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Background Identifying biomarkers to predict kidney transplant failure and to define new therapeutic targets requires more comprehensive understanding of the immune response to chronic allogeneic stimulation. Methods We investigated the frequency and function of CD8+ T cell subsets-including effector memory (EM) and terminally differentiated EM (TEMRA) CD8+ T cells-in blood samples from 284 kidney transplant recipients recruited 1 year post-transplant and followed for a median of 8.3 years. We also analyzed CD8+ T cell reactivity to donor-specific PBMCs in 24 patients who had received living-donor kidney transplants. Results Increased frequency of circulating TEMRA CD8+ T cells at 1 year post-transplant associated with increased risk of graft failure during follow-up. This association remained after adjustment for a previously reported composite of eight clinical variables, the Kidney Transplant Failure Score. In contrast, increased frequency of EM CD8+ T cells associated with reduced risk of graft failure. A distinct TEMRA CD8+ T cell subpopulation was identified that was characterized by expression of FcγRIIIA (CD16) and by high levels of proinflammatory cytokine secretion and cytotoxic activity. Although donor-specific stimulation induced a similar rapid, early response in EM and TEMRA CD8+ T cells, CD16 engagement resulted in selective activation of TEMRA CD8+ T cells, which mediated antibody-dependent cytotoxicity. Conclusions At 1 year post-transplant, the composition of memory CD8+ T cell subsets in blood improved prediction of 8-year kidney transplant failure compared with a clinical-variables score alone. A subpopulation of TEMRA CD8+ T cells displays a novel dual mechanism of activation mediated by engagement of the T-cell receptor or of CD16. These findings suggest that TEMRA CD8+ T cells play a pivotal role in humoral and cellular rejection and reveal the potential value of memory CD8+ T cell monitoring for predicting risk of kidney transplant failure.

International audience; Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature hematopoietic precursors known to suppress immune responses. Interaction of SIRP alpha (SIRPα), expressed by myeloid cells, with the ubiquitous receptor CD47 is an important immune checkpoint of the innate response regulating macrophages and dendritic cells functions. We previously described that MDSC expressing SIRPα accumulated after transplantation and maintained kidney allograft tolerance. However, the role of the SIRPα/CD47 axis on MDSC function remained unknown. Here, we found that blocking SIRPα or CD47 with monoclonal antibodies (mAbs) induced differentiation of MDSC into myeloid cells overexpressing MHC class II, CD86 costimulatory molecule and increased secretion of macrophage-recruiting chemokines (eg, MCP-1). Using a model of long-term kidney allograft tolerance sustained by MDSC, we observed that administration of blocking anti-SIRPα or CD47 mAbs induced graft dysfunction and rejection. Loss of tolerance came along with significant decrease of MDSC and increase in MCP-1 concentration in the periphery. Graft histological and transcriptomic analyses revealed an inflammatory (M1) macrophagic signature at rejection associated with overexpression of MCP-1 mRNA and protein in the graft. These findings indicate that the SIRPα-CD47 axis regulates the immature phenotype and chemokine secretion of MDSC and contributes to the induction and the active maintenance of peripheral acquired immune tolerance.

