IntroductionWe investigated the potential role of HLA molecular mismatches (MM) in achieving stable chimerism, allowing for donor-specific tolerance in patients undergoing combined living donor kidney and hematopoietic stem cell transplantation (HSCT).MethodsAll patients with available DNA samples (N=32) who participated in a phase 2 clinical trial (NCT00498160) where they received an HLA mismatched co-transplantation of living donor kidney and facilitating cell-enriched HSCT were included in this study. High-resolution HLA genotyping data were used to calculate HLA amino acid mismatches (AAMM), Eplet MM, three-dimensional electrostatic mismatch scores (EMS-3D), PIRCHE scores, HLA-DPB1 T-cell epitope group MM, HLA-B leader sequence MM, and KIR ligands MM between the donor and recipient in both directions. HLA MM were analyzed to test for correlation with the development of chimerism, graft vs. host disease (GvHD), de novo DSA, and graft rejection.ResultsFollow-up time of this cohort was 6–13.5 years. Of the 32 patients, 26 developed high-level donor or mixed stable chimerism, followed by complete withdrawal of immunosuppression (IS) in 25 patients. The remaining six of the 32 patients had transient chimerism or no engraftment and were maintained on IS (On-IS). In host versus graft direction, a trend toward higher median number of HLA-DRB1 MM scores was seen in patients On-IS compared to patients with high-level donor/mixed chimerism, using any of the HLA MM modalities; however, initial statistical significance was observed only for the EMS-3D score (0.45 [IQR, 0.30–0.61] vs. 0.24 [IQR, 0.18–0.36], respectively; p=0.036), which was lost when applying the Bonferroni correction. No statistically significant differences between the two groups were observed for AAMM, EMS-3D, Eplet MM, and PIRCHE-II scores calculated in graft versus host direction. No associations were found between development of chimerism and GvHD and non-permissive HLA-DPB1 T-cell epitope group MM, HLA-B leader sequence, and KIR ligands MM.ConclusionOur results suggest an association between HLA-DRB1 molecular mismatches and achieving stable chimerism, particularly when electrostatic quality of the mismatch is considered. The non-permissive HLA-DPB1 T-cell epitope group, HLA-B leader sequence, and KIR ligands MM do not predict chimerism and GvHD in this combined kidney/HSCT transplant patient cohort. Further work is needed to validate our findings.Clinical trial registrationhttps://clinicaltrials.gov/study/NCT00498160, identifier NCT00498160.

While kidney transplantation (KTx) has traditionally required lifelong immunosuppression, an investigational stem cell therapy, FCR001, has been demonstrated to induce tolerance and eliminate the need for immunosuppression through the establishment of persistent mixed chimerism in a phase 2 clinical study. Real‐world evidence (RWE) methods were employed to compare the safety and efficacy of non‐myeloablative conditioning with FCR001 with standard of care [SOC] immunosuppression in a retrospective single‐center analysis of outcomes among propensity score matched living‐donor KTx receiving SOC (n = 144) or FCR001 (n = 36). Among the FCR001 recipients, 26 (72%) developed persistent chimerism allowing durable elimination of all immunosuppression. There was no significant difference in the composite primary endpoint (biopsy‐proven acute rejection [BPAR], graft loss, or death) at 60 months (FCR001 27.8%, n = 10 and SOC 28.5%, n = 41; p = .9). FCR001 recipients demonstrated superior kidney function at 5 years (estimated glomerular filtration rate [eGFR] [mean ± standard deviation]: 64.1 ± 15.3) compared to SOC (51.7 ± 18.8; p = .02). At 5 years, FCR001 recipients experienced fewer complications including new‐onset diabetes post‐transplant, although two patients developed graft versus host disease. In conclusion, RWE demonstrated that KTx combined with non‐myeloablative conditioning and FCR001 resulting in superior kidney function without increasing the risk of rejection, graft loss, or death among patients off immunosuppression.

