HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Degauque, N. Articles by Brouard, S. Search for Related Content PubMed PubMed Citation Articles by Degauque, N. Articles by Brouard, S.

Dominant Tolerance to Kidney Allografts Induced by Anti-Donor MHC Class II Antibodies: Cooperation between T and Non-T CD103⁺ Cells

Nicolas Degauque^*,, David Lair^*,, Alexandre Dupont^*,, Anne Moreau, Gwénaelle Roussey^*,, Frédérique Moizant^*,, François Xavier Hubert^*,, Cédric Louvet^*,, Marcelo Hill^*,, Fabienne Haspot^*,, Régis Josien^*,, Claire Usal^*,, Bernard Vanhove^*,, Jean Paul Soulillou^1,2,*, and Sophie Brouard^2,*,

^* Institut National de la Santé et de la Recherche Médicale–Université de Nantes, Unité Mixte de Recherche 643, Nantes, France; and Immunointervention dans les allo et Xenotransplantations and Institut de Transplantation et de Recherche en Transplantation and Pathology Laboratory, Centre Hospitalier Universitaire-Hotel Dieu, Nantes, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Allograft acceptance can be induced in the rat by pretransplant infusion of donor blood or spleen cells. Although promoting long-term acceptance, this treatment is also associated with chronic rejection. In this study, we show that a single administration of anti-donor MHC class II alloimmune serum on the day of transplantation results in indefinite survival of a MHC-mismatched kidney graft. Long-term recipients accept a donor-type skin graft and display no histological evidence of chronic rejection. The kidney grafts of tolerant animals display an accumulation of TCR C, FoxP3, and IDO transcripts. Moreover, as compared with syngeneic recipients, tolerant recipients harbor a large infiltrate of MHC class II⁺ cells and CD103⁺ cells. In vitro, splenocytes from tolerant recipients exhibit decreased donor-specific proliferation, which is restored by depletion of non-T cells and partially restored by the blockade of IDO. Finally, splenocytes from tolerant recipients, but not purified T cell splenocytes, transfer donor-specific infectious tolerance without chronic rejection, after infusion into naive recipients, over two generations. However, splenocytes depleted of T cells or splenocytes depleted of CD103⁺ cells fail to transfer tolerance. Collectively, these data show that a single administration of anti-donor MHC class II alloimmune serum induces a tolerant state characterized by an infiltration of the kidney graft by regulatory T cells and CD103⁺ cells. These data also show that the transfer of tolerance requires the presence of both T cells and CD103⁺ dendritic cells. The precise mechanism of cooperation of these two cell subsets remains to be defined.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Inducing and understanding the mechanism of donor-specific tolerance, defined as the indefinite survival of a well-functioning graft in an immunocompetent adult host in the absence of immunosuppression, remains the challenge of transplant immunology. Experimentally, a variety of maneuvers can induce donor-specific allograft tolerance, including pretransplantation priming with donor MHC Ags (blood or splenocytes) (1, 2, 3), transfected cells expressing donor MHC Ags (4), soluble MHC molecules or allopeptides (5), MHC gene transfer (6, 7), or DNA vaccination (8). In addition, we have shown that injection of recipients with Abs directed against donor class II MHC can also induce specific tolerance to renal vascularized allografts in adult rats (9, 10). This approach has two potential advantages over donor cell priming (1, 11): it works when the injection is administered at the time of surgery (9) and only donor determinants are targeted. In a model of heart transplantation in the MHC-mismatched LEW.1W to LEW.1A combination, the administration of anti-donor class II Abs induced substantial graft survival prolongation (10). In these experiments, injection of a blocking anti-TGF Ab inhibited the capacity of anti-donor class II Abs to prolong graft survival. Graft survival was also shortened if the graft was depleted of dendritic cells (DC)³ before being transplanted (10). This observation suggested that the tolerance-"inducing" effect of anti-donor Abs was dependent on their interaction with class II-expressing graft resident DC.

Different hypotheses have been proposed to explain the mechanism of action of anti-donor MHC class II Abs. Because the only MHC class II cells in normal rat hearts are DC (12, 13, 14), anti-donor Abs could act in a passive way by inhibiting the direct recognition of LEW.1W DC by LEW.1A T cells. This hypothesis is supported by the absence of CD4⁺ cells in the heart graft (10). Moreover, the interaction of host CD4⁺ cells with donor MHC class II molecules has been reported as critical for the rejection of murine cardiac allografts (15). However, in the same strain combination, removing DC from donor hearts (by irradiation or cyclophosphamide treatment) prolonged survival to a limited extent only (16). Thus, one can hypothesize that anti-donor MHC class II Abs could act in an active manner by inducing a regulatory signal after interaction with DC resident within the heart. Anti-donor MHC class II Abs could opsonize donor resident DC and thus target them for phagocytosis by a specific subset of DC. These tolerogenic DC could lead to the anergy of allogeneic effector cells and/or the conversion of T effector cells into regulatory T cells. Several reports favor this hypothesis and highlight the ability of certain subsets of DC to induce T cells with regulatory functions (for review, see Ref.17).

