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Keywords:

  • Endothelial cells;
  • Foxp3;
  • IFNγ;
  • rat;
  • regulatory T cells;
  • tolerance

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Regulatory T cells have been described to specifically accumulate at the site of regulation together with effector T cells and antigen-presenting cells, establishing a state of local immune privilege. However the mechanisms of this interplay remain to be defined. We previously demonstrated, in a fully MHC mismatched rat cardiac allograft combination, that a short-term treatment with a deoxyspergualine analogue, LF15-0195, induces long-term allograft tolerance with a specific expansion of regulatory CD4+CD25+T cells that accumulate within the graft. In this study, we show that following transfer of regulatory CD4+T cells to a secondary irradiated recipient, regulatory CD25+Foxp3+ and CD25+Foxp3 CD4+T cells accumulate at the graft site and induce graft endothelial cell expression of Indoleamine 2, 3-dioxygenase (IDO) by an IFNγ-dependent mechanism. Moreover, in vivo transfer of tolerance can be abrogated by blocking IFNγ or IDO, and anti-IFNγ reduces the survival/expansion of alloantigen-induced regulatory Foxp3+CD4+T cells. Together, our results demonstrate interrelated mechanisms between regulatory CD4+CD25+T cells and the graft endothelial cells in this local immune privilege, and a key role for IFNγ and IDO in this process.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Functionally specialized subsets of regulatory CD4+T cells have been shown to play an important role in the regulation of both T-cell-mediated and innate immune responses (1). In particular, the CD4+CD25+ subpopulation of T cells has been shown to be crucial in self-tolerance and also to prevent allograft rejection (2–4). It was recently reported that most human and mouse regulatory CD4+CD25+T cells express forkhead box protein 3 (Foxp3), a specific transcription factor that controls both their development and function (5–7). As CD25 is not uniquely expressed by regulatory T cells, but can also be expressed by activated effector cells, the identification of Foxp3 has provided an opportunity to specifically identify regulatory T cells and to track them in vivo so as to better define the mechanisms involved in their regulation of immune responses (8,9).

In models of allograft tolerance in rodents, alloantigen-specific and highly suppressive regulatory CD4+CD25+T cells have been shown to preferentially accumulate within the graft, where they hold effector cells in check by exertion of regulatory mechanisms (10,11). These findings suggest an interplay between regulatory T cells, effector cells and antigen-presenting cells (APC) within the graft itself, leading to local immune privilege and long-term tolerance. The comprehension of the active suppressive mechanisms involved in this phenomenon of local immune privilege may help to generate new therapies in the clinical setting.

We previously demonstrated in a fully MHC mismatched rat cardiac allograft combination, that a short-term treatment with a deoxyspergualine analogue, LF15-0195, induces allograft tolerance with no signs of chronic rejection at long-term and is characterized by a specific expansion of CD4+CD25+T cells that accumulate in the spleen and graft (12–14). Moreover, we observed an accumulation of transcripts coding for numerous cytoprotective molecules in long-term tolerated allografts, such as Indoleamine 2, 3-dioxygenase (IDO), Nitric Oxide synthase (NOS) and Heme-Oxygenase-1 (HO-1) (15). These data suggested that in our model, long-term tolerance is maintained locally by interrelated mechanisms involving regulatory T cells and the graft itself, to regulate local effector cells and to prevent graft damage.

We previously showed that CD4+T cells from the spleen or from the graft are able to transfer tolerance to a subsequent sublethally irradiated host, demonstrating the presence of powerful regulatory T cells (13,14). However, the involvement of Foxp3+ regulatory T cells and the mechanisms of action of tolerance transfer remained to be defined. Here, we demonstrate that following transfer of regulatory CD4+T cells to a secondary recipient, regulatory CD25+Foxp3+ and CD25+Foxp3 CD4+T cells accumulate in the graft and induce the expression of IDO by graft endothelial cells (EC). This induction is strictly dependent on IFNγ, a molecule that plays a crucial role in allograft tolerance in our model. This study thus reveals a key role for IFNγ in the induction of local immune privilege by regulatory CD4+CD25+T cells.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Animals and transplantation

Rats were purchased from the ‘Centre d'Elevage Janvier' (Genest-Saint-Isle-France) and maintained in an animal facility under standard conditions according to our institutional guidelines. LEW.1W (RT1u) or LEW.1A (RT1a) rats served as heart donors and LEW.1A as recipients. Heterotopic cardiac allografts were performed as previously described (16). LF15-0195 (Fournier Laboratories) was administered to recipients by i.p. injection at 3 mg/kg/day for 20 days (12). The model of chronic rejection was induced by two donor blood transfusions (DST) prior to transplantation (15,17). The graft function was assessed daily by scoring heart pulsations through the abdominal wall.

