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Donor-reactive memory cells represent a barrier to long-term kidney graft survival. A better understanding of regulatory mechanisms that counterbalance alloreactive memory responses may help to identify patients with operational tolerance. This prospective study investigated the equilibrium between memory T-cell subsets and regulatory T or B cells (Tregs, Bregs) in peripheral blood of kidney transplant recipients with operational tolerance (N = 8), chronic rejection (N = 8), and different immunosuppressive treatment regimens (N = 81). Patients on hemodialysis and healthy individuals served as controls (N = 50). In addition, the expression of Treg- and Breg-associated molecule genes was analyzed. Patients with chronic rejection showed a disrupted memory T-cell composition with a significantly higher frequency of circulating CD8+ terminally differentiated effector memory (TEMRA) T cells than patients with operational tolerance, patients on hemodialysis, or healthy controls (P < 0.001). Low frequency of CD8+ TEMRA and high frequency of Tregs and transitional Bregs were found in operationally tolerant patients. Consequently, operationally tolerant patients showed, as compared to all other transplant recipients with different immunosuppressive regiments, the lowest ratios between CD8+ TEMRA T cells and Tregs or Bregs (for both P < 0.001). Moreover, a specific peripheral blood transcription pattern was found in operationally tolerant patients with an increased expression of Breg- and Treg-associated genes CD22 and FoxP3 and a decreased FcγRIIA/FcγRIIB transcript ratio (for all P < 0.001). In conclusion, monitoring the balance between circulating CD8+ TEMRA T cells and regulatory cell subsets and their transcripts may help to distinguish transplant recipients with operational tolerance from recipients at risk of graft loss., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: We recently demonstrated that donor-derived modified immune cells (MICs)-PBMCs that acquire immunosuppressive properties after a brief treatment-induced specific immunosuppression against the allogeneic donor when administered before kidney transplantation. We found up to a 68-fold increase in CD19 + CD24 hi CD38 hi transitional B lymphocytes compared with transplanted controls., Methods: Ten patients from a phase 1 clinical trial who had received MIC infusions before kidney transplantation were followed to post-transplant day 1080., Results: Patients treated with MICs had a favorable clinical course, showing no donor-specific human leukocyte antigen antibodies or acute rejections. The four patients who had received the highest dose of MICs 7 days before surgery and were on reduced immunosuppressive therapy showed an absence of in vitro lymphocyte reactivity against stimulatory donor blood cells, whereas reactivity against third party cells was preserved. In these patients, numbers of transitional B lymphocytes were 75-fold and seven-fold higher than in 12 long-term survivors on minimal immunosuppression and four operationally tolerant patients, respectively ( P <0.001 for both). In addition, we found significantly higher numbers of other regulatory B lymphocyte subsets and a gene expression signature suggestive of operational tolerance in three of four patients. In MIC-treated patients, in vitro lymphocyte reactivity against donor blood cells was restored after B lymphocyte depletion, suggesting a direct pathophysiologic role of regulatory B lymphocytes in donor-specific unresponsiveness., Conclusions: These results indicate that donor-specific immunosuppression after MIC infusion is long-lasting and associated with a striking increase in regulatory B lymphocytes. Donor-derived MICs appear to be an immunoregulatory cell population that when administered to recipients before transplantation, may exert a beneficial effect on kidney transplants., Clinical Trial Registry Name and Registration Number: MIC Cell Therapy for Individualized Immunosuppression in Living Donor Kidney Transplant Recipients (TOL-1), NCT02560220., (Copyright © 2022 by the American Society of Nephrology.)

