The Fourth International Workshop on Clinical Transplant Tolerance.

The International Workshop on Clinical Transplant Tolerance is a biennial meeting that aims to provide an update on the progress of studies of immunosuppression minimization or withdrawal in solid organ transplantation. The Fourth International Workshop on Clinical Tolerance was held in Pittsburgh, Pennsylvania, September 5-6, 2019. This report is a summary of presentations on the status of clinical trials designed to minimize or withdraw immunosuppressive drugs in kidney, liver, and lung transplantation without subsequent evidence of rejection. All protocols had in common the use of donor or recipient cell therapy combined with organ transplantation. The workshop also included presentations of mechanistic studies designed to improve understanding of the cellular and molecular basis of tolerance and to identify potential predictors/biomarkers of tolerance. Strategies to enhance the safety of hematopoietic cell transplantation and to improve patient selection/risk stratification for clinical trials were also discussed.

Feasibility, long-term safety, and immune monitoring of regulatory T cell therapy in living donor kidney transplant recipients.

Short-term outcomes in kidney transplantation are marred by progressive transplant failure and mortality secondary to immunosuppression toxicity. Immune modulation with autologous polyclonal regulatory T cell (Treg) therapy may facilitate immunosuppression reduction promoting better long-term clinical outcomes. In a Phase I clinical trial, 12 kidney transplant recipients received 1-10 × 106 Treg per kg at Day +5 posttransplantation in lieu of induction immunosuppression (Treg Therapy cohort). Nineteen patients received standard immunosuppression (Reference cohort). Primary outcomes were rejection-free and patient survival. Patient and transplant survival was 100%; acute rejection-free survival was 100% in the Treg Therapy versus 78.9% in the reference cohort at 48 months posttransplant. Treg therapy revealed no excess safety concerns. Four patients in the Treg Therapy cohort had mycophenolate mofetil withdrawn successfully and remain on tacrolimus monotherapy. Treg infusion resulted in a long-lasting dose-dependent increase in peripheral blood Tregs together with an increase in marginal zone B cell numbers. We identified a pretransplantation immune phenotype suggesting a high risk of unsuccessful ex-vivo Treg expansion. Autologous Treg therapy is feasible, safe, and is potentially associated with a lower rejection rate than standard immunosuppression. Treg therapy may provide an exciting opportunity to minimize immunosuppression therapy and improve long-term outcomes.

Preformed T cell alloimmunity and HLA eplet mismatch to guide immunosuppression minimization with tacrolimus monotherapy in kidney transplantation: Results of the CELLIMIN trial.

Personalizing immunosuppression is a major objective in transplantation. Transplant recipients are heterogeneous regarding their immunological memory and primary alloimmune susceptibility. This biomarker-guided trial investigated whether in low immunological-risk kidney transplants without pretransplant DSA and donor-specific T cells assessed by a standardized IFN-γ ELISPOT, low immunosuppression (LI) with tacrolimus monotherapy would be non-inferior regarding 6-month BPAR than tacrolimus-based standard of care (SOC). Due to low recruitment rates, the trial was terminated when 167 patients were enrolled. ELISPOT negatives (E-) were randomized to LI (n = 48) or SOC (n = 53), E+ received the same SOC. Six- and 12-month BPAR rates were higher among LI than SOC/E- (4/35 [13%] vs. 1/43 [2%], p = .15 and 12/48 [25%] vs. 6/53 [11.3%], p = .073, respectively). E+ patients showed similarly high BPAR rates than LI at 6 and 12 months (12/55 [22%] and 13/66 [20%], respectively). These differences were stronger in per-protocol analyses. Post-hoc analysis revealed that poor class-II eplet matching, especially DQ, discriminated E- patients, notably E-/LI, developing BPAR (4/28 [14%] low risk vs. 8/20 [40%] high risk, p = .043). Eplet mismatch also predicted anti-class-I (p = .05) and anti-DQ (p < .001) de novo DSA. Adverse events were similar, but E-/LI developed fewer viral infections, particularly polyoma-virus-associated nephropathy (p = .021). Preformed T cell alloreactivity and HLA eplet mismatch assessment may refine current baseline immune-risk stratification and guide immunosuppression decision-making in kidney transplantation.

