Multiple infusions of ex vivo-expanded regulatory T cells promote CD163+ myeloid cells and kidney allograft survival in non-lymphodepleted non-human primates

Clinical verification of adoptively transferred regulatory T cell (Treg) efficacy in transplantation remains challenging. Here, we examined the influence of autologous ex vivo-expanded polyclonal Tregs on kidney graft survival in a clinically relevant non-human primate model. Peripheral blood Tregs were isolated and expanded using artificial antigen presenting cells. Immunosuppression was comprised of tapered tacrolimus and CTLA4 immunoglobulin, in five animals each without or with Treg infusions. Escalating Treg doses were administered 6, 10, 13, 16, 20, 23, 27 and 30 days after transplant. Infused Tregs were monitored for Treg signature, anti-apoptotic (Bcl-2) and proliferation (Ki67) marker expression. Treg infusions prolonged median graft survival time significantly from 35 to 70 days. Treg marker (Ki67 and Bcl-2) expression by infused Tregs diminished after their infusion but remained comparable to that of circulating native Tregs. No major changes in circulating donor-reactive T cell responses or total Treg percentages, or in graft-infiltrating T cell subsets were observed with Treg infusion. However, Treg infusion was associated with significant increases in CD163 expression by circulating HLA-DR+ myeloid cells and elevated levels of circulating soluble CD163. Further, graft-infiltrating CD163+ cells were increased with Treg infusion. Thus, multiple Treg infusions were associated with M2-like myeloid cell enhancement that may mediate immunomodulatory, anti-inflammatory and graft reparative effects. Clinical verification of adoptively transferred regulatory T cell (Treg) efficacy in transplantation remains challenging. Here, we examined the influence of autologous ex vivo-expanded polyclonal Tregs on kidney graft survival in a clinically relevant non-human primate model. Peripheral blood Tregs were isolated and expanded using artificial antigen presenting cells. Immunosuppression was comprised of tapered tacrolimus and CTLA4 immunoglobulin, in five animals each without or with Treg infusions. Escalating Treg doses were administered 6, 10, 13, 16, 20, 23, 27 and 30 days after transplant. Infused Tregs were monitored for Treg signature, anti-apoptotic (Bcl-2) and proliferation (Ki67) marker expression. Treg infusions prolonged median graft survival time significantly from 35 to 70 days. Treg marker (Ki67 and Bcl-2) expression by infused Tregs diminished after their infusion but remained comparable to that of circulating native Tregs. No major changes in circulating donor-reactive T cell responses or total Treg percentages, or in graft-infiltrating T cell subsets were observed with Treg infusion. However, Treg infusion was associated with significant increases in CD163 expression by circulating HLA-DR+ myeloid cells and elevated levels of circulating soluble CD163. Further, graft-infiltrating CD163+ cells were increased with Treg infusion. Thus, multiple Treg infusions were associated with M2-like myeloid cell enhancement that may mediate immunomodulatory, anti-inflammatory and graft reparative effects. Translational StatementNonhuman primates are important models for assessment of the feasibility, safety, and efficacy of new therapeutic regimens in transplantation. Here, major histocompatibility complex–mismatched renal allograft survival was prolonged in nonlymphodepleted monkeys given multiple infusions of polyclonal regulatory T cells (Tregs) during the first month after transplant, together with tapered tacrolimus and CTLA4Ig. This therapeutic effect was associated with increased prevalence in blood and the allograft of myeloid cells expressing CD163, which has been associated with M2 macrophage anti-inflammatory/tissue reparative function. This novel observation justifies further investigation of the Treg-M2 macrophage interaction as a potential target to improve transplant survival in preclinical models and the clinic. Nonhuman primates are important models for assessment of the feasibility, safety, and efficacy of new therapeutic regimens in transplantation. Here, major