Secondary Logo

Journal Logo

HSCT-Based Approaches for Tolerance Induction in Renal Transplant

Chhabra, Anita Y. PhD1; Leventhal, Joseph MD, PhD2; Merchak, Andrea R. BS1; Ildstad, Suzanne MD1

doi: 10.1097/TP.0000000000001837
Reviews
Free

Renal transplantation has become the preferred treatment for end stage kidney failure. Although short-term graft survival has significantly improved as advances in immunosuppression have occurred, long-term patient and graft survival have not. Approximately only 50% of renal transplant recipients are alive at 10 years due to the toxicities of immunosuppression and alloimmunity. Emerging research on cell-based therapies is opening a new door for patients to receive the organs they need without sacrificing quality of life and longevity because of drug-based immunosuppression. Research has focused on inducing tolerance, a state in which the body accepts the transplant and graft function is stable. Cell-based therapies to facilitate chimerism and achieve tolerance in major histocompatibility disparate recipients have been developed in mouse, swine, canine, and nonhuman primate models. These findings are now being translated into the clinic in several trials currently underway. Protocols that use a combination of traditional therapeutic agents paired with cell populations including hematopoietic stem cells, regulatory T cells, and facilitating cells are being conducted with the objective to harness the donor immune system to protect the transplanted tissue. The benefits and feasibility of the clinical application of cell-based therapy has been demonstrated, and promising results have been achieved. Here we discuss the preclinical work that has led to the clinical application of the various approaches and a summary of the most current clinical data from groups throughout the world.

The authors provide a timely and balanced review on the current approaches aiming at tolerance in kidney transplantation.

1 Institute for Cellular Therapeutics, University of Louisville, Louisville, KY.

2 Comprehensive Transplant Center, Northwestern Memorial Hospital, Chicago, IL.

Received 22 February 2017. Revision received 16 May 2017.

Accepted 19 May 2017.

S.I. has equity interest in and is the CEO of Regenerex, LLC, a biotech company formed to make FCRx widely available. All other authors declare no conflicts of interest.

A.C. prepared the article. A.M. reviewed the article and prepared the figures. J.L. wrote and edited the article. S.I. oversaw the preparation of the article and finalized the article.

Correspondence: Suzanne T. Ildstad, MD, Institute for Cellular Therapeutics, University of Louisville Louisville, KY. (suzanne.ildstad@louisville.edu).

End-stage renal disease results from numerous etiologies, including diabetes, hypertension, lupus nephritis, IgA nephropathy, polycystic kidney disease, and multiple myeloma. Kidney transplantation and dialysis are the 2 currently used treatments for patients with end-stage renal disease. Renal transplantation provides superior survival and quality of life compared with dialysis.1 However, the benefits of transplantation are limited by the toxicity of the immunosuppressive (IS) agents which are required to prevent allograft rejection. The discovery of cyclosporine A (CyA) in 1978 ushered in a new era in transplantation, resulting in significantly improved short-term graft acceptance and survival of kidney transplants.2 Although IS drugs have reduced early acute rejection, the long-term graft survival beyond 5 years has not dramatically improved.3,4 In fact, there has been little improvement in long-term graft function and patient survival over the past 20 years. The reduced success of long-term graft survival is mainly due to detrimental side effects of IS drugs including hypertension, increased risk of cardiovascular disease, infection-related complications, metabolic syndrome, increased rate of malignancy, and renal compromise.5,6 Fifty percent of renal grafts are lost due to alloimmunity, the toxicity of immunosuppression, and chronic rejection within 11 to 12 years.7 Notably, more than 16.5% of nonrenal recipients of solid organ transplants develop renal failure due to the IS agents.8 The lifetime dependency on IS drugs affects not only quality and quantity of life but is also an economic burden. Approaches to promote “1 transplant for life” therefore remain a high priority. This review focuses on the preclinical underpinnings and translational research now underway using therapeutic cell transfer with hematopoietic stem cell (HSC) to induce transplantation tolerance in the clinic.

Successful engraftment without the long-term requirement for IS drugs has been the goal of transplantation surgeons and physicians for more than half a century. With advances in surgical procedures and increased knowledge of the immune system, scientists have been exploring different strategies to regulate and/or modulate the immune system to specifically accept allografts in both animal models and now humans. The field of transplantation evolved after the landmark contributions by Ray Owen and Sir Peter Medawar in the 1950s. An interesting observation by Ray Owen showed the coexistence of 2 types of red blood cells in freemartin cattle twins who shared a common placenta.9 Notably, this mixed RBC phenotype was not passed on to subsequent progeny, leading to the hypothesis that cells were exchanged via the common shared placenta. Subsequently, Sir Peter Medawar demonstrated that graft rejection was an immunological process and that deletional tolerance to skin grafts could be achieved in presence of donor hematopoietic cells.10 These findings led to the concept of mixed chimerism, a state where the donor and recipient immune systems coexist and prompted research in small and large animal models to develop a safe and effective means to induce tolerance.

There are 2 forms of tolerance observed in transplantation. Operational (spontaneous) tolerance and acquired (induced/deletional) tolerance. An understanding of these is critical to this review. Operational tolerance is a state of stable graft function in the absence of IS drugs for 1 year or longer. Such operational tolerance has been demonstrated in a restricted group of patients that intentionally or accidently discontinued the use of IS drugs and retained their organ transplant. In the entire world experience of renal transplantation comprising 10 of thousands of recipients, less than 100 subjects have fulfilled the criteria for operational tolerance.11 Operational tolerance is based on nondeletional mechanisms where the donor-reactive T cells are present but have undergone exhaustion or are controlled by regulatory mechanisms, such as regulatory T (Treg) cells, as well as other peripheral mechanisms, such as clonal deletion through activation-induced cell death. Such tolerance is not only difficult to achieve but also difficult to maintain because the donor and host cell balance can be lost due to many unforeseeable reasons.12

Deletional tolerance can be intentionally induced using stem cell transplantation. This hematopoietic chimerism-based tolerance is attributed to clonal deletion of alloreactive cells where exposure to donor antigen during reconstitution of the immune system deletes donor-reactive T cells and this along with other peripheral tolerance mechanisms leads to long-term robust tolerance.

