CD4+ Regulatory T Cells Generated in Vitro with IFN-γ and Allogeneic APC Inhibit Transplant Arteriosclerosis
2010; Elsevier BV; Volume: 177; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2010.090292
ISSN1525-2191
AutoresG. Warnecke, Gang Feng, Ryoichi Goto, Satish N. Nadig, Ross S. Francis, Kathryn J. Wood, Andrew Bushell,
Tópico(s)Renal Transplantation Outcomes and Treatments
ResumoWe have developed a method to generate alloreactive regulatory T cells in vitro in the presence of interferon (IFN)-γ and donor antigen presenting cells (APCs). We hypothesized that these IFN-γ–conditioned T cells (Tcon) would reduce transplantation-associated arteriosclerosis. Tcon were generated from mouse (CBA.Ca, H-2k) CD4+ T cells cultured in the presence of IFN-γ for 14 days. These cultures were pulsed with bone marrow–derived B6 (H-2b) APC. 1 × 105 CD25−CD4+ effector T cells from naive H-2k mice were then cotransferred with 4 × 105 Tcon into CBA-rag−/− mice. One day later, these mice received a fully allogenic B6 CD31−/− abdominal aorta transplant. Transfer of CD25−CD4+ effectors resulted in 29.7 ± 14.5% luminal occlusion of allogeneic aortic grafts after 30 days. Cotransfer of Tcon reduced this occlusion to 11.7 ± 13.1%; P < 0.05. In addition, the CD31− donor endothelium was fully repopulated by CD31+ recipient endothelial cells in the absence of Tcon, but not in the presence of Tcon. In some experiments, we cotransplanted B6 skin with aortic grafts to ensure enhanced reactivation of the regulatory cells, which led to an additional reduction in vasculopathy (1.9 ± 3.0% luminal occlusion). In the presence of Tcon, CD4+ T cell infiltration into grafts was markedly reduced by a regulatory mechanism that included reduced priming and proliferation of CD25−CD4+ effectors. These data illustrate the potential of ex vivo generated regulatory T cells for the inhibition of transplant-associated vasculopathy. We have developed a method to generate alloreactive regulatory T cells in vitro in the presence of interferon (IFN)-γ and donor antigen presenting cells (APCs). We hypothesized that these IFN-γ–conditioned T cells (Tcon) would reduce transplantation-associated arteriosclerosis. Tcon were generated from mouse (CBA.Ca, H-2k) CD4+ T cells cultured in the presence of IFN-γ for 14 days. These cultures were pulsed with bone marrow–derived B6 (H-2b) APC. 1 × 105 CD25−CD4+ effector T cells from naive H-2k mice were then cotransferred with 4 × 105 Tcon into CBA-rag−/− mice. One day later, these mice received a fully allogenic B6 CD31−/− abdominal aorta transplant. Transfer of CD25−CD4+ effectors resulted in 29.7 ± 14.5% luminal occlusion of allogeneic aortic grafts after 30 days. Cotransfer of Tcon reduced this occlusion to 11.7 ± 13.1%; P < 0.05. In addition, the CD31− donor endothelium was fully repopulated by CD31+ recipient endothelial cells in the absence of Tcon, but not in the presence of Tcon. In some experiments, we cotransplanted B6 skin with aortic grafts to ensure enhanced reactivation of the regulatory cells, which led to an additional reduction in vasculopathy (1.9 ± 3.0% luminal occlusion). In the presence of Tcon, CD4+ T cell infiltration into grafts was markedly reduced by a regulatory mechanism that included reduced priming and proliferation of CD25−CD4+ effectors. These data illustrate the potential of ex vivo generated regulatory T cells for the inhibition of transplant-associated vasculopathy. Transplant arteriosclerosis is the main cause of allograft loss after cardiac transplantation1Billingham ME Cardiac transplant atherosclerosis.Transplant Proc. 1987; 19: 19-25PubMed Google Scholar and is critically dependent on an inflammatory process mediated by T lymphocytes,2Hruban RH Beschorner WE Baumgartner WA Augustine SM Ren H Reitz BA Hutchins GM Accelerated arteriosclerosis in heart transplant recipients is associated with a T-lymphocyte-mediated endothelialitis.Am J Pathol. 