Donor-specific Unresponsiveness Research Articles

Induction of Long-Lasting Regulatory B Lymphocytes by Modified Immune Cells in Kidney Transplant Recipients.

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. Ten patients from a phase 1 clinical trial who had received MIC infusions before kidney transplantation were followed to post-transplant day 1080. 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. 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. MIC Cell Therapy for Individualized Immunosuppression in Living Donor Kidney Transplant Recipients (TOL-1), NCT02560220.

Tolerance protocol of living kidney transplant for developing countries through basic strategy of lymphocyte depletion.

End-stage kidney failure (ESKD) is a global issue where kidney replacement therapy imposes enormous economic burden to people of developing countries, in addition to the severe limitations to the availability of hemodialysis and peritoneal dialysis technique. The best option of kidney transplantation also requires lifelong combination immunosuppressive medicines, the cost of which is equally comparable to lifelong dialysis. A strategy of achieving transplant tolerance that requires minimum immunosuppressive medicines, although in experimental stage, also requires state-of-art technology with costly medicines and interventions. This is evidently beyond the reach of ESKD patients of developing countries. Hence, globally in developing countries, a need for an innovative but cost-effective tolerance protocol is a burning need for a successful transplant program. In brief, transplant tolerance is defined as a state of donor-specific unresponsiveness to the allograft antigens without the need for ongoing pharmacologic immunosuppression or with a minimal need. Current state-of-art techniques involves: (1) A state of hematological chimera, for complete tolerance; (2) Prope or partial tolerance where immune-reactive T-lymphocytes are inhibited using monoclonal antibodies; and (3) Chimeric antigen receptor for T-regulatory (T-reg) cell therapy using genetically engineered T-reg cells targeting specific T-lymphocyte receptors for inducing anergy. From our real-world experience in transplant management in post-transplant lympho-proliferative disorders (PTLD), we noticed frequently a drastic reduction in the need of immunosuppressive medicines following lympho-ablative therapy for PTLD. We recently published a case study on a real-world experience transplant case where we explained a partial or prope tolerance that developed after lymphocyte ablation therapy, following which the allograft was maintained with low dose dual standard immunosuppressive medicines. Based on this publication, we propose here an innovative tolerance protocol for living related low risk kidney transplantation for developing countries, in this opinion review.

Chimerism-Based Tolerance to Kidney Allografts in Humans: Novel Insights and Future Perspectives

Chronic rejection and immunosuppression-related toxicity severely affect long-term outcomes of kidney transplantation. The induction of transplantation tolerance – the lack of destructive immune responses to a transplanted organ in the absence of immunosuppression – could potentially overcome these limitations. Immune tolerance to kidney allografts from living donors has been successfully achieved in humans through clinical protocols based on chimerism induction with hematopoietic cell transplantation after non-myeloablative conditioning. Notably, two of these protocols have led to immune tolerance in a significant fraction of HLA-mismatched donor-recipient combinations, which represent the large majority of cases in clinical practice. Studies in mice and large animals have been critical in dissecting tolerance mechanisms and in selecting the most promising approaches for human translation. However, there are several key differences in tolerance induction between these models and humans, including the rate of success and stability of donor chimerism, as well as the relative contribution of different mechanisms in inducing donor-specific unresponsiveness. Kidney allograft tolerance achieved through durable full-donor chimerism may be due to central deletion of graft-reactive donor T cells, even though mechanistic data from patient series are lacking. On the other hand, immune tolerance attained with transient mixed chimerism-based protocols initially relies on Treg-mediated suppression, followed by peripheral deletion of donor-reactive recipient T-cell clones under antigenic pressure from the graft. These conclusions were supported by data deriving from novel high-throughput T-cell receptor sequencing approaches that allowed tracking of alloreactive repertoires over time. In this review, we summarize the most important mechanistic studies on tolerance induction with combined kidney-bone marrow transplantation in humans, discussing open issues that still need to be addressed and focusing on techniques developed in recent years to efficiently monitor the alloresponse in tolerance trials. These cutting-edge methods will be instrumental for the development of immune tolerance protocols with improved efficacy and to identify patients amenable to safe immunosuppression withdrawal.