Efforts to minimize and intentionally wean immunosuppression have also been attempted. Most of the studies were terminated because of the inability to predict which subjects could be successfully weaned versus which would experience rejection.13 In a study by Shapiro et al,13 kidney transplant (KTx) recipients underwent induction with Campath or antithymocyte globulin (ATG) followed by immunosuppression with tacrolimus. Spaced/gradual weaning of IS drugs was then performed. Of 174 subjects enrolled, more than 62% were successfully weaned. The remainder either developed donor-specific antibody or experienced rejection. Unfortunately, there was no predictable biomarker to indicate which subjects would fail weaning. The long-term impact of this on renal function remains to be determined because rejection episodes are associated with increased graft loss. The study was therefore discontinued. Feng et al14 showed that operational tolerance can be achieved in 60% of pediatric patients with liver transplants. However, only subjects with stable grafts on immunosuppression monotherapy were included in the study. Notably, subjects with operational tolerance exhibited improved quality of life and fewer toxicities.

T cells play a critical role in graft rejection and have been a main target for current IS regimens for clinical transplantation. Drugs that target T cell deletion, anergy, exhaustion, and senescence are being tested alone or in combination for their efficacy to specifically suppress the immune system and prolong graft survival. A variety of approaches to modulate immune response to induce tolerance, such as clonal deletion using anti–T cell antibodies (Abs; ATG, anti-CD3, CD4, or CD8 Abs), T cell anergy using costimulatory blockade Abs (4Ig),15 targeting T cell signaling using calcineurin inhibitors, regulatory-based mechanisms, such as Treg cell, myeloid-derived suppressor cell (MDSC), and mixed-chimerism–based mechanisms, have been and are being explored.

Establishing chimerism has become increasingly a focus of research because it has the advantage that donor specific tolerance with preservation of immunocompetence occurs. Decades of research in rodent models have shown that chimerism enables solid organ transplant acceptance.16,17 Further testing of HSC transplantation (HSCT)-based tolerance models has more recently been evaluated in large animal models.

Back to Top | Article Outline

PRECLINICAL LARGE ANIMAL MODELS

Miniature Swine Model

Genetically defined miniature swine have provided valuable preclinical data related to enabling the application of chimerism to induce tolerance in the clinic. Four decades ago, Pennington et al18 developed a miniature swine model for chimerism-induced tolerance. They reported that bone marrow (BM) could be engrafted in autologous or swine lymphocyte antigen–matched allografts in recipients conditioned with 900 rads of total body irradiation (TBI). Genetic match between major histocompatibility (MHC) antigens was required for successful engraftment. Genetic mismatch at MHC-II but complete match at MHC-I led to engraftment but was limited by graft-versus-host disease (GVHD). Conversely, MHC-I–mismatched and complete MHC-II–matched BM failed to engraft, suggesting that genetic disparity is a major hurdle in HSCT.19 Sakamoto et al20 further showed that T cells play an important role in GVHD and T cell depletion of BM reduces GVHD without affecting engraftment in a parent to F1 model. Although successful, this protocol had 2 critical challenges that limited its potential use in the clinic. The use of high-dose radiation for conditioning was associated with significant morbidity, and additionally, the transplantation of unmodified BM as a source of stem cells was associated with GVHD. To overcome this, Huang et al21 developed a nonmyeloablative conditioning regimen consisting of 2 doses of 150 cGy TBI, 700 cGy thymic irradiation (TI) and anti-CD3 immunotoxin to deplete host T cells. Long-term chimerism and donor-specific tolerance were achieved with both BM cells and mobilized peripheral blood mononuclear cell (PBMC) infusion.22 Chimerism was successfully established without ablative TBI when host T cells were depleted.23 Lima et al24 showed that TI was not necessary to establish chimerism. The investigators concluded that in vitro donor-specific unresponsiveness, thymic chimerism, peripheral blood chimerism, and the presence of donor colony forming units in bone were predictors of tolerance.25

The efficacy of species-specific cytokines for mobilization was investigated in the swine model. Stem cell factor and IL-3 with and without granulocyte-colony stimulating factor (G-CSF) resulted in optimal mobilization, promoting enhanced engraftment of both lymphoid and myeloid donor cells in conditioned recipients.22 This chimerism resulted in acceptance of donor-specific kidney allografts as well as heart grafts in the absence of IS drugs and GVHD.26 Collectively, these studies supported the feasibility and safety of nonmyeloablative BM-induced tolerance in a large animal model.

Back to Top | Article Outline

Nonhuman Primate Model

Historically, nonhuman primates (NHP) have also been considered important models to assess safety and efficacy for translating HSCT research from mice to humans. Kawai et al27 showed that tolerance to renal allografts could be achieved in MHC mismatched Cynomolgus monkeys using nonmyeloablative conditioning consisting of ATG, TBI (1 dose 300 rad or fractionated 2 × 150 rad), TI (700 rads), short-term cyclosporine A and BM infusion. In another study, the investigators showed that long-term tolerance could be achieved with BM and splenectomy in monkeys. Tolerance did not occur in the presence of a spleen or if delayed splenectomy was performed.28 Because lymphocyte depletion plays an important role in tolerance induction, and splenectomy is not an easily accepted option for humans, alternative approaches to deplete host lymphocytes were tested to develop a more clinically relevant conditioning regimen. Kimikawa et al29 tested the efficacy of a variety of combinations of nonmyeloablative preoperative conditioning reagents including ATG, nonmyeloablative TBI (200 rad), TI (700 rad), short-term CyA induce tolerance in Cynomolgus monkeys. Fractionated irradiation at a dose of 125 cGy in combination with deoxyspergualin was sufficient for the induction of chimerism and long-term tolerance. Overall, these studies further confirmed the safety and feasibility of using low-dose irradiation and lymphocyte depletion to induce long-term graft acceptance free from the requirement for IS drugs.