histocompatibility complex–mismatched renal allograft survival was prolonged in nonlymphodepleted monkeys given multiple infusions of polyclonal regulatory T cells (Tregs) during the first month after transplant, together with tapered tacrolimus and CTLA4Ig. This therapeutic effect was associated with increased prevalence in blood and the allograft of myeloid cells expressing CD163, which has been associated with M2 macrophage anti-inflammatory/tissue reparative function. This novel observation justifies further investigation of the Treg-M2 macrophage interaction as a potential target to improve transplant survival in preclinical models and the clinic. Ex vivo–expanded regulatory T cells (Tregs) have shown considerable promise as innovative cellular therapeutics in experimental organ transplantation (Tx).1Tang Q. Vincenti F. Transplant trials with Tregs: perils and promises.J Clin Invest. 2017; 127: 2505-2512Crossref PubMed Scopus (123) Google Scholar Thus, in rodents, the ability of adoptively transferred autologous polyclonal or more potent donor alloantigen-reactive2Sanchez-Fueyo A. Sandner S. Habicht A. et al.Specificity of CD4+CD25+ regulatory T cell function in alloimmunity.J Immunol. 2006; 176: 329-334Crossref PubMed Scopus (106) Google Scholar,3Sagoo P. Ali N. Garg G. et al.Human regulatory T cells with alloantigen specificity are more potent inhibitors of alloimmune skin graft damage than polyclonal regulatory T cells.Sci Transl Med. 2011; 3: 83ra42Crossref PubMed Scopus (279) Google Scholar Treg (darTreg) to induce Tx tolerance in lymphocyte-deficient/depleted4Hara M. Kingsley C.I. 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Sasaki K. et al.Ex vivo expanded donor alloreactive regulatory T cells lose immunoregulatory, proliferation, and antiapoptotic markers after infusion into ATG-lymphodepleted, nonhuman primate heart allograft recipients.Transplantation. 2021; 105: 1965-1979Crossref PubMed Scopus (14) Google Scholar into lymphodepleted (ATG-treated) heart allograft recipient monkeys failed to prolong graft survival.17Ezzelarab M.B. Zhang H. Guo H. et al.Regulatory T cell infusion can enhance memory T cell and alloantibody responses in lymphodepleted nonhuman primate heart allograft recipients.Am J Transplant. 2016; 16: 1999-2015Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar In these studies, lack of darTreg efficacy was associated with diminished expression of immunoregulatory molecules and proliferation and antiapoptotic markers by the transferred Treg.18Ezzelarab M.B. Zhang H. Sasaki K. et al.Ex vivo expanded donor alloreactive regulatory T cells lose immunoregulatory, proliferation, and antiapoptotic markers after infusion into ATG-lymphodepleted, nonhuman primate heart allograft recipients.Transplantation. 2021; 105: 1965-1979Crossref PubMed Scopus (14) Google Scholar Multiple early phase clinical trials have reported on the safety of Treg infusion in kidney or liver Tx.19Chandran S. Tang Q. Sarwal M. et al.Polyclonal regulatory T cell therapy for control of inflammation in kidney transplants.Am J Transplant. 2017; 17: 2945-2954Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 20Mathew J.M. H-Voss J. LeFever A. et al.A phase I clinical trial with ex vivo expanded recipient regulatory T cells in living donor kidney transplants.Sci Rep. 2018; 8: 7428Crossref PubMed Scopus (158) Google Scholar, 21Sawitzki B. Harden P.N. 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Sawitzki B. et al.Feasibility, long-term safety, and immune monitoring of regulatory T cell therapy in living donor kidney transplant recipients.Am J Transplant. 2021; 21: 1603-1611Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar In these trials, Tregs have been infused once at various times and in different doses after Tx, together with various immunosuppressive (IS) drug regimens, for example, ATG, cyclophosphamide, calcineurin inhibition, and mechanistic target of rapamycin inhibition. However, unequivocal evidence of therapeutic efficacy of Treg transfer has not been established in these trials, which include inconsistent results between liver and kidney Tx.26Koyama I. Bashuda H. Uchida K. et al.A clinical trial with adoptive transfer of ex vivo-induced, donor-specific immune-regulatory cells in kidney transplantation—a second report.Transplantation. 