Cultured thymus tissue implantation promotes donor-specific tolerance to allogeneic heart transplants.

Eighty-six infants born without a thymus have been treated with allogeneic cultured thymus tissue implantation (CTTI). These infants, who lack T cells and are profoundly immunodeficient at birth, after CTTI from an unmatched donor develop T cells similar to those of recipient that are tolerant to both their own major histocompatibility antigens and those of the donor. We tested use of CTTI with the goal of inducing tolerance to unmatched heart transplants in immunocompetent rats. We thymectomized and T cell-depleted Lewis rats. The rats were then given cultured thymus tissue from F1 (Lewis × Dark Agouti ) under the kidney capsule and vascularized Dark Agouti (DA) heart transplants in the abdomen. Cyclosporine was administered for 4 months. The control group did not receive CTTI. Recipients with CTTI showed repopulation of naive and recent thymic emigrant CD4 T cells; controls had none. Recipients of CTTI did not reject DA cardiac allografts. Control animals did not reject DA grafts, due to lack of functional T cells. To confirm donor-specific unresponsiveness, MHC-mismatched Brown Norway (BN) hearts were transplanted 6 months after the initial DA heart transplant. LW rats with LWxDA CTTI rejected the third-party BN hearts (mean survival time 10 days); controls did not. CTTI recipients produced antibody against third-party BN donor but not against the DA thymus donor, demonstrating humoral donor-specific tolerance. Taken together, F1(LWxDA) CTTI given to Lewis rats resulted in specific tolerance to the allogeneic DA MHC expressed in the donor thymus, with resulting long-term survival of DA heart transplants after withdrawal of all immunosuppression.

Trained immunity in organ transplantation.

Consistent induction of donor‐specific unresponsiveness in the absence of continuous immunosuppressive therapy and toxic effects remains a difficult task in clinical organ transplantation. Transplant immunologists have developed numerous experimental treatments that target antigen‐presentation (signal 1), costimulation (signal 2), and cytokine production (signal 3) to establish transplantation tolerance. While promising results have been obtained using therapeutic approaches that predominantly target the adaptive immune response, the long‐term graft survival rates remain suboptimal. This suggests the existence of unrecognized allograft rejection mechanisms that contribute to organ failure. We postulate that trained immunity stimulatory pathways are critical to the immune response that mediates graft loss. Trained immunity is a recently discovered functional program of the innate immune system, which is characterized by nonpermanent epigenetic and metabolic reprogramming of macrophages. Since trained macrophages upregulate costimulatory molecules (signal 2) and produce pro‐inflammatory cytokines (signal 3), they contribute to potent graft reactive immune responses and organ transplant rejection. In this review, we summarize the detrimental effects of trained immunity in the context of organ transplantation and describe pathways that induce macrophage training associated with graft rejection.

Reduced positive selection of a human TCR in a swine thymus using a humanized mouse model for xenotolerance induction.

Tolerance-inducing approaches to xenotransplantation would be optimal and may be necessary for long-term survival of transplanted pig organs in human patients. The ideal approach would generate donor-specific unresponsiveness to the pig organ without suppressing the patient's normal immune function. Porcine thymus transplantation has shown efficacy in promoting xenotolerance in humanized mice and large animal models. However, murine studies demonstrate that T cells selected in a swine thymus are positively selected only by swine thymic epithelial cells, and therefore, cells expressing human HLA-restricted TCRs may not be selected efficiently in a transplanted pig thymus. This may lead to suboptimal patient immune function. To assess human thymocyte selection in a pig thymus, we used a TCR transgenic humanized mouse model to study positive selection of cells expressing the MART1 TCR, a well-characterized human HLA-A2-restricted TCR, in a grafted pig thymus. Positive selection of T cells expressing the MART1 TCR was inefficient in both a non-selecting human HLA-A2- or swine thymus compared with an HLA-A2+ thymus. Additionally, CD8 MART1 TCRbright T cells were detected in the spleens of mice transplanted with HLA-A2+ thymi but were significantly reduced in the spleens of mice transplanted with swine or HLA-A2- thymi. [Correction added on October 15, 2019, after first online publication: The missing superscript values +, -, and bright have been included in the Results section.] CONCLUSIONS: Positive selection of cells expressing a human-restricted TCR in a transplanted pig thymus is inefficient, suggesting that modifications to improve positive selection of cells expressing human-restricted TCRs in a pig thymus may be necessary to support development of a protective human T-cell pool in future patients.