IgG Endopeptidase in Highly Sensitized Patients Undergoing Transplantation.

BACKGROUND: Donor-specific antibodies create an immunologic barrier to transplantation. Current therapies to modify donor-specific antibodies are limited and ineffective in the most highly HLA-sensitized patients. The IgG-degrading enzyme derived from Streptococcus pyogenes (IdeS), an endopeptidase, cleaves human IgG into F(ab')2 and Fc fragments inhibiting complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity, which suggests that IdeS might be useful for desensitization. We report on the combined experience of two independently performed open-label, phase 1-2 trials (conducted in Sweden and the United States) that assessed the efficacy of IdeS with regard to desensitization and transplantation of a kidney from an HLA-incompatible donor. METHODS: We administered IdeS to 25 highly HLA-sensitized patients (11 patients in Uppsala or Stockholm, Sweden, and 14 in Los Angeles) before the transplantation of a kidney from an HLA-incompatible donor. Frequent monitoring for adverse events, outcomes, donor-specific antibodies, and renal function was performed, as were renal biopsies. Immunosuppression after transplantation consisted of tacrolimus, mycophenolate mofetil, and glucocorticoids. Patients in the U.S. study also received intravenous immune globulin and rituximab after transplantation to prevent antibody rebound. RESULTS: Recipients in the U.S. study had a significantly longer cold ischemia time (the time elapsed between procurement of the organ and transplantation), a significantly higher rate of delayed graft function, and significantly higher levels of class I donor-specific antibodies than those in the Swedish study. A total of 38 serious adverse events occurred in 15 patients (5 events were adjudicated as being possibly related to IdeS). At transplantation, total IgG and HLA antibodies were eliminated. A total of 24 of 25 patients had perfusion of allografts after transplantation. Antibody-mediated rejection occurred in 10 patients (7 patients in the U.S. study and 3 in the Swedish study) at 2 weeks to 5 months after transplantation; all these patients had a response to treatment. One graft loss, mediated by non-HLA IgM and IgA antibodies, occurred. CONCLUSIONS: IdeS reduced or eliminated donor-specific antibodies and permitted HLA-incompatible transplantation in 24 of 25 patients. (Funded by Hansa Medical; ClinicalTrials.gov numbers, NCT02224820 , NCT02426684 , and NCT02475551 .).

Understanding Stem Cell Immunogenicity in Therapeutic Applications.

Stem cells and their differentiated progeny offer great hope for treating disease by providing an unlimited source of cells for repairing or replacing damaged tissue. Initial studies suggested that, unlike 'normal' transplants, specific characteristics of stem cells enabled them to avoid immune attack. However, recent findings have revealed that the immunogenicity of stem cells may have been underestimated. Here, we review the current understanding of the mechanisms of immune recognition associated with stem cell immunogenicity, and discuss the relevance of reprogramming and differentiation strategies used to generate cells or tissue from stem cells for implantation in eliciting an immune response. We examine the effectiveness of current strategies for minimising immune attack in light of our experience in the transplantation field and, in this context, outline important challenges moving forward.

Induction of transplantation tolerance through regulatory cells: from mice to men.

Organ transplantation results in the activation of both innate and adaptive immune responses to the foreign antigens. While these responses can be limited with the use of systemic immunosuppressants, the induction of regulatory cell populations may be a novel strategy for the maintenance of specific immunological unresponsiveness that can reduce the severity of the detrimental side effects of current therapies. Our group has extensively researched different regulatory T-cell induction protocols for use as cellular therapy in transplantation. In this review, we address the cellular and molecular mechanisms behind regulatory T-cell suppression and their stability following induction protocols. We further discuss the use of different hematopoietically derived regulatory cell populations, including regulatory B cells, regulatory macrophages, tolerogenic dendritic cells, and myeloid-derived suppressor cells, for the induction of transplantation tolerance in light of new clinical trials developing therapies with some of these populations.

CD8+ Immunosenescence Predicts Post-Transplant Cutaneous Squamous Cell Carcinoma in High-Risk Patients.