Immune cell therapy has several potential applications, including treatment of cancers and autoimmune disease, tissue regeneration, and promotion of immune tolerance. Currently, several regulatory immune cell populations have emerged to be of great interest as a potential therapy in organ transplantation to promote transplant tolerance and prevent graft rejection. These therapies hold the promise of prolonging graft survival while minimizing treatment-related toxicity. In allogeneic hematopoietic stem cell transplantation (HSCT), several cell types, including regulatory T cells, mesenchymal stromal cells, and myeloid derived suppressor cells, have been identified to have the potential to treat acute and chronic graft-versus-host disease (GvHD).1 GvHD is a major transplant-related complication leading to increased morbidity and mortality following allogeneic HSCT.2 GvHD occurs when donor T cells alloreactive to host major histocompatibility antigens attack host tissues and organs. The critical role of T cells in mediating acute GvHD is evidenced by abrogation of GvHD when donor T cells are removed from the graft before transplantation. However, removal of T cells can impair not only HSC engraftment but also T cell–mediated graft-versus-leukemia effects. Conventional immunosuppression does not sufficiently treat GvHD, especially in HLA-mismatched transplant recipients. Therefore, novel therapies are needed for better management of GvHD. Fibroblastic reticular cells (FRCs) are specialized stromal cells notably localized in secondary lymphoid organs that have been demonstrated to interact directly with the immune system. FRCs have been shown to regulate adaptive immune responses through various immune modulating mechanisms,3 including regulation of T-cell migration, survival, and maintenance of peripheral immune tolerance. Tolerogenic macrophages actively participate in T-cell immune regulation.4 The publication by Liu et al5 in this issue of Transplantation sought to define the relationship between FRCs and lymph node macrophages for their tolerogenic roles using in vitro culture system and in vivo mouse models. Their data provide evidence suggesting that lymph nodes FRC-induce regulatory macrophages at an early stage post–allo-HSCT. These FRC-induced macrophages displayed typical macrophage morphology and an immunophenotype based on CD11b+F4/80+ cell surface markers and high expression of PD-L1, an immune checkpoint inhibitor. These findings are consistent with previous studies conducted to define ex vivo–generated regulatory macrophages.6 Liu et al5 further elucidated the role of macrophage colony-stimulating factor (M-CSF) as a critical cytokine for the induction of regulatory macrophages. FRC-induced macrophages inhibited T-cell activation and differentiation toward Th1/Tc1 cells ex vivo. Most importantly, the immunoregulatory function of these induced regulatory macrophages demonstrated an ability to effectively attenuate acute GvHD in vivo. This study has revealed a novel mechanism underpinning the role of the microenvironment in secondary lymphoid organs, specifically highlighting the interactions between FRCs and macrophages for immune tolerogenic regulation. Various aspects of lymph node FRCs and their role in inducing regulatory macrophages to treat or prevent GvHD remains to be fully elucidated. Understanding the mechanisms by which induced regulatory macrophages alleviate GvHD will be critical for clinical implementation of this approach as a regulatory cell therapy product. The mechanisms of GvHD attenuation have been shown directly by FRC-induced macrophages inhibited T-cell activation and differentiation in current study. The indirect mechanism of regulatory macrophages on GvHD may be attributed to their role in the conversion of CD4+ T cells to IL-10+TIGIT+ FoxP3+ regulatory T cells (Treg) as demonstrated by Riquelme et al.7 Their work elegantly demonstrated that human regulatory macrophage can induce Treg, which suppress bystander T-cell proliferation and inhibit dendritic cell maturation. Moreover, preoperative administration of regulatory macrophages results in an increase in circulating TIGIT+ Treg in living-donor kidney transplantation. Additional studies in humans by Hutchinson et al8 further demonstrated the feasibility and safety of regulatory macrophages as a cell therapy product. These clinical