Differential Regulation of Phosphokinases by Anti-HLA Class I and II Antibodies

Purpose: Anti-HLA Zantibodies, both pre-formed and de novo are associated with worse graft survival. Glomerular endothelial cells are the targets for antibody mediated rejection. We hypothesized that there are differences in the mechamsms of endothelial cell activation by of anti-HLA Zclass I and II antibodies. Also, this may explain differences in pathogenicity. Methods: Human primary renal glomerular endothelial cells (HRGEC) were HLA typed. Cells at passage 3-6, were stimulated with Gamma Interferon for 48hours. Cells were then exposed to sera with cell HLA specific anti-HLA Zclass I and II antibodies in separate experiments for a serial time period starting from 2 minutes to maximum of 120mmutes. Cell lysates were collected and studied for Phospho AKT and MAP Kinases by Western Blotting. Results: In these experiments, we demonstrate differential activation of phospho-kinases in the presence of anti-HLA Zclass I and class II antibodies. With anti-HLA Zclass I antibodies progressive activation of phospho AKT and MAP Kinases were noted beginning at 15 minutes [Figure la]. Anti-HLA Zclass II antibodies caused activation phospho AKT for 1 Ommutes beginning at 2 minutes, then activation was not seen till 60 minutes. MAPK were activated from 2-10 minutes with no further activation [Figure lb]. Conclusions: This differential activationmay lead to different downstream pathways that determine the endothelial cell susceptibility. Studies have shown that Phospho AKT activation promotes cell growth and prevents apoptosis. Continuous activation of pAKT by class I antibodies in contrast to class II antibodies may stimulate cell survival signals. Further studies on endothelial cell signaling pathways are needed to elucidate specific markers that are upregulated

Endothelial Cells Do Not Regulate Expression of CD46, CD55 and CD59 in the Presence of Anti-HLA Class I and II Antibodies

Purpose: Anti-HLA antibodies, both pre-formed and de novo are associated with worse graft survival. Complement activation is an important mechanism of antibody mediated immunological injury. We hypothesised that anti-HLA antibodies may differentially induce expression of complement regulatory proteins thus resulting in differential injury. Methods: Human primary renal glomerular endothelial cells (HRGEC) were HLA typed. Cells at passage 3-6, were stimulated with Gamma Interferon for 48hours. Cells were then exposed to sera with cell HLA specific anti-HLA class I, Class II and Class I+n antibodies in separate experiments. Sera with no antibodies acted as a negative control. Expression of mRNA for CD46, CD55, and CD59 were studied by qPCR for all these 4 conditions. Cell lysates collected for Western blot and Flow cytometry were also studied for all the 4 conditions. Results: mRNA isolated from endothelial cells, was quantified for CD46, CD55 and CD59 expression by qPCR and there was no difference in expression levels after 48hours under 4 conditions. There was also no difference in surface expression of CD46, CD55 and CD59 by western blot. In the Figure la overlay graph CD46 expression is not altered under 3 different conditions compared to negative control. Similar findings are presented for CD55 and CD59 expression in Figures lb and lc respectively. Conclusions: In these experiments, we clearly show that CD46, CD55 and CD59 expression on renal glomerular endothelial cells do not change in the presence of anti-HLA Z antibodies. Different susceptibility of anti HLA class l and class II anti-bodies are not explained by this mechanism. Upregulation of these underutilized targets may confer additional endothelial protection in the presence of antibodies, especially if the immune-mediated damage is related to complement activation

HLA-Fatal attraction.

International audienceOpen conformers of the non-classical and monomorphic major histocompatibility complex (MHC) class I molecule HLA-F are ligands for the activating receptor KIR3DS1 and trigger the activation of natural killer (NK) cells

Association of HLA-A and Non-Classical HLA Class I Alleles.

The HLA-A locus is surrounded by HLA class Ib genes: HLA-E, HLA-H, HLA-G and HLA-F. HLA class Ib molecules are involved in immuno-modulation with a central role for HLA-G and HLA-E, an emerging role for HLA-F and a yet unknown function for HLA-H. Thus, the principal objective of this study was to describe the main allelic associations between HLA-A and HLA-H, -G, -F and -E. Therefore, HLA-A, -E, -G, -H and -F coding polymorphisms, as well as HLA-G UnTranslated Region haplotypes (referred to as HLA-G UTRs), were explored in 191 voluntary blood donors. Allelic frequencies, Global Linkage Disequilibrium (GLD), Linkage Disequilibrium (LD) for specific pairs of alleles and two-loci haplotype frequencies were estimated. We showed that HLA-A, HLA-H, HLA-F, HLA-G and HLA-G UTRs were all in highly significant pairwise GLD, in contrast to HLA-E. Moreover, HLA-A displayed restricted associations with HLA-G UTR and HLA-H. We also confirmed several associations that were previously found to have a negative impact on transplantation outcome. In summary, our results suggest complex functional and clinical implications of the HLA-A genetic region