Most morbidity associated with malignancy in long-term renal transplant recipients is due to cutaneous squamous cell carcinoma (SCC). Previously identified measures to stratify SCC risk have limited use, however. We hypothesized that an increased proportion of senescent, terminally differentiated CD8(+) T cells would identify renal transplant recipients at elevated SCC risk. Peripheral blood lymphocytes were isolated from 117 stable transplant recipients at high risk of SCC and analyzed phenotypically by flow cytometry. Participants were followed up prospectively for SCC development. The predictive value of variables was assessed using Cox regression. Age at transplant and enrollment, dialysis duration, and previous disease were predictive of SCC development during follow-up. Previously published clinical phenotype-based risk scores lost predictive value with the removal of age as a covariate. The percentage of CD57-expressing CD8(+) T cells was the strongest immunologic predictor of future SCC and correlated with increasing CD8(+) T cell differentiation. We dichotomized participants into those with a majority (CD57hi) and a minority (CD57lo) of CD8(+) T cells expressing CD57; CD57hi participants were more likely to develop SCC during follow-up (hazard ratio, 2.9; 95% confidence interval, 1.0 to 8.0), independent of potential confounders, and tended to develop earlier recurrence. The CD57hi phenotype was stable with time and associated with increasing age and cytomegalovirus seropositivity. Our results show that the CD57hi phenotype is a strong predictor of SCC development and recurrence in this cohort of long-term, high-risk renal transplant recipients. This information may allow identification of recipients who may benefit from intensive dermatologic screening and immunosuppression reduction.

Alloreactive regulatory T cells generated with retinoic acid prevent skin allograft rejection.

CD4(+) CD25(+) Foxp3(+) regulatory T (Treg) cells mediate immunological self-tolerance and suppress immune responses. Retinoic acid (RA), a natural metabolite of vitamin A, has been reported to enhance the differentiation of Treg cells in the presence of TGF-ß. In this study, we show that the co-culture of naive T cells from C57BL/6 mice with allogeneic antigen-presenting cells (APCs) from BALB/c mice in the presence of TGF-ß, RA, and IL-2 resulted in a striking enrichment of Foxp3(+) T cells. These RA in vitro-induced regulatory T (RA-iTreg) cells did not secrete Th1-, Th2-, or Th17-related cytokines, showed a nonbiased homing potential, and expressed several cell surface molecules related to Treg-cell suppressive potential. Accordingly, these RA-iTreg cells suppressed T-cell proliferation and inhibited cytokine production by T cells in in vitro assays. Moreover, following adoptive transfer, RA-iTreg cells maintained Foxp3 expression and their suppressive capacity. Finally, RA-iTreg cells showed alloantigen-specific immunosuppressive capacity in a skin allograft model in immunodeficient mice. Altogether, these data indicate that functional and stable allogeneic-specific Treg cells may be generated using TGF-ß, RA, and IL-2. Thus, RA-iTreg cells may have a potential use in the development of more effective cellular therapies in clinical transplantation.

The where and when of T cell regulation in transplantation.

Multiple cell types contribute to the peripheral regulation of T cell alloresponses in haematopoieitc cell transplantation (HCT) and solid organ transplantation (SOT). Of these, regulatory T cells (Tregs) are the principal players and have shown the greatest success in the therapeutic control of detrimental immune responses. Investigations into the induction, location, and mechanism of suppression utilised by Tregs to control alloreactive responses are ongoing. The activation and homing characteristics of Tregs are important to their regulatory capabilities, with activation and homing occurring in the same time and space as conventional T cells. This review discusses these characteristics and recent advances in the field as we move closer to the ultimate goal of utilising Tregs as treatment for allograft rejection and graft-versus-host disease (GvHD).

Regulatory T cells: first steps of clinical application in solid organ transplantation.

Solid organ transplantation is the treatment of choice for patients with end-stage organ failure. To prevent rejection of the transplanted organ continuous treatment with immunosuppressive medication is needed. Immunosuppression may be harmful to the transplant recipient, increasing the risk of cancer, infections and cardiovascular disease. To improve transplant and patient survival, there is a need for an immune-modulatory regimen that is not only potent in preventing rejection of the transplanted organ, but has less side effects compared to current immunosuppressive regimens. Increasingly, transplantation research focusses on regulatory T cell (Treg) therapy to achieve this aim, in which Treg are used as a strategy to allow reduction of immunosuppression. Currently, the first clinical trials are underway investigating the safety and feasibility of Treg therapy in renal transplantation. This review gives an overview of the rationale of using Treg therapy in transplantation, previous experience with Treg therapy in humans, and the expected safety, potential efficacy and cost-effectiveness of Treg therapy in solid organ transplantation.