studies in humans represent a promising frontier in preventing or reducing GvHD and tolerance induction in solid organ transplant recipients. However, numerous hurdles will first need to be overcome for the therapy of FRC-induced regulatory macrophages to become a clinical reality. Several preclinical studies and clinical trials have demonstrated reduction and/or prevention of GvHD when immune regulatory cells are coinfused with HSC following conditioning. However, a current unmet medical need is the development of a cellular therapy that can be used to treat GvHD when it occurs. One hurdle that will need to be overcome for the clinical application of induced regulatory macrophages in treating GvHD is in obtaining sufficient number of cells for therapy. The current study by Lui et al5 demonstrated that FRC macrophages induced ex vivo from BMC possesses the potential for immunosuppressive capacity and M-CSF was the critical cytokine for FRC to induce macrophages.5 M-CSF was found to be highly expressed in FRC, but further study needs to define the accurate source of M-CSF at local stromal environment in lymph nodes including endothelial cells, follicular dendritic cells, marginal reticular cells, and fibroblastic FRCs. The ex vivo generation of tolerogenic macrophages may help ensure sufficient cells for the use of this therapy in the clinic. Although the use of ex vivo–generated regulatory macrophages represent a promising prospect for cell therapy, several additional challenges will need to be overcome.9 Among these challenges is the need to evaluate ex vivo-generated regulatory macrophages for their immunogenicity because they should not initiate proinflammatory responses or cause sensitization in allogeneic recipients. Furthermore, it will be critical to ensure manufacturing and release testing control to maintain cellular phenotype and immunomodulatory function. The immunosuppressive function will also require additional assessment to avoid nonspecific effects leading to increased infection or tumor formation. The homing of induced regulatory macrophages has been studied clinically and these cells after central venous administration were observed to traffic from lungs to liver, spleen, and hematopoietically active bone marrow.10 The feasibility of translating FRC-induced macrophages to a clinical cell therapy product for allogeneic solid organ transplantation has been underpinned by several clinical pilot studies.8 Taken together, the study by Liu et al5 provides important preclinical insight into the role FRCs play in promoting regulatory macrophages and their role in alleviating the risk of GvHD in allogeneic HSC transplantation.

Background. Thirty-seven patients have received a living-donor kidney transplant in a phase 2 study designed to induce tolerance with facilitated allogeneic hematopoietic stem cell transplant. The study protocol is based on tolerogenic CD8 + /T-cell receptor − facilitating cells (FCR001; also including hematopoietic stem cells and αβ-T-cell receptor + T cells) and low-dose, nonmyeloablative conditioning. Persistent chimerism allowing full immunosuppression (IS) withdrawal was achieved in 26 patients (time off IS 36–123 mo). Methods. We evaluated biomarkers of tolerance through urinary cell mRNA profiling and immunocompetence to respond to vaccination in these patients. We also assessed kidney function and metabolic parameters compared with standard-of-care patients on IS. Results. Persistently chimeric patients retained chimerism after removal of IS and remained rejection free without donor HLA–specific antibody development. The presence of donor chimerism at >50% correlated with a signature of tolerance in urinary cell mRNA profiles, with a uniquely elevated increase in the ratio of cytotoxic T lymphocyte–associated protein 4 to granzyme B mRNA. Tolerance was associated with protection from recurrence of immune-mediated causes of kidney disease. Tolerant participants were safely vaccinated, developed protective immune responses, and did not lose chimerism after vaccination. When compared with kidney transplant recipients treated with standard IS, tolerant participants showed stable kidney function and reduced medication use for hypertension and hyperlipidemia. Conclusions. These results suggest that elimination of IS has distinct advantages in living-donor kidney allograft recipients.