1990; 137: 871-882PubMed Google Scholar, 3Wang Y Burns WR Tang PC Yi T Schechner JS Zerwes HG Sessa WC Lorber MI Pober JS Tellides G Interferon-gamma plays a nonredundant role in mediating T cell-dependent outward vascular remodeling of allogeneic human coronary arteries.FASEB J. 2004; 18: 606-608Crossref PubMed Scopus (60) Google Scholar especially CD4+ T cells.4Shi C Lee WS He Q Zhang D Fletcher Jr, DL Newell JB Haber E Immunologic basis of transplant-associated arteriosclerosis.Proc Natl Acad Sci U S A. 1996; 93: 4051-4056Crossref PubMed Scopus (189) Google Scholar, 5Ensminger SM Spriewald BM Witzke O Pajaro OE Yacoub MH Morris PJ Rose ML Wood KJ Indirect allorecognition can play an important role in the development of transplant arteriosclerosis.Transplantation. 2002; 73: 279-286Crossref PubMed Scopus (41) Google Scholar We have previously shown that CD4+ T cell–mediated rejection of skin allografts can be successfully inhibited in a mouse adoptive transfer model by CD25+CD4+ regulatory T cells generated in vivo by donor-specific blood transfusion under the cover of an anti-CD4 antibody.6Hara M Kingsley CI Niimi M Read S Turvey SE Bushell AR Morris PJ Powrie F Wood KJ IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo.J Immunol. 2001; 166: 3789-3796PubMed Google Scholar, 7Kingsley CI Karim M Bushell AR Wood KJ CD25+CD4+ regulatory T cells prevent graft rejection: cTLA-4- and IL-10-dependent immunoregulation of alloresponses.J Immunol. 2002; 168: 1080-1086PubMed Google Scholar This pretreatment protocol is also successful in inducing tolerance to heterotopic cardiac allografts in primary immunocompetent recipients.8Pearson TC Madsen JC Larsen CP Morris PJ Wood KJ Induction of transplantation tolerance in adults using donor antigen and anti-CD4 monoclonal antibody.Transplantation. 1992; 54: 475-483Crossref PubMed Scopus (143) Google Scholar Further, we have shown that CD25+CD4+ regulatory T cells generated to alloantigen in vivo using donor-specific blood transfusion and anti-CD4 antibody regulate transplant arteriosclerosis of allogeneic mouse abdominal aorta transplants, both in adoptive transfer and primary recipient systems.9Warnecke G Bushell A Nadig SN Wood KJ Regulation of transplant arteriosclerosis by CD25+CD4+ T cells generated to alloantigen in vivo.Transplantation. 2007; 83: 1459-1465Crossref PubMed Scopus (28) Google Scholar However, the development of protocols to generate regulatory T cells in vivo may be much more difficult in the clinical situation than in rodent models. An alternative approach emerging as an attractive way of exploiting T cell regulation in man is the potential transfer of in vitro generated or expanded recipient-derived regulatory T cell populations as a cellular therapy. Several different methods for in vitro expansion/generation of Tregs have been described, including polyclonal expansion of naturally occurring Tregs,10Hoffmann P Eder R Kunz-Schughart LA Andreesen R Edinger M Large-scale in vitro expansion of polyclonal human CD4(+)CD25high regulatory T cells.Blood. 2004; 104: 895-903Crossref PubMed Scopus (429) Google Scholar generation of Tregs using allogeneic antigen presenting cells (APCs), interleukin-2, and tumor growth factor (TGF)-b,11Yamagiwa S Gray JD Hashimoto S Horwitz DA A role for TGF-beta in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood.J Immunol. 2001; 166: 7282-7289PubMed Google Scholar, 12Godfrey WR Spoden DJ Ge YG Baker SR Liu B Levine BL June CH Blazar BR Porter SB Cord blood CD4(+)CD25(+)-derived T regulatory cell lines express FoxP3 protein and manifest potent suppressor function.Blood. 2005; 105: 750-758Crossref PubMed Scopus (251) Google Scholar ectopic expression of the key transcription factor Foxp3,13Hori S Nomura T Sakaguchi S Control of regulatory T cell development by the transcription factor Foxp3.Science. 