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Transplant arteriosclerosis (TA) is the hallmark of chronic allograft dysfunction (CAD) affecting transplanted organs in the long term [1,2]. These fibroproliferative lesions lead to neointimal thickening of arteries in all transplanted allografts . Luminal narrowing then leads to graft ischemia and organ demise. To date, there are no known tolerance induction strategies that prevent TA [3,4]. Therefore, this study was designed to test the hypothesis that human regulatory T cells (Treg cells) expanded ex vivo could prevent TA. Here we show the comparative capacity of Treg cells, sorted via two separate strategies, to prevent TA in a clinically relevant chimeric humanized mouse system. We found that the in vivo development of TA in human arteries was prevented with the treatment of ex vivo expanded human Treg cells. Additionally, we show that Treg cells sorted based on the low expression of CD127 (IL-7Rα) provide a more potent therapy to conventional Treg cells. Our results demonstrate, for the first time, that human Treg cells can inhibit TA by impairing effector function and graft infiltration. We anticipate our findings to serve as a foundation for the clinical development of therapeutics targeting TA in both allograft transplantation and other immune-mediated causes of vasculopathy .
Investigating the mechanisms driving the development of TA in situ in transplant recipients is challenging. While protocol biopsies and intravascular imaging provide snapshots of the process and enable the evolution of CAD to be described[6,7], the development of a chimeric humanized mouse system has enabled the mechanisms and impact of novel interventions to be studied in vivo. Allogeneic peripheral blood mononuclear cells (PBMC) were found to elicit immune-mediated rejection of transplanted human arterial segments mimicking the TA seen in human transplantation. Interestingly, IFN-γ elicited the same response in the absence of leukocytes, supporting the concept that both immune and non-immune mechanisms contribute to the process. In this report, we investigated the possibility of attenuating the fibroproliferative lesions seen with TA using ex vivo expanded human Treg cellular therapy, compared the capacity of CD25hiCD4+ vs. CD127loCD25+CD4+ cells to suppress allogeneic immune responses, and evaluated the impact of Treg cells on IFN-γ production.
Human Treg cells were FACS-sorted from healthy donor PBMC as CD25hiCD4+ cells (designated CD25hi) or CD127loCD25+CD4+ cells (designated CD127lo) to greater than 94% purity (n=5 independent experiments; Supplementary Fig. 1a). After sorting, the cells were expanded in vitro using beads coated with CD3 and CD28-specific antibodies and recombinant human IL-2 (Fig. 1a)[9,10]. Both populations consistently expanded to greater than 600-fold (n=5 expansions; Supplementary Fig. 1b). After expansion, both subsets retained expression of Treg cell markers including CD25, FoxP3, GITR, CTLA-4 (Fig. 1b,c and Supplementary Fig. 1c). Interestingly, significant differences were observed in the expression of CD127, CD62L and CD27. Moreover, in vitro suppression assays consistently revealed a higher suppressive capacity in CD127lo sorted cells towards allo-stimulated autologous PBMC (n = 5) as well as CD25−CD4+ effector cells (n = 3) after expansion (Fig. 1d,e). Expanded CD127lo cells expressing CD62L and CD27 suppressed PBMC much more efficiently than cells expressing low levels of CD62L, CD27, or both (Fig. 1f). The molecular mechanism of suppression by expanded Treg cells in vitro was not dependent on IL-10 or CTLA-4 (Supplementary Fig. 2a) and required cell-cell contact (Supplementary Fig. 2b). Moreover, both CD25hiCD4+ and CD127loCD4+ expanded Treg cells inhibited dendritic cell maturation as demonstrated by the reduced increase in CD86 expression (Supplementary Fig 2c,d). Importantly, the expanded Treg populations could be frozen for storage without loss of functional activity (Supplementary Fig. 3a) and exerted their suppressive ability even over HLA-mismatched PBMC (Supplementary Fig. 3b).
Next, the functional activity of the CD25hiCD4+ and CD127loCD4+ expanded cells, from five separate PBMC donors, was tested in vivo. Human internal mammary arterial (IMA) sidebranches, procured from patients undergoing coronary artery bypass surgery, were transplanted into the infrarenal aorta of BALB/c.Rag2−/−.Il2rg−/− mice deficient in T, B, and NK cells. The following day 10 × 106 allogeneic or autologous PBMC were used to inoculate mice bearing human arterial grafts. Transplanted human arterial grafts were maximally dilated and harvested 30 days after transplantation (Fig. 2a). In mice reconstituted with allogeneic PBMC (defined as >1% splenic human leukocyte chimerism; Supplementary Fig. 4) a significant vascular intimal hyperplasia and a decrease in luminal diameter was observed in the transplanted vessels as compared to that of vessels transplanted into animals that either did not receive human PBMC, or those reconstituted with PBMC autologous to the arterial graft (Fig. 2b–e; Supplementary Table 1). Importantly, the neointimal proliferation seen in rejecting arterial grafts was allo-specific to the human vessel as the native mouse aorta prior to the suture line was not affected (Supplementary Fig. 5a). Moreover, xeno-responses were confirmed to play only a minor role in this system as evidenced by in vitro mixed lymphocyte reactions (MLR); wherein, allo-human responses predominated over any and all xeno-responses (Supplementary Fig. 5b). In vitro analysis of precursor frequency, measured by IFN-γ ELISPOT or proliferation, demonstrated increased frequency of donor alloantigen specific responding cells upon restimulation, irrespective of the presence or absence of mouse splenocytes (Fig. 2f,g).