Antibodies

Some anti-rat monoclonal antibodies (Abs) (European collection of Cell Culture [Salisbury, UK]) were labeled with FITC, Allophycocyanin-Cyanin7 (APC-Cy7), PE, AlexaFluor-647 or-488 (Bioatlantic, Nantes, France). Commercial Abs: anti-rat CD3, Foxp3 (Clinisciences, Montrouge, France), CD31 (Serotec, Oxford, UK); Rabbit polyclonal anti-rat iNOS (Sigma-Aldrich, Saint Quentin, France), IDO (Neosystems, Strasbourg, France) and HO-1 (Stressgen Biotechnologies, Canada). Biotin-anti-mouse IgG, AlexaFluor-568 anti-mouse, FITC anti-rabbit, HRP-streptavidin and PE-streptavidin purchased from Vector Laboratories (Burlingame, CA). HRP-conjugated goat anti-rabbit or anti-mouse IgG (from Pierce, Rockford, IL). The hybridomas producing anti-rat CD28 antibody was kindly provided by T. Hüning (University of Würzburg, Germany) and anti-rat IFNγ (DB-1) by P.H. Van der Meide (Rijswijk, The Netherlands).

Western blot

Total lysate from naïve hearts or from syngeneic, chronically rejected or tolerant allografts (day 100 or from transferred recipients [day 35]) were prepared. Nitrocellulose membrane blocked with TBS (Tween-20-Tris buffered saline)-5% milk, were incubated with rabbit anti-IDO, anti-HO-1 or mouse anti-tubulin (1 μg/mL) diluted in TBS-5% milk (overnight) and then with HRP-goat anti-rabbit (1:6000) or anti-mouse IgG (1:3000). Bands were revealed by enhanced chemiluminescence (Amersham, Little Chalfont, UK), exposed to Kodak film and quantified with Kodak Digital Science Image Analysis 1D Software.

Immunohistology

Cardiac tissue was snap-frozen in liquid nitrogen after embedding in OCT compound (Tissue Tek, Miles Laboratories, Elkhart, IN). Cryostat sections (7 m) fixed in acetone were incubated overnight with anti-IDO, iNOS or HO-1 Abs (1.5 μg/mL), followed by a FITC-anti-rabbit, by anti-rat CD31 or class II MHC (5μg/mL) and by AlexaFluor-568 anti-mouse Abs, and DAPI and mounted in Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA), and observed by fluorescence microscopy (Axioskop2 plus, Carl Zeiss Inc., Göttingen, Germany). Images were visualized (×400) and processed using the AxionVision Viewer program (Carl Zeiss Inc.).

Cell purification

Graft-infiltrating cells (GIC):  GIC from tolerant recipients (day 100) or from recipients that had received cell transfers (day 5 or day 35) were recovered, pressed through a stainless steel mesh and isolated by density gradient centrifugation on Ficoll-Paque (Amersham, Biosciences, Sweden) and then stained with mAbs and analyzed with a FACS LSR-II flow cytometer (Becton Dickinson, Mountain View, CA).

T-cell purification:  Spleen-derived CD4+, CD8+, CD4+CD25 and CD4+CD25+ T cells from naïve rats or from tolerant recipients were purified by positive selection using a FACSAria flow cytometer (Becton Dickinson). Splenocytes were stained with R7-3-FITC, Ox35-Cy7, Ox8-PE or Ox39-APC Alexa-647 for CD4+, CD8+, CD4+CD25 or CD4+CD25+ T-cell purification. Purity was >99%.

Endothelial cell-T-cell coculture

EC of LEW.1W or LEW.1A origin were isolated as previously described (18) and plated into 6 well plates (NUNC™Merck, Eurolab, France) (1 × 106 cells/well) in complete medium (RPMI 1640 supplemented with 2 mM l-glutamine, 5 × 10−5 M-2-mercaptoethanol, 1 mM sodium pyruvate, 1% nonessential amino acids, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 10% heat-inactivated FCS [GIBCO]). After overnight culture (37°C, 5% CO2), highly purified CD4+CD25 or CD4+CD25+T cells from naïve rats or from tolerant recipients were added (5 × 105 cells/well) to the EC cultures (with irrelevant mAb [3G8] or anti-IFNγ at 5 μg/mL). Twenty-four h later, the lymphocytes were discarded and the adherent EC were washed several times and lysed with Trizol (Invitrogen) for RT/PCR analysis of hypoxanthine phosphoribosyltransferase (HPRT) and IDO expression.