2003; 299: 1057-1061Crossref PubMed Scopus (51) Google Scholar, 14Fontenot JD Gavin MA Rudensky AY Foxp3 programs the development and function of CD4+CD25+ regulatory T cells.Nat Immunol. 2003; 4: 330-336Crossref PubMed Scopus (6089) Google Scholar, 15Jaeckel E von Boehmer H Manns MP Antigen-specific FoxP3-transduced T-cells can control established type 1 diabetes.Diabetes. 2005; 54: 306-310Crossref PubMed Scopus (187) Google Scholar and selection of Tregs using T cell receptor (TCR) stimulation in the presence of rapamycin.16Battaglia M Stabilini A Roncarolo MG Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells.Blood. 2005; 105: 4743-4748Crossref PubMed Scopus (950) Google Scholar We have developed an additional novel method to generate alloreactive regulatory T cells in vitro in which naïve recipient CD4+ T cells are stimulated with bone marrow–derived donor APC in the presence of interferon (IFN)-γ. This conditioning protocol results in the emergence of a dominant CD25+CD62L+FoxP3+ regulatory T cell population (conditioned T cells, Tcon) by initiating apoptosis of potential effectors, inhibiting Th17 responses, and promoting Tregs development by expansion of naturally occurring Tregs and conversion of FoxP3− precursors.17Feng G Wood KJ Bushell A Interferon-gamma conditioning ex vivo generates CD25+CD62L+Foxp3+ regulatory T cells that prevent allograft rejection: potential avenues for cellular therapy.Transplantation. 2008; 86: 578-589Crossref PubMed Scopus (86) Google Scholar, 18Feng G Gao W Strom TB Oukka M Francis RS Wood KJ Bushell A Exogenous IFN-gamma ex vivo shapes the alloreactive T-cell repertoire by inhibition of Th17 responses and generation of functional Foxp3+ regulatory T cells.Eur J Immunol. 2008; 38: 2512-2527Crossref PubMed Scopus (89) Google Scholar The resultant population inhibits the rejection of donor-specific skin grafts mediated by naïve CD25−CD4+ effector T cells in a sensitive adoptive transfer mouse allograft model.17Feng G Wood KJ Bushell A Interferon-gamma conditioning ex vivo generates CD25+CD62L+Foxp3+ regulatory T cells that prevent allograft rejection: potential avenues for cellular therapy.Transplantation. 2008; 86: 578-589Crossref PubMed Scopus (86) Google Scholar The emergence of this population appears to be independent of endogenous interleukin-10 as none is detected in the cultures but is critically dependent on IFN-γ because cells driven under identical conditions in the absence of exogenous cytokine lack regulatory activity and contribute directly to allograft rejection.17Feng G Wood KJ Bushell A Interferon-gamma conditioning ex vivo generates CD25+CD62L+Foxp3+ regulatory T cells that prevent allograft rejection: potential avenues for cellular therapy.Transplantation. 2008; 86: 578-589Crossref PubMed Scopus (86) Google Scholar, 18Feng G Gao W Strom TB Oukka M Francis RS Wood KJ Bushell A Exogenous IFN-gamma ex vivo shapes the alloreactive T-cell repertoire by inhibition of Th17 responses and generation of functional Foxp3+ regulatory T cells.Eur J Immunol. 2008; 38: 2512-2527Crossref PubMed Scopus (89) Google Scholar Here we demonstrate that these Tregs also have the ability to impact the development of transplant associated vasculopathy and explore some of the mechanisms involved. CBA.Ca (CBA, H2k), CBA.Ca rag1−/− (CBA-rag−/−, H2k), CBA.Ca CP-1 (CP-1, H2k), C57BL/6 (B6, H2b), and C57BL/6 CD31−/− (B6 CD31−/−, H2b) mice were obtained from and housed in the Biomedical Services Unit of the John Radcliffe Hospital (Oxford, UK). CBA-rag−/− mice were originally kindly provided by Dr. D. Kioussis (National Institute for Medical Research, Mill Hill, London, U.K.). CP-1 mice are transgeneic for human CD52, a pan-leukocyte surface antigen, and were originally kindly provided by H. Waldmann.19Gilliland LK Walsh LA Frewin MR Wise MP Tone M Hale G Kioussis D Waldmann H Elimination of the immunogenicity of therapeutic antibodies.J Immunol. 