In 2004, Kawai et al30 showed that a short course of anti-CD154 monoclonal antibody for costimulatory blockade eliminated the need for splenectomy in NHP. However, late chronic rejection was observed in 3 of 8 NHP recipients. The investigators then evaluated the ability of mobilized PBMCs to induce chimerism using similar conditioning. Cynomolgus monkeys were conditioned with TBI, TI, and ATG plus a short course of anti-CD154 antibody and CyA followed by infusion of PBMC mobilized using either G-CSF or SCF or high dose of G-CSF, this resulted in mixed chimerism.31 Kean et al32 used stem cells to produce high level donor chimerism in Rhesus macaques conditioned with busulfan and sirolimus with CD28/CD154 costimulatory molecule blockade. Subjects received BM and renal transplant or only renal transplant followed by sirolimus and CD28/CD40 blockade as maintenance IS drugs. Most subjects exhibited whole blood but not T cell chimerism. This was not sufficient to induce tolerance to renal allografts.33 It was found that even the MHC-matched transplants were rejected after withdrawal of IS drugs. It was hypothesized that this was potentially due to the activated phenotype of the antigen experienced recipient T cells.32 The authors concluded that in NHPs transient, low levels of mixed T cell chimerism is insufficient to induce allograft tolerance, and chimerism per se cannot be used as biomarker of tolerance.

Most of the studies described above used either low-dose TBI or TI as conditioning along with T cell depleting/costimulatory signal blocking Abs and short-term administration of CyA. To determine if chimerism is achievable without irradiation, Sogawa et al34 compared different doses of cyclophosphamide (CyP) to irradiation and found that although hematopoietic reconstitution was more rapid in CyP-based conditioning, there was reduced long-term graft and animal survival due to rejection, B cell lymphoma, and toxicities associated with CyP. Taken together, the above studies demonstrate that chimerism established using clinically applicable nonmyeloablative conditioning can provide long-term protection to allografts in NHPs.

Back to Top | Article Outline

Canine Model

Dogs have also provided critically important preclinical findings to confirm the feasibility and safety of HSCT-based tolerance approaches. Storb et al35 demonstrated that large quantities of mobilized HSCT successfully engrafted in dogs conditioned with 1200 cGy TBI. Engraftment was more sustained in dog leukocyte antigen (DLA)-matched dogs compared with mismatched dogs. The mobilized apheresis product was cryopreserved in dimethyl-sulfoxide (DMSO) and stored at −80°C. This important finding demonstrated that frozen BM cells retain the function of hematopoietic progenitor cells.36 The fact that successful engraftment was achieved using frozen BM cells has expanded possibilities for tolerance-inducing strategies using organs/grafts from deceased donors. Although low-dose irradiation (450 cGy) was sufficient to induce chimerism in DLA-identical dogs, increased irradiation was associated with superior engraftment.37 Unlike in NHP, splenectomy did not enhance engraftment of DLA-mismatched BM transplants in dogs conditioned with 950 cGy irradiation.38

To reduce TBI-related toxicity, CyP was added to the conditioning regimen. It had previously been shown in mice that conditioning with 200 cGy TBI plus T cell depletion of the recipient plus a single dose of posttransplant CyP on day +2 resulted in durable engraftment.39,40 These findings were successfully translated to the dog model where it was shown that 200 cGy TBI plus mycophenolate mofetil (MMF) and CyA resulted in mixed chimerism in DLA-matched dogs without GVHD.41 Conditioning of canine recipients with ATG plus 100 cGy TBI, MMF, and CyA did not allow engraftment.42 Chimeric dogs were tolerant to renal and vascularized composite tissue allografts.43,44 Importantly, cryopreserved BM was functional to engraft and induce tolerance to renal allografts. In a follow-up study, it was found that dogs preconditioned with antilymphocyte serum (D−6 to D+7) that received a kidney transplant (D0), followed by an infusion of freshly fractionated BM (D+13/+14) or freeze/thawed BM, demonstrated chimerism and significantly prolonged allograft function compared to dogs treated with antilymphocyte serum only.45 The fact that successful engraftment occurs using freeze/thawed BM infused 2 weeks after a kidney transplant widens the potential of application of this regimen in deceased donors. This nonmyeloablative 200 cGy TBI-based approach is now widely used in the clinic in patients with hematologic malignancies and comorbidities that prevent ablative HSCT. Subjects are conditioned with 200 cGy TBI plus 2 or 4 doses of CyP pre- and post-BM infusion and managed as outpatients.46 This nonmyeloablative conditioning has significantly reduced the morbidity for HSCT.

To date, chimerism-based tolerance approaches for tissue and organ transplant have been shown to be safe and achievable in all species tested. These diverse methods to avoid toxicity of ablative conditioning suggest a promising future for expanding HSCT-based tolerance induction to nonmalignant diseases.

Back to Top | Article Outline

CLINICAL TRANSLATION TO HUMANS AND THEIR OUTCOMES

Based on the promising results of therapeutic cell transfer in preclinical models for establishing tolerance and long-term allograft survival, several clinical trials have been initiated. The following section discusses outcomes of 4 clinical approaches to induce tolerance in HLA-matched and HLA-mismatched renal transplant recipients.

Back to Top | Article Outline

Clinical Trials at Massachusetts General Hospital

Over the past 12 years, the group at Massachusetts General Hospital (MGH) has developed and continues to refine conditioning approaches to induce tolerance to renal allografts. The first approach addressed HLA-matched donor and recipients of a combined iliac crest marrow and kidney in 10 subjects with multiple myeloma and end-stage renal failure. Nonmyeloablative conditioning using CyP (60 mg/kg D-6, D-5), ATG and TI (700 cGy) was used followed by posttransplant CyA.47 Six patients were alive as of 2016, with 2 in complete remission ranging from 5 to 13 years.48 Two of the other 4 patients underwent a second HSCT from the same donor after relapse of disease. Two subjects experienced acute GVHD and 4 developed chronic GVHD. Although chimerism was transient in 5 of the 10 subjects, the renal allografts were not rejected and 4 of the 5 transiently chimeric patients were successfully weaned off IS drugs. The patients with full donor chimerism remained on IS drugs due to the development of GVHD.49 One subject with transient chimerism remained off IS with normal renal function for 3 years, but subsequently expired due to relapse 7.7 years posttransplantation. One subject with durable chimerism also died due to acute myeloid leukemia. Of the 2 patients who died, only 1 had graft failure. Three of the 6 remaining living patients have been off IS drugs for more than 3 years (Table 1).50 It was concluded from this pioneering work that durable chimerism did not appear to be a requisite for the induction of tolerance to renal allografts. This protocol has recently been resumed and expanded to include other hematologic malignancies, haploidentical donors, and the CyP has been replaced by 400 cGy TBI.48