Background: In heart allograft in the rat, a sustained costimulation blockade with CTLA4Ig prevents alloreactive T-cell activation and promotes a long-term graft survival through the action of tolerogeneic dendritic cells. It is unclear whether similar mechanisms might occur after xenotransplantation. To test that hypothesis, we have analyzed the action of CTLA4Ig in a model of CD4+T cell-mediated xenograft rejection. Methods: Hamster hearts were transplanted into LEW.1A rats receiving an accommodation-inducing treatment consisting of a short course administration of LF15-0195 and a daily administration of cyclosporine A (CSA). To achieve long-term delivery of CTLA4Ig, an intravenous administration of an adenovirus vector coding for mouse CTLA4Ig (Ad-CTLA4Ig) was added to the accommodation induction protocol. On day 40 post-transplantation, rejection was induced by CSA withdrawal. In other xenograft recipients, CD28/B7 costimulation was inhibited at that time only by injections of CTLA4Ig or anti-CD28 antibodies. Graft survival, immunohistology, as well as development of antibodies and regulatory cells were examined. Results: Xenografts survived 6 days after CSA withdrawal in controls and were rejected, as previously described, through the action of CD4+ xenoreactive T cells. Interfering with CD28/B7 costimulation inhibited this xenoreactive T cell response and delayed rejection to day 10. In recipients that had received Ad-CTLA4Ig, survival was prolonged to day 19 and this was accompanied by the appearance of regulatory cells exhibiting non-donor-specific suppressive activity dependent on IL-2, NO, and IDO. These regulatory cells were different from those previously identified after Ad-CTLA4Ig administration in heart allograft in the rat. In these recipients, rejection occurred as a consequence of an evoked anti-donor IgM response and complement activation and not of a cellular rejection as complement inhibition with cobra venom factor further prolonged xenograft survival. Conclusion: CD28/B7 blockade delays CD4+ T cell-mediated rejection after CSA withdrawal in accommodated recipients of hamster heart xenografts. In addition, a sustained expression of CTLA4Ig has the potential of inducing cellular regulatory mechanisms. However, such treatment does not prevent the development of xenoreactive IgM antibodies that participate in vascular rejection processes in a complement-dependent manner.

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Material, methods and results In rat recipients of kidney allografts where tolerance was induced by costimulation blockade, we observed a 2-fold decrease of plasma CCL5 levels as compared to control syngeneic grafted animals (0.60 ± 0.17 vs. 1.17 ± 0.48 ng/ml; p

Background Antagonist antibodies targeting CD28 have been proposed as an alternative to the use of CD80/86 antagonists to modulate T cell responses in autoimmunity and transplantation. Advantages would be the blockade of CD28mediated co-stimulatory signals without impeding co-inhibitory signals depending on CD80 interactions with CTLA-4 and PD-L1 that are important for the control of immune responses and for the function of regulatory T cells. Anti-CD28 antibodies are candidate antagonists only if they prevent access to the CD80/86 ligands without simultaneously stimulating CD28 itself, a process that is believed to depend on receptor multimerization.

Background . Preliminary studies showed that heat treatment of glutaraldehyde preserved valvular bioprostheses mitigates calcification. This study was carried out to define the physicochemical characteristics of the heat-treated tissues to elucidate the mechanism involved in the mitigation. Methods . Glut bovine pericardium or porcine valve samples were treated at 50°C in a 0.625% glutaraldehyde solution for 2 months. Some samples underwent assay for shrinkage temperature, moisture content, ninhydrin test, and acid hydrolysis, and other samples were incubated in human serum for 3 days and then analyzed by electrophoresis to study protein adsorption. Results . Heat treatment mitigated calcification without adversely affecting shrinkage temperature (84.81°C versus 83.95°C) and moisture content (78.68% versus 78.71%). A significant reduction in free amino groups (0.15 versus 0.37 mol NH 2 /mol collagen) and a significant increase in resistance to acid hydrolysis were observed. Total protein content was similar, but significant differences were found for four proteins adsorbed in the tissues (167, 45, 11.6, and 10 kDa). Conclusions . The anticalcification effect of heat treatment may be attributed to structural changes, lipid extraction, increased resistance, and modifications of the type and concentration of the proteins adsorbed in the tissue.