1999; 162: 3663-3671PubMed Google Scholar B6 CD31−/− mice were generously provided by Drs. G. Duncan and T. Mak (Amgen Institute, Toronto, Canada). All animals were housed and treated in accordance with the Animals (Scientific Procedures) Act 1986. Sex-matched mice between 6 and 12 weeks of age at the time of the first experimental procedure were used in all experiments. CD4+ T cells were isolated from naive CBA spleens using Miltenyi CD4 (L3T4) MicroBeads. On reanalysis the CD4+ population was more than 95% pure. Bone marrow–derived dendritic cells (DCs) were generated from B6 donors using a modification of published methods.20Inaba K Inaba M Romani N Aya H Deguchi M Ikehara S Muramatsu S Steinman RM Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.J Exp Med. 1992; 176: 1693-1702Crossref PubMed Scopus (3309) Google Scholar, 21Yamaguchi Y Tsumura H Miwa M Inaba K Contrasting effects of TGF-beta 1 and TNF-alpha on the development of dendritic cells from progenitors in mouse bone marrow.Stem Cells. 1997; 15: 144-153Crossref PubMed Scopus (184) Google Scholar Briefly, bone marrow was harvested from mouse femurs, RBCs were lysed, and B cells, T cells, and MHC class II–positive cells depleted using RA3.6B2 (rat-anti-mouse B220; hybridoma obtained from ATCC, Manassas, VA), YTA 3.1 (rat-anti-mouse CD4), and YTS 169 (rat-anti-mouse CD8; hybridomas kindly provided by Prof. H. Waldmann, Sir William Dunn School of Pathology, University of Oxford, Oxford, U.K.), TIB120 (rat-anti-mouse MHC class II; ATCC), followed by negative selection using sheep-anti-rat IgG-coated Dynabeads (Dynal Biotech, Oslo, Norway). 1 × 106 enriched DCs precursor cells were cultured with 2 ng/ml each of rmGM-CSF and rhTGF-β 1 (PeproTech, London, UK), and at day 6, conditioned DCs were harvested, washed, and counted before use. 5 × 105 purified naive CBA CD4+ T cells were cocultured with 5 × 106 conditioned B6 DCs for 7 days in the presence of 5 ng/ml IFN-γ (IFN-γ conditioning). On day 7 the T cells were restimulated with conditioned B6 DCs under identical conditions and harvested after a further 7 days for adoptive transfer into immune deficient mice. CD25−CD4+ T cells for use as an effector population were purified from naïve wild-type CBA or CP1-CBA spleens. CD4+ T cells were enriched using TIB 120, M1/70 (rat-anti-mouse monocyte/macrophage, ATCC), RA3–6B2, and YTS 169 followed by incubation with anti-rat Dynabeads and magnetic separation. The enriched cells (>85% CD4+) were incubated with anti-CD25-PE antibody followed by anti-PE microbeads and CD25− cells were recovered using the MACS-system (Miltenyi Biotech Ltd., Bisley, UK). CBA-rag−/− mice were injected intravenously with 1 × 105 CD25−CD4+ T cells with or without coinjection of 4 × 105 conditioned T cells. Each experiment contained animals reconstituted with CD25−CD4+ T cells only to validate the efficacy of the effector population. One day after adoptive transfer, transplantation of a section of abdominal aorta from B6 CD31−/− donors was performed using a technique described by Koulack and colleagues.22Koulack J McAlister VC Giacomantonio CA Bitter-Suermann H MacDonald AS Lee TD Development of a mouse aortic transplant model of chronic rejection.Microsurgery. 