TABLE 1

TABLE 1

In the interim, the MGH group focused their efforts on haploidentical donor/recipient pairs. Ten HLA-disparate haploidentical living donor patients with renal failure without underlying malignancy were enrolled. The first 3 patients received conditioning (NKD03) consisting of 700 cGy TI (D-1), 2 doses of CyP (60 mg/kg on D−5 and D−4) and anti-CD2 T cell depleting antibody (D−2, −1, 0, +1). One patient (3) developed irreversible acute humoral rejection and experienced graft loss. That subject was retransplanted using standard IS drugs. The conditioning was modified for the next 2 patients with addition of 2 doses of rituximab (D−7, −2). However, both subjects developed donor-specific antibody. This regimen was further modified (modified 2 NKD03) by adding 2 additional doses of rituximab for the next 5 patients (D−7, −2, +5, +12). All patients received posttransplant prednisone (D4 to D20) and methylprednisolone (D10, 11, 12) (Figure 1). Tacrolimus was administered starting 1 day pretransplant for 8 weeks and then slowly tapered. All patients developed transient chimerism and all but 1 exhibited reversible capillary leak syndrome termed engraftment syndrome. Seven of the 10 subjects achieved operational or functional tolerance with successful tapering and cessation of immunosuppression without renal allograft rejection for more than 3 years (Table 1). Subsequently 1 of the 7 subjects experienced disease recurrence and 2 of the 7 subjects experienced chronic humoral rejection for which immunosuppression was resumed. One subject experienced graft loss due to thrombotic microangiopathy. The remaining 4 subjects remain off immunosuppression from 5 to 13 years.48

FIGURE 1

FIGURE 1

In summary, the MGH study in HLA-mismatched kidney transplantation experienced 3 graft losses due to rejection and 3 of the remaining 7 subjects developed signs of chronic rejection and were placed on back on IS drugs. Four patients have been off IS drugs for 3 years or longer with stable renal function.51 The results from these studies are promising and demonstrate the feasibility of the approach but there is still need for improvement in the pretransplant conditioning approach.

This study has recently resumed in subjects with hematologic malignancy with 4 subjects enrolled and transplanted. The following modifications in conditioning were implemented: fludarabine (24 mg/m2 per day for 5 days followed by dialysis) replaced ATG and 200 cGy of TBI replaced the TI.48 The first patient received ATG instead of fludarabine and experienced a renal allograft rejection episode at 10 to 14 days. She remains on IS drugs. She had only transient chimerism.48 The ATG was then replaced with fludarabine in the next 2 subjects. Durable donor chimerism was achieved. One subject is off IS drugs, and the second had severe fludarabine neurotoxicity resulting in death 6 months after transplantation. The next patient received a reduced dose of fludarabine (24 mg/m2 per day for 3 days) and increased length of dialysis. The patient has normal renal function with full donor chimerism and is now being slowly weaned off IS drugs.48,52 Together, these studies show that long-term tolerance can be achieved by combined BM and kidney transplantation in patients with HLA-matched and haploidentical donors.

Back to Top | Article Outline

Clinical Trials at Stanford University

Investigators at Stanford University have been using a total lymphoid irradiation (TLI)-based approach first developed in a mouse model, then tested in NHP. After success in establishing tolerance in NHP, this regimen was applied to renal transplant patients in a clinical trial involving 6 HLA-mismatched patients. G-CSF mobilized CD34+ selected cells (ranging from 3 to 11 million/kg) to which varying numbers of CD3+ cells were added was administered on day 11 posttransplant. The TLI- and ATG-based protocol included 5 daily doses of ATG starting on the day of kidney transplant (1.5 mg/kg) followed by 10 doses of TLI (80 cGy/dose for first 4 and 100 cGy/dose for the remaining 2 patients) from day 1 to day 14. Prednisone was administered as premedication drug for ATG and discontinued on day 10, and CyA was administered beginning day 0. Two of 6 patients developed transient chimerism, showed no signs of GVHD, were tolerant to donor cells in mixed lymphocyte reaction, and therefore were weaned from IS drugs. Unfortunately, these patients developed Banff I rejection 3.5 and 5.5 months after immunosuppression was discontinued and immunosuppression was therefore resumed. The other 4 patients did not develop chimerism and remained on IS drugs (Table 1).53

The protocol was subsequently modified to focus on HLA-identical related donor/recipients’ pairs. Twenty-two patients were conditioned with 10 doses of TLI (120 cGy/dose) and 5 doses of ATG and transplanted with cryopreserved G-CSF mobilized CD34-selected PBMC (5-16 million CD34+/kg). Based on murine models that showed the requirement for donor T cells for engraftment,54 a defined dose of CD3+ T cells (1 million/kg) was included in the stem cell product. All patients received prednisone (D0 tapering to D10), MMF for 30 days after transplantation, and CyA (Figure 2). Patients that showed stable chimerism for at least 6 months without signs of GVHD or clinical rejection were weaned from IS drugs. All but 5 patients were successfully tapered off IS drugs between 6 and 14 months (Table 1). Six patients exhibited durable chimerism with 65% or greater donor and the remaining 10 patients lost donor chimerism during or after discontinuation of CyA. All patients have exhibited stable renal function with follow-up from 10 to 101 months.