Immunomodulatory effect of a decellularized skeletal muscle scaffold in a discordant xenotransplantation model.

Decellularized (acellular) scaffolds, composed of natural extracellular matrix, form the basis of an emerging generation of tissue-engineered organ and tissue replacements capable of transforming healthcare. Prime requirements for allogeneic, or xenogeneic, decellularized scaffolds are biocompatibility and absence of rejection. The humoral immune response to decellularized scaffolds has been well documented, but there is a lack of data on the cell-mediated immune response toward them in vitro and in vivo. Skeletal muscle scaffolds were decellularized, characterized in vitro, and xenotransplanted. The cellular immune response toward scaffolds was evaluated by immunohistochemistry and quantified stereologically. T-cell proliferation and cytokines, as assessed by flow cytometry using carboxy-fluorescein diacetate succinimidyl ester dye and cytometric bead array, formed an in vitro surrogate marker and correlate of the in vivo host immune response toward the scaffold. Decellularized scaffolds were free of major histocompatibility complex class I and II antigens and were found to exert anti-inflammatory and immunosuppressive effects, as evidenced by delayed biodegradation time in vivo; reduced sensitized T-cell proliferative activity in vitro; reduced IL-2, IFN-γ, and raised IL-10 levels in cell-culture supernatants; polarization of the macrophage response in vivo toward an M2 phenotype; and improved survival of donor-derived xenogeneic cells at 2 and 4 wk in vivo. Decellularized scaffolds polarize host responses away from a classical TH1-proinflammatory profile and appear to down-regulate T-cell xeno responses and TH1 effector function by inducing a state of peripheral T-cell hyporesponsiveness. These results have substantial implications for the future clinical application of tissue-engineered therapies.

A diametric role for OX40 in the response of effector/memory CD4+ T cells and regulatory T cells to alloantigen.

OX40 is a member of the TNFR superfamily that has potent costimulatory properties. Although the impact of blockade of the OX40-OX40 ligand (OX40L) pathway has been well documented in models of autoimmune disease, its effect on the rejection of allografts is less well defined. In this article, we show that the alloantigen-mediated activation of naive and memory CD4(+) T cells results in the induction of OX40 expression and that blockade of OX40-OX40L interactions prevents skin allograft rejection mediated by either subset of T cells. Moreover, a blocking anti-OX40 had no effect on the activation and proliferation of T cells; rather, effector T cells failed to accumulate in peripheral lymph nodes and subsequently migrate to skin allografts. This was found to be the result of an enhanced degree of cell death among proliferating effector cells. In clear contrast, blockade of OX40-OX40L interactions at the time of exposure to alloantigen enhanced the ability of regulatory T cells to suppress T cell responses to alloantigen by supporting, rather than diminishing, regulatory T cell survival. These data show that OX40-OX40L signaling contributes to the evolution of the adaptive immune response to an allograft via the differential control of alloreactive effector and regulatory T cell survival. Moreover, these data serve to further highlight OX40 and OX40L as therapeutic targets to assist the induction of tolerance to allografts and self-Ags.

Moving to tolerance: clinical application of T regulatory cells.

Decreasing the incidence of chronic rejection and reducing the need for life-long immunosuppression remain important goals in clinical transplantation. In this article, we will review how regulatory T cells (Treg) came to be recognized as an attractive way to prevent or treat allograft rejection, the ways in which Treg can be manipulated or expanded in vivo, and the potential of in vitro expanded/generated Treg for cellular therapy. We will describe the first regulatory T cell therapies that have been or are in the process of being conducted in the clinic as well as the safety concerns of such therapies and how outcomes may be measured.

Regulatory T cells in transplantation tolerance.