2020; 104: 2415-2423Crossref PubMed Scopus (21) Google Scholar Furthermore, selective decreases in autologous darTreg after Tx can limit clinical applicability of Treg therapy.27Tang Q. Leung J. Peng Y. et al.Selective decrease of donor-reactive T(regs) after liver transplantation limits T(reg) therapy for promoting allograft tolerance in humans.Sci Transl Med. 2022; 14eabo2628Crossref Scopus (16) Google Scholar Here, we investigated the therapeutic efficacy of multiple polyclonal Treg infusions and underlying mechanisms in nonlymphodepleted nonhuman primate kidney allograft recipients. We validated the fate of infused Tregs given during the first month after Tx and assessed their influence on host lymphoid and myeloid cell populations. Treg infusions resulted in significant prolongation of graft survival associated with increased incidences of Tregs in secondary lymphoid tissue and CD163+ (M2-like) myeloid cells in the circulation and graft. These novel observations suggest that multiple Treg infusions early after Tx in nonlymphodepleted recipients may enhance graft survival through the promotion of anti-inflammatory and reparative M2-like cells. Healthy rhesus monkeys (Macaca mulatta; 5–7 kg) from the National Institute of Allergy and Infectious Disease–sponsored colony (Yemassee, SC) were maintained in the Department of Laboratory Animal Resources, University of Pittsburgh School of Medicine. All procedures were performed in accordance with the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication no. 86-23, revised 1985) and under a University of Pittsburgh Institutional Animal Care and Use Committee–approved protocol. Ten randomly selected monkeys received grafts from ABO-compatible, major histocompatibility complex–mismatched donors (Supplementary Table S1). Bilateral nephrectomy of recipient native kidneys was performed before graft insertion. Serum creatinine levels, blood urea nitrogen, urine protein/creatinine ratios, weight loss, and percent change in estimated glomerular filtration rate (Supplementary Methods) were measured to assess graft function. Animals were divided into 2 groups: control (no cell infusion; n = 5) and experimental (Treg infusion; n = 5). Experiment endpoint was defined as either creatinine levels >5 mg/dl, or increased creatinine levels >3.0 mg/dl combined with increased blood urea nitrogen levels >70 mg/dl. Recipients were euthanized due to either weight loss >20% or failure to thrive (loss of appetite and emaciation). Immunosuppression (Figure 1a) comprised costimulation blockade (CTLA4Ig), 20 mg/kg on days −1 and 0 and postoperative days (PODs) 3, 6, 10, 13; and 20 mg/kg/wk until POD60; 12.5 mg/kg/wk until POD125; and 5 mg/kg/wk until POD180; and tacrolimus (target whole blood trough levels: 10–15 ng/ml POD−2 to POD27, 5–10 ng/ml POD28–41, and 1–5 ng/ml POD42–60) via i.m. injection. Blood samples were collected before and weekly after Tx to evaluate T-cell subsets. Serum samples were collected before Tx, weekly after Tx, and at euthanasia (M264 euthanized on POD15). Recipient peripheral blood mononuclear cells (PBMCs) were isolated before Tx, POD28, POD35, and at euthanasia. Recipient mesenteric lymph nodes (LNs) and PBMCs were obtained on POD0 and POD35 (day of biopsy). Donor PBMCs, spleen, and LNs were collected on the day of Tx. Recipient LN, spleen, and kidney graft were harvested at euthanasia. Tissue samples were collected for histologic analysis and immune cell isolation, as described in Supplementary Methods. After live/dead staining with Zombie Aqua Fixable Viability Kit (BioLegend) (4 °C) for 15 minutes, cell suspensions were surface-stained. For intracellular staining, cell suspensions were fixed/permeabilized for 45 minutes (4 °C) using fixation/permeabilization buffer (eBioscienceTM; Invitrogen), followed by intracellular staining (40 minutes at 4 °C). Data were acquired on an LSRFortessa (BD Bioscience) and analyzed by the FlowJo software (version 10; TreeStar Inc.). For cytokine staining, T cells were activated for 6 