FIGURE 2

FIGURE 2

With the remarkable success in HLA-matched recipients, the investigators at Stanford recently initiated another study in haploidentical living related and unrelated donor/recipient pairs. Ten subjects have been reported. Recipient conditioning is like that for the HLA-identical subjects, with 10 doses of TLI (120 cGy/dose) and 5 doses of ATG. CD34+ selected mobilized PBMC (mPBMC) with cell doses varying from 8 to 22 × 106 CD34+ cells/kg were transplanted. A dose escalation of CD3+ cells was added to the CD34 product. Patient 1 received 3 × 106 CD3/kg, patients 2 to 5 10 × 106 CD3/kg, patients 6 and 7 20 × 106 CD3/kg, and the most recent 3 were administered 50 × 106 CD3+ T cells/kg recipient weight. Per the latest report by Scandling et al,55 patients infused with high-dose CD34+ (>15 million/kg) and T cell (>10 million/kg) showed high levels of lymphocyte chimerism. The last 3 patients received almost half of the CD34+ dose but a high T cell dose. All 3 showed high levels of chimerism at the day 60 timepoint. To date, none of the subjects have been completely tapered off IS drugs (Table 1). It will be interesting to see if these patients maintain durable chimerism after tapering of immunosuppression. Only 2 patients have been weaned off MMF at 9 months posttransplant to date, with tacrolimus tapering initiated at 9 months. Serum creatinine levels were reported to be normal in these patients.

Collectively, the investigators have successfully demonstrated the safety and feasibility of CD34+ cell-based therapy in inducing tolerance in HLA-matched kidney transplant patients. The challenge remains to develop approaches to induce tolerance in HLA-mismatched donors to effectively use the available donor organs including organs from deceased donors. In a recent review of the chimerism-based experience, the authors suggest that durable chimerism may be a prerequisite to durable tolerance in light of the late rejection episodes seen in the transiently chimeric subjects.53

Back to Top | Article Outline

Clinical Trials at Northwestern University

Two studies are taking place at Northwestern University (NW). Researchers have been conducting a pilot trial in HLA-identical sibling renal transplantation using multiple infusions of CD34-selected HSCT of donor iliac crest and/or CD34 selected G-CSF-mobilized cells with the underlying hypothesis that a Treg cell feedback loop will be augmented.56 The patients were not preconditioned. Two doses of alemtuzumab (D0 and D4) plus tacrolimus and MMF were administered. Three months posttransplant, tacrolimus was converted to Sirolimus. MMF was discontinued between 12 and 18 months, and immunosuppression was tapered and completely withdrawn by 24 months. Donor cryopreserved CD34+ cells (0.3-1.0 × 106 CD34+ cells/kg) collected from iliac crest were infused on day 5. Additionally, cryopreserved CD34+ cells from mPBMCs were infused at 3, 6, and 9 months postkidney transplant (Figure 3). Five of the 10 patients treated had normal protocol biopsies at 36 months after complete IS withdrawal (Table 1). Two nontolerant patients had recurrent renal disease and were maintained on IS drugs. The other 3 patients resumed IS after showing signs of subclinical rejection on protocol biopsy although their serum creatinine and panel-reactive antibodies remained low.56 Transient low levels of donor chimerism were present during the first year (3%) in the tolerant subjects. Prolonged CD4+ and CD8+ T cell depression, with an increase in Treg cells (CD4+ CD25+ CD127Foxp3+) cells was observed in both operationally tolerant and nontolerant patients. Global RNA profiling of whole blood and urine shows differential downregulation of genes associated with inflammatory pathways in tolerant patients compared with nontolerant patients.56 This study suggests that durable chimerism may not be required to induce operational tolerance between HLA-identical related donor/recipient pairs.56

FIGURE 3

FIGURE 3

In 2009, NW and the University of Louisville embarked on a trial of combined living donor kidney/G-CSF mobilized engineered donor stem cell transplantation to induce tolerance. As of June 2016, 31 subjects have been transplanted in this phase 2 protocol (IDE 13947). The protocol is based on the engineering of the donor stem cell transplant to enrich for tolerogenic CD8+/T cell receptor (TCR)-facilitating cells (FCRx), a heterogeneous population of cells in mice and humans that has been extensively characterized and described.57-60 Recipients are conditioned nonmyeloablatively with fludarabine (30 mg/m2 per dose, days −5, −4, −3), CyP (50 mg/kg per dose, day −3 and +3), 200 cGy TBI (day −1) followed by a living donor kidney transplant (day 0). Dialysis is performed in all subjects 8 to 10 hours after each dose of fludarabine. The conditioning regimen is modeled after the pioneering work by Fuchs et al, using G-CSF mobilized HSCT in subjects conditioned with fludarabine (D −6 to −2), 4 doses of CyP (D −6, −5, +3, +4), with 200 cGy TBI that was shown to significantly reduce transplant-related morbidity and the risk of GVHD in patients with hematologic malignancy (Figure 4).61 A G-CSF mobilized peripheral blood mononuclear cell product is apheresed from the donor at least 2 weeks before the kidney transplant, processed to remove graft-versus-host disease (GVHD)–producing cells yet retain CD34+ cells and FCRx, and cryopreserved until administration day +1 postkidney transplant. The subjects are discharged postoperative day 2 and managed thereafter as outpatients.

FIGURE 4

FIGURE 4

Subjects have ages ranging from 18 to 65 years and were from 6 of 6 HLA-matched related to 0 of 6 HLA-matched unrelated. Twelve subjects had unrelated and 19 had related donors. Thirty of 31 subjects exhibited donor chimerism at 1 month post-FCRx. The 1 subject without chimerism was highly sensitized (PRA > 50%). Subjects are maintained on MMF and tacrolimus SOC immunosuppression for the first 6 months. At 6 months, a protocol biopsy is performed. If the biopsy is free from rejection, and if renal function is stable and greater than 50% donor chimerism present, the MMF is discontinued. At 9 months, the tacrolimus is reduced to trough levels of 3 to 6. Tacrolimus is discontinued at 1 year if chimerism, normal renal function and normal KTx biopsy are present. There was a learning curve in the earliest phases of this study. Subjects NW1 and NW4 received a suboptimal cell dose and were only transiently chimeric (<6 months). Another subject (NW11) with a high PRA (33%, maximum historic 64%) demonstrated only transient chimerism. Exclusion criteria were subsequently modified after this to exclude recipients with a PRA greater than 20 and to target a minimum cell dose. Transiently chimeric subjects resumed endogenous hematopoiesis, and most of them are on low-dose tacrolimus monotherapy with stable renal function.

