Regulation of FasL expression, and delineation of differentiation states of cytotoxic lymphocytes

Abstract

CD8+ T cells are vital cytotoxic lymphocytes against intracellular pathogens and tumor cells. CD8+ T cells utilize a fast-acting degranulation of granzyme/perforin and slow-acting receptor:ligand, FasL:Fas, pathway of inducing target cell lysis. The importance of degranulation based target cell killing by CD8+ T cells has been well established while the FasL:Fas based mechanism of killing has been far less characterized. Additionally, it remains unclear as to the purpose of possessing the slow-acting FasL:Fas mechanism of killing in CD8+ T cells. In this thesis, I investigated the differential expression patterns of intracellular FasL and GrB in stimulated CD8+ T cells to further delineate the roles between degranulation and FasL. Interestingly, while I found GrB expression in CD8+ T cells to be proportional to the level of stimulation, weak stimulation is more favorable for FasL expression. In ex vivo stimulated CD8+ T cells, while strong stimulation induced expression of intracellular FasL, only weakly stimulated cells were able to sustain FasL expression over an extended period of time. Using allogeneic tumors, I further demonstrated that high antigen availability stimulates CD8+ T cells to express GrB and FasL while low antigen availability favors intracellular expression of FasL expression. Collectively, these results suggest two strategies of effector molecule expression: a low antigen level strategy favoring FasL expression and high antigen level strategy of GrB and FasL expression. In addition to CD8+ T cells, NK cells are also potent cytotoxic lymphocytes. As part of the innate immune system, NK cells were not believed to possess adaptive immune cell features. However, recent studies have demonstrated that following stimulation, NK cells may possess a number of memory cell-like features such as longevity, antigen specificity and enhanced secondary responses. While no definitive memory marker(s) have been found for memory NK cells, Ly-6C, a memory marker for CD8+ T cells, is a promising candidate. In my study, I first demonstrated that Ly-6C positive NK cells are mature NK cells based on effector function and phenotype. Additionally, based on Ly-6C staining with monoclonal AL-21 and iMAP antibodies, two subsets of Ly-6C positive NK cells are visible, an AL-21 only reactive subset, Ly-6CAL-21, and an AL-21/iMAP reactive subset, Ly-6CAL-21/iMAP. I further demonstrated that Ly-6C positive NK cells develop from Ly-6C negative NK cells and that Ly-6CAL-21/iMAP NK cells develop from Ly-6CAL-21 NK cells. In long term studies, this progression of Ly-6C expression suggests that Ly-6CAL-21/iMAP NK cells may represent a previously undescribed differentiation state of long-lived NK cells. Collectively, AL-21 and iMAP co-reactivity towards Ly-6C may further define the population of memory NK cells.