The unmet need in clinical transplantation. Despite its crucial role in promoting improved outcomes in organ transplantation, conventional long-term immunosuppressive (IS) drug therapy is associated with potential life-threatening side effects. These include infection, certain types of malignancy, diabetes and increased risk of cardiovascular disease. In addition, use of calcineurin inhibitors (tacrolimus/cyclosporine) can cause kidney injury and a significant rate of chronic renal insufficiency by 5 years post-transplant. Therefore, an important unmet clinical need and a major goal in transplantation research is to reduce, or better, safely eliminate the burden of immunosuppressive therapy within the first few years after transplant surgery, without sacrificing long-term therapeutic benefits. Presently, complete withdrawal/elimination of anti-rejection therapy (transplant tolerance) is only possible in a small proportion (approx. 10%) of stable liver transplant patients during the first few years post-transplant (and only very rarely in recipients of kidney grafts), when IS drug withdrawal would be most beneficial. Successful translation from preclinical models to humans of a safe and effective regimen that can achieve a state of stable, drug-free tolerance, i.e. donor-specific immunological unresponsiveness in the absence of graft tissue injury, has remained elusive. Elucidation of mechanisms that underlie the development of transplant tolerance, testing of innovative strategies to promote transplant tolerance, and identification of biomarkers that may reliably predict tolerance are major research goals at the STI.
The potential of cell therapy. In recent years, extensive research has focused on the therapeutic potential of naturally-occurring regulatory immune cells. Although present in only small numbers in normal, healthy individuals, these cells are thought to preserve tolerance to self antigens (Ags), preventing the potential development of autoimmune disorders. Importantly, regulatory immune cells can now be generated in large numbers in cell culture facilities. They include regulatory myeloid cells (dendritic cells (DCs); macrophages), regulatory T lymphocytes and mesenchymal stem cells. Their evaluation in transplant recipients as highly-promising, novel cellular therapies may lead to reduced patient dependence on IS drugs and the achievement of donor-specific tolerance. With continuous NIH funding for over 25 years, Dr. Angus Thomson, Distinguished Professor of Surgery & Immunology, has pioneered the development and testing of regulatory dendritic cell (DCreg) therapy, and in collaboration with Dr. Abhinav Humar, Starzl Professor of Transplant Surgery and Clinical Director of the STI and key co-investigators,- Drs. Lillian Tran, Camila Macedo, Beth Elinoff, Adriana Zeevi, Jake Demetris, Diana Metes and Fadi Lakkis, has instigated a first-in-human trial of DCreg therapy in living donor liver transplantation (LDLT).
Regulatory dendritic cells (DCregs). Dendritic cells are highly-specialized, bone marrow-derived Ag-presenting myeloid cells (first described almost 50 years ago) that induce, integrate and regulate innate and adaptive immune responses. Many years of innovative research instigated in the STI have established that DCregs, in contrast to stimulatory DCs, can subvert naïve and also memory T cell responses (that are resistant to conventional anti-rejection therapy) by various mechanisms. They can induce or restore T cell tolerance in organ transplantation or autoimmune disease, respectively. In experimental transplantation, both allogeneic (donor-derived) DCregs and donor Ag-pulsed host (autologous) DCregs are effective. Our work has also confirmed that adoptive transfer of allogeneic (donor-derived) DCregs can safely and effectively regulate host T cell responses and prolong graft survival in a clinically-relevant, major histocompatibility complex (MHC)-mismatched nonhuman primate organ allograft model -an important bridge to clinical testing.
Initial clinical testing of DCregs. Human DCregs can be generated in culture from precursors (monocytes) in peripheral blood, using a variety of pharmacologic or biologic agents that enhance their tolerogenicity. In volunteers, adoptive transfer of Ag-pulsed DCregs via local administration can control T cell responses to model Ags,- flu matrix peptide or keyhole limpet hemocyanin. Taken together with compelling laboratory evidence of the therapeutic efficacy of DCregs in preclinical models, these findings led to the initial testing of clinical grade DCreg in phase I safety trials in autoimmune disease. In these trials, autologous DCreg (unpulsed or autoAg-pulsed) have been administered locally in type-1 diabetes and rheumatoid arthritis, intraperitoneally in refractory Crohn’s disease, or intravenously in multiple sclerosis and neuromyelitis optica patients. The results have emphasized the feasibility, safety and potential efficacy of autologous DCreg therapy.