Introduction: Urinary cell mRNA profiling provides an unparalleled window into the intra-graft events in kidney transplant recipients. Utilizing this powerful molecular tool, we aimed to characterize mRNA signatures associated with human kidney allograft tolerance induced with tolerogenic CD8+/TCR- facilitating cells (FCR001) and a nonmyeloablative conditioning regimen. Methods: We developed a custom panel of mRNAs encoding immunoregulatory proteins implicated in allograft tolerance and rejection and measured absolute levels of urinary cell mRNAs using preamplification enhanced real time quantitative PCR assays. We quantified urinary cell mRNA levels in 28 biopsy-matched urines from 14 FCR001-tolerant patients with durable chimerism and off immunosuppression (FCR Durable Cohort); 43 biopsy-matched urines from 34 kidney allograft recipients prospectively enrolled in the Clinical Trials in Organ Transplantation-04 (CTOT-04) and with biopsies with acute cellular rejection (TCMR Cohort); and 161 biopsy-matched urines from 124 kidney transplant recipients prospectively enrolled in CTOT-04 and with biopsies without rejection features (No Rejection Cohort). Results: Urinary cell mRNA profile of the FCR Durable Cohort was unique and discriminated the tolerant patients from the patients with TCMR biopsies and the patients with No Rejection biopsies. Absolute levels of urinary cell mRNAs, summarized in Table 1, show that the median level of CTLA-4 mRNA is significantly higher in the FCR Durable Cohort (median 439 copies per microgram of RNA) than in the TCMR cohort (135 copies) or the No Rejection Cohort (13 copies) (Kruskal Wallis [KW] P= 8x10-21). In contrast, urinary cell level of mRNA encoding cytotoxic attack molecule granzyme B was significantly lower in the FCR Durable Cohort (median 106 copies) than in the TCMR cohort (3304 copies) or the No Rejection Cohort (337 copies) (KW P= 1x10-11). The differential gene expression pattern resulted in the ratio of CTLA-4 mRNA to granzyme B mRNA to be significantly higher in the FCR Durable Cohort (5.62) than in the TCMR Cohort (0.03) or the No Rejection Cohort (0.04) (KW P= 1x10-14). Urinary cell levels of mRNA for CD3E, perforin, IP-10, FoxP3, and TGFB1 in the FCR Durable Cohort were significantly lower in the TCMR Cohort and mostly like the profile in the No Rejection Cohort. Comparison to an FCR Transient Cohort (11 biopsy-matched urines from 3 patients conditioned with the FCR001 regimen but with transient chimerism and on immunosuppression) revealed a higher ratio of CTLA-4 mRNA to granzyme B mRNA in the FCR Durable Cohort compared to the FCR Transient Cohort (5.62 vs. 1.99,P=0.21). Conclusion: Kidney allograft tolerance induced with CD8+/TCR- facilitating cells and a nonmyeloablative conditioning regimen is associated with a tolerogenic signature of enhanced expression of CTLA-4 mRNA and decreased expression of mRNA for granzyme B and perforin. The research was supported, in part, by NIH grants U01AI63589 and R37AI051652.

Methods: 37 subjects were transplanted in a phase 2 protocol based upon tolerogenic CD8+/TCR-facilitating cells (FCR001) to induce tolerance in recipients of living donor renal allografts (KTx). Recipients were conditioned with fludarabine (30mg/m2/dose, days -5,-4,-3), cyclophosphamide (50mg/kg/dose, day-3 and+3), 200 cGy TBI (day-1) followed by KTx (day0). A G-CSF mobilized product was apheresed from the donor, processed to remove graft-versus-host disease (GVHD)-producing cells yet retain CD34 + cells and FC, and cryopreserved until administration day+1 post-KTx. Follow up is 60 - 154 months. Pts ranged in age from 18-64 yrs and were 6/6 HLA matched related to 0/6 matched unrelated. MMF and tacrolimus immunosuppression (IS) was weaned and discontinued at 1 yr if post-Tx chimerism, normal renal fcn and normal KTx biopsy were noted. Results: Durable chimerism allowing for full IS withdrawal developed in 26 pts (time off IS 48- 136 months); the majority (23/26) showed full (>95%) donor whole blood/T cell chimerism. 6 subjects have been IS free for > 10 years. Transient chimerism was seen in 8 pts. All stable chimeric subjects retained chimerism after removal of IS and remain rejection-free. Long term chimeric subjects off IS have no evidence of immune defect: they show robust T, B, and NK cell reconstitution, can be safely vaccinated and develop protective immunity. Transiently chimeric pts resumed endogenous hematopoiesis and were maintained on low-dose IS. There were two cases of GVHD. 1 subject exhibited grade 1-2 acute GI GVHD that responded to corticosteroids, followed by mild chronic GVHD. The second pt presented late and died of treatment resistant GI GVHD with CMV 11 months post-Tx. There have been three graft losses, related to infections in subjects on IS. There have been three subject deaths. Over all patient survival is 91.8% and death censored graft survival 94.1%. Tolerant FCR001 subjects have significantly better renal function than comparable KTx on SOC IS. Hypertension and hyperlipidemia is more common in SOC than tolerant FCR001 pts. Conclusion: High levels of durable chimerism and tolerance with a low (5.5%) incidence of GVHD has been achieved in mismatched recipients of KTx. There are significant long term medical benefits to establishing tolerance in KTx recipients using the FCR001 approach. Funding support for study from Regenerex and Talaris Therapeutics.