RNA extraction and real-time quantitative RT/PCR

RNA extraction and real-time quantitative RT/PCR was performed as previously described in an Applied Biosystems-GenAmp 7700 Sequence Detection System using SYBR-Green PCR Core Reagents (19). The oligonucleotides used were: rHPRT (Up-CCTTGGTCAAGCAGTACAGCC, Lo-TTC-GCTGATGACACAAACATGA), rIDO (Up-ATCCAGACACCTTTTTCCACG, Lo-CAGCAGATCCTTCACCAACG) and rIFNγ (Up-TGGATGCTATGGAAGG-AAAGA, Lo-GATTCTGGTGACAGCTGGTG). HPRT was used as an endogenous control gene to normalize for varying starting amounts of RNA, data were expressed in arbitrary units (AU).

Stimulation assays

Anti-CD3/CD28:  CD4+T cells from naive rats or from rejecting or tolerant recipients (5 × 104 cells/well) were stimulated in 96 well flat-bottom plates (NUNC™) coated with anti-CD3 (0.75 μg/mL) and with addition of soluble anti-CD28 (0.6 μg/mL) in a final volume of 200 μL of complete medium.

Mixed Leukocyte Reaction (MLR):  APC-enriched cell populations from donor-type isolated by a Nicodenz gradient served as stimulator cells. Responder (CD4+T cells) (2 × 105) were labeled with CFSE (Carboxyfluorescein Diacetate, Succinimidyl Ester) (Molecular Probes, Eugene, OR) as previously described (20) and plated with stimulator cells (5 × 104) in 96 well round-bottomed plates in 200 μL of complete medium. For intracellular cytokine staining, CD4+T cells were cultured in the presence of brefeldin A (5 μg/mL, last 4 h). Cells were stained with anti-TCRαβ and CD4 mAbs, fixed in 2% paraformaldehyde, incubated with PBS/0.2% FCS/0.5% saponin (15 min) and then with anti-IFNγ or anti-Foxp3-APC (5 μg/mL) for 1 h, washed and analyzed using a FACS LSR II after gating on the CD4+T-cell population.

In vivo transfer experiments and IFNγ and IDO neutralization

A total of 20 × 106 CD4+T cells from tolerant recipients or from naïve rats (in some cases stained with CFSE) were injected i.v. (in 500 μL of PBS) on the day of cardiac transplantation into secondary syngeneic irradiated recipients (4 Grey, whole body irradiation (IFR 26, Nantes, France) 1 day before transplantation). A neutralizing anti-rat IFNγ mAb was injected i.p. (2.5 mg/kg) every day. 1-Methyl-DL-Tryptophan (Sigma) neutralizing IDO enzymatic activity (competitive inhibitor) was prepared by dilution in NaOH, adjusted to pH 9 with HCl, diluted in water, and administered orally at 0.2 mg/kg twice daily as previously described (21).

Statistical analysis

The Kaplan–Meier method was used to establish survival curves. Survival was analyzed with a log-rank test, and differences were considered significant when p-values were <0.05. Statistical evaluation was performed using the Student's t-test for unpaired data and results were considered significant if p-values were <0.05. Data were expressed as mean ± SEM.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Foxp3+CD4+CD25+ regulatory T cells accumulate in the spleen and graft of tolerant recipients

We previously demonstrated an accumulation of CD4+CD25+T cells expressing high levels of Foxp3 mRNA transcripts in the spleen and graft of long-term tolerant recipients and that Foxp3 could be considered as a marker of regulatory CD4+CD25+T cells in rats (13,14). Here we studied the expression of Foxp3 at the protein level in CD4+T cells from tolerant recipients. We found a significant increase in the percentage of Foxp3+CD4+CD25+T cells in the spleen of tolerant recipients (8.6%) compared to naïve rats (3.9%), long-term syngeneic recipients (4.4%) and recipients that had rejected their allografts (3.6%)(n = 4, *p < 0.05, **p < 0.01, Figure 1A[a]; representative dot plot Figure 1B). We also observed numerous CD4+CD25+T cells that did not express Foxp3 in the spleen of tolerant recipients (16.9%) compared to naïve rats (4.6%), long-term syngeneic recipients (8%) and recipients that had rejected their allografts (7%) (n = 4, **p < 0.01, Figure 1A[a]; representative dot plot Figure 1B). The same results were observed in absolute number (n = 4, *p < 0.05, **p < 0.01, ***p < 0.001, Figure 1A[b]). Moreover, up to 40% of the CD4+T cells expressed CD25 within the graft-infiltrating cells of tolerant recipients, with about half of them expressing Foxp3 (n = 4, Figure 1A[a]; representative dot plot Figure 1B).