The identification and characterization of regulatory T (T(Reg)) cells that can control immune responsiveness to alloantigens have opened up exciting opportunities for new therapies in transplantation. After exposure to alloantigens in vivo, alloantigen-specific immunoregulatory activity is enriched in a population of CD4+ T cells that express high levels of CD25. In vivo, common mechanisms seem to underpin the activity of CD4+CD25+ T(Reg) cells in both naive and manipulated hosts. However, the origin, allorecognition properties and molecular basis for the suppressive activity of CD4+CD25+ T(Reg) cells, as well as their relationship to other populations of regulatory cells that exist after transplantation, remain a matter of debate..

Immunologic unresponsiveness to alloantigen in vivo: a role for regulatory T cells.

Exposure to alloantigen in vivo or in vitro induces alloantigen reactive regulatory T cells that can control transplant rejection. The mechanisms that underpin the activity of alloantigen reactive regulatory T cells in vivo are common with those of regulatory T cells that prevent autoimmunity. The identification and characterization of regulatory T cells that control rejection and contribute to the induction of immunologic unresponsiveness to alloantigens in vivo has opened up exciting opportunities for new therapies in transplantation. Findings from laboratory studies are informing the design of clinical protocols using regulatory T cells as a cellular therapy.

Novel biomarkers and functional assays to monitor cell-therapy-induced tolerance in organ transplantation.

PURPOSE OF REVIEW: Cell-based immunotherapy offers a novel approach to minimize the need for immunosuppressive drugs and to promote a state of immunological tolerance to a transplanted organ. We review the most promising biomarkers and functional assays able to identify patients tolerant to their graft. Such a signature of tolerance is essential in the assessment of the efficacy with which trials of cellular therapies promote immunoregulation and minimize graft rejection. RECENT FINDINGS: A multitude of novel cellular therapies have entered early-phase clinical trials in solid-organ transplant patients. Recent multicentre collaborations have enabled the determination of distinct tolerance profiles for both liver and kidney transplant recipients. These have been shown to be highly predictive of tolerance in certain settings and show utility in identifying patients in whom immunosuppressive drugs can be weaned or discontinued. SUMMARY: In order to become a viable treatment option in solid-organ transplantation, the latest large, multicentre clinical trials of cellular therapies must utilize, validate and discover the biomarkers with the capacity to reliably identify a signature of immune tolerance.

The potential role for regulatory T-cell therapy in vascularized composite allograft transplantation.

PURPOSE OF REVIEW: Vascularized composite allograft (VCA) transplantation restores defects to a degree not possible by conventional techniques. However, it is limited by the need for long-term immunosuppression and high rates of acute rejection directed against skin. There is therefore a need for a therapy that may shift the risk-benefit ratio in favour of VCA transplantation. Regulatory T cells (Tregs) are a subset of T cells with potent immunoregulatory properties and the potential to promote immunosuppression-free allograft survival. In this review, we consider the evidence for Treg therapy in VCA transplantation. RECENT FINDINGS: CD4 Tregs are the best-studied immunoregulatory cell type, and a large amount of experimental and clinical data is emerging to endorse their use in VCA transplantation. Data from animal and humanized models are particularly encouraging and demonstrate the potent efficacy of Treg at preventing skin allograft rejection. Moreover, central tolerance induction techniques in VCA transplantation models are demonstrating a dependence on Tregs for graft survival. SUMMARY: An improvement in outcomes after VCA transplantation has the potential to revolutionize the field. Several effective therapeutic strategies have demonstrated great promise experimentally, and there is now a need to assess their safety and efficacy in a clinical setting.

Late Treatment With Autologous Expanded Regulatory T-cell Therapy After Alemtuzumab Induction Is Safe and Facilitates Immunosuppression Minimization in Living Donor Renal Transplantation.