hours with phorbol 12-myristate-13-acetate (Sigma) and ionomycin (Sigma) in the presence of GolgiStop (BD Bioscience), followed by incubation with anti–interferon-γ, anti–interleukin-17A, and anti–tumor necrosis factor-α. The phenotype of ex vivo–expanded Treg, peripheral blood, and LN lymphocyte subsets were assessed. Antibodies used are listed in Supplementary Methods. Serum samples collected before and after Tx were stored at −20 °C. Complement was heat-inactivated by incubation at 56 °C for 30 minutes. Donor LN CD3+ T cells were used as targets for quantitation of antidonor IgM and IgG alloantibodies by flow cytometry. Briefly, 0.5 × 106 donor CD3+ T cells were incubated with serum samples for 30 minutes at room temperature. Next, cells were incubated with either phycoerythrin-conjugated antimonkey IgM for 30 minutes or antimonkey IgG Biotin for 30 minutes (4 °C), followed by incubation with phycoerythrin-antistreptavidin for 30 minutes (4 °C). Finally, cells were stained with BUV395 anti-CD3. Six days before Tx, peripheral blood Tregs were isolated from prospective recipient using nonhuman primate CD4+CD25+ cell isolation kits (Miltenyi Biotech), followed by polyclonal expansion using artificial antigen-presenting cells (L-cells), together with recombinant human interleukin-2 (2000 U/ml). Half of the media was changed every 2 to 3 days. Freshly expanded Tregs were infused at designated doses (Figure 1b) on POD6, 10, 13, and 16. After Treg infusion on POD10, remaining expanded Tregs were stored in liquid N2 for subsequent harvest and further expansion for infusion on POD20, 23, 27, and 30. Tregs were suspended in 2% v/v human AB serum in sterile saline (106/ml) before infusion, and their phenotype and purity were validated by flow cytometry. Expanded Tregs of kidney allograft recipient M25 were labeled with 1 μM DeepRed for 10 minutes (37 °C) before infusion. Labeled cells were infused at designated doses shown in Figure 1b. Serum samples were collected at designated times before and after Tx. Soluble(s) CD16328Moller H.J. Peterslund N.A. Graversen J.H. et al.Identification of the hemoglobin scavenger receptor/CD163 as a natural soluble protein in plasma.Blood. 2002; 99: 378-380Crossref PubMed Scopus (192) Google Scholar was measured by the enzyme-linked immunosorbent assay (LSBio, Cat#LS-F32525). Biopsies (needle or wedge) were formalin-fixed and paraffin-embedded. Standard 4 μm sections were stained with hematoxylin and eosin, trichrome, periodic acid–Schiff, periodic acid–silver methenamine stain, and C4d. Sections were scored by board-certified pathologist (AJD) blinded to the treatment group using the Banff classification of human renal allograft pathology.29Roufosse C. Simmonds N. Clahsen-van Groningen M. et al.2018 reference guide to the Banff classification of renal allograft pathology.Transplantation. 2018; 102: 1795-1814Crossref PubMed Scopus (412) Google Scholar The details of immunohistochemical methods are in Supplementary Methods. Actuarial survival analysis was performed by Kaplan-Meier analysis. Analysis of covariance was performed to compare outcome percentages, adjusted for baseline values for CD4+CD25hiFoxp3hi Treg percentages and CD163+ cells before and after Tx. Repeated-measures 2-way analyses of variance were performed for sCD163 analysis. Statistical analyses were conducted using the JMP Pro 17.1.0 Software (JMP Statistical Discovery LLC). A P value of < 0.05 was considered significant. The phenotype and purity of expanded Tregs were assessed on the day of infusion. For each infusion, the percentage of total CD4+T cells was 99%, whereas the percentages of CD8+T cells, CD20+, and CD14+ cells were all <1% to 2%. Escalating dosage and timing of Treg infusions were predetermined as follows: 10 × 106/kg on POD6 and 10, 30 × 106/kg on POD13 and 16, and 50 × 106/kg on POD20, 23, 27, and 30 to ensure uniformity of cell dose (Figure 1b). Total infused Treg numbers (per recipient) are shown in Table 1 (range: 1.125–1.815 × 109).Table 1Summary of polyclonal Treg doses and time of infusions in kidney allograft recipientsInfusionsDay (after transplantation)×106 cells/kgM24M29M208M260M25aM25 received DeepRed-labeled