Characterizing the role of mammalian SIDT1 in T cells

Abstract

The C. elegans SID-1 protein is essential for the phenomenon known as systemic RNA interference. Mammals possess two S1D-1 homologs, SIDT1 and SIDT2, which are highly conserved but have not been extensively characterized.;In this thesis, we have generated several new reagents, including monoclonal antibodies and mice, to comprehensively characterize murine SIDT1. We found that SIDT1 expression was widely variable across mouse tissues and cell types, and was largely restricted to secondary lymphoid organs. Within the hematopoietic cell population, mature naive T cells - especially CD8+ T cells - expressed particularly high levels of SIDT1.;Yet, despite at least one previous report that mammalian SIDT1 can enable passive, efficient and functional uptake of naked siRNA, we have shown that this does not appear to be the case with respect to primary murine CD8 + T cells, even at high concentrations of siRNA and incubation at 37°C. Wild-type CD8+ T cells could take up only low levels of siRNA, and the level of uptake was indistinguishable from SIDT1deficient and SIDT1-overexpressing CD8+ T cells. Moreover, co-culture of CD8+ T cells and siRNAs did not result in RNAi-mediated gene silencing.;Instead, we found SIDT1 to be a novel marker of memory in CD8 + T cells. In response to primary infections with either Listeria monocytegenes (LM) or herpes simplex virus type 2 (HSV 2), S1DT1 expression was essentially abrogated in terminally-differentiated, short-lived effector CD8+ T cells (SLECs). In contrast, SIDT1 expression was maintained at high - but heterogeneous - levels in memory precursor effector CD8+ T cells (MPECs) throughout effector and memory stages. Within the population of long-lived memory cells, central memory cells (T CM) exhibited the highest levels of SIDT1, while effector memory cells (TEM) expressed somewhat lower levels (at least in the context of infection by LM).;In sum, SIDT1 can be used to distinguish MPECs from SLECs, as well as to distinguish TCM cells from TEM ceps, and possibly to identify new effector and/or memory subpopulations. Therefore, we believe SIDT1 is a novel and unique marker with potential applications for the study of effector and memory CD8+ T cell responses, including vaccine development.