Teff ratio or an enhanced T cell‐mediated response. Our study included 17 de‐identified primary melanoma specimens previously stained and scored for Treg, Teff, CD8, MHC class II and GILT. Scoring was performed through identification of four areas per specimen with highest Treg and Teff cell density. These four areas were then averaged with ± standard deviation (SD). With use of landmark association, these four areas were identified and scored for MHC class II and GILT in APC’s and tumor cells with consideration to presence/absence, intensity and frequency of staining. Statistical significance was not reached relative to our hypothesized relationship of a decreased Treg to Teff cell ratio in the presence of GILT+ MHC class II. Similarly, we did not reach statistical significance when comparing individual cell types to GILT, MHC class II and GILT + MHC class. In our study, we were unable reach statistical significance relative to our proposed correlation between MHC class II and GILT presence leading to a decreased Treg to Teff cell ratio or enhanced T‐cell mediated immune response. A major limitation of our study included the small sample size leading to a probable type II error, prompting the need for further investigation of the factors influencing the Treg to Teff cell ratio within the melanoma tumor microenvironment on a larger scale.