Upregulation of CD80 on glomerular podocytes plays an important role in development of proteinuria following pig-to-baboon xeno-renal transplantation - an experimental study.

We have previously reported that co-transplantation of the kidney with vascularized donor thymus from α-1,3-galactosyltransferase gene knockout pigs with an anti-CD154 with rituximab-based regimen led to improved xenograft survival in baboons with donor-specific unresponsiveness. However, nephrotic syndrome emerged as a complication in which the glomeruli showed mild mesangial expansion with similarities to minimal change disease (MCD) in humans. Since MCD is associated with CD80 expression in glomeruli and elevated urinary excretion, we evaluated a potential role for CD80 in xenograft nephropathy. Study 1 confirmed high urinary CD80 excretion in nephrotic animals with renal xenografts showing CD80 expression in glomeruli. In Study 2, baboons receiving xenografts received CTLA4-Ig once a week from the second postoperative week or no CTLA4-Ig. The non-CTLA4-Ig group developed severe proteinuria with modest mesangial expansion with high urinary excretion of CD80 and documented CD80 expression in glomerular podocytes. All of the recipients in non-CTLA4-Ig groups had to be euthanized before POD 60. In contrast, CTLA4-Ig group showed a marked reduction in proteinuria and survived significantly longer, up to 193 days. These results demonstrate that anti-CD80 targeted therapy represents a promising strategy for reduction of proteinuria following renal xeno-transplantation with improved survival.

Nanoparticle-Based Modulation and Monitoring of Antigen-Presenting Cells in Organ Transplantation.

Donor-specific unresponsiveness while preserving an intact immune function remains difficult to achieve in organ transplantation. Induction of tolerance requires a fine modulation of the interconnected innate and adaptive immune systems. Antigen-presenting cells (APCs) predominate during allograft rejection and create a highly inflammatory context where allospecific T cells are primed. Currently, the available protocols to prevent allograft rejection include a cocktail of drugs that are efficient in the short-term, but with severe long-term side effects and considerable toxicity. Consequently, better and less burdensome strategies are needed to promote indefinite allograft survival. Targeted delivery of immunosuppressive drugs that prevent the alloimmune response may address some of these problems. Nanoparticle-based approaches represent a promising strategy to negatively modulate the alloresponse by specifically delivering small compounds to APCs in vivo. Nanoparticles are also used as integrating imaging moieties to monitor inflammation for diagnostic purposes. Therefore, nanotechnology approaches represent an attractive strategy to deliver and monitor the efficacy of immunosuppressive therapy in organ transplantation with the potential to improve the clinical treatment of transplant patients.

Tolerance in xenotransplantation.

This review describes recent progress in tolerance-inducing strategies across xenogeneic immunological barriers as well as the potential benefit of a tolerance strategy for islets and kidney xenotransplantation. Using advanced gene editing technologies, xenotransplantation from multitransgenic alpha-1,3-galactosyltransferase knockout pigs has demonstrated marked prolongation of renal xenograft survival, ranging from days to greater than several months for life-supporting kidneys, and more than 2 years in a heterotopic nonlife-supporting cardiac xenograft model. Continuous administration of multiple immunosuppressive drugs has been required and attempts to taper immunosuppression have been unsuccessful. It appears likely that low levels of T cell dependent antibodies and activation of innate responses are responsible for xenograft loss. Mixed chimerism and thymic transplantation approaches have achieved xenogeneic tolerance in pig-to-mouse models and both have recently been extended to pig-to-baboon models. Encouraging results have been reported, including persistence of macrochimerism, prolonged pig skin graft survival, donor-specific unresponsiveness in vitro and detection of recent T cell emigrants in vivo. Although tolerance induction in vivo has not yet been achieved in pig-to-baboon models, recent results are encouraging that this goal will be attainable through genetic engineering of porcine donors.