Allospecific memory T cells are a barrier against long-term graft survival. Production of multiple cytokines by a single T cell is considered a sign of an active ongoing immune response, the presence of these polyfunctional cells has not been addressed in transplanted patients accordingly to graft outcome. Memory phenotype, based on the expression of CD45RO and CD27, and polyfunctional T cells were evaluated in long-term graft survival patients (LTS), short-term survival patients (STS), chronic rejection patients (ChrRx), dialysis patients (DIAL) and healthy controls (Ctrls). Memory T cells were quantified ex vivo, after allogeneic and anti-CD3 plus anti-CD28 stimulation, in cells proliferating or not to these stimuli. The percentages of cells producing IFNγ, IL-2 and/or TNFα after allogeneic stimulation and the memory phenotype of single cytokine producing cells were evaluated. Ex vivo CD8+CD45RO-CD27- effector cells were decreased in transplanted patients compared to non-transplanted individuals. After allogeneic stimulation, CD4+CD45RO+CD27+, central memory cells in LTS and CD4+CD45RO-CD27- effector cells in Dial were augmented compared to Ctrls and ChrRx, and CD8+CD45RO-CD27- effector cells were increased in ChrRx. There were no differences in the percentage of single cytokine producing cells among the groups. IFNγ+TNFα+CD4 and CD8 cells were detected in Ctrls, STS and ChrRx and no cells positive for the three cytokines were found. The phenotype of cytokine producing cells was mainly effector memory. Interestingly, in LTS there was an increase in effector cells producing IFNγ and IL-2. Changes in subpopulation distribution in patients with different outcomes may be a reflection of the graft acceptance or rejection status., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The mechanisms underlying maintenance of renal allografts in humans under minimal or conventional immunosuppression are poorly understood. There is evidence that CD4(+) CD25(+) regulatory T cells and clonal deletion, among other mechanisms of tolerance, could play a key role in clinical allograft survival. Twenty-four TCR-Vbeta families were assessed in CD4(+) CD25(-), CD4(+) CD25(low) and CD4(+) CD25(high) T cells from patients with long-term renal allograft survival (LTS), patients exhibiting chronic rejection (ChrRx), patients on dialysis (Dial) and healthy controls (HC) by flow cytometry. LTS patients presented a higher variability in their TCR-Vbeta repertoire, such decreased percentage of Vbeta2(+), Vbeta8a(+) and Vbeta13(+) in CD4(+) CD25(low) and (high) compared with CD4(+) CD25(-) subset and increased Vbeta4 and Vbeta7 families in CD4(+) CD25(high) T cells exclusively. Additionally, LTS patients, particularly those that were not receiving calcineurin inhibitors (CNI), had increased percentages of CD4(+) CD25(high) T cells when compared with Dial (P < 0.05) and ChrRx (P < 0.05) patients. Our results suggest that a differential expression of particular TCR-Vbeta families and high levels of circulating CD4(+) CD25(high) T cells in long-term surviving renal transplant patients could contribute to an active and specific state of immunologic suppression. However, the increase in this T cell subset with regulatory phenotype can be affected by CNI.

Background: Naturally occurring regulatory T cells have been associated with long-term allograft survival. We investigated whether gene transcripts of Treg-related molecules are upregulated or downregulated in kidney transplant recipients with different clinical outcomes and may serve as markers of operative tolerance., Methods: Expression levels of transcription factor (forkhead box P3 [FOXP3], t-bet, and GATA3), regulatory molecule (cytotoxic T-lymphocyte antigen-4, glucocorticoid-induced tumor necrosis factor receptor-related protein, tribbles protein-1, and transforming growth factor-beta), and chemokine receptor (CCR7 and CXCR4) genes were measured in kidney graft recipients with long-term (> or = 9 years) stable renal function (LTS) or chronic rejection (ChrRx). Patients on dialysis and healthy individuals served as controls., Results: The level of FOXP3 transcripts was lower in ChrRx patients than in LTS patients (P<0.01). The highest transforming growth factor-beta transcripts were observed in ChrRx and the highest CCR7 and CXCR4 transcripts were observed in LTS patients. In LTS patients, FOXP3 gene expression was associated with CXCR4 gene expression (P=0.015). FOXP3 and CCR7 transcript levels were higher in LTS patients without calcineurin inhibitor therapy than in LTS patients with calcineurin inhibitors., Conclusion: Our results suggest that high expression of FOXP3 and chemokine receptor genes in LTS patients are possible indicators of a regulatory process that contributes to long-term allograft acceptance. Markers that were increased in LTS patients were found to be decreased in ChrRx patients, suggesting that rejection may partly be the result of a lack of this regulatory process. FOXP3 and CCR7 and CXCR4 transcripts might be used as markers to distinguish patients who developed long-term allograft acceptance from patients who are prone to ChrRx.

The immune response elicited by an allogenic transplant usually leads to an effector response resulting in allograft rejection; however, some individuals maintain a long-term functioning transplant without signs of rejection (operational tolerance) even in the absence of immunosuppression. It has been suggested that the same mechanisms are responsible for tolerance to self-antigens and alloantigens. One of such mechanisms is immune regulation and several cell subsets with regulatory properties have been identified. Among them, the best characterized cell populations are the regulatory T cells (Treg). Although Treg in mice are CD4+CD25+, in humans the Treg phenotype is restricted to CD4 T cells with high expression of CD25 (CD25high) and Foxp3. Phenotypic and functional analysis of circulating regulatory or suppressor T cells in transplant patients may be useful for detection of operationally tolerant patients. Moreover, future in vitro manipulation of these cells with therapeutic purposes could lead to accomplish induction of in vivo tolerance in clinical transplantation. Herein, we review the experimental and clinical evidence for the role of regulatory cells in transplant biology.