Background . Several techniques have been proposed to mitigate calcification of glutaraldehyde-preserved bioprostheses. None has been fully satisfactory. Knowing that heat induces significant molecular changes, we investigated the potential benefit of the heat treatment of glutaraldehyde-fixed tissues. Methods . Samples of porcine valves and bovine pericardium treated in 0.625% glutaraldehyde were submitted to temperatures from 37° to 70°C for 2 to 12 weeks and then implanted subcutaneously in newborn rats for 3 months. In a second protocol, nine heat-treated porcine valve bioprostheses and seven control porcine valve bioprostheses were implanted in the mitral position in sheep for 20 weeks. Results . Spectrophotometry and x-ray analysis comparing heat-treated versus non–heat-treated tissues showed the following: porcine valve: 6.7 ± 2.3 μg Ca/mg (n = 8) versus 239.9 ± 2.9 μg Ca/mg (n = 8); bovine pericardium: 19.5 ± 8 μg Ca/mg (n = 6) versus 108 ± 10.3 μg Ca/mg (n = 8); and porcine valve bioprostheses: 0 versus ++. Conclusions . Heat treatment of glutaraldehyde-treated bioprostheses significantly mitigated calcification in both subcutaneous and mitral position.

Background . The methods currently used to analyze the process of calcification of bioprostheses give only global information on calcium deposition. We investigated the potential advantage of infrared spectroscopy, which makes it possible to analyze the various components of the calcification process, ie, lipids, proteins, and calcium deposits. Methods . Sixty porcine aortic leaflets were fixed in 0.6% glutaraldehyde and then subsequently implanted in 10-day-old Wistar rats. The valve leaflets were removed 2, 7, 14, 21, 35, and 56 days after implantation. Results . Before implantation infrared spectroscopic analysis revealed the presence of proteins only. On day 2 after implantation, all valves showed minor lipid deposits. On day 7, amorphous calcium phosphate was detected. Between days 7 and 14, crystalline forms of calcium phosphate appeared and amorphous calcium phosphate progressively changed into carbapatite over the 56-day period. Conclusions . Infrared spectroscopy yields valuable additional information on the nature and kinetics of the various components of glutaraldehyde-treated tissues after implantation. It may prove to be important in the evaluation of new techniques of calcium mitigation.

Background . The incidence of calcification of porcine valve bioprostheses shows important, and as yet unexplained, variations. Previous studies by others showed that osteopontin and osteocalcin are expressed in calcified porcine valve bioprostheses. However, no study has yet explored other proteins that could also be involved. Methods . Twelve porcine valve bioprostheses were retrieved from 12 patients and were separated into two groups. Group 1 (n = 6) had early calcification after 4 to 9 years (mean, 6 ± 2.3 years). The mean age of the patients at the time of implantation was 46 ± 9 years. Group 2 (n = 6) had no calcification after 8 to 14 years (mean, 12 ± 2.8 years). The mean age was 47 ± 13.4 years. These valves were analyzed by electrophore-sis, and the bands were quantified by densitometry. Results . A 14-kd protein showed a 50% increase in the calcified group. A 31-kd protein found in the calcified group was not detected in the noncalcified group. Three other proteins (45, 39, and 28 kd) showed reduced adsorption in the calcified group. Conclusions . Important differences were found in the proteins adsorbed in calcified and noncalcified bioprostheses after implantation in patients. Besides osteopontin and osteocalcin, several other proteins may play a role in the process of calcification of valvular bioprostheses.

Background . Proteins adsorbed by bioprosthetic tissues after implantation play a major role in the process of calcification. We investigated whether there are differences in protein adsorption between bovine pericardial and porcine valvular tissues that could correlate with the differences observed clinically. Methods . Glutaraldehyde-treated bovine pericardial and porcine valve samples were implanted subcutaneously in rats and retrieved 1 month after implantation. Total protein content was assessed by Lowry's method. Qualitative analysis was performed by polyacrylamide gel electrophoresis. Quantitative analysis was performed by densitometry. Results . Total protein content showed a higher protein concentration in porcine valve tissue than in pericardialtissue: 149 ± 22.6 μg/mg dry tissue versus 108 ± 12.7 μg/mg dry tissue (38% increase). In pericardial tissue, four protein bands (17, 16, 15.5, and 13.5 kd) showed decreased concentration when compared with porcine valve tissue, whereas one band (11 kd) showed increased concentration. Conclusions . Significant differences were found in protein content between bovine pericardial and porcine valve tissues. Correlations with clinical findings may lead to a better understanding of the mechanism involved in the process of calcification, particularly the role played by the structure of the tissues.