One hurdle of long-term manned spaceflight is the reduction in cellular immunity experienced by astronauts in the microgravity environment. T lymphocytes, the primary effectors of cellular immunity, are unresponsive to mitogenic lectins or T cell receptor (TCR) ligation when cultured in vitro during spaceflight. Responsiveness can be partially rescued by directly activating the diacyl glycerol- (DAG) and calcium-signaling pathways that lie downstream of the TCR. Studies using rotating wall vessel (RWV) bioreactors as ground-based microgravity analogs have shown that sub-mitogenic doses of PMA, a DAG mimetic, can rescue T cell activation in response to mitogenic-lectins and -antibodies. Based on these findings we hypothesized that RWV-culture impairs T cell activation by affecting the generation of or response to the DAG second-messenger. This hypothesis was investigated by making comparative analyses of TCR-induced signal transduction in primary human CD4+ T cells in RWV- and 1-g (static)-culture.Our results show that TCR-induced phospholipase Cγ1 (PLCγ1) activation and the subsequent generation of DAG is unimpaired in CD4+ T lymphocytes cultured in the RWV. Furthermore, the primary targets of the DAG second-message, the guanylnucleotide replacing protein for RAS (RASGRP) and protein kinase cθ (PKCθ), were both activated by cells cultured in the RWV. Gene expression downstream of these enzymes, however, was only induced by T cells cultured in static conditions. A closer inspection of these pathways indicated that during culture in the RWV the extracellular regulated kinase (ERK), which is downstream of RASGRP, may become sequestered in the cytosol and thus prevented from mediating gene expression in the nucleus. Based on these results we conclude, that altered compartmentalization of ERK prevents T cells from becoming activated in the RWV by limiting the activation of key transcription factors required for the cell to become activated and enter the cell-cycle.Ph.D., Biomedical Engineering -- Drexel University, 200