Introduction We previously reported the induction of kidney transplant tolerance by the establishment of durable whole blood and T-cell chimerism and withdrawal of immunosuppression (IS) in 26 of 37 highly mismatched recipients of combined stem cell and living donor kidney transplant recipients (KTx) with low risk of graft versus host disease (GvHD), using FCR001, an investigational cell therapy. The focus of the current analysis is to evaluate the impact of degree of bidirectional donor/recipient mismatching using high resolution allele typing at HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1, on the ability to establish durable chimerism, allowing full immunosuppression (IS) withdrawal and the induction of transplant tolerance. Methods In this phase 2 trial, recipients received nonmyeloablative conditioning with fludarabine 30mg/m2 (Days -5, -4, -3), cyclophosphamide 50mg/kg (Days-3 and +3), 200 cGy TBI (Day-1) followed by KTx (Day 0). G-CSF mobilized peripheral blood mononuclear cells were apheresed from the donor >2 weeks prior to KTx, processed to remove GvHD-producing cells yet retain CD34 +cells and tolerogenic CD8+/TCR facilitating cells (FC) and cryopreserved until infusion day+1 after KTx. IS consisted of mycophenolate and tacrolimus. IS was weaned and discontinued in the presence of sustained donor T-cell chimerism (>50%), stable renal function and no evidence of biopsy-proven rejection. Results Samples from 32 of the 37 subject pairs were available for analysis; of these, three subjects did not have sufficient DNA to test for locus DPB1.All 32 recipients, ranging from age 18 - 65 years, have reached at least 4.5 years of follow-up up to 12 years. Two recipients were re-transplants. Of the 29 D/R pairs with data from all 12 alleles, 21 were mismatched between 6 to 12 alleles (6 related, 15 unrelated), 8 were mismatched between 2 and 5 alleles (all related). Of the three D/R pairs missing DP data, one was mismatched 6 of 10, another 10 of 10, and a third 2 of 10 alleles. Despite the high degree of mismatch, durable chimerism allowed for full IS withdrawal in 25 of these 32 subjects (time off IS from 3.5 - 11 years). 12/25 off IS were from unrelated D/R pairs and ≥ 8 HLA mismatches; the majority showed >95% donor whole blood/T cell chimerism. Three have exhibited stable mixed chimerism ranging between 40% - 60%. Of the subjects not off IS, two failed to engraft their cells; four lost chimerism by 4 months, and one developed GVHD. Durably chimeric patients retained chimerism after removal of IS, remain rejection-free without donor-specific antibody (DSA) with up to 12 years following KTx and have not resumed IS. Transiently chimeric subjects resumed endogenous hematopoiesis and are maintained on low dose IS with stable renal function. There have been two cases of GvHD: one grade 2 lower GI acute GvHD that developed during conversion from tacrolimus to sirolimus and responded to steroids; this patient has developed moderate chronic GvHD of the skin. He is off IS with normal renal function. The second presented late following development of severe gastrointestinal symptoms and manifested treatment-resistant lower GI GvHD with associated tissue-invasive CMV colitis that proved fatal at 11 months post-transplant. Conclusions In summary, high levels of durable chimerism and tolerance with a low (5.5%) incidence of GvHD has been achieved in highly mismatched related and unrelated recipients of FCR001 + kidney transplant. Figure 1 Figure 1. Krieger: Talaris Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Tambur: Viela Bio and Sanofi: Consultancy; ThermoFisher: Speakers Bureau. Schneider: Talaris Therapeutics, Inc.: Current Employment. Olsen: Talaris Therapeutics, Inc.: Current Employment. Ildstad: Talaris Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

Background. Mobilization of hematopoietic stem cells (HSCs) has become the preferred approach for HSC transplantation. AMD3100, a competitive inhibitor of C-X-C motif chemokine receptor-4, has been found to be a rapid mobilizing agent. The present study evaluated approaches to optimize the product collected. Methods. Mobilized peripheral blood mononuclear cells (mPBMCs) from B6 mice were transplanted to recipient BALB/c mice conditioned with ablative or nonmyeloablative approaches. Results. The optimal dose of AMD3100 was found to be 5.0 mg/kg. Optimal HSC mobilization was observed when AMD3100 (day 10) was coadministered with Flt3 ligand (FL) (days 1–10) and granulocyte colony-stimulating factor (GCSF) (days 