There have been 2 cases of GVHD in the Northwestern/Louisville experience (article in preparation). One subject (NW27) developed grade 1 to 2 skin and colonic GVHD during conversion from tacrolimus to sirolimus due to calcineurin inhibitors toxicity. This was successfully treated with corticosteroids. He is now off all IS drugs without severe active GVHD but has experienced mild cutaneous and ocular GVHD. He has returned to work, and his activity level is excellent. He has been off all immunosuppression for 15 months and has high levels of donor chimerism. A second subject (NW33) developed concomitant grade 2 to 3 colonic GVHD and severe cytomegalovirus (CMV) colitis at day 134 posttransplant. He initially presented to a community hospital after 5 days of bloody diarrhea where the working diagnosis was GVHD colitis. He was treated with corticosteroids. There was significant delay in diagnosis and treatment of his CMV Colitis, resulting in severe ulceration of his colonic mucosa. His disease proved to be treatment-resistant, and he ultimately expired due to septic complications. The protocol has been modified to require that the subjects be treated at a transplant center and not a local community hospital for any symptoms that develop. The subjects are also contacted weekly by a study nurse coordinator.

There have been 2 renal allograft losses. Both graft losses occurred in the first year posttransplant and were most likely attributable to the immunosuppression. Subject NW2 developed viral sepsis at month 3 and thrombosed his renal vein. He was successfully retransplanted off study. Subject 19 developed severe antibiotic resistant Klebsiella recurrent infections of his native polycystic kidneys and liver that contributed to KTx loss at 9 months.62

Durable chimerism allowing for full IS drug withdrawal has been achieved in 19 subjects (time off immunosuppression ranging from 3 to 65 months) (Table 1).62 The majority of chimeric subjects have “full” (>98% donor) chimerism, and 3 subjects mixed chimerism. All stable chimeric subjects retained chimerism after removal of IS and remain rejection-free, as demonstrated by normal protocol biopsy, whereas 3 of 5 who were transiently chimeric had subclinical rejection on protocol biopsy. In summary, high levels of durable chimerism and tolerance with a low incidence of GVHD have been achieved in mismatched related/unrelated recipients of combined FCRx and living donor KTx.

None of the fully chimeric patients developed recurrent autoimmune disease, whereas 2 subjects who were transiently chimeric developed recurrent disease. These small numbers support previously reported experiences in subjects with hematologic malignancy and underlying autoimmunity who experienced disease remission after HSCT.63 This novel FCRx-mediated transplant tolerance induction using reduced intensity conditioning is safe and has the potential to treat a variety of autoimmune diseases. The chimeric subjects demonstrated rapid T cell reconstitution with a diverse de novo T cell repertoire and did not develop any serious infections after being off IS drugs. Also, they were successfully vaccinated, had memory to hepatitis and responsive to pneumococcal vaccine, suggesting immune-competence.64-67

It is important to keep in mind the rationale for pursuing the goal for tolerance induction. Although short-term success in renal transplant recipients has significantly improved, long-term patient and graft survival have not. There is a significant risk of death and a fixed rate of graft loss over time as reported by Organ Procurement and Transplantation Network.68 It must be recognized that the up-front management and risk to cell-based therapies are greater than standard of care renal transplant but the potential lifelong benefit both in improved quality of life and survival is significant. Collectively, these pioneering studies have placed cell-based therapies at the forefront and as the approaches are tested and refined to ensure continued success chimerism-based tolerance may evolve to become standard of care and the preferred management for solid organ transplants with a goal to have 1 transplant for life.