Rationale for a first-in-human DCreg trial in liver transplantation. Donor-derived DCregs inherently express donor MHC Ags with the potential to induce tolerance specifically to these Ags against which immune-mediated rejection responses are directed. Encouraging preclinical testing of donor-derived (allogeneic) DCregs by Drs. Mohamed Ezzelarab and Thomson in collaboration with Drs. Humar and Wijkstrom at the STI provided a strong impetus for testing the potential of these cells to induce tolerance in clinical organ transplantation. Biologically, the liver is considered the transplanted organ most likely to induce tolerance (32-35) and many liver transplant patients can tolerate reduced doses of IS drugs, with some going on to complete IS drug withdrawal, depending on the number of years post-transplant and other factors (36). However, early complete withdrawal of IS drugs lasting for great than 1 year has been documented in only 13% of stable adults 1-2 years post-transplant (39). Given this background, the living donor liver transplantation program at the STI (the largest in North America) provided an excellent opportunity to plan and conduct first-in-human early phase trials of donor-derived DCreg therapy (clinicaltrials.gov NCT 03164265). The underlying hypothesis is that donor-derived DCreg administration a week before transplant to prospective adult living donor liver transplant recipients treated will be safe and induce immunological changes conducive to development of donor-specific hypo-responsiveness, and the likelihood of safe withdrawal of all IS by approximately 24 months post-transplantation.
Trial objectives. The primary objective of the initial phase I/IIa single center, prospective, open label, non-controlled, non-randomized trial (NCT 03164265) is to determine the feasibility, safety and preliminary efficacy of donor-derived DCreg infusion one week before transplantation to achieve IS withdrawal in LDLT. The secondary objective is to assess the influence of DCreg and IS drug withdrawal on anti-donor immune reactivity, renal function and cardiovascular risk factors.
Trial protocol. Low-risk LDLT recipients are given a single dose infusion of donor-derived DCregs (2.5-10^6/kg) with concurrent mycophenolic acid therapy (1/2 dose) one week before transplantation. They are maintained on standard of care MPA and tacrolimus for the first 12 months post-transplant. They are then evaluated for tacrolimus weaning on the basis of a protocol biopsy. Those who meet the criteria of no rejection, permissive liver function tests and graft biopsy proceed to tacrolimus weaning. Successfully weaned participants who remain off all IS for 1 year with a clean liver biopsy are deemed operationally tolerant. Successfully weaned participants who remain rejection-free undergo 3 years of follow-up after the last dose of IS. They undergo a subsequent graft biopsy 3 yrs after IS withdrawal. Detailed immunological monitoring is performed over the first year post-transplant and data compared with data from and a matched reference/comparison group of standard-of-care (SOC) patients, to address our hypothesis that DCreg infusion may induce changes in host immune cell populations conducive to regulation of anti-donor immune reactivity and subsequent safe withdrawal of IS therapy.
Clinical and mechanistic observations. Between September 2017 and January 2020 fifteen prospective LDLT recipients received a single infusion of DCreg one week before transplant. No cell manufacturing problems were encountered and no adverse effects were associated with DCreg infusion. Following DCreg infusion, donor MHC Ags and immune regulatory molecules expressed by DCreg-derived exosomes are acquired by host DCs that likely mediate the function of the infused regulatory cells. In addition, during the interval between cell infusion and transplant surgery, T cell subset changes are observed that are potentially conducive to modulated anti-donor immune reactivity. Two participants were withdrawn from study for reasons unrelated to DCreg infusion. No significant differences in post-operative complications were observed compared with a propensity-matched reference group of 40 STI standard-of-care patients that, unlike the study group, received basilixumab induction in addition to triple drug IS. In the first 12 months post-transplant incidences of infections and biopsy-proven acute cellular rejection were lower in the study group, although differences were not statistically significant. At 12 months, there were significantly lower levels of circulating immune effector cells (CD8 T effector cells and NK cells) and pro-inflammatory cytokines in the DCreg group. Single cell RNAseq analysis further revealed reduced expression of cytolytic and effector genes and inflammatory pathways in graft-infiltrating CD8+ T cells. By contrast, circulating levels of a putative tolerogenic DC subset were elevated in the DCreg but not the SOC reference group. No evidence of de novo DSA was detected at 12 months.
At 12 months post-transplant, 8/13 patients were eligible for IS drug weaning. Four patients have been successfully weaned, although one returned to IS therapy following a COVID booster 8.5 months after withdrawal of all IS. Three patients remain stable, off all IS for greater than 1 year (2 years 9 months, 1 year 6 months and 1 year 4 months), all with a clean biopsy at 1 year biopsy off all IS. Thus, operational tolerance has been achieved in 3/8 (37.5%) of patients eligible for drug weaning, or in 3/13 (23%) of total enrolled patients available for analysis. This compares with 10/77 (13%) of eligible and 10/257 (4%) of total enrolled patients in a recent multi-center, non-interventional trial of early IS drug withdrawal in adult liver transplantation. While patient numbers are small, these initial clinical and mechanistic observations justify further evaluation of the potential of DCreg therapy for promotion of tolerance induction in liver transplantation, with a randomized trial being the appropriate next step.
The clinical trial of DCreg in LDLT described above is supported by UPMC Enterprises and STI research development funds. A separate IS drug weaning trial of delayed donor-DCreg infusion in LDLT recipients (NCT 04208919), in which DCregs are administered to stable graft recipients with a permissive graft biopsy 1-3 years post-transplant, is also being conducted at UPMC. In addition, an NIH-funded trial of donor-derived DCregs in living donor kidney transplantation (NCT 03726307) is currently enrolling patients.
