The long-term survival of kidney grafts continues to be hampered by two reasons. First, conventional immunosuppressive therapy is still unable to prevent long-term kidney graft loss, and a significant proportion of patients lose their grafts due to chronic (antibody-mediated) rejection (1). Second, immunosuppressive drugs have significant side effects such as infections and cancers that lead to the death of transplanted patients with functioning allografts (2). For this reason, one of the main goals in the field of solid organ transplantation is to create durable graft acceptance without immunosuppressive therapy, i.e., tolerance. Tolerance can be achieved by inducing durable chimerism in the setting of combined kidney and bone marrow transplantation (3). However, this approach is associated with the side effects of conditioning therapy and carries the risk of graft-versus-host disease (4). For example, in a Northwestern University trial on the induction of mixed chimerism using FCRx therapy in 37 HLA-mismatched living-donor kidney transplant recipients, two patients suffered graft-versus-host disease, two patients experienced graft loss and a total of up to 3 patients ultimately died (4, 5). The subsequent FREEDOM-1 and FREEDOM-2 clinical trials, using the same therapy, were recently terminated prematurely presumably for the same reasons (6, 7). Rather than transplanting the entire immune system, there have been recent efforts to modify the recipient’s immune system through cellular therapies to accept the transplanted organ (8–12). In the recently published The One study, patients receiving different cellular therapies, e.g., regulatory T lymphocytes and tolerogenic dendritic cells, along with reduced conventional immunosuppressive therapy were compared with a reference group of patients receiving regular immunosuppressive therapy after living donor kidney transplantation (10). This study demonstrated the safety and feasibility of regulatory T lymphocyte therapies together with tacrolimus alone in preventing kidney graft rejection while reducing infectious complications (11, 12). Currently, efforts are underway to further improve the efficacy of such an approach by introducing a chimeric antigen receptor (CAR) into regulatory T lymphocytes that recognizes HLA-A2 on transplanted organs. In this way, regulatory T lymphocytes accumulate in an HLA-A2 positive graft when introduced into an HLA-A2 negative recipient (13).

We have previously published the results of a phase I trial using modified donor peripheral blood mononuclear cells (MIC) together with reduced standard immunosuppressive therapy to prevent rejection in 10 living donor kidney transplant recipients (14, 15). Four of the 10 patients who had received the highest cell dose of 1.5 × 10⁸ MIC per kg body weight 7 days prior to surgery showed donor-specific immunosuppression, as evidenced by a lack of cellular stimulatory reactivity after transplantation when tested against their respective donors in vitro. These patients also had higher numbers of IL-10-producing CD19⁺CD24^hiCD38^hi transitional B lymphocytes as well as of other regulatory B lymphocyte subsets at various stages of maturation which are thought to have regulatory properties. The T-cell response against the donor was increased after depletion of B lymphocytes in vitro, suggesting that regulatory B lymphocytes were pathophysiologically relevant to establish donor-specific unresponsiveness. These results were also supported by a gene expression signature consistent with the COMBINED-g7 consensus signature of operational tolerance in three of the four patients, suggesting that we actively induced an operationally tolerant phenotype by high-dose MIC treatment one week before surgery (14, 15). In the current analysis, we wanted to investigate how this cell therapy approach together with reduced immunosuppressive therapy affected the continued clinical course of treated patients. We compared the clinical outcomes of the 10 MIC patients to outcomes of 15 matched transplanted controls. In addition, we provide immunological outcomes of MIC patients up to year 5 after surgery.

We have recently shown that a single pretransplant infusion of MIC led to long-lasting donor-specific unresponsiveness (14, 15, 20, 21). This was accompanied by an increased frequency of regulatory B lymphocytes and a consensus gene expression signature of operational tolerance. The question arises whether this intervention, together with reduced immunosuppression, was sufficient to effectively prevent the occurrence of an alloimmune response while reducing the deleterious side effects of conventional drug therapy. To test this hypothesis, the clinical outcomes of the 10 MIC patients were compared to the outcomes of 15 standard-risk recipients who received regular triple-drug maintenance therapy. Neither acute rejections nor DSA were detected in MIC patients, although they had received mononuclear donor blood cells 2 or 7 days before surgery. We also found fewer opportunistic infections in MIC patients when compared to transplanted controls, most likely as a result of the reduced immunosuppressive therapy. These results held strong when comparing MIC patients only to ten transplanted controls who had no formal contraindication for inclusion into the TOL-1 study. Most strikingly, MIC patients continued to show absent anti-donor T cell reactivity and high transitional B lymphocyte numbers out to year 5 which might be considered a proof of continued donor-specific unresponsiveness.