1995; 16: 110-113Crossref PubMed Scopus (105) Google Scholar In some mice, full-thickness B6 tail skin allografts were transplanted onto graft beds on the flanks of reconstituted mice immediately following aorta transplantation. Tcon generated from total CD4+ T cells from B6 (H2b) Foxp3-GFP reporter mice were stained for CD4 and TCR-β then flow sorted using a BD FACSAria instrument. GFP+ and GFP− cells were then transferred independently into B6.Rag mice together with syngeneic CD25− cells as an effector population. One day later, these reconstituted mice were transplanted with CBA (H2k) aorta allografts. Purity of enriched T cell populations was evaluated by flow cytometry. Cells were stained for 30 minutes at 4°C with the flourochrome-coupled antibodies 145-2C11 (anti-CD3-FITC), RM4-5 (anti-CD4-PerCP), and 7D4 (anti-CD25-PE; all BD Biosciences). For analyses from mouse spleens, single cell suspensions were prepared, RBCs were osmotically lysed, and the cells incubated with anti-CD4 FITC (clone GK1.5) and anti-TCR-β PE (clone H57-597; both BD Pharmingen, Oxford, UK). Staining of intracellular Foxp3 was performed according to the manufacturer’s instructions (clone FJK-16s, eBioscience, San Diego, CA). In other assays, cells were incubated with anti-human CD52, anti-CD4-PerCP, anti-CD25-PE, and then stained for intracellular Foxp3. Data were acquired using a FACSort flow cytometer (Becton Dickinson, San Jose, CA) and analyzed using Cellquest software. 96-well MultiScreenHTS-IP filter plates (Millipore, Billerica, MA) were coated with anti-mouse IFN-γ capture antibody (AN-18, Mabtech AB, Sweden). B6 T cell depleted stimulators were prepared using rat anti-mouse CD4 (YTA3.1) and rat anti-mouse CD8 (YTS169) followed by Dynabeads and magnetic separation. Responder cells were titrated from 1 × 105 cells per well and incubated with or without 3 × 105 stimulators at 37°C and 5% CO2 for 14 hours. Spots were developed by sequential incubations with biotinylated anti-mouse IFN-γ detection antibody (R4-6A2), streptavidin-alkaline phosphatase, and BCIP/NCT substrate (all Mabtech AB, Sweden) and plates read and analyzed on an AID Bio-Tek reader (AID, Winooski, VT). The frequency of donor-reactive IFN-γ secreting CD4+ cells was calculated using the following formula:[(number of spots in wells with stimulators)−(number of spots in wells without stimulators)]. Aortic grafts were harvested 30 days after transplantation, flushed with saline then OCT-compound (Tissue-Tek, Sakura, The Netherlands) and snap-frozen embedded in OCT-compound in liquid nitrogen. Transverse sections of 5 to 6 μm thickness were cut and stained with Hematoxylin and Eosin (H&E) or Elastin van Giesson (EvG). Morphometric analysis of transplant arteriosclerosis was performed on EvG-stained sections. Digital photographs of three EvG-stained sections from each graft were taken at ×40 original magnification using a light microscope (Nikon, Tokyo, Japan) and a Coolpix digital camera (Nikon). Digitized images were then analyzed using Photoshop software (version 6.0, Adobe, San Jose, CA). The areas within the lumen of the vessel and within the internal elastic lamina were circumscribed and the respective absolute pixel counts recorded. From these measurements, the quotient for the thickness of the intima was calculated (Qint). The Qint indicates relative thickness (%) of the intima or % luminal occlusion [Qint = intima/ (lumen + intima) × 100]. Biotinylated or unconjugated antibodies against CD31, CD3, CD4, CD8, CD11b, and GR1 (BD Biosciences, Oxford, UK) were used for immunohistochemistry. Transverse 6-μm sections were air-dried, fixed, and stored at −80°C until further analyses. Primary antibodies were applied for 1 hour at room temperature or overnight at 4°C, followed by secondary streptavidin-HRP (Vector Laboratories, Burlingame, CA) for biotinylated primary antibodies or by mouse-anti-rat-IgG-HRP (Jackson Immunoresearch, West Grove, PA) for unconjugated primary antibodies for 45 minutes at room temperature. Staining was visualized using dimethylaminoazobenzene (Sigma, St. Louis, MO) and hematoxylin counterstain. For enumeration of CD4+ T cell infiltration, five random high-power fields were examined by area-counting in sections taken from