Tregs (for each Treg infusion).Total Tregs (×106) per infusion#16104356717053#2104153677051#31330123159195170153#416123156192180147#52050200250320305240#623200245320300245#726200260325285245#830195250325285245Total Tregs infused (×106)11251429181516651379Treg, regulatory T-cell.Treg infusions #1, #2, #3, and #4 were obtained from freshly isolated Tregs (6 days before transplant) followed by expansion for 22 days.On day 16 of expansion, after harvesting Tregs for infusion #2 (day 10 after transplant), remaining Tregs were stored in liquid N2 for harvest and expansion for later infusions (#5, #6, #7, and #8).a M25 received DeepRed-labeled Tregs (for each Treg infusion). Open table in a new tab Treg, regulatory T-cell. Treg infusions #1, #2, #3, and #4 were obtained from freshly isolated Tregs (6 days before transplant) followed by expansion for 22 days. On day 16 of expansion, after harvesting Tregs for infusion #2 (day 10 after transplant), remaining Tregs were stored in liquid N2 for harvest and expansion for later infusions (#5, #6, #7, and #8). Expanded Treg for all infusions demonstrated high forkhead box P3 (Foxp3) and CD25, and low levels of CD127 expression. In addition, expanded Treg expressed higher levels of the Treg markers CTLA4 and Helios than autologous effector CD4+CD25−T cells. Variable levels of phospho–signal transducer and activator of transcription 3, phospho–signal transducer and activator of transcription 5, and the transcription factors T box expressed in T cells and retinoic acid receptor–related orphan nuclear receptor γt expression and low levels of CD45RA and transcription factor GATA binding protein 3 expression were observed (Figure 1c). In addition, expanded Treg expressed variable levels of the chemokine receptors CCR4, CCR7, and CXCR3. Collectively, these observations demonstrated that expanded polyclonal Tregs exhibited comparable levels of Treg markers to host autologous Tregs when infused. All graft recipients exhibited increased levels of creatinine and blood urea nitrogen at various time points after Tx, in addition to increased urine protein/creatinine ratios and weight loss. However, the experiment endpoint (creatinine >5, or creatinine >3.0 + blood urea nitrogen >70) occurred earlier in controls (Figure 2a) compared with Treg-infused recipients (Figure 2b). In correlation, Treg-infused recipients demonstrated a higher percent change in estimated glomerular filtration rate after Tx compared with controls (Supplementary Figure S1). One control recipient (M27) was euthanized on POD38 due to intussusception, whereas one recipient (M25) in the Treg group was electively euthanized on POD49 for tracking of infused Tregs. In the control group (no Treg), the experiment endpoint was reached in a median of 34 days compared with a median of 49 days in the Treg group (Table 2), as determined by Kaplan-Meier analysis (Figure 2c, left panel) (P < 0.05). The median graft survival time was 35 days in the control group compared with 70 days in the Treg group (Table 2; Figure 2c, right panel).Table 2Summary of kidney allograft survival in the control and experimental groupsControl group (no Treg infusion; n = 5)Experimental group (Treg infusion; n = 5)Treg infusion, rangeN/A1.13–1.82 × 109/kgExperiment endpoint, d13, 16, 34, 38aEuthanized due to intussusception., 4137, 41, 49bElectively euthanized for tracking of labeled Tregs., 58, 234Median, dcMedian of experiment endpoint and euthanasia were determined by Kaplan-Meier analysis (long-rank test), as shown in Figure 2c.3449Euthanasia, d15, 35, 35, 38aEuthanized due to intussusception., 5938, 49bElectively euthanized for tracking of labeled Tregs., 70, 86, 234Median, dcMedian of experiment endpoint and euthanasia were determined by Kaplan-Meier analysis (long-rank test), as shown in Figure 2c.3570N/A, not applicable; Treg, regulatory T cell.a Euthanized due to intussusception.b Electively euthanized for tracking of labeled Tregs.c Median of experiment endpoint and euthanasia were determined by Kaplan-Meier analysis (long-rank test), as shown in Figure 2c. Open table in a new tab N/A, not applicable; Treg, regulatory T cell. Overall, these clinical observations