In this article, using the same LEW.1W to LEW.1A genetic combination but a kidney graft model in which anti-class II Abs induce indefinite graft survival, we further characterize the mechanisms involved in the maintenance phase of tolerance (100 days after transplantation). We show that anti-donor class II Abs induce a donor-specific tolerance, which could be transferred to naive recipients. The grafts of tolerant recipients are characterized by an infiltration of regulatory T cells, suggesting that regulatory T cells may be instrumental within the graft environment. However, we show that transfer of tolerance in this model cannot be achieved by large numbers of purified T cells but require the presence of both non-T CD103⁺ DC and T cell splenocytes. The cooperative mechanisms between these two cell subsets remains to be defined.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Animals and surgical procedures

Inbred male adult rats (200–250g) of the LEW.1A (RT1a) and LEW.1W (RT1u) congenic strains were purchased from Janvier. All animals were maintained under standard conditions according to European and Institutional Guidelines. Orthotopic kidney transplantations were performed aseptically, as previously described (9). Recipients underwent an initial nephrectomy on the day of transplantation and a second nephrectomy was performed 7 days later. Rejection, indicated by the death of the binephrectomized rat, was confirmed by histology. In parallel, modifications in renal function were monitored in the urine (total protein and creatinine) and serum (urea, creatinine). The protein:creatinine ratio was considered normal when below 0.2 and pathological (proteinuria) when above this level. Tolerant recipients (>100 days) were challenged with donor (LEW.1W), third-party (Brown Norway (BN)), or recipient (LEW.1A) MHC-type expressing skin grafts. Skin grafts were transplanted onto the lateral thoracic wall and were inspected daily for rejection. Graft function was monitored by measuring the blood urea (mmol/L), creatinemia (µmol/L) levels, and the urine protein (g/L):creatinine (mmol/L) ratios (proteinuria, g/mmol) in tolerant recipients (n=5) compared with naive rats (n=4).

Experimental groups and adoptive transfer experiments

Group 1 consisted of unmodified LEW.1A recipients of LEW.1W kidneys (n=6). In group 2, LEW.1A recipients were injected i.v. with 0.5 ml of anti-LEW.1W class II alloimmune serum (n=15) at the end of the surgical procedure (9) (see Table I). Additional groups of recipients were used to test the effect of cell transfer from tolerant recipients to naive nonirradiated recipients of a LEW.1W kidney. Adoptive transfers were performed i.v. on the day of transplantation. Group 3 received 8 x 10⁷ splenocytes from tolerant recipients (n=12). Group 4 received 8 x 10⁷ purified T cells from the spleens of tolerant recipients (n=5). Group 5 received 5–8 x 10⁷ T cell-depleted splenocytes from tolerant recipients (n=6). Group 6 received 8 x 10⁷ CD103⁺ cell-depleted splenocytes from tolerant recipients (n=5). Group 7 received 8 x 10⁷ splenocytes from naive LEW.1A rats (n=3). Group 8 received 8 x 10⁷ T cell-depleted splenocytes from naive LEW.1A rats (n=2).

LEW.1A rats (n=10) were immunized with LEW.1W skin grafts and injected i.v. 3 days later and biweekly for 2 mo with 10⁸ mononuclear cells purified from LEW.1W spleen cells. Depletion of anti-class I Abs was performed as described elsewhere (10).

Cell purification

APC purification. APC from tolerant recipients, naive LEW.1A, LEW.1W, or BN rats were enriched from spleens digested with collagenase D (2 mg/ml; Boehringer Mannheim) for 20 min at 37°C. Ten micromolar EDTA was added for 5 min and cells were washed and resuspended in 5 µM EDTA/PBS/2% FCS. The resulting suspension was deposited on a Nicodenz gradient (14.5%; Nycomed Pharma), centrifuged, and adjusted to the appropriate concentration.