three transverse planes from each individual aorta. Data were compared between groups by the Mann–Whitney U test or by analysis of variance followed by Bonferroni post hoc analyses if multiple groups were compared. P values 80% recipient-derived (Figure 3, A and B). However, this effect was almost completely inhibited by the cotransfer of IFN-γ–conditioned T cells where the vast majority of the endothelium remained donor-derived, demonstrating clearly the efficiency of regulation in this system. To maximize the chances of detecting informative phenotypic changes in tissue histology, we sought to optimize regulation in this model given the fact that cotransfer reduced rather than abolished transplant arteriosclerosis. It has been shown previously that regulation mediated by in vivo generated regulatory cells can be enhanced by antigen-specific reactivation.29Karim M Feng G Wood KJ Bushell AR CD25+CD4+ regulatory T cells generated by exposure to a model protein antigen prevent allograft rejection: antigen-specific reactivation in vivo is critical for bystander regulation.Blood. 2005; 105: 4871-4877Crossref PubMed Scopus (119) Google Scholar, 30Sawitzki B Kingsley CI Oliveira V Karim M Herber M Wood KJ IFN-gamma production by alloantigen-reactive regulatory T cells is important for their regulatory function in vivo.J Exp Med. 2005; 201: 1925-1935Crossref PubMed Scopus (250) Google Scholar We took advantage of the observation that allogeneic skin allografts are accepted in the presence of conditioned T cells in rag−/− hosts,17Feng G Wood KJ Bushell A Interferon-gamma conditioning ex vivo generates CD25+CD62L+Foxp3+ regulatory T cells that prevent allograft rejection: potential avenues for cellular therapy.Transplantation. 2008; 86: 578-589Crossref PubMed Scopus (86) Google Scholar, 18Feng G Gao W Strom TB Oukka M Francis RS Wood KJ Bushell A Exogenous IFN-gamma ex vivo shapes the alloreactive T-cell repertoire by inhibition of Th17 responses and generation of functional Foxp3+ regulatory T cells.Eur J Immunol. 2008; 38: 2512-2527Crossref PubMed Scopus (89) Google Scholar and hypothesized that skin but not aorta grafts contain sufficient donor antigen presenting cells for full reactivation of the IFN-γ conditioned Tregs. Therefore, mice were reconstituted with naïve CD25−CD4+ cells plus IFN-γ–conditioned cells and simultaneously transplanted with skin and aorta grafts. This resulted in a further reduction in transplant arteriosclerosis (1.9 ± 3.0% luminal occlusion), indicating improved suppressive function mediated by the IFN-γ–conditioned Tregs, consistent with enhanced reactivation in vivo (Figure 4, A–C versus Figure 2C). Importantly, the presence of an additional skin graft resulted in luminal obstruction that was virtually identical to that seen in mice transplanted with aortic allografts but without cell reconstitution (Figure 1C, P = 0.55). Thus, additional alloantigen challenge reduced vasculopathy to a level that resulted from the surgery itself. A clear impact of the additional skin graft was also seen in terms of the number of CD4+ T cells found within the aorta itself. As shown in representative photomicrographs (Figure 4D), CD4+ T cells were readily detected in aorta allografts of mice reconstituted with effectors only and in mice reconstituted with effectors plus Tcon and correlated closely with CD3 staining (data not shown). However, in aorta allograft recipients reconstituted with T effectors plus Tcon and cotransplanted with an additional skin graft, CD4+ T cells were virtually absent. Enumeration showed that this effect is statistically significant (Figure 4E) indicating that at least part of the mechanism of regulation mediated by reactivated Tregs involves an inhibition of effector cell recruitment to the graft i
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