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Figure 1. High percentage and absolute number of Foxp3+CD4+CD25+ and Foxp3CD4+CD25+T cells in the spleen and allograft of long-term tolerant recipients. (A) (a) Percentage, (b) Absolute number and (B) representative dot plots of Foxp3+CD25+ and Foxp3CD25+ in spleen-derived CD4+T cells from naïve rats, from long-term recipients with syngeneic grafts, from long-term recipients that had rejected their allografts (untreated), from long-term tolerant allograft recipients (day 100 posttransplantation), and graft infiltrating CD4+T cells from long-term tolerant recipients (day 100 posttransplantation). Cells were stained with anti-TCR, anti-CD4 and anti-CD25, fixed, permeabilized and intracellular stained with anti-Foxp3 mAb as described in the Materials and Methods. Cells were analyzed by FACS with gating on CD4+T cells and results are expressed as percentage and absolute number of CD4+T cells (n = 4), * p < 0.05, ** p < 0.01, *** p < 0.001.

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These data demonstrate a specific accumulation of Foxp3+CD4+CD25+T cells in the spleen and graft of tolerant recipients, and also the presence of numerous Foxp3CD4+CD25+T cells.

Endothelial cells from long-term-tolerated allografts express IDO, iNOS and HO-1

We previously demonstrated a high expression of transcripts coding for IDO, iNOS and HO-1 in long-term-tolerated allografts compared to allografts that had developed chronic rejection and syngeneic grafts (15). In order to determine which cells express these molecules, we performed immunofluorescence on allografts. Interestingly, only EC (CD31+) from tolerated allografts expressed HO-1, iNOS or IDO (Figures 2A[b], [c] and [d], respectively). Staining was attributed mostly to medium–sized vessels for iNOS and HO-1, and to small vessels for IDO. In long-term allografts that had developed chronic rejection or in syngeneic grafts, no staining for HO-1 or iNOS was detectable by immunofluorescence and staining for IDO was observed in only a few EC. The higher expression of IDO in tolerated allografts compared to syngeneic grafts and of HO-1 compared to allografts displaying signs of chronic rejection was confirmed at the protein level by Western blot (Figure 2B, n = 3, *p < 0.05). These results demonstrate a high and specific expression of HO-1, iNOS and IDO by EC in long-term-tolerated allografts.

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Figure 2. Endothelial cells from long-term-tolerated allografts express high levels of HO-1, iNOS and IDO. (A) Representative pictures of immunofluorescence analysis of long-term-tolerated allografts (Tol.), syngeneic grafts (Syng.) or chronically rejected allografts (C.R.) (day 100 posttransplantation) as described in Material and Methods for (a) Dapi (blue) and isotypic controls rabbit IgG-anti-rabbit FITC (green) and mouse IgG-anti-mouse Alexa 647 (red), respectively (b) HO-1, (c) iNOS and (d) IDO (green) with Dapi (blue) and CD31 (red) staining and merged. Original magnification: ×400 for HO-1 and iNOS and ×600 for IDO. (B) Representative Western Blot analysis of IDO and HO-1 expression in naive hearts (Naive), long-term syngeneic grafts (Syng.), chronically rejected allografts (C.R.) or tolerated allografts (Tol.) (day 100 posttransplantation). The histogram represents IDO/Tubulin and HO-1/Tubulin signal quantification in three samples for each group *p < 0.05.

Following transfer, CD4+T cells from tolerant recipients induce accumulation of regulatory Foxp3+CD25+ and IFNγ+ CD4+T cells and the production of IDO by donor graft EC

The accumulation of regulatory T cells within the tolerated allografts and the high expression of cytoprotective molecules by graft EC suggested an interplay between these cells. To test this hypothesis, we transferred CFSE-labeled spleen-derived CD4+T cells from tolerant recipients or from naïve rats to secondary, sub lethally irradiated heart allograft recipients and analyzed their proliferation, homing and phenotype. Five days after transfer of CFSE-labeled CD4+T cells from naïve rats or tolerant recipients, some CFSE+CD4+T cells were detected in the new allograft and were proliferating (3%, n = 4), but none of them were Foxp3+ (Figure 3A[a] and [b]). However, interestingly, a higher percentage of CFSECD4+Foxp3+ was observed in the allografts following transfer of CD4+T cells from tolerant recipients compared with transfer of CD4+T cells from naïve rats (16% vs. 10%, n = 4, Figure 3A[a] and [b]). These data demonstrate a specific accumulation of Foxp3+CD4+T cells in the allografts following transfer with CD4+T cells from tolerant recipients that seem to be newly induced regulatory T cells from host origin. Moreover, similar to the observations in long-term-tolerant recipients, 35 days after transfer of CD4+T cells from tolerant recipients, numerous Foxp3+CD4+CD25+ and Foxp3CD4+CD25+ T cells accumulated in the spleen (9% and 13%, respectively) and within the graft (14% and 27%, respectively) compared to the spleen of recipients that have been transferred with naïve CD4+T cells and that received a syngeneic graft (6% and 3%, respectively)(n = 3 or 4, Figure 3B[a] and [b], **p < 0.01, ***p < 0.001). Interestingly, in the allografts, the Foxp3CD4+CD25+T cells contained numerous IFNγ+-producing cells (11%; n = 3, Figure 3B[b]). These data demonstrate that following transfer with regulatory CD4+T cells from tolerant recipients, newly induced Foxp3+ and IFNγ+ CD4+CD25+T cells accumulated in the allografts.