4–10). There was a 228.8-fold increase of HSC with FL/GCSF/AMD3100 compared with AMD3100 treatment alone. When unmodified mPBMCs were transplanted into ablated allogeneic recipients, all recipients expired by day 40 from severe acute graft versus host disease (GVHD). When T cells were depleted from mPBMC, long-term survival and engraftment were achieved in majority of the recipients. When PBMC mobilized by FL/GCSF/AMD3100 were transplanted into recipients conditioned nonmyeloablatively with anti-CD154/rapamycin plus 100, 200, and 300 cGy of total body irradiation, 42.9%, 85.7%, and 100% of mice engrafted, respectively. Donor chimerism was durable, multilineage, and stable. Lymphocytes from mixed chimeras showed no response to host or donor antigens, suggesting functional bidirection T-cell tolerance in vitro. Most importantly, none of the engrafted mice exhibited clinical features of GVHD. Conclusions. FL/GCSF/AMD3100 is an efficient treatment to maximally mobilize HSC. Durable engraftment and donor-specific tolerance can be achieved with mPBMC in nonmyeloablative conditioning without GVHD.

37 patients have been transplanted in a phase 2 protocol to establish chimerism to induce tolerance in up to 0 of 6 matched related and unrelated recipients of living donor renal allografts (KTx). The protocol is based upon tolerogenic CD8+/TCR- facilitating cells (FC) and nonmyeloablative conditioning. Recipients were conditioned with fludarabine (30mg/m2 days -5,-4,-3), cyclophosphamide (50mg/kg day-3 and+3), 200 cGy TBI (day-1) followed by KTx (day0). G-CSF mobilized peripheral blood mononuclear cells were apheresed from the donor >2 weeks before kidney transplantation, processed to remove graft-versus-host disease (GVHD)-producing cells yet retain CD34 +cells and FC, and cryopreserved until transplantation on day+1 after kidney transplantation. Immunosuppression consisted of mycophenolate mofetil and tacrolimus. 36 patients have reached at least 1 year of follow up (range 12-105 months) and are the focus of this analysis. Patients ranged in age from 18-65 yrs. Enrollment was agnostic to degree of HLA match; 2 were 6/6 and 3 5/6 matched related using high resolution allele level typing, with the remainder 4/6 to 0/6 matched related (n=15) or unrelated (n=16) Two of the recipients were renal re-transplants. Tacrolimus/MMF immunosuppression (IS) was weaned and discontinued at 1 year if chimerism (>50% whole blood and T cell), normal renal function and normal kidney transplant biopsy were noted. 34 of 36 subjects exhibited peripheral blood donor chimerism at one month post KTx. Durable chimerism allowing for full IS withdrawal developed in 26 (time off IS from 1- 88 months); the majority (23/26) showed >95% donor whole blood/T cell chimerism. Three have exhibited stable-mixed chimerism ranging between 40% - 60%. Three patients failed to develop chimerism. Transient chimerism occurred in 8 patients. Durably chimeric patients retained chimerism after removal of IS, remain rejection-free without donor-specific antibody (DSA) and show immunocompetence to vaccinations. None have had to resume immunosuppression. Transiently chimeric subjects resumed endogenous hematopoiesis and are maintained on low-dose IS with stable renal function. There have been two cases of GVHD: one grade 2 lower GI acute GVHD that developed during conversion from tacrolimus to sirolimus that responded to steroids; this patient has developed moderate chronic GVHD of the skin. He is off IS. The second presented late following development of severe gastrointestinal symptoms and manifested treatment-resistant lower GI GVHD with associated tissue-invasive CMV colitis that proved fatal at 11 months post-Tx. There have been two additional kidney graft losses, both related to early infections. A second subject death occurred in a heavy (>100 pack yr) smoker who developed advanced stage lung cancer 4.5 years after Tx. A third subject developed pneumococcal sepsis > 4 years after transplant. He had not undergone the recommended pneumococcal vaccination post HSCT. Overall patient survival is 91.8% and death censored graft survival 94.1%. These rates compare favorably to treatment-related mortality rates after standard of care renal transplant. In summary, high levels of durable chimerism and tolerance with a low (5.5%) incidence of GVHD has been achieved in up to 0 of 6 matched related and unrelated recipients of FCR001 + kidney transplant. Figure Ildstad: Talaris Therapeutics Inc: Employment, Equity Ownership, Other: Founder and CSO.

