Overexpression of miR-24 in HeLa cells did not significantly alter the relative distribution of miR-24 on polysome gradients, nor did it influence the levels of p16 mRNA in the Ctrl

ls, human PBL was used to test the caspase activity after 5 h of TIRC7 targeting using sHLA-DRa2. Caspase 9 was activated as was indicated by an increase of the cleaved product in the presence of sHLA-DRa2 whereas no activation of caspase 8 was observed in PBL. Analysis of caspase 7 showed an increase of the cleaved product of 20kDa size upon TIRC7 ligation, while an activation of caspase 3 was not observed. To analyze whether TIRC7-dependent apoptosis can be reversed by adding inhibitor of caspase 9 to the cultures, incubation of cells 23863710 with the caspase 9 inhibitor z-LEH-fmk reduced TIRC7-dependent apoptosis to normal levels. Although no activation of caspase 8 was observed after sHLA-DRa2 ligation, modulation of the FasL expression on T cells was analyzed. Flow cytometry analysis of PBL activated with anti-CD3/CD28 antibody for 24 h in the presence of the sHLA-DRa2 revealed a remarkably reduced expression of FasL at the cell surface of human T cells. These results are in agreement with a down-regulatory effect of TIRC7 on ZAP70 activity, since ZAP70 was shown to be essential for the regulation of FasL expression on the surface of activated T cells and indicate that TIRC7 mediated modulation of the proliferative response in PBL involves the intrinsic, mitochondrial pathway via caspase 9. Interaction of sHLA-DRa2 with TIRC7 expressed in CD4+ and CD8+ T cells results in cell cycle arrest and apoptosis in lymphocytes In addition to the induction of anergy and inhibition of proliferative response, cell cycle arrest as well as apoptosis are further Lonafarnib important mechanisms to control the activation of lymphocytes. To examine whether the antiproliferative effect induced by sHLA-DRa2 crosslinking to TIRC7 involve these mechanisms as well human PBL were stimulated with anti-CD3/ CD28 antibodies in the presence of sHLA-DRa2 or control protein, and analyzed for cell cycle arrest and apoptosis using flow HLA-DR co-localizes with TIRC7 at the APC-T cell interface and soluble HLA-DRa2 prevents APC mediated T cell cytokine release in vitro and in vivo Upon activation of T lymphocytes both, HLA proteins and TIRC7 cluster at the site of T cell/APC junction. To examine HLA-DR Alpha 2 the distribution of both molecules at the 1828342 site of cell cell interaction, human PBL were activated with recall antigens for 72 h. Confocal microscopy showed that in recall antigen activated human lymphocytes both, TIRC7 and HLA-DR, concentrated at the site of the cell cell interaction. An overlay of both immunostains suggested a co-localization of both molecules . Functional importance of T cell activation through crosslinking with MHC class II molecules bearing APC is the induction of IL12 expression which leads to an increased IFN-c expression in T cells. IFN-c acts itself as an activator of APC which in turn induces IL-12 subsequently resulting in inflammation. Data obtained from our studies indicates that TIRC7 and HLA DR alpha 2 interaction controls APC – T cell interaction by induction of negative regulatory signals and prevents an excess of APC – T cell interaction during immune activation. We therefore hypothesize that triggering TIRC7 signals via sHLA-DRa2 should suppress the APC – T cell interaction and prevent associated cytokine release upon stimulation. To prove this hypothesis in vitro, we used the model of LPS induced APC activation. Macrophages exposed 48 h to either sHLA-DRa2 or control protein in the presence of LPS and subsequently subjected to analysis of cyt