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Figure 3. Following transfer to a secondary recipient, CD4+T cells from tolerant recipients induce the accumulation of Foxp3+ and IFNγ+ CD4+T cells. (A) (a) Percentage and (b) representative dot plots of CFSE+ versus Foxp3+ cells in CD4+T cells in the allografts of transferred recipients. 20 × 106 CFSE-labeled spleen CD4+T cells from long-term-tolerated allografts or from naïve rats were transferred to a secondary irradiated host as described in the Materials and Methods. Five days after transfer, graft infiltrating cells were purified, stained with anti-TCR and anti-CD4, fixed, permeabilized and intracellular stained with anti-Foxp3 mAb as described in the Materials and Methods. Cells were analyzed by FACS with gating on CD4+T cells (n = 3 or n = 4), *p < 0.05. (B) (a) Percentage of Foxp3+CD25+ and Foxp3CD25+ in spleen-derived CD4+T cells of transferred recipients that have received naïve CD4+T cells (20 × 106) and syngeneic grafts (Transf. syng., n = 5) and from recipients that have received CD4+T cells from tolerant recipients (20 × 106) and allogeneic grafts (Transf. tol., n = 3). (b) Percentage of Foxp3+CD25+, Foxp3CD25+ and IFNγ+CD25+ in CD4+T cells in the allografts of recipients that have received CD4+T cells from tolerant recipients (20 × 106) and allogeneic grafts (GIC Transf. tol., n = 3). Thirty-five days after transfer, spleen-derived or graft infiltrating cells were purified stained with anti-TCR, anti-CD4 and anti-CD25, fixed, permeabilized and intracellular stained with anti-Foxp3 or anti-IFNγ mAb as described in the Materials and Methods. Cell were analyzed by FACS with gating on CD4+T cells and results are expressed as percentage of CD4+T cells.

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Moreover, allografts from recipients that had received CD4+T cells from tolerant recipients expressed higher levels of protein for IDO than syngeneic grafts from recipients that had received CD4+T cells from naïve rats (day 35, n = 3, **p < 0.01, Figure 4A[a]). By immunohistology, IDO was found to be expressed exclusively by EC of small-size vessels (CD31+) and not by any MHC Class II+ leukocytes (Figure 4A[b]). However, HO-1 was not expressed by EC but by other leukocytes, and no expression of iNOS protein was detected in the allografts (data not shown). These data suggested that CD4+T cells from recipients that had received cell transfers were able to induce graft EC to produce IDO. To test this hypothesis in vitro, we coincubated highly purified CD4+CD25+ or CD25T cells from tolerant recipients or from naïve rats with allogeneic (donor origin) or syngeneic adherent EC and assessed expression of IDO. As shown in Figure 4B, only the CD25+ subpopulation of CD4+T cells from tolerant recipients was able to induce significant expression of IDO transcripts in allogeneic EC (n = 3, **p < 0.01). This induction was moderate compared to that obtained with a high dose of IFNγ. Moreover, the induction was donor-specific and strictly dependent on IFNγ. These data demonstrate that CD4+CD25+T cells from tolerant recipients directly induce donor-type EC to rapidly express IDO through an IFNγ-dependent mechanism.

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Figure 4. Regulatory CD4+T cells from tolerant recipients induce donor-type EC to express IDO by an IFNγ-dependent mechanism. (A) (a) Representative Western Blot analysis of IDO expression in naive hearts (Naïve, n = 3), syngeneic grafts from recipients that had received CD4+T cells from naive rats (Transf. syng., n = 5) (day 35 posttransplantation) or allografts from recipients that had received CD4+T cells from tolerant recipients (Transf. tol, n = 3) (day 35 posttransplantation). The histogram represents IDO/Tubulin signal quantification, **p < 0.01. (b) Representative pictures of immunofluorescence analysis of transferred allografts (day 35 posttransplantation) as described in Material and Methods for IDO (green) with Dapi (blue) and CD31 or MHC Class II (red) staining (merged). Original magnification: ×600. (B) CD4+CD25 or CD4+CD25+T cells from naïve or tolerant recipients were coincubated for 24 h with allogeneic LEW.1W or syngeneic LEW.1A EC lines in the presence of anti-IFNγ or control 3G8 (5 μg/mL), and IDO was assessed specifically in EC by quantitative RT/PCR as described in the Materials and methods. EC were incubated with a high dose of IFNγ (50 ng/mL) to induce IDO expression. Results are expressed in AU of IDO/HPRT ± SEM (n = 3, **p < 0.01).