BACKGROUND: The TWO Study (Transplantation Without Overimmunosuppression) aimed to investigate a novel approach to regulatory T-cell (Treg) therapy in renal transplant patients, using a delayed infusion protocol at 6 mo posttransplant to promote a Treg-skewed lymphocyte repopulation after alemtuzumab induction. We hypothesized that this would allow safe weaning of immunosuppression to tacrolimus alone. The COVID-19 pandemic led to the suspension of alemtuzumab use, and therefore, we report the unique cohort of 7 patients who underwent the original randomized controlled trial protocol. This study presents a unique insight into Treg therapy combined with alemtuzumab and is therefore an important proof of concept for studies in other diseases that are considering lymphodepletion. METHODS: Living donor kidney transplant recipients were randomized to receive autologous polyclonal Treg at week 26 posttransplantation, coupled with weaning doses of tacrolimus, (Treg therapy arm) or standard immunosuppression alone (tacrolimus and mycophenolate mofetil). Primary outcomes were patient survival and rejection-free survival. RESULTS: Successful cell manufacturing and cryopreservation until the 6-mo infusion were achieved. Patient and transplant survival was 100%. Acute rejection-free survival was 100% in the Treg-treated group at 18 mo after transplantation. Although alemtuzumab caused a profound depletion of all lymphocytes, including Treg, after cell therapy infusion, there was a transient increase in peripheral Treg numbers. CONCLUSIONS: The study establishes that delayed autologous Treg therapy is both feasible and safe, even 12 mo after cell production. The findings present a new treatment protocol for Treg therapy, potentially expanding its applications to other indications.

Single cell tracking of gadolinium labeled CD4+ T cells by laser ablation inductively coupled plasma mass spectrometry.

Cellular therapy is emerging as a promising alternative to conventional immunosuppression in the fields of hematopoietic stem cell (HSC) transplantation, autoimmune disease, and solid organ transplantation. Determining the persistence of cell-based therapies in vivo is crucial to understanding their regulatory function and requires the combination of an extremely sensitive detection technique and a stable, long-lifetime cell labeling agent. This paper reports the first application of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to perform single cell detection of T cell populations relevant to cellular immunotherapy. Purified human CD4(+) T cells were labeled with commercially available Gd-based magnetic resonance imaging (MRI) contrast agents, Omniscan and Dotarem, which enabled passive loading of up to 10(8) Gd atoms per cell. In mixed preparations of labeled and unlabeled cells, LA-ICP-MS was capable of enumerating labeled cells at close to the predicted ratio. More importantly, LA-ICP-MS single cell analysis demonstrated that the cells retained a sufficient label to remain detectable for up to 10 days post-labeling both in vitro and in vivo in an immunodeficient mouse model.

Conversion to sirolimus in kidney transplant recipients with squamous cell cancer and changes in immune phenotype.

BACKGROUND: Conversion to sirolimus from calcineurin inhibitor- (CNI), azathioprine- (AZA) and mycophenolate-based regimens reduces the risk of development of squamous cell carcinoma of the skin (SCC) in kidney transplant recipients (KTRs). Sirolimus conversion may also be protective by permitting beneficial changes in immune phenotype. It is not known how sirolimus will affect immune phenotype in KTRs with SCC. METHODS: Thirty-two KTRs with SCC were enrolled into this single-blinded randomized study and 13 KTRs randomized to sirolimus (4-10 ng/mL) and prednisolone 5 mg/day. RESULTS: Six-month post conversion to sirolimus FOXP3(+) CD127(low)CD25(high)CD69(-), the number of T cells (putative Treg) increased significantly (P = 0.008). Natural killer (NK) and CD56(bright) NK cells also increased significantly (P = 0.039 and 0.02). T-cell number only significantly increased in those KTRs where CNI was ceased as part of the conversion to mammalian target of rapamycin inhibitors (mTORi's) (P = 0.031) implying CNI cessation rather than mTORi initiation induced an increase in T-cell number. Increases in the NK cell number was only significant in those KTRs where AZA was ceased (P = 0.040), implying AZA cessation rather than mTORi initiation caused the NK cell number to increase. At 6 months, sirolimus conversion reduces new SCC/year, rate ratio 0.49 (95%CI: 0.15-1.63), P = 0.276. On therapy analysis and intention-to-treat analysis over 24 months, the rate ratios were 0.84 and 0.87, respectively, and did not reach significance. CONCLUSIONS: Conversion to mTORi from CNI may reveal a pre-existing high Treg phenotype by unmasking CNI inhibition of FOXP3 expression. Cessation of AZA leads to increased NK cell number. High FOXP3(+) T-cell number on conversion to mTORi may predict those KTRs who continue to accrue SCC.