We then reconstituted mice transplanted with human vessels with human PBMC and ex vivo expanded CD25hi or CD127lo cells from the same allogeneic blood donor at a 1:1 ratio. Here we show that both CD25hi and CD127lo populations have a significant impact on the development of TA in vivo without inhibiting the rate of reconstitution (Fig. 2h–j and Supplementary Fig. 4b–d). Analysis of vessels transplanted into mice reconstituted with human Treg cells only was not feasible as the level of reconstitution achieved using fractionated CD4+ T cells alone was <1% (data not shown). The vasculopathy seen in chimeric humanized mice treated with CD25hi Treg cellular therapy was significantly less than that in mice reconstituted with allo-PBMC alone (P = 0.013); however, TA is still evident in these grafts at varying degrees (Fig. 2h,j). In contrast, animals treated with CD127lo ex vivo expanded Treg cells reveal almost complete abrogation of TA without such variation between different PBMC and vessel donors, corroborating our in vitro results (Fig. 1 and Fig. 2 i,j). Moreover, in the presence of Treg cell therapy IFN-γ production is markedly diminished when compared to stimulation of PBMC alone with alloantigen either in vivo (Fig. 3a,b and Supplementary Fig. 6a) or in vitro (Fig. 3c and Supplementary Fig. 6b).
The ability of Treg cells to inhibit the secretion of IFN-γ in vivo, thought to be integral to the development of TA[8,12,13], was confirmed by testing serum samples procured from human leukocyte engrafted mice; upregulation of human IFN-γ by human PBMC alone was substantially inhibited by CD127lo Treg cellular therapy (Fig. 3a). Importantly, the expression of IFN-γ within the graft itself was also significantly inhibited by treatment with CD127lo Treg cells as indicated by real-time PCR analysis (Fig. 3b; P < 0.01). Suppression of IFN-γ expression within the allograft correlated with increased detection of intragraft FOXP3 mRNA in animals treated with CD127lo Treg cells (Fig. 3b), indicating that CD127lo Treg cells may infiltrate the graft. Histological assessment revealed that virtually all human graft infiltrating cells were CD3+ T cells (854 ± 280 hCD45+ versus 845 ± 318 hCD3+ in the neointima in the PBMC group; mean ± SD). Notably, human CD4 and CD8 infiltration of both the graft adventitia and neointima was markedly decreased in mice treated with PBMC + CD127lo Treg cell therapy compared to that in mice reconstituted with PBMC alone or with PBMC + CD25hi cells (Fig. 3d). Taken together, these data suggest that treatment with Treg cells may inhibit the development of TA by preventing the effector response of PBMC and mobilization of IFN-γ producing cells to an allograft [15,16].
We then investigated the potency of CD127lo Treg cells on the development of TA in the human-mouse chimera. Interestingly, when 2 × 106 CD127lo Treg cells were transferred with 10 × 106 PBMC, we also observed a significant decrease in the development of arteriosclerotic disease when compared to transplanted animals inoculated with PBMC alone (Fig. 4a–c,e; P < 0.05). However, the efficacy of regulation was lost when 1 × 106 CD127lo Treg cells were co-transferred with 10 × 106 PBMC (Fig. 4d). The level of TA seen in animals receiving five-fold less CD127lo Treg cells was similar to that of animals receiving PBMC + CD25hi Treg cells at a 1:1 ratio (Fig. 4f). Additionally, CD4+ and CD8+ lymphocytes produced less IFN-γ in vitro when co-cultured with CD127lo Treg cells at a 1:1 and a 1:1/5 ratio confirming the increased potency of CD127lo Treg cells (Fig. 4g). Taken together, these data suggest that titrated levels of ex vivo expanded Treg cells sorted based on CD127lo expression are five fold more potent than that of CD25hi Treg cells.