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In vivotransfer of tolerance is dependent on IFNγ and IDO

In order to determine whether IDO or IFNγ, which is a potent inducer of IDO expression (22), play a role in the mechanisms of transfer of tolerance, we used neutralizing anti-rat IFNγ mAb and 1-Methyl-DL-Tryptophan. Transfer of tolerance with CD4+T cells from tolerant recipients was abrogated in 3 out of 4 recipients by in vivo treatment with anti-IFNγ mAb (n = 4, *p < 0.05, Figure 5A) and in all recipients treated with 1-Methyl-DL-Tryptophan (n = 4, **p < 0.01, Figure 5A). However, 1-Methyl-DL-Tryptophan treatment do not break the tolerant state of tolerated allografts induced by LF15-0195 treatment suggesting other mechanisms of regulation at long-term (n = 4, Figure 5A). No effect of control irrelevant mAb 3G8 or vehicle was observed (data not shown). Graft recipients that had received cell transfers and that had promptly rejected their allografts following anti-IFNγ treatment (day 10 after transplantation, n=2) contained numerous CD4+CD25+low T cells in their allografts (40%) and few CD4+CD25+highFoxp3+ T cells (7%) (n = 2, Figure 5B) compared to the controls at day 35 (14%, Figure 3B, and representative dot plot Figure 5B). These data suggest that anti-IFNγ treatment induces cellular rejection with numerous effector CD4+T cells and few regulatory Foxp3+CD4+T cells infiltrating the allografts. Moreover, rejected allografts from transferred recipients treated with anti-rat IFNγ had considerably less IDO transcripts than untreated recipients (Figure 5C, n = 3, *p < 0.05). These results demonstrate that IFNγ plays a crucial role in the mechanisms of tolerance transfer in our model that is mediated by the induction of IDO expression by graft EC.

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Figure 5. Anti-IFNγ treatment abrogates transfer of tolerance by blocking induction of IDO expression. (A) Long-term LF15-0195-treated recipients or secondary recipients that have been transferred with no cells (untreated) or 20 × 106 spleen CD4+T cells from long-term-tolerated allografts were treated as described in the Materials and Methods. A neutralizing mouse anti-rat IFNγ mAb (clone DB1, IgG1) was injected i.p. at day 0 (2.5 mg/kg) every day until rejection. 1-Methyl-DL-Tryptophan neutralizing IDO enzymatic activity (competitive inhibitor) was administered orally at 0.2 mg/kg twice daily. Graft survival was monitored daily by abdominal palpation, and rejection was defined as cessation of heartbeat (n = 4 in each group, **p < 0.01, *p < 0.05). (B) Representative Dot plots of Foxp3 versus CD25 in CD4+T cells from allografts of transferred recipients that had received no treatment and that tolerated their allografts (Transf. tol.) (day 35 posttransplantation) or that had rejected the allograft by anti-IFNγ treatment (Transf. tol. + anti-IFNγ) (day 10). Graft-infiltrating cells were purified stained with anti-TCR, anti-CD4 and anti-CD25, fixed, permeabilized and intracellular stained with anti-Foxp3 as described in the Materials and Methods. Cell were analyzed by FACS with gating on CD4+T cells and results are expressed as percentage. (C) IDO transcripts were assessed by quantitative RT/PCR as described in the Materials and Methods in syngeneic grafts from recipients that had received CD4+T cells from naïve rats (Transf. syng., n = 5) (day 35 posttransplantation), in allografts from recipients that had received CD4+T cells from tolerant recipients (Transf. tol, n = 3) and in allografts from recipients that had received CD4+T cells from tolerant recipients that have rejected their allografts following anti-IFNγ treatment (Transf. tol. + anti-IFNγ, n = 3); *p < 0.05,** p < 0.01. Results are expressed in AU of IDO/HPRT ± SEM.