Abstract 44: Tolerance Induction with Post Transplantation High-Dose Cyclophosphamide relies on Recipient-derived Immune Regulation and Donor Bone Marrow in a Murine Model of Vascularized Composite Allotransplantation

PURPOSE: Developing novel treatment concepts to minimize/avoid immunosuppression by induction of immune tolerance represents the prime task in the field of transplantation. In the clinic to date, this was achieved in highly selected patients in clinical trials of kidney transplantation combined with concomitant stem cell or bone marrow transplantation. Vascularized composite allotransplantation (VCA) is evolving as viable treatment modality for patients suffering from functional and esthetic defects to craniofacial structures, extremities and urogenital tissues. In certain VCAs transplantation of vascularized bone marrow may be inherently part of the allograft designs (i.e. hands and faces) and thereby representing a unique opportunity in combining bone marrow and VCA. METHODS: Murine skin, heart, and hind limb (VCA) transplants were performed across a full MHC mismatch barrier. Recipients treatment comprised non-myeloablative TBI and T-cell depletion and a single dose of post-transplant cyclophosphamide (PTCy). Donor BM and splenocytes (DBM) were injected at the time of transplantation. Post-transplant multi-lineage and, Foxp3 chimerism as well as Vβ-TCR staining was performed. Donor-specific unresponsiveness was tested by MLR and by 2° skin and solid organ transplantation (SOT). Mechanistic studies were undertaken using transgenic mice (DEREG and Foxp3-DTR) to investigate the role of regulatory T cells (Tregs). VCA was performed in animals thymectomized prior to receiving PTCy and the transplant to highlight the role of the thymus in induction and maintenance of tolerance in this murine model. RESULTS: Untreated animals rejected skin grafts, SOT and VCA acutely within 14 ± 1 days, 9 ± 2 days, and 8 ± 1 days, respectively. The treatment regimen extended skin and SOT graft survival (32 ± 8; 65 ± 4, respectively). Additional DBM augmentation lead to allograft survival of >150 days in skin and SOT. However, indefinite graft survival of >150 days was observed in all animals receiving the induction regimen and a VCA ± DBM. In groups receiving a VCA ± DBM, donor chimerism was detected at 22.51% ± 5.96% and 30.17% ± 8.72%, respectively. Prior or post transplantation depletion of recipient-derived Tregs in VCA recipients did not lead to abrogation of tolerance. In recipients of skin graft + DBM, chimerism was not achieved after recipient-derived Treg depletion prior to transplantation (POD-2) and consequently the allografts were rejected (MST = 23.5 days, N=4). In-vitro, Vβ-T cell receptor staining indicates clonal deletion as an additional central tolerance mechanism. However, transplantation into a thymectomized host did not abrogate long-term allograft survival (N=6). All long-term survivors showed donor-specific T cell unresponsiveness in-vitro (MLR) while demonstrated proliferation against 3rd party stimulators. In-vivo, tolerant animals accepted donor-matched secondary skin, while 3rd party FVB/N skin was acutely rejected. Donor-matched hearts were accepted long-term. CONCLUSION: Taken together, robust tolerance and immunosuppression-free long-term allograft survival can be achieved with PTCy in stringent fully MHC mismatched murine models of skin, heart, and vascularized composite allotransplantation. Stable multi lineage chimerism and recipient derived regulatory T cells play a critical role in maintaining tolerance in this model, in particular in the peritransplant time period.

Cell therapeutic approaches to immunosuppression after clinical kidney transplantation.