In our current analysis, none of the 10 MIC patients showed evidence of DSA against HLA-A, -B, -DR, or -DQB antigen mismatches during the 5-year follow-up period. The absence of DSA after but also on day -1 before transplantation is even more remarkable considering that MIC patients had received up to 1.34 x10¹⁰ nucleated donor blood cells 2 or 7 days before surgery. Due to the introduction of the donor antigen already 7 days before transplantation, one would normally expect a sensitizing effect. A graft-protective effect similar to the one observed in our study was also observed by Marti et al. when infusing unmodified mononuclear cells twice before living donor kidney transplantation, but at the expense of sensitizing 6 of 61 (10%) patients by administering cells bearing donor HLA on their surface before surgery (22). In our study, no such sensitization was detected before transplantation, which is most likely due to the modification process of the cells, but also to the infusion timing at day -7 before transplantation, which did not allow sufficient time for allosensitization. In comparison, a higher proportion of 5 of 15 (33%) transplanted controls developed DSA during 5-year follow-up, which is considerably higher than in MIC patients but also slightly higher than reported in the literature for transplanted patients at comparable immunologic risk (23, 24). In a study by Wiebe et al, only 15% of patients overall developed DSA at a median of 6.2 years post-transplant (25). These DSA, especially when capable of activating complement, bind to the endothelium of the allograft and lead to antibody-mediated allograft injury (25, 26). Explanations for the observed differences between transplanted controls in our study and findings from the literature could be the use of cyclosporine A (instead of tacrolimus) or the stringent routine screening in our patients, in which DSA were detectable only in a low range of up to 2,052 MFI in 3 of 5 patients.

Infections, malignancies, and cardiovascular events are the major causes of premature death in immunocompromised kidney transplant recipients (27–30). In the Swiss Transplant Cohort Study, a total of 1964 clinically relevant infections, mostly bacterial and viral, were identified in 1612 kidney transplant recipients during the first year after surgery (28). Overall, 81% of patients had an infectious event, and 53% of patients had at least one clinically relevant infection. A total of 12 patients eventually died from infection. The One study recently demonstrated that cell therapy approaches combined with reduced immunosuppression are capable of effectively reducing infectious episodes (10). The incidence of serious adverse events related to infection and infestation was nearly six times higher in the control trial than in the combined cell therapy trials, and this increase was primarily due to a higher number of viral infections in the control trial (10). Likewise, we found in our study an increased frequency of opportunistic infections, mostly viral, and a trend towards more pneumonia episodes from any cause (opportunistic and non-opportunistic combined) in matched control patients when compared to MIC patients on reduced immunosuppressive therapy. We cannot judge on the effect of our approach on malignancy and cardiovascular events since only 1 episode of each was detected during the study, notably both in transplanted control patients on full immunosuppressive therapy.

We admit that our study has limitations. We show 5-year follow-up of only 10 patients from a 30-day single-arm phase I clinical trial, and only 4 patients had received the highest MIC number 7 days before surgery, which was considered most effective. The 15 transplanted control patients were thoroughly matched but were selected retrospectively, and a potential selection bias toward better outcomes in MIC patients cannot be excluded. In addition, the control patients had basiliximab induction therapy and, because of the nature of the study, stronger immunosuppressive maintenance therapy, at least compared with MIC patients of group C. Another limitation arises from the fact that the anti-donor T-cell response and flow cytometry analyses were available only in MIC patients but not in transplanted controls. Nevertheless, the concomitant lower infection rates and a lower incidence of DSA in MIC patients suggest a potential clinical relevance of this cell therapy product when administered together with reduced conventional immunosuppressive therapy.

In conclusion, MIC infusions prior to living donor kidney transplantation were shown in this small series to result in donor-specific unresponsiveness that effectively controls alloimmune responses. One would expect that this treatment would offer an opportunity to reduce the side effects of conventional chemical immunosuppression. These results are very promising and suggest that MIC therapy is a potential new effective treatment option for kidney transplant recipients. A Phase IIb study is currently underway to test the hypothesis that a larger series of patients treated with MIC and a reduced immunosuppressive drug regimen will show the same positive effects (EudraCT No. 2021-000561-33, ClinicalTrials.gov Identifier: NCT05365672) (31).

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Ethics committee of the University of Heidelberg, Heidelberg, Germany. The patients/participants provided their written informed consent to participate in this study.

MSa, CMo, CK, GO, CS, DC, MZ, MSm, PT, and AS designed the study. MSa, CMo, and MZ recruited participants. CK, LW, AH-K, MSm, and AS produced MIC under GMP conditions and performed quality controls of the products. MSa, CMo, FK, CSp, LB, CN, CMa, LS, and MSa collected the data. MSa, CMo, LW, CK, TT, SS, LP, NE, CSü, GP, AM, CSc, RW, PS, UM, VS, MK, SK, MF, MSt, IH, A-IK, BK, GB, CM-T, JR, MSm, PT, AS, and VD analyzed the data. MSa, CMo, GO, TT, CSü, MZ, MSm, PT, AS, and VD interpreted the data. MSa and CMo designed the figures. MSa, CMo, GO, and VD wrote the first draft of the manuscript, and all authors revised it critically and approved the final version. All authors contributed to the article and approved the submitted version.