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Expression of IFNγ is detected in the Foxp3 subpopulation of CD4+CD25+T cells but is important for the survival of regulatory Foxp3+CD4+CD25+T cells

In order to determine whether regulatory Foxp3+CD4+ CD25+T cells expressed IFNγ, we assessed intracellular IFNγ production following alloantigen specific or polyclonal stimulation. We were unable to detect intracellular IFNγ protein by FACS in Foxp3+CD4+T cells from either naïve or tolerant recipients following either types of stimulation (data not shown). However, we observed expression of IFNγ by Foxp3CD4+T cells from tolerant recipients following alloantigen-specific or polyclonal stimulation (Figure 6A, 2.6% and Figure 6B, 6.4%, n = 3, respectively) that was similar to that observed with Foxp3CD4+T cells from naïve rats (Figure 6A, 3.4% and Figure 6B, 5.5%, n = 3, respectively) but reduced compared to that observed with Foxp3CD4+T cells from rejecting recipients (Figure 6A, 10.3%, ***p < 0.001 and Figure 6B, 12.9%, n = 3, **p < 0.01, respectively). In order to determine whether spleen-derived CD4+CD25+T cells from tolerant recipients expressed quantitatively more IFNγ than those from naïve rats, we performed quantitative RT/PCR and observed no difference in the level of expression of IFNγ transcripts (Figure 6C). However, the level of IFNγ transcripts in the CD4+CD25+T cells was relatively low compared to that in the CD4+CD25 and CD8+T-cell subpopulations.

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Figure 6. IFNγ is expressed at high levels by nonFoxp3+CD4+T cells. T cells from naïve rats, from long-term tolerant recipients (day 100) or from recipients that had rejected their allografts (untreated) (A) were cultured with allogeneic (LEW.1W) APC or (B) underwent polyclonal stimulation (plate bound anti-CD3 plus soluble anti-CD28) for 3 days as described in the Materials and Methods. Cells were recovered and stained with anti-TCR and anti-CD4, fixed, permeabilized and intracellular stained with anti-Foxp3 and anti-IFNγ mAb as described in the Materials and Methods. Cell were analyzed by FACS with gating on the CD4+T cells and results are expressed as percentage of CD4+T cells (n = 3). **p < 0.01, ***p < 0.001. (C) IFNγ transcripts were assessed by quantitative RT/PCR as described in the Materials and Methods in highly purified CD4+CD25+, CD4+CD25 and CD8+T cells from naïve rats or from long-term tolerant recipients; n = 3 in each group. Results are expressed in AU of IFNγ/HPRT ± SEM.

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Interestingly, we noted that the addition of anti-IFNγ during in vitro alloantigen-specific stimulation of CD4+T cells from tolerant recipients strongly reduced the percentage of recovered Foxp3+CD4+T cells (Figure 7A, 1.6% vs. 4.1%, n = 3, **p < 0.01). We also observed a reduction, albeit to a lesser degree, in recovered Foxp3+CD4+T cells with CD4+T cells from naïve rats in the presence of anti-IFNγ (Figure 7A, 1.3% vs. 2.9%, n = 3, **p < 0.01). However, anti-IFNγ treatment did not reverse the hypoproliferative state of CD4+T cells from tolerant recipients despite the reduction in Foxp3+CD4+T cells (27% of proliferation vs. 25% with control antibody 3G8 and 41% with CD4+T cells from naïve rats) (Figure 7B). These results demonstrate that IFNγ is detected by intracellular FACS staining only in the Foxp3CD4+T-cell subpopulation but seems to play a role in the survival/expansion of regulatory Foxp3+CD4+T cells.

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Figure 7. IFNγ is important for survival/expansion of alloantigen-induced Foxp3+CD4+ regulatory T cells. CFSE-labeled T cells from naïve rats or from long-term tolerant recipients (day 100) were stimulated by allogeneic (LEW.1W) APC for 5 days in the presence of anti-IFNγ (DB1) or irrelevant Ab (3G8) (5 μg/mL) as described in the Materials and Methods. Cells were recovered and stained with anti-TCR and anti-CD4, fixed, permeabilized and intracellular stained with anti-Foxp3 as described in the Materials and Methods. Cell were analyzed by FACS after gating on CD4+T cells. (A) Results are expressed as percentage of Foxp3+ cells in the CD4+T-cell population (n = 3, **p < 0.01). (B) Representative histograms of CFSE profile. Results represent the percentage of proliferation of CD4+T cells and the Mean Fluorescence of the proliferating cells. Data are representative of three independent experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