Spleen and blood cell purification. One hundred days after transplantation, spleen cells from tolerant recipients and naive LEW.1A rats were isolated by passing spleen tissue through a stainless steel mesh and depleting erythrocytes by osmotic shock. The splenocytes isolated were then washed twice in PBS. One portion was enriched for T cells using rat T cell-enrichment columns (R&D Systems) and another portion was depleted of T cells using an anti-CD3 Ab followed by Dynal beads (Dynal Biotech). Cell purifications and depletions were systematically checked by flow cytometric analysis (>95%). PBL from tolerant recipients and naive LEW.1A rats were enriched by a Ficoll gradient procedure and washed twice in PBS.

CD103⁺ splenocyte depletion. CD103⁺ splenocytes were depleted by positive selection of anti-_E-chain integrin-expressing cells (CD103⁺) using OX62 mAb followed by incubation with Pan Mouse IgG Dynabeads according to the manufacturer’s recommendations (Dynal Biotech); cell purity was systematically assessed using flow cytometry (>95%).

Graft-infiltrating cell extraction. Kidneys were digested with collagenase D (2 mg/ml; Boehringer Mannheim) twice for 10 min at 37°C. Cells were then extracted by passing kidney tissue through a stainless steel mesh. The resulting suspension was deposited on a Ficoll gradient, centrifuged, and depleted of erythrocytes by osmotic shock. The kidney-infiltrating cells isolated were then washed twice in PBS. One part was used for flow cytometry analysis and the other for MLR.

MLRs and inhibition assays

Standard one-way MLR were performed. Splenocytes were isolated from naive rats and tolerant recipients 100 days after transplantation. Donor-type LEW.1W and third-party BN-enriched APC were irradiated. Triplicate samples of responder (10⁵ cells/well) and stimulator cells (2 x 10⁴ cells/wells) were plated in 96-well round-bottom plates in a final volume of 200 µl of RPMI 1640 medium supplemented with 2 mM L-glutamine, 5 x 10^–5M 2-ME, 1 mM sodium pyruvate (Invitrogen Life technologies), 1% nonessential amino acids, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10% heat-inactivated FCS (Invitrogen Life Technologies). The cells were then cultured at 37°C in 5% CO₂. Proliferation of the responder population was assessed on day 3 and/or 5 by measuring the incorporation of [³H]TdR (0.5 µCi/well; Amersham Biosciences) during the final 8 h of culture. Cells were then harvested onto glass fiber filters and [³H]TdR incorporation was measured by standard scintillation procedures (Packard Instrument). In certain experiments, MLR were performed in the presence of the IDO antagonist 1-methyltriptophan (500 µg/well; Sigma-Aldrich). The results were expressed as specific cpm.

To test the suppressive activity of TCR^–CD103⁺ DC from tolerant recipients, 2 x 10⁴ enriched DC suspensions from LEW.1W rats were cultured in triplicate in U-bottom 96-well plates (0.2 ml) with 1 x 10⁵ T cells purified from naive LEW.1A and decreasing numbers of enriched TCR^–CD103⁺ DC from tolerant anti-class II-treated LEW.1A rats irradiated (used as control) or not for 5 days at 37°C in 7% CO₂. Cultures were pulsed with [³H]TdR for the last 8 h of culture. To test the suppressive activity of T cells from tolerant recipients, 1 x 10⁵ splenocytes or whole T cells purified from naive or tolerant LEW.1A rats were cultured in triplicate in U-bottom 96-well plates (0.2 ml) with 2 x 10⁴ enriched DC suspensions from either LEW.1W or BN rats for 5 days at 37°C in 7% CO₂. For inhibition assessment, increasing numbers of splenocytes purified from naive or tolerant LEW.1A rats were added to the MLR using naive T cells as responders. Cultures were pulsed with [³H]TdR for the last 12 h of culture. The results were expressed as specific cpm.

Abs and flow cytometric analysis

Splenocytes and graft-infiltrating cells were isolated and prepared as previously described. Analysis was performed using CellQuest software (BD Biosciences). The following hybridomas were obtained from the American Type Culture Collection and produced by Bioatlantic or in our laboratory and were used to phenotype rat leukocytes from the spleen and blood of naive LEW.1A rats and anti-donor class II-treated recipients 100 days after transplantation: W3/25 (anti-CD4), OX1 and OX30 (anti-CD45), OX3 (anti-RT1.u), OX6 (anti-RT1.u/RT1.a), OX7 (anti-Thy 1–1), OX8 (anti-CD8), OX19 (anti-CD5), OX22 (anti-CD45RC), OX26 (anti-CD71), OX33 (anti-CD45 present on B cells), OX34 (anti-CD2), OX35 (anti-CD4), OX39 (anti-CD25), 0X41 (anti-GRP), OX62 (anti-₁-chain integrin; CD103), OX85 (anti-CD62L), WT1 (anti-CD11A), WT5 (anti-CD11B), 8A2 (anti-CD11C), JJ319 (anti-CD28), anti-CD80 (anti-B7.1, 3H5), anti-CD86 (anti-B7.2, 24F), 3.2.3 (anti-NKRP1), and R7.3 (anti-TCR).

