Degradation of NF-kappa B in T cells by gangliosides expressed on renal cell carcinomas

T cells from cancer patients are often functionally impaired, which imposes a barrier to effective immunotherapy. Most pronounced are the alterations characterizing tumor-infiltrating T cells, which in renal cell carcinomas includes defective NF-kappaB activation and a heightened sensitivity to apoptosis. Coculture experiments revealed that renal tumor cell lines induced a time-dependent decrease in RelA(p65) and p50 protein levels within both Jurkat T cells and peripheral blood T lymphocytes that coincided with the onset of apoptosis. The degradation of RelA/p50 is critical for SK-RC-45-induced apoptosis because overexpression of RelA in Jurkat cells protects against cell death. The loss of RelA/p50 coincided with a decrease in expression of the NF-kappaB regulated antiapoptotic protein Bcl-xL at both the protein and mRNA level. The disappearance of RelA/p50 protein was mediated by a caspase-dependent pathway because pretreatment of T lymphocytes with a pan caspase inhibitor before coculture with SK-RC-45 blocked RelA and p50 degradation. SK-RC-45 gangliosides appear to mediate this degradative pathway, as blocking ganglioside synthesis in SK-RC-45 cells with the glucosylceramide synthase inhibitor, PPPP, protected T cells from tumor cell-induced RelA degradation and apoptosis. The ability of the Bcl-2 transgene to protect Jurkat cells from RelA degradation, caspase activation, and apoptosis implicates the mitochondria in these SK-RC-45 ganglioside-mediated effects.     

NF-kappa B RelA (p65) is essential for TNF-alpha-induced fas expression but dispensable for both TCR-induced expression and activation-induced cell death

The Fas death receptor plays a key role in the killing of target cells by NK cells and CTLs and in activation-induced cell death of mature T lymphocytes. These cytotoxic pathways are dependent on induction of Fas expression by cytokines such as TNF-alpha and IFN-gamma or by signals generated after TCR engagement. Although much of our knowledge of the Fas death pathway has been generated from murine studies, little is known about regulatory mechanisms important for murine Fas expression. To this end, we have molecularly cloned a region of the murine Fas promoter that is responsible for mediating TNF-alpha and PMA/PHA-induced expression. We demonstrate here that induction of Fas expression by both stimuli is critically dependent on two sites that associate with RelA-containing NF-kappaB complexes. To determine whether RelA and/or other NF-kappaB subunits are also important for regulating Fas expression in primary T cells, we used CD4 T cells from RelA(-/-), c-Rel(-/-), and p50(-/-) mice. Although proliferative responses were significantly impaired, expression of Fas and activation-induced cell death was unaffected in T cells obtained from these different mice. Importantly, we show that unlike fibroblasts, which consist primarily of RelA-containing NF-kappaB complexes, T cells have high levels of both RelA and c-Rel complexes, suggesting that Fas expression in T cells may be dependent on redundant functions of these NF-kappaB subunits.     

Overexpression of heme oxygenase (HO)-1 renders Jurkat T cells resistant to fas-mediated apoptosis: involvement of iron released by HO-1

We recently demonstrated that heme oxygenase (HO)-1 is constitutively expressed in human CD4+CD25+ regulatory T cells and induced by anti-CD28 or anti-CD28/anti-CD3 stimulation, even in CD4+CD25- responder T cells. To study the effects of HO-1 expression on lymphocyte survival, we transfected the HO-1 gene or induced the gene to express HO-1 protein with cobalt protoporphyrin (CoPP) in Jurkat T cells. Consistently, anti-Fas antibody triggered apoptotic cell death in wild-type Jurkat T cells. Surprisingly, however, HO-1-overexpressing Jurkat T cells showed strong resistance to Fas-mediated apoptosis. In contrast, abrogation of HO-1 expression by antisense oligomer against HO-1 gene from CoPP-treated cells or depletion of iron by desferrioxamine from HO-1-transfected cells abolished the resistance. In addition, exogenously added iron rendered wild-type Jurkat T cells resistant. The resistance involved IkappaB kinase (IKK) activation via iron-induced reactive oxygen species formation, NF-kappaB activation by activated IKK, and c-FLIP expression by activated NF-kappaB. Primary CD4+ T cells induced by CoPP to express HO-1 also showed more resistance to Fas-mediated apoptosis than untreated cells. Our findings suggest that HO-1 plays a critical and nonredundant role in Fas-mediated activation-induced cell death of T lymphocytes.