We previously demonstrated, in a fully MHC mismatched rat cardiac allograft combination, that a short-term treatment with a deoxyspergualin analog, induces long-term tolerance with a specific expansion of CD4+CD25+T cells that accumulate in the spleen and graft and are able to transfer tolerance to a secondary recipient (12–14). In this study, we demonstrated that both Foxp3+ and Foxp3 subpopulations of CD4+CD25+T cells accumulated in the spleen and the graft of tolerant recipients. The Foxp3CD4+CD25+T cells contained IFNγ-secreting cells that could represent other kinds of regulatory T cells or allogeneic-activated T cells. In fact, it has been shown in a model of skin allograft transplantation in mice that regulatory T cells represent only about one-half of the T cells that are present in tolerated allografts and that these nonregulatory T cells are able to reject the grafts when the regulatory CD4+T subpopulation is depleted (11,23). It has also been shown, in a model of colitis, that regulatory T cells accumulate in the colonic lamina propria and are in direct contact with dendritic cells, as well as effector T cells (24). Therefore, tolerated grafts could contain effector T cells, being held in check directly or indirectly by CD4+ regulatory T cells.

Here, we show that following transfer, CD4+T cells from long-term-tolerated recipients induce the accumulation of Foxp3+ and IFNγ+ CD4+T cells in the graft of a secondary recipient and induce EC to express high levels of IDO. These data demonstrate a state of local immune privilege within the graft with an interplay between regulatory T cells and graft EC to regulate T-cell responses and maintain long-term tolerance. Moreover, we show in vitro and in vivo that the induction of IDO expression in EC by CD4+T cells is IFNγ-dependent and that we were able to abrogate the transfer of tolerance by blocking IFNγ or IDO in vivo. Therefore, IFNγ plays a key role in the mechanisms of tolerance in this model. IFNγ has been shown to be a potent inducer of IDO in APC (22). IDO has been described as being expressed primarily by monocytes/macrophages and dendritic cells, but also by EC (25–27). A role for IDO in T-cell anergy and apoptosis has been demonstrated in several studies by depleting tryptophan and tryptophan metabolites (25,28–30), and over-expression of IDO in human EC has been shown to prevent T-cell activation (31).

We found that anti-IFNγ led to a reduction of regulatory Foxp3+CD4+T cells and to the abrogation of tolerance transfer. These data suggest an important role for IFNγ in the survival/expansion of alloantigen-induced regulatory Foxp3+CD4+T cells and in the induction of IDO by graft EC. IFNγ is usually considered as a pro-inflammatory cytokine, driving Th1-type cell-mediated immune responses (32,33). However, recent data have demonstrated a paradoxical function for IFNγ, depending on the concentration and the microenvironment in which it is expressed. Along these lines, IFNγ treatment has been shown to downregulate experimental autoimmune encephalomyelitis by enhancing NO in local microglia and astrocytes in the target tissue (34). Moreover, a population of autoantigen-specific regulatory CD4+T cells, secreting low levels of IFNγ, was shown to directly stimulate APC to produce NO and to inhibit the proliferation of pathogenic T cells and the development of diabetes (35). In the transplantation setting, the generation and function of alloantigen-reactive regulatory T cells in a model of anti-CD4 therapy was impaired dramatically in IFNγ-deficient mice (36). The authors demonstrated that alloantigen-induced regulatory CD4+CD25+T cells rapidly and transiently express low levels of IFNγ and that induction of tolerance following adoptive transfer is IFNγ-dependent (36). Moreover, in rat, donor alloantigen-specific regulatory CD8+T cells have previously been shown to promote local graft immune privilege through IFNγ-dependent IDO expression (37).

We were unable to detect intracellular production of IFNγ protein by spleen-derived or graft-infiltrating Foxp3+CD4+ CD25+T cells from long-term tolerant recipients. It is possible that IFNγ expression is transient and/or too low to enable detection by intracellular staining. Another explanation could be that the source of IFNγ is not the Foxp3+ subpopulation but the Foxp3 subpopulation of CD4+CD25+T cells. Indeed, in our model, we demonstrated numerous Foxp3CD4+CD25+T cells that accumulate within the graft and spleen and that express quantitatively low levels of IFNγ. These cells could be regulatory T cells not expressing Foxp3. Alternatively, a part of these cells could be effector cells expressing low levels of IFNγ that are held in check by regulatory Foxp3+CD4+CD25+T cells. The IFNγ produced by these cells may be necessary for the survival, expansion and functional properties of the regulatory Foxp3+CD4+CD25+T cells. Along these lines, it has been shown, in both the human and mouse setting, that in vitro IFNγ treatment of CD4+CD25T cells leads to their conversion into regulatory CD4+T cells, as characterized by increased expression of Foxp3 and enhanced regulatory function (38).

In conclusion, we show here a key role for IFNγ and IDO in the induction of local immune privilege in allograft tolerance with an interplay between regulatory T cells and graft EC. Further studies will help to define the paradoxical role of IFNγ in the immune response and its regulation, with prospects for developing immunotherapies in a clinical setting.

References

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  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References
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