Kidneys were minced and digested in 2 mg/ml collagenase D (Roche) in RPMI 1640/1% FCS for 15 min at 37°C. EDTA (10 mM) was added for the last 5 min, and the cell suspension was then pipetted up and down several times and filtered. Cells were washed once in PBS/2mM EDTA/1% FCS, and mononuclear cells were isolated by centrifugation over Ficoll-Hypaque (Amersham Biosciences). For cytofluorometric analyses, 1 x 10⁶ cells were incubated for 20 min at 4°C with R7.3-PE, CD4-PE-cyanin 7 (Cy7), OX62-allophycocyanin, or OX6-allophycocyanin-Cy7 mAb along with different FITC-conjugated mAb: TCR, TCR, CD25, CD161, CD11b, CD62L, CD172a, CD45RC, or CD8. Cells were washed twice and analyzed on a FACSAria (BD Biosciences). CD161a and CD172a were used to discriminate plasmacytoid DC (OX62⁺CD4⁺ DC) and OX62⁺CD4^– DC subsets (18). Data were analyzed using FlowJo software (Tree Star).

Histology and immunohistology

Kidney tissue samples were placed in 10% formol. Hematoxylin-phloxin-Safran (H&S) staining was performed on paraffin-embedded sections. Vascular lesions (percentage of obstruction, leukocyte infiltration, and medium lesions) were analyzed in at least 10 medium-size vessels. Snap-frozen graft sections from tolerant recipients (>100 days) embedded in Tissue Tek (OCT compound; Bayer Diagnostics) were cut into 5-µm sections and fixed in acetone for 10 min at room temperature for immunochemistry. The cell infiltrate was measured by immunochemistry using a three-step indirect immunoperoxidase technique with primary Ab and the corresponding biotin-conjugated anti-mouse Ig-Ab, HRP-conjugated streptavidin, and vasoactive intestinal peptide substrate. The area of each immunoperoxidase-labeled tissue section infiltrated by cells was determined by quantitative morphometric analysis as previously described (19). Briefly, the number of positively stained cells on each slide was counted by morphometric analysis using the point-counting technique, with a 121 intersection squared grid. The number of positively stained cells under the grid intersection was counted in a minimum of 10 adjacent fields (at a magnification of x 400). The percentage area of each section occupied by cells of a particular phenotype was calculated as follows: [number of positive cells under grid intersection/(total number of grid intersections = 121)] x 100.

The results are expressed as the mean ± SD of positive cells per area of the tissue section. The following primary Abs were used: OX1/OX30 (anti-CD45), ED2 (anti-CD68 like), R7-3 (anti-TCR), OX62 (anti-₁-chain integrin), OX6 (anti-RT1.u/RT1.a), W3/25 (anti-CD4), OX8 (anti-CD8), and 3.2.3 (anti-NKRP-1). Anti-donor-specific IgM and IgG isotypes were examined in the kidney grafts of tolerant recipients (>100 days posttransplantation) and syngeneic rats. Rejected grafts from untreated recipients harvested on day 7 were used as a positive control. Deposits of the different isotypes of rat Ig in the kidneys were studied by immunochemistry. Nontransplanted kidneys from LEW.1W rats were used as controls. Sections were incubated for 45 min with FITC-conjugated mouse mAbs directed against rat IgM (MARM-4) (1/100) and IgG (MARG1) (1/100) (University of Louvain, Brussels, Belgium) and rinsed three times with PBS.

mRNA transcript analysis

C, IDO, HO-1, foxP3, and cytokine (IL-2, IFN-, IL-13, TGF1, IL-10, IL-4, and TNF-) transcript analysis was performed by real-time PCR as described elsewhere (20) on kidney grafts from tolerant recipients and syngeneic LEW.1A grafts on day 100 after transplantation and on kidneys from naive nontransplanted LEW.1W rats. HPRT was used as an endogenous control to normalize for RNA levels. The results were expressed as the intrasample target:HPRT mRNA copy number ratio.

