CD4+ FoxP3+regulatory T (Treg) cells modulate autoimmune and alloimmune responses.1-5 Induction of kidney allograft tolerance, via transient mixed hematopoietic chimerism and nonmyeloablative conditioning, has been achieved in large animal models,6 and humans.7 However, kidney allograft tolerance was achieved in only 60% to 70% of cynomolgus monkeys (cynos) and humans, and tolerance could not be readily extended to islet, heart or lung allografts in monkeys.8-10 Although durable mixed chimerism has been achieved with total lymphoid radiation, antithymocyte globulin and donor kidney transplantation in the HLA-identical transplant setting, this approach has not yet succeeded in achieving durable chimerism or tolerance across HLA barriers.11-14 Another approach achieves renal allograft tolerance with development of full donor chimerism across extensive HLA barriers,15,16 but the full donor chimerism likely reflects the more rigorous and potentially toxic host conditioning and/or graft-versus-host reactivity of the infused donor T cells, which eliminates recipient hematopoiesis, and high rates of opportunistic infection were observed.17 Mixed chimerism, in contrast, provides a steady supply of recipient-derived antigen presenting cells (APCs), conferring superior ability to mount cytotoxic T cell responses that clear viral infections compared to full chimeras.17-20 Thus, the reliable achievement of durable mixed chimerism across HLA barriers, with its potential to induce tolerance to any type of donor organ and to cure congenital hematologic disorders, remains an important and elusive goal in humans.21,22
In mice, adoptive transfer of recipient blood-derived natural Treg cells at the time of bone marrow transplant (BMT) with minimal conditioning regimen permitted the establishment of permanent hematopoietic mixed chimerism and skin allograft tolerance.23-26 We have adapted the use of Treg cells for the abovementioned cyno model that otherwise achieves only transient mixed hematopoietic chimerism and which has been extensively characterized.8-10 We tested the hypothesis that the addition of expanded recipient Treg cells to the “standard” conditioning protocol would promote durable chimerism and allow acceptance of a donor kidney after a marked delay of 4 months, when donor kidneys are uniformly rejected by transient chimeras prepared with this protocol.27
MATERIALS AND METHODS
Male adult cynos (Charles River Primates, Wilmington, MA and Sanofi-Synthelabo, Bridgewater, NJ) were used. All procedures were approved by the IACUC of Columbia University and Massachusetts General Hospital (MGH). Both are AAALAC international accredited institutions.
Cynomolgus Major Histocompatibility Complex Genotyping
Peripheral blood mononuclear cell (PBMCs) were genotyped at the University of Wisconsin Primate Research Center Laboratory http://www.primate.wisc.edu/wprc/services/genetics.html.28-31
Recipients of major histocompatibility complex (MHC) mismatched donor BMT (Table 1 and Figure S1, SDC,https://links.lww.com/TP/B373) underwent the “standard” conditioning regimen as previously described6,32 +/− Treg cells (Figure 1A). Cyclosporine levels were maintained between 200-400 ng/mL.
Treg Cell Sorting and Expansion
The 1.0% of CD4+ T cells expressing the highest levels of CD25 were sorted (FACSAria or Influx, BD Biosciences, Billerica, MA) and plated (1 × 105 cells/cm2) on fibroblasts (L929) (10 × 105 cells/cm2) expressing human CD32 (FcR), CD58 (LFA-3) and CD8033,34 (referred as artificial APCs [aAPC]) in combination with human recombinant IL-2 (200 U/mL), anti-CD3 (SP-34) 100 ng/mL, and rapamycin 100 μg/mL (Sigma-Aldrich, St Louis, MO) for 7 days. Growth medium consisted of RPMl-1640 (Gibco), fetal calf serum (Gibco), L-glutamine, penicillin/streptomycin, and nonessential amino acids. After 7 days, cells were replated with irradiated donor PBMCs (1 PBMC to 1 Treg cell) and IL-2 (200 U/mL), anti-CD3 (SP-34) 1 μg/mL or alternatively in combination with aAPCs (3 × 105 to 5 × 105 cells per cm2) (Table 2). Cells were cultured for another 7 days, then split and cultured for another 7 days. When irradiated aAPCs were used, Treg cells were cultured for an additional 5 days in the presence of rapamycin 100 ng/mL (Table 2). After expansion, cells were cryopreserved in fetal calf serum (Gibco) with 5% dimethyl sulfoxide for future use.