Refinement of immunosuppressive strategies has led to further improvement of kidney graft survival in recent years. Currently, the main limitations to long-term graft survival are life-threatening side effects of immunosuppression and chronic allograft injury, emphasizing the need for innovative immunosuppressive regimens that resolve this therapeutic dilemma. Several cell therapeutic approaches to immunosuppression and donor-specific unresponsiveness have been tested in early phase I and phase II clinical trials in kidney transplantation. The aim of this overview is to summarize current cell therapeutic approaches to immunosuppression in clinical kidney transplantation with a focus on myeloid suppressor cell therapy by mitomycin C-induced cells (MICs). MICs show great promise as a therapeutic agent to achieve the rapid and durable establishment of donor-unresponsiveness in living-donor kidney transplantation. Cell-based therapeutic approaches may eventually revolutionize immunosuppression in kidney transplantation in the near future.

Induction of donor-specific immune tolerance using bioengineered thymus organoid

Abstract Extended use of immunosuppression is a major setback in organ transplantation. Hence, establishment of donor specific immune unresponsiveness, without the need of immunosuppression, is critical and remains a major challenge. Immunological tolerance is known to occur through two main mechanisms: central (thymus) and peripheral. Our lab has recently reported that bioengineered thymus organoids, made of de-cellularized thymus scaffolds populated with thymic epithelial cells (TECs), can successfully support T-cell generation in athymic nude mice. Here, we aim to test the hypothesis that transplantation of bioengineered thymus, incorporated with donor TECs, can induce central tolerance to donor antigens. Thymus scaffold were reconstructed with either TECs harvested from F1 offspring of B6 (H-2b) and NOD (H-2b/g7) or TECs from both B6 and CBA/J (H-2k) mixed in 1:1 ratio. These reconstructed thymus organoids are transplanted to the B6. Nude recipient, that are subsequently challenged with syngeneic, allogeneic and third party skin graft. Results from recipients with transplanted bioengineered thymus consisting F1 TECs demonstrated successful engraftment of skin transplants from both syngeneic (B6) and allogeneic (B6.H-2b/g7) whereas, clear rejection of the third party (CBA/J). Similarly, we observed prolonged survival of allograft skin (CBA/J) than third party (Balb/C) in recipient mice with reconstructed thymus organoids containing TECS from both donor (B6) and recipient (Balb/c). Furthermore, immune unresponsiveness to allograft was shown in MLR assay. Overall, our findings suggest reconstructed thymus-containing donor TECs could potentially have clinical application to induce-donor specific immune tolerance.

Effects of Lung Cotransplantation on Cardiac Allograft Tolerance Across a Full Major Histocompatibility Complex Barrier in Miniature Swine.

A 12-day course of high-dose tacrolimus induces tolerance of major histocompatibility complex-mismatched lung allografts in miniature swine but does not induce tolerance of heart allografts unless a kidney is cotransplanted. To determine whether lungs share with kidneys the ability to induce cardiac allograft tolerance, we investigated heart-lung cotransplantation using the same induction protocol. Hearts (n = 3), heart-kidneys (n = 3), lungs (n = 6), and hearts-lungs (n = 3) were transplanted into fully major histocompatibility complex-mismatched recipients treated with high-dose tacrolimus for 12 days. Serial biopsy samples were used to evaluate rejection, and in vitro assays were used to detect donor responsiveness. All heart-kidney recipients and five of six lung recipients demonstrated long-term graft survival for longer than 272 days, while all heart recipients rejected their allografts within 35 days. Tolerant recipients remained free of alloantibody and showed persistent donor-specific unresponsiveness by cell-mediated lympholysis/mixed-lymphocyte reaction. In contrast, heart-lung recipients demonstrated rejection of both allografts (days 47, 55, and 202) and antidonor responsiveness in vitro. In contrast to kidneys, lung cotransplantation leads to rejection of both heart and lung allografts, indicating that lungs do not have the same tolerogenic capacity as kidneys. We conclude that cells or cell products present in kidney, but not heart or lung allografts, have a unique capacity to confer unresponsiveness on cotransplanted organs, most likely by amplifying host regulatory mechanisms.