These data demonstrate the unique ability of ex vivo expanded human Treg cells to successfully abrogate the development of TA in vivo. Over the past decade, both thymus derived and antigen induced Treg cells, initially identified by Sakaguchi et. al., as CD25+CD4+, have been highlighted for their suppressive capabilities in vitro and in vivo[11,17,18] and therapeutic potential in the regulation of autoimmunity, allergy and immune-mediated transplant rejection. Although, the results presented here are promising and exciting, they must also be interpreted with caution. As seen in Supplementary Fig. 7, mice treated with Treg cell therapy lose medial architecture in the graft when compared to native arterial sections or animals treated with PBMC alone.
Therefore, translation to the clinic, is likely dependent upon the isolation of pure populations of suppressive cells that can reproducibly and safely abrogate disease. Critical to this process is the identification of markers that permit the isolation and enrichment of viable Treg cells for therapeutic transfer. The recent observation of the inverse correlation between the IL-7 Rα (CD127) subunit and the suppressive capacity of CD25hiCD4+ Treg cells allows for an additional potential target to isolate and expand cells with increased suppressive function ex vivo[19-21]. In mouse models, only the subpopulation of CD25+CD4+ Treg cells expressing CD62L+ were found responsible for protection against lethal acute GVHD. Therefore, the higher expression of CD62L on the ex vivo expanded CD127lo Treg cells used here may be advantageous in clinical situations allowing for more efficient homing to secondary lymph nodes, as well as in the chimeric humanized in vivo model used here, based on the high conservation of the CD62L molecule between species. Additionally, the downregulation of the IL-7Rα along with the upregulation of CD27 (Fig. 1) have been correlated previously to a highly suppressive subset of Treg cells believed to exert their effects locally (i.e. allograft or draining lymph node) rather than re-circulating systemically[19,24]. The paradigm of disease that has been proposed to explain development of TA is one of local effector cell-mediated recruitment of rapidly proliferating vascular smooth muscle cells leading to neointimal formation and arterial luminal occlusion. With our findings, it seems that immunoregulation of these locally destructive effector cells may be feasible with the use of Treg cellular therapy. However, further tests of the efficacy and safety of these cells are essential.
We obtained BALB/c.rag2−/−.Il2rg −/−(H2d) mice from Charles River Laboratories and housed the animals under specific pathogen-free conditions. We performed all experiments using protocols approved by the Committee on Animal Care and Ethical Review at Oxford University and in accordance with the UK Animals (Scientific Procedures) Act 1986. We used mice between the ages of 6-10 weeks.
The vast majority of mice (over 75%) reconstituted with human PBMC showed >1% splenic engraftment defined as fully reconstituted. The level of reconstitution was routinely over 10% and did not correlate with amount of arteriosclerosis seen (SNN, GW, KJW unpublished observations).
We obtained perforating side branches of the IMA during procurement of the left and right IMA in a skeletonized fashion and immediately placed in a 4.0% phenoxybenzamine and normal saline solution. Human tissue used for this study was obtained under the ethical reference number 04/Q1605/89. Study was approved by UK National Research Ethics Service, Oxfordshire REC B. Informed consent was obtained from all patients.
Aortic interposition grafts were performed with human IMA sidebrancehes into the abdominal aorta of BALB/c.rag2−/−.Il2rg −/− mice with an end-to end anastomosis using a technique described by Koulack et al.
We isolated PBMC from buffy coats provided by the National Blood Service (NBS) by Ficoll-Paque (GE Healthcare) gradient centrifugation. CD4+ T cells were then enriched using the Human CD4+ Lymphocyte Enrichment Set (BD), stained with appropriate antibodies (CD4 APC and CD25 PE or CD127 PE, CD25 APC and CD4 PerCP, all BD) and sorted using a BD FacsARIA cell sorter (BD). CD4+ T cells expressing high levels of CD25 (CD25hiCD4+) and low levels of CD127 (CD127lo CD25+CD4+) were subsequently sorted and recovered. In a limited number of experiments we stained whole PBMC for sorting giving a similar purity after each sort (more than 96%). Cells were cultured in RPMI 1640 media supplemented with L-glutamine, penicillin/streptomycin, sodium pyruvate and 10% of human AB pooled serum. For expansion, sorted cells were cultured in the presence of recombinant human IL-2 (1,000 U ml−1) (Chiron) and Human T-Expander CD3/CD28 Dynabeads (Invitrogen) in a 1:2 cell per bead ratio for two rounds of expansion, 7 days each. After the second round of expansion, beads were removed and cells were silenced for 2 days in medium containing 200 U ml−1 of IL-2 (Fig. 1a). After each expansion, the phenotype of cells were assessed by staining for FoxP3 (eBiosciences), GITR (R&D Systems) CD25, CTLA-4, CD127, CD27 and CD62L (BD).
We generated single cell suspensions from spleen and performed a red cell lysis was performed. For phenotypic analysis, antibodies against human CD3 (eBioscience), CD4 (Caltag), CD8, CD25, CD45, and CD127 (BD) were used.