Bone marrow (BM) was harvested aseptically from donor iliac bones by multiple percutaneous aspirations or surgically from the vertebrae. BM cells (1.3-3.0 × 108 mononuclear cells/kg) were infused intravenously. CD34+ content was 1% (+/− 0.4%), as determined by flow cytometry.
The details of the kidney transplant procedure were reported previously.35 Kidneys were transplanted between days 119 and 134 post-BMT. Recipients underwent unilateral native nephrectomy and ligation of the contralateral ureter on the day of transplant. The remaining native kidney was removed ~100 days after transplantation.
Flow Cytometric Analyses, Detection of Chimerism, and Cell Sorting
Whole blood was lysed and labeled with a combination of the following mAbs: CD3 PerCPCy5.5 (SP34.2), CD4-APC (L200), CD4-PE (L200), CD8-APC (SK1), CD11b-PE (ICRF44). CD20-PE (2H7), CD25-PE (BC96), CD31-PE (WM59), CD56-PE (MY31), pan-MHC A.B.C-PE (W6/32), FOXP3-PE (236A/E7). For chimerism analysis, we used H38 (anti-BW6; One Lambda, Inc., Canoga Park, CA). The recipient and donor pairs were chosen based on their MHC haplotypes and H38 expression. The fluorescence of the stained samples was analyzed using FACS Calibur and FlowJo software.
Mixed Lymphocyte Reactions and Treg Cell Suppression Assays
Mixed lymphocyte reactions (MLRs) were performed as previously described.6 In addition, Treg cells were titrated for their specificity in suppressing host antidonor versus third party and donor antihost versus third party responses. Donor or host PBMC responders were stimulated with irradiated host, donor, or third-party PBMCs. Host nonirradiated Treg cells were added to the culture and pulsed with tritiated thymidine 4 days after initiation of culture and read in a beta counter as previously described.6 Treg cells were also tested for suppression of anti–CD2-, anti–CD3-, and anti–CD28-coated NHP activation bead-mediated activation (Miltenyi Biotec) at 1 bead to every 2 PBMCs.
Expansion, Phenotype, and Suppression of Cyno CD4+ CD25high
An average of 118 907 ± 9588 CD4+ CD25high cells were sorted from each blood draw. Usually a 10- to 100-fold expansion was achieved within the first 7 (0.695 × 106 ± 0.175 × 106) to 14 (22.47 × 106 ± 4.3 × 106) days of culture (Figure 1B, representative lines). At the end of culture, Treg cells were analyzed for phenotype (Figure 1C) and function (Figures 2A, B) before cryopreservation.
Infused Treg cell expressed high levels of FOXP3 and CD25 (Figure 1C) (Table S1, SDC,https://links.lww.com/TP/B373). Inhibition of the proliferation of bead- (anti-CD2/CD3/CD28) stimulated autologous (cryopreserved pretransplant) PBMCs generally revealed greater than 95% suppression at a 1:1 ratio of PBMCs/Treg cell (Figures 2A and B). The infused Treg cells varied in suppressive potency but all achieved 50% suppression at or above a 1:2 Treg cell/PBMC ratio (Figures 2A and B) (Table S1, SDC,https://links.lww.com/TP/B373).
We aimed to generate polyclonal, nonspecifically suppressive Treg cell lines with our expansion protocol. While donor PBMCs were added during the expansion period as a source of APCs, specificity studies on 2 different Treg cell lines (Figures 2C-F) revealed similar suppression of host antidonor, host anti-third–party, antihost, and donor anti–third-party responses.
Proof of Concept that Treg Cell Infusion Can Prolong Multilineage Donor Cell Chimerism
We tested whether polyclonal Treg cells could prolong donor hematopoietic chimerism compared to controls, which historically achieved transient (30-60 days) chimerism.6 Three control animals were treated as previously described,6 except they did not receive a donor kidney graft on Day 0. Five animals received the same treatment plus Treg cell infusions posttransplant. These 5 animals (M5210, 90-39, 6c64, 6c1, 90-15) received expanded polyclonal autologous Treg cells (15-53 × 106 per infusion) during the first week posttransplant (days 0, 2, 5, 7) and on day +50 (Table S1, SDC,https://links.lww.com/TP/B373). Total dose was 88-96 × 106/kg. Two Treg cell recipients, M5210 and 90-39, developed significant multilineage chimerism (Figure 3, top row). Although M5210 survived long-term, animal 90-39 died of cytomegalovirus (CMV) disease on day 43 with significant donor chimerism in all lineages (Figure 3). The chimerism in M5210 (Figure 3, top left panel) persisted longer than ever observed in this model, remaining detectable in the lymphoid, monocyte and granulocyte lineages until days 292, 224, and 335, respectively.
We monitored CMV viremia, and when it exceeded 10 000 copies/mL (initially) or 1000 copies/mL (after our experience in the first few animals), we treated animals with Ganciclovir and/or Foscarnet. Animal 6c64 was given antiviral prophylaxis to prevent CMV reactivation and developed only low and short-lived chimerism (Figure 3) with prolonged pancytopenia, suggesting that BM-toxic effects of the antiviral treatment may have impaired both donor and recipient hematopoiesis. Two additional Treg cell recipients, 6c1 and 90-15, experienced CMV reactivation with high viral loads and required treatment with antivirals at high doses within the first week posttransplant. These animals developed only short-lived and low levels of chimerism (6c64, 6c1, 90-15 shown in Figure 3, middle row) in association with protracted cytopenias, often requiring transfusions. These results suggest that CMV reactivation and/or the bone marrow toxic effects of early antiviral therapy may have potentially interfered with initial engraftment of the donor marrow.
Of the 3 control animals (90-47,90-7,90-1) receiving BMT without Treg cell infusion, 1 (animal 90-47) died of CMV before the development and implementation of the CMV surveillance and treatment protocol, without showing any significant chimerism (Figure 3, bottom row, left panel). The 2 other controls survived long term. Animal 90-7 also developed low-level, short-lived chimerism. The third control animal, 90-1, was unique in that both it and the donor were CMV-negative and chimerism lasted over 100 days before disappearing.6,32
In summary, recipients that reactivated CMV, regardless of Treg cell infusion, succumbed to disease if not treated promptly with antivirals. Early CMV reactivation and its treatment or prophylaxis were associated with very short-lived chimerism. Only 2 animals survived without antiviral treatment (1 Treg cell recipient and 1 CMV-negative non-Treg cell control.) The Treg cell recipient, M5210, had only a very low level CMV viremia (<1000 copies/mL) and exhibited the longest documented donor chimerism ever seen with this or related protocols over a period of more than twenty years.
Only Treg Cell Recipients Developed T Cell Chimerism
The 3 evaluable (ie, that were not treated early with antivirals) animals that had measurable lymphoid chimerism included Treg cell recipients M5210 and 90-39 and CMV(−) control recipient 90-1 (Figure 4). However, in the non–Treg cell recipient 90-1 (the control animal in which donor and recipient were CMV negative), lymphoid chimerism included NK cells (data not shown) and B cells (eg, Figure 4B, left), but did not include significant donor T cell chimerism. In contrast, both evaluable Treg cell recipients had not only B cell and NK cell chimerism, but also significant CD4 and CD8 T cell chimerism (Figures 4B, center and C, right). In M5210, the long-lived Treg cell recipient, T cell chimerism first appeared 45 days post-BMT (2.5 weeks after cyclosporine had been discontinued) and increased significantly on day +60 post-BMT. Similarly, Treg cell recipient 90-39 (which died of CMV on day + 43) had a spike in donor T cell chimerism in the peripheral blood 1 month after BMT (at the time immunosuppression was discontinued) (Figure 4B, right) and still had peripheral blood T cell chimerism (5%, mostly in CD4 T cells) on the day of euthanasia. These results represent the first time that T cell chimerism has been observed using this nonmyeloablative monkey BMT model and suggest that Treg cells promote T cell chimerism.
We then investigated, in the only long-lived Treg cell recipient chimera, the phenotype of host and donor T cells, including CD31, a marker expressed on new thymic emigrants36 and CD45RA, a marker of naïve T cells, among both donor and recipient T cells (Figure 5). Almost all donor CD4 and CD8 T cells in M5210 expressed CD31 throughout follow-up (Figures 5A and B). Consistent with de novo origin in the recipient thymus, the expression of CD45RA was also very high on donor CD4+ T cells (Figure 5C), peaking close to 90%. For recipient T cells, expression of CD31 in both CD4 and CD8 cell populations was significantly less. However, CD31 expression increased markedly in host T cells (80%) early after the transplant (Figures 5A-B), suggesting that a wave of new host T cells was released from the thymus after transplant. The percentage of host-derived CD31+ T cells slowly decreased from day+/−20 until day +50. CD45RA expression on recipient CD4+ T cells peaked at 50% at about 1 month posttransplant. The expression of CD31 and CD45RA was lower in animals that did not develop donor T cell chimerism, as shown in Figures 5A to C (bottom rows) for animal 90-7, a control BMT recipient that did not receive Treg cells. In summary, donor T cells exhibited high levels of CD45RA and CD31, suggesting de novo development from the thymus in animal M5210.
Kinetics of CD3+ CD4+ FOXP3+ Cells in the Circulation After Infusion
Peripheral Treg cell counts and percentages were similar in the 3 controls and the 2 evaluable Treg cell recipients that developed high levels of chimerism (M5210 and 90-39) (Figure 5D). Treg cells were largely of recipient origin (Figure S2, SDC,https://links.lww.com/TP/B373). A peak was observed on day +50 in M5210 after the infusion of 28 million Treg/kg, which was given in an effort to reverse a sudden increase in the absolute number of CD95+CD28− effector CD8 T cells (Figure 5E) of mostly recipient origin (Figure 4B, middle panel) 20 days after the discontinuation of cyclosporine A (levels on day + 48 were subtherapeutic at 118 ng/mL and low on day +53 at 35 ng/mL). CD8 T cell counts declined after the infusion of Treg cells on day +50 (Figure 5E). Both the T cell chimerism (Figure 4B) and the myeloid chimerism (Figure 3, top left panel) increased shortly after the Treg cell infusion and remained stable for an additional 80 days (Figure 3, top left). Of note, on day 80 post-BMT, there was a second increase in the absolute CD8 T cell count that was followed by a subsequent spontaneous Treg cell increase, after which CD8 counts normalized (Figure 5E).
In summary, infusion of Treg cells was associated with the development of T cell chimerism, prolonged multilineage chimerism, and reversal of increasing recipient effector CD8+ T cell counts in animal M5210.
Proof of Principle: Persistent Chimerism Induced by Treg Cell Treatment Was Associated With In Vitro Donor-Specific Hyporesponsiveness and Allograft Tolerance
M5210 showed donor-specific unresponsiveness in MLR at day 106, before kidney transplantation was performed (Figure 6B), whereas strong proliferative responses to the donor were present pre-BMT (Figure 6A). Similar responses to third party were observed pretransplantation and posttransplantation.
Animals were challenged with a solid organ allograft (a kidney from the same BMT donor) 4 months after the original BMT, without immunosuppression. Only 1 Treg cell recipient (M5210) was evaluable at the 4-month timepoint. At the time of kidney transplant, M5210 remained chimeric in all lineages (Figures 3 and 4). The recipient's contralateral ureter was ligated on day 0 and on day 100 postkidney transplant the recipient's contralateral kidney was removed. Serum creatinine levels (Figure 6E) remained normal and stable until the day of euthanasia 293 days postkidney transplant, demonstrating tolerance to the donor kidney. Histopathology on day +294 postkidney transplant showed no evidence for rejection in M5210 (Figure 6F).
Two control animals that underwent the same protocol (without Treg cells) were also grafted with a kidney from their same BM donor 4 months post-BMT. In contrast to M5210, the 2 controls rejected their donor kidneys within a month (Figure 6E), in line with previous results.27 The donor kidneys in nonchimeric control animals showed Banff grade 3 rejection (Figures 6F and G) at the time of euthanasia. Nonchimeric Treg cell recipients (ie, animals that received Treg cells but had short-lived hematopoietic chimerism in association with early CMV reactivation and treatment) retained antidonor proliferative responses (Figures 6C and D) and rejected donor kidneys on day +120 post-BMT (n = 2), similar to controls (data not shown). These results are proof of concept and suggestive of the importance of mixed chimerism in tolerance induction in this model. No or minimal antidonor alloantibody was detected in animals that rejected their donor kidneys (Figure S3, SDC,https://links.lww.com/TP/B373).
In summary, control recipients rejected the donor kidneys uniformly, whereas the only evaluable long-term surviving Treg cell recipient M5210 maintained normal kidney function until termination of the experiment. This result provides proof-of-principle that prolongation of chimerism using expanded Treg cells can promote more robust tolerance than that achieved in previous studies using this model.6,27,32
Our studies provide proof-of-concept that expanded recipient-derived polyclonal Treg cells can increase and extend donor hematopoietic chimerism and promote robust allograft tolerance across MHC barriers in a NHP nonmyeloablative BMT model without an increased risk of GVHD. Infusion of Treg cell is an attractive approach to overcoming HvG responses, because it may further reduce the risk of GVHD37 rather than increasing this risk or the overall toxicity of the conditioning regimen like most other approaches.
Phase I clinical trials using Treg cells have shown safety,38 but efficacy remains to be proven. Both induced and natural Treg cell promoted engraftment, stable mixed chimerism and tolerance in mice under a minimal conditioning protocol in which the BM is otherwise rejected.24,26 We demonstrate in a monkey model that host Treg cells improved the level and duration of chimerism, extending it to the T cells. Moreover, robust donor-specific tolerance was achieved in 1 evaluable animal such that a donor kidney grafted at 4 months posttransplant was accepted without immunosuppression. Previous studies using this protocol without Treg cells, in which donor BM and kidney were co-transplanted on day 0, were associated with long-term kidney graft survival in about 60% of animals6,39 and donor hematopoietic chimerism (in some animals reaching +/−85%) consistently disappeared by day 60 post-BMT.32 A delay in grafting a donor kidney to more than 3 months post-BMT was always associated with rejection of the donor graft.27
Previous work in NHPs (rhesus macaques) using a nonmyeloablative BMT regimen with co-stimulatory blockade achieved prolonged levels of donor chimerism as long as basiliximab and belatacept were infused. Chimerism was lost after discontinuation of this treatment,40 and allograft tolerance was not achieved. In contrast, an animal in our study retained chimerism to 335 days and accepted a donor kidney grafted at 4 months, despite stopping immunosuppressive monotherapy at 28 days post-BMT.
In mice, we have shown that the presence of T cell chimerism is associated with early and long-term deletional tolerance, because thymic engraftment of donor T cell progenitors reflects successful ablation of intrathymic alloreactivity, permitting intrathymic engraftment of both thymocyte progenitors and donor APCs that contribute to negative selection of donor-reactive T cells.22,41–43 For the first time in the more than 20 years using this monkey nonmyeloablative BMT regimen, we have obtained evidence of de novo donor thymopoiesis, with T cell chimerism consisting of recent thymic emigrants in the peripheral blood. CD45RA and CD31 expressions suggested that almost all donor cells were newly-developed, whereas recipient T cells were a mix of new thymic emigrants and naive or memory T cells that evaded host conditioning.
CMV reactivation presented a major impediment to achieving the goals of our studies. Although this complication has not been described in previous studies using the model we adopted, uniform CMV reactivation has been reported in cynomolgus monkeys receiving Thymoglobulin.44 CMV reactivation was not directly caused by the infusion of Treg cells because control animals had a similar rate of reactivation. Only 1 animal, M5210, was able to control CMV without antiviral treatment. Because CMV itself can directly affect BM function45-47 and antiviral treatments are known to be BM toxic, modifications to the protocol are needed. The increased duration and level of chimerism observed in the 1 CMV-negative (non–Treg cell recipient) transplant (albeit without T cell chimerism or tolerance) supports a direct role for CMV and/or its treatment in limiting hematopoietic engraftment. We are currently exploring substitution of the mTOR inhibitor rapamycin for CSA to better control CMV reactivation and enhance Treg cell function, expansion, and survival.24,48
In summary, we provide proof-of-concept that BM plus expanded cryopreserved polyclonal recipient Treg cell can prolong donor chimerism, promote T cell chimerism and induce robust tolerance without an increase of toxic conditioning intensity or GVHD risk in a preclinical monkey model. Successful refinement of this protocol has the potential to be translated to the clinic.
The authors thank Dr. Remi Creusot for the critical review of the article.
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