Progressive Loss of Donor-Reactive CD4+ Effector Memory T Cells due to Apoptosis Underlies Donor-Specific Hyporesponsiveness in Stable Renal Transplant Recipients.

Following kidney transplantation, donor-specific hyporesponsiveness (DSH) may develop, defined as a lowered response of alloreactive T cells, specifically directed to donor Ag. This study aimed to characterize the nature of DSH through multiparameter flow cytometric assays measuring changes in phenotype and function of donor-reactive T cells after transplantation. This study characterized donor-reactive T cells, identified by CD137 expression, from the peripheral blood of stable human kidney transplant recipients (n = 47) before, at 3-5 y after, and >5 y after transplantation. The phenotype (T cell subset, differentiation status, and transcription factor expression) and function (proinflammatory cytokine production) of CD4+ and CD8+ donor-reactive CD137+ T cells was evaluated by both supervised and unsupervised analyses. Results demonstrated a decline in CD4+ donor-reactive T cells within the first 3-5 y after transplantation. Predominantly, the population of effector memory T cells capable of producing two or more proinflammatory cytokines was affected. This decline was strongly correlated with reduced proliferation of CD4+ T cells to donor Ag. The donor-reactive CD8+ T cells declined substantially only after >10 y. The frequency of T cells reactive to unrelated alloantigens did not alter significantly after transplantation, excluding an aspecific effect of immunosuppressive medication. After transplantation, an increase in donor Ag-induced apoptosis was found, specifically within the donor-reactive CD4+ memory T cell subsets. In conclusion, a significant decrease in donor-reactive polyfunctional effector memory CD4+ T cells underlies the development of DSH in kidney transplant recipients, which is likely mediated by specific activation-induced cell death.

Polyfunctional donor-reactive T cells are associated with acute T-cell-mediated rejection of the kidney transplant.

Acute T-cell-mediated rejection (aTCMR) still remains a clinical problem after kidney transplantation despite significant improvements in immunosuppressive regimens. Polyfunctional T cells, i.e. T cells producing multiple pro-inflammatory cytokines, are believed to be the most relevant T cells in an immune response. The aim of this study was to determine whether polyfunctional donor-reactive T cells are associated with aTCMR. In a case-control study, 49 kidney transplant recipients with a biopsy-proven aTCMR in the first year after transplantation were included, as well as 51 controls without aTCMR. Circulating donor-reactive T cells were identified by the expression of CD137 after short-term co-culture with donor antigen-presenting cells. Polyfunctional donor-reactive T cells were further characterized by dissection into different T-cell subsets encompassing the spectrum of naïve to terminally differentiated effector T cells. Prior to kidney transplantation, proportions of donor-reactive CD4+ (0.03% versus 0.02%; P < 0.01) and CD8+ (0.18% versus 0.10%; P < 0.01) CD137++ T cells were significantly higher in recipients with a biopsy-proven aTCMR versus non-rejectors. Polyfunctionality was higher (P = 0.03) in this subset of CD137-expressing T cells. These cells were predominantly of the EM/EMRA-phenotype, with polyfunctional donor-reactive CD137++CD4+ T cells predominantly co-expressing CD28 whereas approximately half of the polyfunctional CD137++CD8+ T cells co-expressed CD28. In addition, at the time of aTCMR, polyfunctional donor-reactive CD137++ CD4+, but not CD8+, T cells, were specifically decreased by 75% compared to before transplantation in recipients with as well as those without an aTCMR. Prior to transplantation, the proportion of polyfunctional donor-reactive CD137++ T cells is associated with the occurrence of a biopsy-proven aTCMR within the first year after transplantation.

Older age is associated with a distinct and marked reduction of functionality of both alloreactive CD4+ and CD8+ T cells.

Introduction: Older recipient age is associated with a significant decreased risk for rejection after kidney transplantation which is incompletely understood. Methods: In a longitudinal study, circulating alloreactive T cells were assessed of young (≤45 years) and older (≥55 years) stable kidney transplant recipients. Alloreactive T-cells were identified by CD137-expression and phenotype, cytokine producing and proliferative capacity, were evaluated using multiparameter flowcytometry. Results: The results show that before transplantation frequencies of alloreactive CD4+ and CD8+ T-cells in older KT-recipients are significantly higher and shifted towards an effector memory-phenotype. However, the frequency of polyfunctional (≥2 pro-inflammatory cytokines) CD4+ T-cells was significantly lower and less IL2 was produced. The frequency of polyfunctional alloreactive CD4+ T-cells and proliferation of alloreactive T-cells donor-specifically declined after transplantation reaching a nadir at 12 months after transplantation, irrespective of age. A striking difference was observed for the proliferative response of alloreactive CD8+ T-cells. This was not only lower in older compared to younger recipients but could also not be restored by exogenous IL2 or IL15 in the majority of older recipients while the response to polyclonal stimulation was unaffected. Conclusion: In conclusion, older age is associated with a distinct and marked reduction of functionality of both alloreactive CD4+ and CD8+ T-cells.

A Novel Technique for the Generation of Substantial Numbers of Functional Resident T Cells from Kidney Tissue.

Studying functionality and antigen-specificity of resident kidney T cells derived from a kidney biopsy is hampered by the lack of sufficient numbers of T cells obtained by the standard method of enzymatic tissue dissociation. Enzymatic dissociation of kidney tissue was compared to a novel method of whole kidney tissue culture allowing T cells to migrate into the medium in the presence of exogenous IL-2 and IL-15. T cell numbers were quantified and phenotype of resident T cells (CD69+CD103+/−), TCR Vß repertoire and functional characteristics were analyzed with multi-parameter flow cytometry. Renal tissue culture for four weeks in the presence of exogenous IL-2 and IL-15 yielded significantly higher numbers of T cells (1.3 × 104/mm3) when compared to cultures without exogenous cytokines (71/mm3) or direct isolation by enzymatic dissociation (662/mm3 T cells, p < 0.05). The proportion of T cells with a resident phenotype did not change in the tissue culture; percentages amounted to 87.2% and 85.1%, respectively. In addition, frequencies of CD4+, CD8+, CD4−CD8−, T cells and MAIT T cells remained similar. For both CD4+ and CD8+, T cells had a more differentiated memory phenotype after tissue culture, but the distribution of TCR Vß families did not change. In addition, the predominant Th1 cytokine secretion profile and poly-functionality of resident kidney T cell remained intact. T cell proliferation potential was not affected, excluding exhaustion and enrichment of BKV- and CMV-reactive resident T cells was observed. In conclusion, the kidney tissue culture method yields significantly increased numbers of resident T cells without major effects on composition and functionality.