Back to Top | Article Outline

REFERENCES

1. Russell JD, Beecroft ML, Ludwin D, et al. The quality of life in renal transplantation—a prospective study. Transplantation. 1992;54:656–660.
2. Merion RM, White DJ, Thiru S, et al. Cyclosporine: five years' experience in cadaveric renal transplantation. N Engl J Med. 1984;310:148–154.
3. Hariharan S, Johnson CP, Bresnahan BA, et al. Improved graft survival after renal transplantation in the United States, 1988 to 1996. N Engl J Med. 2000;342:605–612.
4. Meier-Kriesche HU, Schold JD, Kaplan B. Long-term renal allograft survival: have we made significant progress or is it time to rethink our analytic and therapeutic strategies? Am J Transplant. 2004;4:1289–1295.
5. Engels EA. Cancer in solid organ transplant recipients: there is still much to learn and do. Am J Transplant. 2017. [published online April 10 2017]. doi: 10.1111/ajt.14140.
6. Schaefer HM. Long-term management of the kidney transplant recipient. Blood Purif. 2012;33:205–211.
7. Lodhi SA, Meier-Kriesche HU. Kidney allograft survival: the long and short of it. Nephrol Dial Transplant. 2011;26:15–17.
8. Ojo AO, Held PJ, Port FK, et al. Chronic renal failure after transplantation of a nonrenal organ. N Engl J Med. 2003;349:931–940.
9. Owen RD, Davis HP, Morgan RF. Quintuplet calves and erythrocyte mosaicism. J Hered. 1946;37:290–297.
10. Billingham RE, Brent L, Medawar PB. Actively acquired tolerance of foreign cells. Nature. 1953;172:603–606.
11. Orlando G, Hematti P, Stratta RJ, et al. Clinical operational tolerance after renal transplantation: current status and future challenges. Ann Surg. 2010;252:915–928.
12. Auchincloss H Jr. In search of the elusive Holy Grail: the mechanisms and prospects for achieving clinical transplantation tolerance. Am J Transplant. 2001;1:6–12.
13. Shapiro R, Basu A, Tan H, et al. Kidney transplantation under minimal immunosuppression after pretransplant lymphoid depletion with thymoglobulin or campath. J Am Coll Surg. 2005;200:505–515.
14. Feng S, Ekong UD, Lobritto SJ, et al. Complete immunosuppression withdrawal and subsequent allograft function among pediatric recipients of parental living donor liver transplants. JAMA. 2012;307:283–293.
15. Yamada A, Konishi K, Cruz GL, et al. Blocking the CD28-B7 T-cell costimulatory pathway abrogates the development of obliterative bronchiolitis in a murine heterotopic airway model. Transplantation. 2000;69:743–749.
16. Zeng YJ, Ricordi C, Tzakis A, et al. Long-term survival of donor-specific pancreatic islet xenografts in fully xenogeneic chimeras (WF rat—B10 mouse). Transplantation. 1992;53:277–283.
17. Colson YL, Zadach K, Nalesnik M, et al. Mixed allogeneic chimerism in the rat. Donor-specific transplantation tolerance without chronic rejection for primarily vascularized cardiac allografts. Transplantation. 1995;60:971–980.
18. Pennington LR, Sakamoto K, Popitz-Bergez FA, et al. Bone marrow transplantation in miniature swine. I. Development of the model. Transplantation. 1988;45:21–26.
19. Popitz-Bergez FA, Sakamoto K, Pennington LR, et al. Bone marrow transplantation in miniature swine. II. Effect of selective genetic differences on marrow engraftment and recipient survival. Transplantation. 1988;45:27–31.
20. Sakamoto K, Sachs DH, Shimada S, et al. Bone marrow transplantation in miniature swine. III. Graft-versus-host disease and the effect of T cell depletion of marrow. Transplantation. 1988;45:869–875.
21. Huang CA, Fuchimoto Y, Scheier-Dolberg R, et al. Stable mixed chimerism and tolerance using a nonmyeloablative preparative regimen in a large-animal model. J Clin Invest. 2000;105:173–181.
22. Colby C, Chang Q, Fuchimoto Y, et al. Cytokine-mobilized peripheral blood progenitor cells for allogeneic reconstitution of miniature swine. Transplantation. 2000;69:135–140.
23. Fuchimoto Y, Huang CA, Yamada K, et al. Mixed chimerism and tolerance without whole body irradiation in a large animal model. J Clin Invest. 2000;105:1779–1789.
24. Lima B, Gleit ZL, Cameron AM, et al. Engraftment of quiescent progenitors and conversion to full chimerism after nonmyelosuppressive conditioning and hematopoietic cell transplantation in miniature swine. Biol Blood Marrow Transplant. 2003;9:571–582.
25. Horner BM, Cina RA, Wikiel KJ, et al. Predictors of organ allograft tolerance following hematopoietic cell transplantation. Am J Transplant. 2006;6:2894–2902.
26. Schwarze ML, Menard MT, Fuchimoto Y, et al. Mixed hematopoietic chimerism induces long-term tolerance to cardiac allografts in miniature swine. Ann Thorac Surg. 2000;70:131–138.
27. Kawai T, Cosimi AB, Colvin RB, et al. Mixed allogeneic chimerism and renal allograft tolerance in cynomolgus monkeys. Transplantation. 1995;59:256–262.
28. Kawai T, Poncelet A, Sachs DH, et al. Long-term outcome and alloantibody production in a non-myeloablative regimen for induction of renal allograft tolerance. Transplantation. 1999;68:1767–1775.
29. Kimikawa M, Kawai T, Sachs DH, et al. Mixed chimerism and transplantation tolerance induced by a nonlethal preparative regimen in cynomolgus monkeys. Transplant Proc. 1997;29:1218.
30. Kawai T, Sogawa H, Boskovic S, et al. CD154 blockade for induction of mixed chimerism and prolonged renal allograft survival in nonhuman primates. Am J Transplant. 2004;4:1391–1398.
31. Nadazdin O, Abrahamian G, Boskovic S, et al. Stem cell mobilization and collection for induction of mixed chimerism and renal allograft tolerance in cynomolgus monkeys. J Surg Res. 2011;168:294–300.
32. Kean LS, Adams AB, Strobert E, et al. Induction of chimerism in rhesus macaques through stem cell transplant and costimulation blockade-based immunosuppression. Am J Transplant. 2007;7:320–335.
33. Ramakrishnan SK, Page A, Farris AB 3rd, et al. Evidence for kidney rejection after combined bone marrow and renal transplantation despite ongoing whole-blood chimerism in rhesus macaques. Am J Transplant. 2012;12:1755–1764.
34. Sogawa H, Boskovic S, Nadazdin O, et al. Limited efficacy and unacceptable toxicity of cyclophosphamide for the induction of mixed chimerism and renal allograft tolerance in cynomolgus monkeys. Transplantation. 2008;86:615–619.
35. Storb R, Epstein RB, Bryant J, et al. Marrow grafts by combined marrow and leukocyte infusions in unrelated dogs selected by histocompatibility typing. Transplantation. 1968;6:587–593.
36. Storb R, Epstein RB, Le Blond RF, et al. Transplantation of allogeneic canine bone marrow stored at −80 degrees C in dimethyl sulfoxide. Blood. 1969;33:918–923.