indicate that infusion of ex vivo–expanded polyclonal Tregs was associated with delayed graft injury and improved graft function compared with kidney recipients with no Treg infusion. Memory T-cell subsets in peripheral blood were analyzed based on CD45RA/CCR7 or CD28/CD95 expression, as described in Supplementary Figure S2. For CD4+ memory T-cell subsets, no major differences were observed between the control and Treg groups, before and after Tx. For CD8+ memory T-cell subsets, in some recipients, there was a trend toward increased percentages of CD28–CD95+T cells; however, there were no marked differences between the control and Treg groups. These data suggest that Treg infusion did not influence circulating CD4+ or CD8+ memory T-cell subsets quantitatively. Memory and isotype-switched B cells in blood were analyzed based on CD27/CD21 and CD27/IgD expression, respectively (Supplementary Figure S3). No marked differences were observed between the groups. Serum antidonor IgM and IgG Ab levels (Supplementary Figure S4A) were measured before and after Tx. In the control group, 2 recipients (2/5) exhibited a modest increase in IgM levels, whereas 2 recipients (2/5) exhibited increased IgG levels. M264 (with the shortest survival time; 15 days) exhibited the highest percent increase in both IgM and IgG Ab levels in the control group. In the Treg group, 1 recipient (1/5) exhibited increased IgM levels and 3 recipients (3/5) exhibited transient increases in IgG levels. IgM and IgG levels were not markedly different between the groups. Hence, no significant changes were observed in donor-specific Ab levels, with or without Treg infusion. Recipient PBMCs obtained before and after Tx were cocultured with donor cells for 5 days. As shown in Figure 3a, no marked differences in CD4+ and CD8+T-cell proliferative responses to donor antigen were observed in either group. Similarly, no marked changes were observed in percentages of interferon-γ+tumor necrosis factor-α+ and interleukin-17+ cells among total CD4+ and CD8+T cells after stimulation with donor cells in both groups (Figure 3b). On the other hand, percentages of CD4+CD25hiFoxp3hi Tregs in total donor-reactive CD4+T cells were reduced in both groups after Tx compared with before Tx (Figure 3c), and no statistical differences were observed between the groups. These data suggest that no major changes in phenotype and function of circulating donor-reactive T cells were observed with or without Treg infusion, likely due to a predominant influence of tacrolimus and CTLA4Ig (i.e., reduced Foxp3 expression) on recipient donor-reactive T cells. These data suggest that, although no major changes in phenotype and function of circulating donor-reactive T cells were observed in both groups, likely due to a predominant influence of tacrolimus and CTLA4Ig (reduced effector function and Foxp3 expression), Treg infusion may alleviate reductions in donor-reactive CD4+CD25hiFoxp3hi percentages after Tx. Next, we evaluated CD4+CD25hiFoxp3hi Treg percentages in peripheral blood and mesenteric LNs on POD0 (day of Tx) and POD35 (day of biopsy). In both groups, percentages of Tregs in blood were reduced on POD35 compared with POD0. Similarly, CD4+CD25hiFoxp3hi Treg percentages in mesenteric LNs were reduced on POD35 compared with POD0 in both groups. Higher percentages of Tregs in mesenteric LNs were observed in Treg-infused recipients, although the increase was not statistically significant (analysis of covariance adjusted to POD0 percentages; P = 0.0731). These data suggest a predominant influence of immunosuppression (tacrolimus and CTLA4Ig) on Treg incidences in both groups (Figure 4). To assess their fate, expanded Tregs were labeled with DeepRed immediately before each infusion in 1 recipient (M25). Sequential blood samples were collected, followed by PBMC isolation and storage. Next, percentages of DeepRed-labeled Tregs were evaluated (Figure 5a). Labeled Tregs remained detectable in blood after their infusion, albeit at minimal levels. On POD35, infused Tregs were observed in mesenteric LN sections, and their presence was confirmed by flow cytometry (Supplementary Figure S5). After elective e