Cell therapy for immunosuppression after kidney transplantation.

To give an overview over cell therapeutic approaches to immunosuppression in clinical kidney transplantation. A focus is on myeloid suppressor cell therapy by mitomycin C-induced cells (MICs). Literature review with an emphasis on already existing therapies. Several cell therapeutic approaches to immunosuppression and donor-specific unresponsiveness are now being tested in early phase I and phase II trials in clinical kidney transplantation. Cell products such as regulatory T cells or regulatory macrophages, or other myeloid suppressor cell therapies, may either consist of donor-specific, third-party, or autologous cell preparations. Major problems are the identification of the suppressive cell populations and their expansion to have sufficient amount of cells to achieve donor unresponsiveness (e.g., with regulatory T cells). We show a simple and safe way to establish donor unresponsiveness in living-donor kidney transplantation by MIC therapy. A phase I clinical trial is now under way to test the safety and efficacy of this cell therapeutic approach. Cell therapeutic approaches to immunosuppression after kidney transplantation may revolutionize clinical transplantation in the future.

Tolerance, Mixed Chimerism, and Graft Survival in HLA Matched and Mismatched Recipients of Kidney and Hematopoietic Cell Transplants.

Purpose: The establishment of persistent mixed chimerism, immune tolerance and the withdrawal of maintenance immunosuppressive (IS) drugs (mycophenolate mofetil and a calcineurin inhibitor) without kidney graft loss or dysfunction in clinical kidney transplantation. Methods: Thirty-seven patients were conditioned with a post-kidney transplant regimen of total lymphoid irradiation and antithymocyte globulin followed by an infusion of purified CD34+ hematopoietic progenitor cells mixed with a defined number of CD3+ T cells collected from G-CSF “mobilized” donor blood mononuclear cells. Results: Twenty-two HLA-matched and 15 HLA-mismatched patients were enrolled, and all patients have maintained good graft function at the last observation point, with follow-up to 13 years. All HLA-matched patients received an infusion of 1x106 T cells/kg, along with at least 4x106 CD34+ cells/kg, from the donors. The criteria for IS drug withdrawal were persistent mixed chimerism for at least 6 months, no clinical rejection and no subclinical rejection on surveillance biopsy, and no evidence of graft versus host disease. Of these patients, 16 have discontinued IS drugs for up to 5 years (median 3 years) without subsequent evidence of acute or chronic rejection. Five are maintained on IS drugs due to failure to meet drug withdrawal criteria (4) or relapse of lupus (1), and one is in IS drug taper. One patient off IS drugs with a history of coronary disease died during vigorous exercise. Immune monitoring of these patients showed a marked transient decrease in conventional T and B cells, an increase in the percentage of CD11b+ myeloid cells, and increase of Tregs and NKT cells among all T cells in the blood, and donor-specific unresponsiveness in the MLR indicative of tolerance. In the HLA-mismatched patients, the dose of donor T cells was escalated in order to achieve the persistent mixed chimerism observed in the HLA-matched patients. This required a marked increase in the infused dose of cells in comparison to the matched patients. Tapering of IS drugs in the HLA-mismatched patients with mixed chimerism is underway. Conclusion: Persistent mixed chimerism was achieved in both HLA-matched and HLA-mismatched patients, all kidney grafts are functioning, and thus far IS drugs were successfully withdrawn in the majority of HLA-matched patients.

Endogenous Stem Cell Mobilization Via CXCR4 Antagonism Allows for Drug Free Kidney Transplantation in a Large Animal Preclinical Model.