Facilitating cells (FC) are a CD8+ TCR- bone marrow subpopulation that enhance engraftment of purified hematopoietic stem cells (HSC) and induce antigen-specific CD4+ CD25+ FoxP3+ regulatory T cell (Treg) in vivo. The major subpopulation in FC resembles plasmacytoid precursor dendritic cells (p-preDC) both phenotypically and functionally. Here, we report that the number of FC was significantly reduced in Fms-like tyrosine kinase 3-ligand-knockout (Flt3-L-KO) mice. Specifically, there was a selective decrease in the B220+ CD11c+ CD11b- p-preDC FC subpopulation. The p-preDC FC subpopulation in FC total is restored after Flt3-L administration to Flt3-L-KO mice. FC from Flt3-L-KO donors exhibit impaired facilitation of allogeneic HSC engraftment in ablatively conditioned mice (B6 → NOD) as well as in mice conditioned with reduced intensity conditioning (B6 → BALB/c). In addition, the number of CD4+ CD25+ Foxp3+ Treg from Flt3-L-KO mice is significantly decreased. This was associated with the expression of chemokine receptor CXCR3+ or CCR5+ on Treg. Treg from the spleen of Flt3-L-KO mice showed impaired facilitation of engraftment of allogeneic HSC compared to wild-type Treg. Flt3-L treatment significantly expanded Treg, and restored their facilitating function. These results suggest that Flt3-L is an important growth factor in the development and homeostasis of p-preDC FC and in the role of FC inducing generation of Treg. Flt3-L provides potent immunoregulatory properties that may be clinically useful to improve tolerance induction and enhance the function of allogeneic cell therapies. Stem Cells 2018;36:1567-1577.

Eliminating the long term costs and side effects of immunosuppression (IS) is a compelling reason to pursue transplant tolerance. Tolerance in kidney transplant (KTx) pts has been achieved through the infusion of donor HSC under some form of conditioning. The increased upfront cost and intensive treatment required by tolerance protocols demands that long term outcomes be superior to that achieved with SOC IS regimens. Since 2009 we have conducted a Phase 2 trial of combined HSC/living donor KTx in mismatched and unrelated pts where the goal has been establishment of durable donor macrochimerism. Our protocol is based upon tolerogenic CD8+/TCR-facilitating cells (FCRx). Recipients were conditioned with fludarabine (30mg/m2 days -5,-4,-3), cyclophosphamide (50mg/kg day-3 and+3), 200 cGy TBI (day-1) followed by KTx (day0). A G-CSF mobilized donor leukopheresis product was performed >2 weeks pre-KTx, processed to remove graft-versus-host disease (GVHD)-producing cells yet retain CD34 + cells and FC, and cryopreserved until use day+1 post-KTx. TAC + MMF based IS was weaned and discontinued at 1 year if chimerism, normal renal fcn and normal KTx biopsy were noted. The 1st 20 FCRx subjects with > 5 years of follow up were compared to a cohort of SOC pts from 2009-2012 who would have been eligible for our Phase 2 trial(n=166). SOC KTx received Alemtuzumab induction and TAC + MMF IS w.o oral steroids. 34 SOC underwent planned CNI conversion and were excluded from analysis. In the remaining 132 SOC there were 13 graft losses and 9 deaths with resultant overall patient survival (PS) of 93.2 % and graft survival (GS) of 83.3%. In the FCRx cohort there were 2 graft losses and 1 death, with PS of 95% and GS of 85%. Current mean eGFR (CKD-EPI) for tolerant (tol) FCRx with durable chimerism off all IS (n=11, 75.8ml/min/1.73m2) was superior to that of transiently chimeric FCRx (n=6, 62.3 ml/min/1.73m2) or SOC (53.1 ml/min/1.73m2, p = 0.0017, tol vs SOC ). Med Rx for hypertension and hyperlipidemia was common in SOC KTx(82.8% and 43%) but uncommon in tol FCRx (18% and 9%). In summary PS and GS at five yrs was comparable between FCRx and SOC. Tol FCRx had significantly better renal function than SOC. Med Rx for hypertension and hyperlipidemia was more common in SOC than tol FCRx. We conclude there are significant long term medical benefits to establishing tolerance in KTx using the FCRx approach.