The mechanisms underlying long-term acceptance of kidney allografts in humans under minimal or no maintenance immunosuppression are poorly understood. We analyzed the T-cell receptor (TCR) repertoires in circulating T cells of patients with long-term (> or = 9 years) renal allograft survival with (LTS-IS) and without immunosuppression (LTS-NoIS). T cells of LTS patients exhibited strongly altered TCR Vss usage, including an increased frequency of oligoclonality and a decreased frequency of polyclonality. All 3 LTS-NoIS and 12 of 16 LTS-IS patients demonstrated oligoclonality in at least three or more TCR V beta families, and the frequency of oligoclonality in these patients was significantly higher as compared to patients with well-functioning grafts at 3 years (p < 0.005 both), an uncomplicated course during the first year (p < 0.0001, both), acute rejection (p < 0.0001, both), chronic allograft nephropathy at 7 (p < 0.0001, both) or 13 years (p < 0.0001, both), dialysis patients (p < 0.0001, both) or healthy controls (p < 0.0001, both). In contrast to LTS patients, all other studied patient groups exhibited a polyclonal TCR repertoire. Our data indicate that TCR alteration is a common feature of long-term allograft outcome, which might be explained by clonal deletion, exhaustion of alloreactive T cells or predominant expression of particular T-cell subpopulations, such as regulatory T cells.

Background: Patients with long-term functioning organ allografts may have developed different mechanisms that explain the lack of graft rejection. However, it is not known how these mechanisms interplay or whether one of them predominates in such situations., Methods: The authors analyzed the expression of T-cell surface molecules involved in alloantigen recognition, signal transduction, co-stimulation, and activation markers on circulating T cells from patients with normal kidney function 10 or more years after transplantation, short-term survival (1-4 years), and chronic rejection and from healthy adults. The percentage and the median fluorescent intensity of each marker were determined by flow cytometry. Proliferative response against specific and third-party donors and mitogenic stimulation were also determined., Results: Peripheral blood lymphocytes from patients with long-term surviving kidneys had decreased expression of T-cell receptor (TCR)-alphabeta (P < 0.01), CD3epsilon (P < 0.05), and CD3 zeta-chains (P < 0.001); diminished percentages of CD4(+)CD28(+) (P < 0.001) cells; and increased expression of CTLA-4(+) (P < 0.01) on CD3(+) cells. CD4(+)CD25(+)CD69(+) cells were also increased in long-term surviving patients. Long-term surviving patients had decreased donor-specific proliferative responses., Conclusions: The decreased expression of TCR-alphabeta and epsilon- and zeta-chains on circulating T cells of long-term surviving patients suggests that these cells may have defects in alloantigen recognition or signal transduction that may result in decreased numbers of T cells expressing co-stimulatory molecules and activation markers as well as a decreased specific proliferative response. The decrease in the percentage of CD28(+) cells and the increase in CD4(+)CD25(+)CD69(+) cells suggest that regulatory mechanisms of the immune response are still active in such patients.

Abstract: High serum levels of soluble CD30 (sCD30) are associated with poor renal allograft survival, and regulatory T cells (Tregs) influence allograft survival depending on CD30 signaling. However, how sCD30 modulates alloimmune responses remains poorly understood. We measured the level of Tregs and sCD30 in patients with end-stage renal failure (ESRF) and analyzed whether allo- or polyclonal stimulation of the patients’ T cells results in the expression and release of CD30. ESRF patients showed increased serum sCD30 levels and lower percentages of circulating Tregs as compared to healthy controls (HC) (p <0.001 and 0.024). Polyclonal and allogeneic stimulation resulted in higher expression of CD30, and after polyclonal stimulation, ESRF patients showed higher percentages of CD30-expressing T cells than HC (p <0.001). Compared to autologous stimulation, allogeneic stimulation induced significantly higher expression of CD30 on T cells of ESRF patients only. After polyclonal as well as allogeneic stimulation, an increased sCD30 content was found in culture supernatants of both ESRF patients and HC (p <0.001). Together with decreased Tregs, high serum sCD30 and increased induction of CD30 on T cells after polyclonal stimulation may explain exacerbated alloimmune responses and poor allograft survival in ESRF patients in whom immunosuppression is not able to control the alloimmune response. [Copyright &y& Elsevier]