Organ Transplantation is lifesaving therapy but requires immunosuppresion (IS) with resultant morbidity. Current IS strategies focus on inhibition of T cell activation and seek to avoid donor-recipient engagement. Marrow derived endogenous stem cells have been shown to participate in organ regeneration and recovery in injury scenarios and also demonstrate immunomodulatory properties. However a role for endogenous stem cells post transplant has not been described, though recent small animal data is suggestive. Here we show that pharmacologic mobilization of endogenous stem cells immediately post kidney transplant leads to host repopulation of the graft and donor specific immunologic unresponsiveness allowing for subsequent immunosuppressive-free long term graft and recipient survival. We found in a series of maximally mismatched SLA defined miniature swine renal transplants indefinite survival and relatively normal graft histology after animals had received a short course of stem cell mobilization with the CXCR4 antagonist plerixafor and low dose tacrolimus. This ten-day treatment was repeated at 1 and 2 months after transplant, after which no further treatment was given. Three animals displayed long term normal graft function, minimal inflammatory changes on biopsy, and donor immunologic unresponsiveness, in contrast to control animals which expired of rejection and renal insufficiency relatively rapidly. To determine kidney allograft replacement by host-derived cells, Y-chromosome (Sry) expression in genomic DNA of graft biopsies was analyzed by qPCR in sex-mismatched transplants and revealed 50% of genomic DNA from kidney biopsies to be of host genotype at 6 months post transplantation indicating host repopulation of the graft. Our results demonstrate that mobilization of endogenous stem cells in the post transplant setting allows for graft repopulation and immunoregulation in the absence of further immunosuppression. We anticipate that endogenous stem cell mobilization via CXCR4 antagonism may represent a novel post transplant strategy which would avoid the morbidities of current conventional IS.

Induction of Cardiac Allograft Tolerance Across a Full MHC Barrier in Miniature Swine by Donor Kidney Cotransplantation

We have previously shown that tolerance of kidney allografts across a full major histocompatibility complex (MHC) barrier can be induced in miniature swine by a 12-day course of high-dose tacrolimus. However, that treatment did not prolong survival of heart allografts across the same barrier. We have now tested the effect of cotransplanting an allogeneic heart and kidney from the same MHC-mismatched donor using the same treatment regimen. Heart allografts (n = 3) or heart plus kidney allografts (n = 5) were transplanted into MHC-mismatched recipients treated with high-dose tacrolimus for 12 days. As expected, all isolated heart allografts rejected by postoperative day 40. In contrast, heart and kidney allografts survived for >200 days with no evidence of rejection on serial cardiac biopsies. Heart/kidney recipients lost donor-specific responsiveness in cell-mediated lympholysis and mixed-lymphocyte reaction assays, were free of alloantibody and exhibited prolonged survival of donor, but not third-party skin grafts. Late (>100 days) removal of the kidney allografts did not cause acute rejection of the heart allografts (n = 2) and did not abrogate donor-specific unresponsiveness in vitro. While kidney-induced cardiac allograft tolerance (KICAT) has previously been demonstrated across a Class I disparity, these data demonstrate that this phenomenon can also be observed across the more clinically relevant full MHC mismatch. Elucidating the renal element(s) responsible for KICAT could provide mechanistic information relevant to the induction of tolerance in recipients of isolated heart allografts as well as other tolerance-resistant organs.

The Successful Induction of Systemic Tolerance to a Class-I MHC Disparity through Adoptive Transfer in a Large Animal Model

