UVM Theses and Dissertations
Format:
Print
Author:
Saligrama, Phan Thai
Dept./Program:
Cell and Molecular Biology Program
Year:
2013
Degree:
Ph. D.
Abstract:
The ability to control the levels of active caspases is crucial to the life and death of activated Tcells. Caspases, which are translated as inactive zymogens and subsequently activated upon proteolytic cleavage, are cysteine proteases that cleave substrates after an aspartic acid residue following a specific amino acid sequence, and are involved in the initiation (e.g. caspase-8, -9, -10) and execution (e.g. caspase-3, -6, -7) of apoptosis. Apoptosis occurs through cleavage of death substrates, including the Inhibitor of Caspase-activated DNAse (ICAD) and poly-ADP ribose polymerase (PARP) by active caspase-3. It was found that caspases, particularly caspase-8, are also required to initiate T cell proliferation. This was confirmed by studies in caspase-8-deficient murine T cells and mutant caspase-8 human T cells. However, little is known about what determines the level of caspase activity in cycling T lymphocytes. Thus, a critical question central to this work considered how caspas~ activity is regulated to promote death or survival of T cells. Specifically, this dissertation examined how differences in the T cell subsets, [upsilon, sigma] and the cytokine environment impact the levels of active caspase-3 and therefore T cell survival.
[Upsilon sigma] Tcells function between the innate and adaptive immune responses. Their numbers often increase during infections and at sites of chronic inflammation. Previous studies suggest that [upsilon sigma] T cell signaling is stronger than [alpha beta] T cells, as reflected by increased intracellular calcium and phosphorylated-ERK following TCR stimulation. It is conceivable that differences in TCR signaling may lead to differences in caspase activity. Actively cycling human Lyme arthritis synovial [upsilon sigma] T cell clones of the V[sigma]1 subset manifested higher overall caspase activity, particularly of effector caspase-3, as well as ICAD cleavage compared with [alpha beta] T cell clones. The increased caspase activity was reproducible in primary synovial fluid [upsilon signma] T cells from patients with Lyme arthritis. As such, the presence of active caspase-3 likely explains why [upsilon sigma] T cells have increased spontaneous cell death and are more susceptible to activation induced cell death (AICD).
In the next phase, the cytokine environment of the inflamed synovium was modeled using IL-15, and compared to T cells cultivated in IL-2. Although IL-2 and IL-15 share [upsilon]c and IL2R[beta] (CD122) receptor subunits and are capable of stimulating proliferation and activation of T cells, AICD occurs upon TCR stimulation in the presence of IL-2 but not with IL-15. IL-2-induced AICD is mediated by high levels of caspase activity, particularly active caspase-3. Despite similar levels of active caspase-8 and -9 in IL-15-cultured and IL-2-cultured T cells, active caspase-3 levels are decreased in the presence of IL-15. This was true for both [alpha beta] and [upsilon sigma] T cells. Caspase-3 activity also closely paralleled the type of metabolism. IL-15 supports limited glucose utilization compared to IL-2, as evidenced by decreased surface glucose transporter 1 expression, reduced hexokinase II protein, lower lactate -levels, and diminished NADP(H) and NAD(H) levels. The reduction in glucose consumption in T cells under IL-15 conditions paralleled the levels of reactive species, including reactive nitrogen and oxygen species. In turn, this results in S-Nitrosylation-mediated inactivation of caspase-3. The novel finding that IL-15 promotes S-Nitrosylation of caspase-3 in primary murine T cells suggests that IL-15 acts as a survival cytokine through maintaining a large pool of caspase-3 in its inactive form.
Reports have indicated increased levels of IL-15 as well as autoreactive T lymphocytes at sites of inflammation in autoimmune diseases. The findings provide a potential explanation why T cells might survive better at inflamed synovial sites where regional cytokines might drive non-glycolytic metabolism and hence survival. The data presented within this dissertation also suggest that promoting caspase-3 inactivation, which is downstream of IL-15 signaling, may be central to the pathogenesis of certain autoimmune disorders.
[Upsilon sigma] Tcells function between the innate and adaptive immune responses. Their numbers often increase during infections and at sites of chronic inflammation. Previous studies suggest that [upsilon sigma] T cell signaling is stronger than [alpha beta] T cells, as reflected by increased intracellular calcium and phosphorylated-ERK following TCR stimulation. It is conceivable that differences in TCR signaling may lead to differences in caspase activity. Actively cycling human Lyme arthritis synovial [upsilon sigma] T cell clones of the V[sigma]1 subset manifested higher overall caspase activity, particularly of effector caspase-3, as well as ICAD cleavage compared with [alpha beta] T cell clones. The increased caspase activity was reproducible in primary synovial fluid [upsilon signma] T cells from patients with Lyme arthritis. As such, the presence of active caspase-3 likely explains why [upsilon sigma] T cells have increased spontaneous cell death and are more susceptible to activation induced cell death (AICD).
In the next phase, the cytokine environment of the inflamed synovium was modeled using IL-15, and compared to T cells cultivated in IL-2. Although IL-2 and IL-15 share [upsilon]c and IL2R[beta] (CD122) receptor subunits and are capable of stimulating proliferation and activation of T cells, AICD occurs upon TCR stimulation in the presence of IL-2 but not with IL-15. IL-2-induced AICD is mediated by high levels of caspase activity, particularly active caspase-3. Despite similar levels of active caspase-8 and -9 in IL-15-cultured and IL-2-cultured T cells, active caspase-3 levels are decreased in the presence of IL-15. This was true for both [alpha beta] and [upsilon sigma] T cells. Caspase-3 activity also closely paralleled the type of metabolism. IL-15 supports limited glucose utilization compared to IL-2, as evidenced by decreased surface glucose transporter 1 expression, reduced hexokinase II protein, lower lactate -levels, and diminished NADP(H) and NAD(H) levels. The reduction in glucose consumption in T cells under IL-15 conditions paralleled the levels of reactive species, including reactive nitrogen and oxygen species. In turn, this results in S-Nitrosylation-mediated inactivation of caspase-3. The novel finding that IL-15 promotes S-Nitrosylation of caspase-3 in primary murine T cells suggests that IL-15 acts as a survival cytokine through maintaining a large pool of caspase-3 in its inactive form.
Reports have indicated increased levels of IL-15 as well as autoreactive T lymphocytes at sites of inflammation in autoimmune diseases. The findings provide a potential explanation why T cells might survive better at inflamed synovial sites where regional cytokines might drive non-glycolytic metabolism and hence survival. The data presented within this dissertation also suggest that promoting caspase-3 inactivation, which is downstream of IL-15 signaling, may be central to the pathogenesis of certain autoimmune disorders.