For cytokine analysis, cells were stimulated with irradiated allogeneic PBMC for 5 days and re-stimulated for 5 h with phorbol 12-myristate 13-acetate (PMA, 100 ng ml−1, Sigma), ionomycin (1 μg ml−1, Sigma) and monensin (GolgiStop, 1 μl ml−1, BD) before being stained with antibodies to CD4, CD8, CD3 (BD), and the viability dye 7-AAD (eBiosciences). We then fixed and permeabilized cells and stained with antibody to intracellular IFN-γ (BD). .
We tested the suppressive activity of expanded CD25hi or CD127lo cells by using an in vitro suppression assays. Autologous PBMC (1 × 105) were incubated with irradiated allogeneic PBMC (1 × 105) and varying numbers of expanded CD25hi or CD127lo cells. We then assessed proliferation after 7 days by addition of 3H-thymidine for the last 16 h of culture.
In all experiments, human PBMC were obtained from random donors and the data pooled. 10 × 106 Ficoll-Paque purified human PBMC were injected intraperitoneally into recipient mice. Alternatively, human PBMC were adoptively transferred in the same fashion with an equal number of CD25hi or CD127lo ex vivo expanded human T cells. In the described titration experiments, 2 × 106 CD127lo or 1 × 106 CD127lo were co-transferred with 10 × 106 PBMC.
As per manufacturer's protocol (eBioscience ELISA Ready-SET-Go! Kit)
Cytokine multiplex for the following TH1 and TH2 cytokines were stained for using two color (PE and FITC) bead array systems manufactured by Bender Medsystems (Vienna, Austria). The assay was carried out using manufacturer's protocols. Cytokines detected were: IL-6, IL-8, IL-10, IL-2, IFN-γ, IL-12p70, TNF-β, TNF-α, IL-5, and IL-4.
We performed IFN-γ ELISPOT (BD Biosciences) in 3–6 repeats with 5 × 104 responder cells and 1 × 105 irradiated stimulatory cells per well incubated for 15 h. The assay was carried out following manufacturer's protocol.
Total RNA was isolated from arterial grafts using NucleoSpin RNA XS kit (Fisher Scientific) and followed by cDNA synthesis using High Capacity RNA-to-cDNA kit (Applied Biosystems). Real-time quantification was performed using Stratagene Mx3000P (Agilent Technologies) using the HPRT probe and primers as described in  and TaqMan Gene Expression Assays (Applied Biosystems) for IFN-γ and FOXP3. Quantification of the gene of interest was given by ΔCt method.
After thirty days post transplantation, we procured aortic grafts which were then snap- frozen in OCT embedding medium prior to cryostat sectioning at a thickness of 6–10 μm. After drying, sections were fixed in 100% acetone for 10 min at 4 °C and stored in −80 °C until further use. We then blocked all sections with 10% BSA and 5% mouse serum at room temperature for 30 min. Primary antibodies, mouse anti-human CD4 (BD) and mouse anti-human CD8 (BD) were then applied. Following several washes, HRP-conjugated secondary antibody (DAKO Cytomation) was applied, and sections were visualized with diaminobenzidene (Sigma).
We analysed data from mice displaying >1% human CD45+ live leukocytes in the spleen as measured by flow cytometry. Morphometric analysis of transplant arteriosclerosis on EvG-stained sections were measured as described. Briefly, percentage intimal expansion (IE) was calculated using the following formula: % intimal expansion = (AI- AL/ AI) × 100; where AI is the area within the internal elastic lamina and AL is the luminal area.
Statistical evaluations were performed using Graphpad Prism software. Statistical significance was determined using Mann-Whitney U test. P < 0.05 was considered statistically significant.
We thank the staff of the Biomedical Services Unit at the John Radcliffe Hospital for expert animal care; M. Barnardo and the staff at the Clinical Transplant Immunology Laboratory, The Oxford Transplant Centre for molecular HLA typing; M. Carvalho-Gaspar for advice on real-time PCR; F. Issa and R. Goto for assistance with some experiments; A. Bushell and N. Jones for valuable discussions. The cardiac surgical registrars and operating theatre staff for assistance with the procurement of vessels. This work was supported by grants from The Wellcome Trust, European Union Integrated Project, RISET (www.RISETfp6.org), Medical Research Council UK and Garfield Weston Trust. SNN is an International Society for Heart and Lung Transplantation Research Fellow; JW is a RISET investigator, GW is a DFG Fellow; WZ was supported by an unrestricted grant from Becton Dickinson; AS was supported by the Swedish Heart and Lung Foundation and the Swedish Research Council.
Competing financial interest policy – Authors declare no competing financial interest