Background: We have previously reported that 12 days of high dose CyA uniformly leads to tolerance of MHC class I-mismatched renal grafts in MHC inbred miniature swine. We have now attempted to induce tolerance in secondary recipients by the adoptive transfer of a long-term tolerant kidney with or without tolerated cells. Methods: (1) Adoptive Transfer Donors: Three MHC inbred SLAdd miniature swine received life-supporting MHC class I-mismatched kidneys (SLAgg) with 12 days of high dose CyA to induce tolerance. Three to four months after kidney transplant (KTx) these animals received a Donor Specific Transfusion (DST) to expand T regulatory cells. (2) Adoptive Transfer Recipients: Three recipients, MHC-matched to the adoptive transfer donors (SLAdd), received 150 rad whole body irradiation (WBI) one day before receiving class I mismatched kidneys from the donors. Two of these animals also received 4-5 × 10ˆ9 cells/kg cells, obtained by leukapheresis from the adoptive transfer donors, on day -1. Tolerated kidneys (SLAgg) were transplanted (day 0) without further immunosuppression. Second donor matched kidneys from naïve SLAgg animals were transplanted >100 days later to confirm tolerance. (3) Controls: Three SLAdd animals received 150 rad WBI on day -1 followed by a class I MHC mismatched KTx. (4) Graft function was assessed by serum creatinine and histology. Immunologic responses were assessed by Cell Mediated Lysis (CML) assays. Anti-Donor MHC antibody was assessed by FACS. Results: The three controls rejected the naïve Class I mismatched kidney by day 9. All three recipients accepted tolerated kidneys >100 days and did not reject second donor matched naïve kidneys transplanted without immunosuppression, confirming systemic tolerance. All three animals maintained donor specific unresponsiveness by cellular assays and two animals did not develop antibody to donor. Conclusions: To our knowledge, these results provide the first evidence for the successful transfer of systemic tolerance in a large animal model by the adoptive transfer of tolerated kidneys into 150-rad irradiated miniature swine.

Mechanisms of Donor-Specific Tolerance in Recipients of Haploidentical Combined Bone Marrow/Kidney Transplantation

We recently reported long-term organ allograft survival without ongoing immunosuppression in four of five patients receiving combined kidney and bone marrow transplantation from haploidentical donors following nonmyeloablative conditioning. In vitro assays up to 18 months revealed donor-specific unresponsiveness. We now demonstrate that T cell recovery is gradual and is characterized by memory-type cell predominance and an increased proportion of CD4⁺ CD25⁺ CD127⁻ FOXP3⁺ Treg during the lymphopenic period. Complete donor-specific unresponsiveness in proliferative and cytotoxic assays, and in limiting dilution analyses of IL-2-producing and cytotoxic cells, developed and persisted for the 3-year follow-up in all patients, and extended to donor renal tubular epithelial cells. Assays in two of four patients were consistent with a role for a suppressive tolerance mechanism at 6 months to 1 year, but later (≥ 18 months) studies on all four patients provided no evidence for a suppressive mechanism. Our studies demonstrate, for the first time, long-term, systemic donor-specific unresponsiveness in patients with HLA-mismatched allograft tolerance. While regulatory cells may play an early role, long-term tolerance appears to be maintained by a deletion or anergy mechanism.

Rapamycin-conditioned, alloantigen-pulsed myeloid dendritic cells present donor MHC class I/peptide via the semi-direct pathway and inhibit survival of antigen-specific CD8 + T cells in vitro and in vivo

Dendritic cells (DC) are “professional” bone marrow-derived antigen (Ag)-presenting cells of interest both as therapeutic targets and potential cellular vaccines due to their ability to regulate innate and adaptive immunity. Harnessing the inherent tolerogenicity of DC is a promising and incompletely explored approach to the prevention of allograft rejection. Previously, we and others have reported the ability of pharmacologically-modified DC, that resist maturation, to inhibit CD4 + T cell responses and prolong allograft survival. Here we evaluated the ability of murine myeloid DC conditioned with the immunosuppressive pro-drug rapamycin (RAPA) to acquire and directly present alloAg to syngeneic CD8 + T cells. RAPA-conditioned DC (RAPA-DC) pulsed with allogeneic splenocyte lysate acquired and expressed donor MHC class I and enhanced the apoptotic death of directly-reactive donor Ag-specific CD8 + T cells in vitro. Moreover, following their adoptive transfer, they reduced the survival of these T cells in vivo. The ability of RAPA-DC to inhibit the survival of alloAg-specific CD8 + T cells provides a potential mechanism by which host-derived DC may act as negative regulators of T cell alloreactivity and support donor-specific unresponsiveness. Adoptive cell therapy with alloAg-pulsed RAPA-DC may offer an effective approach to suppression of alloimmunity, with reduced dependence on systemic immunosuppression.