In this review, we summarized the latest results from the interventional clinical trials on inducing clinical tolerance in the recipients of human leukocyte antigen (HLA)-matched or mismatched living donor kidney transplantation via establishment of either transient or durable donor chimerism by hematopoietic stem cell (HSC)-based cell therapies. The protocols utilized by the three medical centers in the United States are different in the donor cells composition of hematopoietic stem cells transplantation (HSCT) and its timing, and the conditioning regimen prepared for the transplantation. Tolerant recipients from the clinical trials benefited from better long-term outcome, improved quality of life and reductions in lifetime healthcare expenses compared with Standard of Care recipients on conventional immunosuppression. Durable chimerism is indispensable to achieve immunologic tolerance to kidney transplantation and to protect against recurrence of the original renal disease in HLA-mismatched kidney transplant recipients by concomitant HSCT.

Purpose of review This review discusses the role and mechanisms by which facilitating cells promote stem cell engraftment and induce tolerance in HLA-disparate kidney transplant recipients. Recent finding Facilitating cells in both mice and human are heterogeneous, consisting of several subpopulations. They have been shown to enhance stem cell engraftment in allogeneic recipients. They also increase hematopoietic stem cells (HSC) clonogenicity, enhance migration and homing of stem cells via secretion of cytokines/chemokines/growth factors, prevent apoptosis of stem cells and induce regulatory cells. This review summarizes the findings that led to the development of chimerism-based induction of tolerance using FCRx (a mobilized blood product enriched in stem cells and facilitating cells) in allogenic kidney transplant patients. Summary A phase-2 clinical trial based on FCRx therapy has been successful in inducing tolerance to living donor kidney allografts, leading to withdrawal of immunosuppression in over 70% of patients transplanted. The ultimate goal of establishing tolerance in the absence of immunosuppresive drugs can be achieved using FCRx therapy.

The development of safe conditioning protocols has reduced the morbidity and mortality of hematopoietic stem cell transplantation (HSCT), allowing broader application for treatment of a variety of non-malignant conditions including autoimmune diseases, hemoglobinopathies, metabolic disorders etc., as well as inducing tolerance to solid organ transplants. One of the most successful clinical trials using a facilitating cell enhanced HSCT paired with a kidney transplant has effectively induced tolerance in the absence of immunosuppressive drugs, maintaining the function of the transplanted kidney, and reconstituting the immunocompetence of the recipient. This novel protocol eliminates the need for immunosuppressive drugs, the key source of kidney and liver toxicity, increased malignancy, and shortened life span. CD8+ TCR facilitating cells (FC) are a population of tolerogenic cell which promote hematopoietic stem cell engraftment across human leukocyte antigen (HLA) barriers. In this review, we discuss the bench to bedside journey of FC, from discovery in mouse models, characterization of the subpopulations of FC, the mechanisms by which FC induce tolerance and clinical application. As a novel personalized medicine, FC may change the approach to overcoming HLA barriers for both HSCT and solid organ transplant recipients.