37. Storb R, Raff RF, Appelbaum FR, et al. What radiation dose for DLA-identical canine marrow grafts? Blood. 1988;72:1300–1304.
38. Raff RF, Loughran TP Jr, Graham T, et al. Splenectomy does not enhance engraftment of DLA-nonidentical marrow transplants. Exp Hematol. 1993;21:385–387.
39. Colson YL, Wren SM, Schuchert MJ, et al. A nonlethal conditioning approach to achieve durable multilineage mixed chimerism and tolerance across major, minor, and hematopoietic histocompatibility barriers. J Immunol. 1995;155:4179–4188.
40. Xu H, Chilton PM, Huang Y, et al. Addition of cyclophosphamide to T-cell depletion based nonmyeloablative conditioning allows donor T-cell engraftment and clonal deletion of alloreactive host T-cells after bone marrow transplantation. Transplantation. 2007;83:954–963.
41. Storb R, Yu C, Wagner JL, et al. Stable mixed hematopoietic chimerism in DLA-identical littermate dogs given sublethal total body irradiation before and pharmacological immunosuppression after marrow transplantation. Blood. 1997;89:3048–3054.
42. Diaconescu R, Little MT, Leisenring W, et al. What role is there for antithymocyte globulin in allogeneic nonmyeloablative canine hematopoietic cell transplantation? Biol Blood Marrow Transplant. 2005;11:335–344.
43. Mathes DW, Hwang B, Graves SS, et al. Tolerance to vascularized composite allografts in canine mixed hematopoietic chimeras. Transplantation. 2011;92:1301–1308.
44. Graves SS, Mathes DW, Georges GE, et al. Long-term tolerance to kidney allografts after induced rejection of donor hematopoietic chimerism in a preclinical canine model. Transplantation. 2012;94:562–568.
45. Hartner WC, De Fazio SR, Maki T, et al. Prolongation of renal allograft survival in antilymphocyte-serum-treated dogs by postoperative injection of density-gradient-fractionated donor bone marrow. Transplantation. 1986;42:593–597.
46. Luznik L, O'Donnell PV, Symons HJ, et al. HLA-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide. Biol Blood Marrow Transplant. 2008;14:641–650.
47. Spitzer TR, Delmonico F, Tolkoff-Rubin N, et al. Combined histocompatibility leukocyte antigen-matched donor bone marrow and renal transplantation for multiple myeloma with end stage renal disease: the induction of allograft tolerance through mixed lymphohematopoietic chimerism. Transplantation. 1999;68:480–484.
48. Chen YB, Kawai T, Spitzer TR. Combined bone marrow and kidney transplantation for the induction of specific tolerance. Adv Hematol. 2016;2016:6471901.
49. Fudaba Y, Spitzer TR, Shaffer J, et al. Myeloma responses and tolerance following combined kidney and nonmyeloablative marrow transplantation: in vivo and in vitro analyses. Am J Transplant. 2006;6:2121–2133.
50. Spitzer TR, Sykes M, Tolkoff-Rubin N, et al. Long-term follow-up of recipients of combined human leukocyte antigen-matched bone marrow and kidney transplantation for multiple myeloma with end-stage renal disease. Transplantation. 2011;91:672–676.
51. Kawai T, Sachs DH, Sykes M, et al. HLA-mismatched renal transplantation without maintenance immunosuppression. N Engl J Med. 2013;368:1850–1852.
52. Kawai T, Chen YB, Sykes M, et al. HLA identical or haploidentical combined kidney and bone marrow transplantation for multiple myeloma with end-stage renal failure. Am J Transplant. 2016;16(Suppl 3):270.
53. Strober S. Use of hematopoietic cell transplants to achieve tolerance in patients with solid organ transplants. Blood. 2016;127:1539–1543.
54. Palathumpat V, Dejbakhsh-Jones S, Strober S. The role of purified CD8+ T cells in graft-versus-leukemia activity and engraftment after allogeneic bone marrow transplantation. Transplantation. 1995;60:355–361.
55. Scandling J, Busque S, Shizuru J, et al. Chimerism, graft survival, and withdrawal of immunosuppressive drugs in HLA matched and mismatched patients after living donor kidney and hematopoietic cell transplantation. Am J Transplant. 2015;15:695–704.
56. Leventhal JR, Mathew JM, Salomon DR, et al. Genomic biomarkers correlate with HLA-identical renal transplant tolerance. J Am Soc Nephrol. 2013;24:1376–1385.
57. Kaufman CL, Colson YL, Wren SM, et al. Phenotypic characterization of a novel bone-marrow derived cell that facilitates engraftment of allogeneic bone marrow stem cells. Blood. 1994;84:2436–2446.
58. Fugier-Vivier I, Rezzoug F, Huang Y, et al. Plasmacytoid precursor dendritic cells facilitate allogeneic hematopoietic stem cell engraftment. J Exp Med. 2005;201:373–383.
59. Colson YL, Shinde Patil VR, Ildstad ST. Facilitating cells: novel promoters of stem cell alloengraftment and donor-specific transplantation tolerance in the absence of GVHD. Crit Rev Oncol Hematol. 2007;61:26–43.
60. Huang Y, Elliott MJ, Yolcu ES, et al. Characterization of human CD8(+)TCR(-) facilitating cells in vitro and in vivo in a NOD/SCID/IL2rγ(null) mouse model. Am J Transplant. 2016;16:440–453.
61. Brodsky RA, Luznik L, Bolanos-Meade J, et al. Reduced intensity HLA-haploidentical BMT with post transplantation cyclophosphamide in nonmalignant hematologic diseases. Bone Marrow Transplant. 2008;42:523–527.
62. Leventhal J, Galvin J, Stare D, et al. Seven year follow-up of a phase 2 clinical trial to induce tolerance in living donor renal transplant recipients. Am J Transplant. 2016;16(Suppl 3):269–270.
63. Leventhal J, Miller J, Abecassis M, et al. Evolving approaches of hematopoietic stem cell-based therapies to induce tolerance to organ transplants: the long road to tolerance. Clin Pharmacol Ther. 2013;93:36–45.
64. Leventhal J, Abecassis M, Miller J, et al. Chimerism and tolerance without GVHD or engraftment syndrome in HLA-mismatched combined kidney and hematopoietic stem cell transplantation. Sci Transl Med. 2012;4:124ra28.
65. Leventhal JR, Abecassis M, Miller J, et al. A phase 2 clinical trial of donor-specific tolerance induction in recipients of HLA disparate living donor kidney allografts by donor stem cell infusion. Am J Transplant. 2012;12(Suppl s3):27.
66. Leventhal JR, Mathew JM, Ildstad S, et al. HLA identical non-chimeric and HLA disparate chimeric renal transplant tolerance. Clin Transpl. 2013:145–156.
67. Leventhal JR, Elliott MJ, Yolcu ES, et al. Immune reconstitution/immunocompetence in recipients of kidney plus hematopoietic stem/facilitating cell transplants (under review). Transplantation. 2015;99:288–298.
68. US Department of Health & Human Services, Health Resources & Services Administration. Organ Procurement and Transplantation Network. https://optn.transplant.hrsa.gov/. Updated 2017.
Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved.