CD4+ FoxP3+ regulatory T (Treg) cells modulate autoimmune and alloimmune responses.1-5 6 7 8-10 11-14 15,16 17 17-20 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 8-10 27
MATERIALS AND METHODS
Animals
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
Conditioning Regimen
Recipients of major histocompatibility complex (MHC) mismatched donor BMT (Table 1 and Figure S1, SDC, https://links.lww.com/TP/B373 6,32 Figure 1 A). Cyclosporine levels were maintained between 200-400 ng/mL.
TABLE 1: Donor: recipient MHC mismatches (refer to
Figure S1, SDC, https://links.lww.com/TP/B373 )
FIGURE 1: Transplant scheme and Treg cell expansion. A, Transplant protocol. B, Expansion of Treg cell lines from 4 animals over 4 weeks. The average number of cells for each animal at each expansion timepoint is graphed (SEM) (bars). C, A representative phenotype of Treg cells at the end of culture, with high levels of CD25 and of FOXP3.
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 5 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.
TABLE 2: Treg cell protocols
BMT
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.
Kidney Transplantation
The details of the kidney transplant procedure were reported previously.35
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 6
RESULTS
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 1 B, representative lines). At the end of culture, Treg cells were analyzed for phenotype (Figure 1 C) and function (Figures 2 A, B) before cryopreservation.
FIGURE 2: Culture of cynomolgus regulatory T cells. A, Highest quality Treg cell suppressed over 50% the proliferation of bead-stimulated (anti-CD2CD3CD28) PBMCs at 1:32 Treg cell/PBMC ratio. B, All Treg cell lines achieved at least 50% suppression of proliferation at a 1:2 Treg cell/PBMC ratio. Microsuppression assays shown. (C-F) MLRs assessing the specificity of host Treg cell. Host (C, D) and donor (E, F) PBMC responders were plated with either host, donor, or third-party stimulators. Host Treg cells were added to the cultures at the indicated PBMC/Treg cell ratios (1:1, 1:2 and 1:4) and assessed for suppressive activity. All data points represent means of triplicates. Error bars indicate SE. Similar results were obtained in a repeat experiment (not shown).
Infused Treg cell expressed high levels of FOXP3 and CD25 (Figure 1 C) (Table S1, SDC, https://links.lww.com/TP/B373 Figures 2 A 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 2 A 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 2 C-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 6 6 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 6 /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.
FIGURE 3: Summary of percent donor chimerism. Granulocyte (black circles), monocyte (blue triangles) and lymphocyte (red squares) lineages of each animal are shown. Animals with boxed identification numbers (M5210, 90-39, and 90-1) received no antiviral treatments. Animals whose figures have a cross died or were euthanized due to untreated or unmanageable CMV disease. Animal 90-47 was serologically CMV− pre-Tx, but developed CMV after BMT from a CMV+ donor that had been serologically negative on initial screen. Animal 90-1, who was CMV− pre-Tx and received a BMT from a CMV− donor, never developed CMV. The 5 animals shown on the top part of the figure received BMT + Treg cells, whereas the 3 below the dotted line received BMT without Treg cells.
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 4 B, 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 4 B, 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 4 B, 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.
FIGURE 4: Chimerism analysis of BMT recipients. A, Representative flow cytometry of animal M5210 on day + 99 post-BMT. Donor chimerism is measured with the Bw6+ (MHC-I) marker. B cell (CD20+ ), monocyte (CD11b+ ) and T cell (CD3, CD4 and CD8) chimerism measured among cells with low/medium forward and side scatter (not shown). Granulocytes (CD11b+) were analyzed among cells with high forward and side scatter (not shown). B, B cell (black circle), CD4 (red triangle) and CD8 (blue square) T cell chimerism in the 3 animals with the highest and most prolonged chimerism. CMV− control animal 90-1 developed high levels B cell chimerism, but no T cell chimerism and chimerism declined after discontinuation of immunosuppression and was completely lost by day 110. Treg cell recipient M5210 developed high B cell chimerism and delayed, prolonged T cell chimerism in both CD4 and CD8 T cell lineages. Chimerism lasted over 300 days post-BMT. Treg cell recipient 90-39 was euthanized at day 43 due to CMV disease. At the time of euthanasia B cell and CD4 T cell chimerism was detectable in the peripheral blood.
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 Figure 5 ). Almost all donor CD4 and CD8 T cells in M5210 expressed CD31 throughout follow-up (Figures 5 A 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 5 C), 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 5 A-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 5 A 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.
FIGURE 5: Immune reconstitution of animal M5210 Post-BMT (+Treg cell) and 90-7 (control). (A-C, top row M5210 and bottom row 90-7 control). Dotted lines indicate first detection of donor chimerism in CD4 or CD8 T cells. (A, top) Total CD4 T cells (black circle) expressed CD31 at increased levels posttransplant. Donor CD4 T cells (blue squares) maintained high CD31 expression. Recipient-derived CD4 T cells (red triangles) expressed lower levels of CD31. (B, top) Total CD8 T cells expressed high CD31 levels after transplant (black circle). CD31 expression in host CD8 T cells (red triangles) decreased, however donor-derived cells (blue squares) maintained high CD31 expression long after transplant. (C, top) Donor-derived CD4 T cells (blue squares) expressed higher levels of CD45RA compared to recipient CD4 T cells (red triangles). In contrast, animal 90-7 never exhibited any donor chimerism (A-C, bottom row) and the levels of CD31 and CD45 were lower than those observed in M5210 donor (blue) T cell populations. D, Average percentage of CD4 T cells expressing FOXP3 after transplant in Treg cell recipients (red triangles) (n = 2 animals) compared to control animals (blue squares) (n = 3). (E) Animal M5210 absolute Treg cell numbers (red triangles) compared with the absolute number of CD8 T cells (blue squares).
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 5 D). Treg cells were largely of recipient origin (Figure S2, SDC, https://links.lww.com/TP/B373 + CD28− effector CD8 T cells (Figure 5 E) of mostly recipient origin (Figure 4 B, 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 5 E). Both the T cell chimerism (Figure 4 B) 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 5 E).
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 6 B), whereas strong proliferative responses to the donor were present pre-BMT (Figure 6 A). Similar responses to third party were observed pretransplantation and posttransplantation.
FIGURE 6: Tolerance to the donor in M5210 Treg cell recipient but not in control. A, Pretransplant MLR in animal M5210 demonstrates strong proliferative responses to donor (diagonal stripes) and weaker response to third party (horizontal stripes). B, Day 106 post-Tx MLR (before kidney allograft from the same BM donor). Proliferation is maintained to third party but not to donor. C, Animals that lost chimerism never became tolerant nor showed decreased antidonor proliferation. An example is shown in (C) pretransplant and (D) posttransplant day 78. (E) After kidney transplant, creatinine levels in M5210 (black circle) stayed in the normal range while that in 2 control (non-Treg cell) animals (dashed grey lines) showed increases 2 to 3 weeks after kidney transplant. F, G, Kidney histopathology. Biopsies were taken from transplanted donor kidneys at the time of euthanasia. Shown is day +28 and day +294 (day of euthanasia) post-kidney transplant in animals 90-1 (F) (control, left) and M5210 (G) (+Treg cell, right) respectively. 90-1 had extensive lymphocyte infiltrates scored as a Banff grade 3 rejection, while M5210 showed no signs of rejection.
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 6 E) 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 6 F).
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 6 E), in line with previous results.27 Figures 6 F 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 6 C 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
DISCUSSION
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
Phase I clinical trials using Treg cells have shown safety,38 24,26 6,39 32 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
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
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 45-47 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.
ACKNOWLEDGMENTS
The authors thank Dr. Remi Creusot for the critical review of the article.
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