Statistical analysis

Data were analyzed using the Mann-Whitney U test (analysis of two groups) or the Kruskal-Wallis test followed by a Dunn’s post hoc test (analysis of more than two groups). A two-way ANOVA followed by a Bonferroni post hoc test was used to analyze the effect of the addition of 1-methyltryptophan (1-MT) and the effect of anti-donor class II treatment. Survival curves were analyzed using the Kaplan-Meier test. Differences were defined as statistically significant when p < 0.05 (_*) and p < 0.01 (_**).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Anti-donor class II Abs induce tolerance of kidney grafts in recipients that reject third-party skin grafts

MHC-mismatched LEW.1W kidney graft survival in untreated LEW.1A recipients was 11 ± 1 days (n = 6) (Table I). Administration of a single dose of 0.5 ml of anti-donor class II alloimmune serum (Table I) induced long-term graft survival in all LEW.1A recipients (n = 15). The same serum, however, did not prolong the survival of BN (RT1ⁿ) kidneys (data not shown). Furthermore, long-term surviving kidney allograft recipients accepted a LEW.1W skin graft but rejected a third-party BN skin graft (Fig. 1A). Tolerated kidney allografts displayed no histological or clinical signs of chronic rejection (Fig. 1B). Finally, these results were supported by normal 100-day protein:creatinine ratios in urine samples (0.16 ± 0.02 g/mmol; n = 5) and by low and stable blood urea (6.42 ± 2.6 mmol/L) and creatinemia (35.4 ± 7 µmol/L) levels (naive rat urea, 5.5 ± 0.3 mmol/L; creatinemia, 27.5 ± 4.4 µmol/L; protein:creatinine ratio, <0.2 g/mmol; Fig. 1C). Kidneys from tolerant recipients displayed only barely detectable levels of IgM and IgG deposition, similar to that observed in syngeneic grafts harvested 100 days after transplantation (Fig. 1D).

View larger version (46K): [in this window] [in a new window]   FIGURE 1. Anti-donor class II Abs induce donor-specific tolerance. A, Long-term surviving LEW.1A recipients of a kidney allograft accepted a donor-Ag (LEW.1W) skin graft but rejected a third-party (BN) skin graft. Tolerant recipients accepted a donor-matched skin graft. B, Kidney grafts from tolerant recipients did not show pathological changes indicative of chronic rejection, but only slight local fibrosis and lymphoid infiltration, only moderate acute tubular necrosis, and no vascular or glomerular lesions. C, Blood urea and creatinine and urine protein:creatinine ratios were measured in tolerant recipients 1 year posttransplantation and compared with normal rats. D, Kidney grafts from tolerant recipients displayed barely detectable levels of IgM (a) and IgG (d) deposition, similar to that observed in syngeneic grafts (b and e, respectively) unlike rejected grafts from untreated recipients harvested on day 7 after transplantation and used as a positive control (c and f, respectively).

The grafts of anti-donor class II-treated tolerant recipients displayed an increased number of MHC-II and ₁-integrin⁺ (CD103⁺) infiltrating cells

One hundred days after transplantation, the grafts of tolerant rats exhibited a larger infiltrate than those from syngeneic recipients (81 ± 46 vs 12.5 ± 2.9 CD45⁺ cells per graft surface area in tolerant rat kidneys and syngeneic kidneys, respectively) (p < 0.05) or normal LEW.1W rats (9.7 CD45⁺ cells per graft surface area) (Fig. 2). As assessed by immunochemistry, the grafts from tolerant rats were mainly infiltrated by MHC class II⁺ cells (66 ± 27 vs 15 ± 10 cells per graft surface area in tolerant rat kidneys and syngeneic kidneys, respectively) and CD103⁺ cells (27.8 ± 8.3 vs 4.2 ± 0.5 cells per graft surface area in tolerant rat kidneys and syngeneic kidneys, respectively; p < 0.05). Grafts from tolerant recipients presented a T cell infiltrate (CD4⁺ or CD8⁺) similar to that observed in the grafts of syngeneic recipients (Fig. 2). Graft-infiltrating cells from tolerant rats were then analyzed by five-color flow cytometry to characterize the CD103⁺ cells in more detail. Fitting with the immunochemistry analysis, the graft-infiltrating cells contained relatively high numbers of CD103⁺ cells compared with naive animals (Fig. 3). Eighty percent (78 ± 4.8%) of these CD103⁺ cells coexpressed TCR, among which 76 ± 3.5% were CD8⁺ with a CD45RC^–/+ CD62L^lowCD25^– phenotype, 19% ± 2 were CD4⁺ T cells characterized by a CD25^lowCD45RC^–CD62L^– phenotype (Fig. 3), and 7% ± 5 were double-positive CD4⁺CD8⁺ cells. One-third of the remaining 20 ± 4.2% of the CD103⁺TCR^– cells could be further divided into MHC II⁺CD4⁺ (45 ± 3%) and MHC II⁺CD4^– (55 ± 3%) DC (21), and two-thirds were MHC II^–, among which 59 ± 3.1% were T cells with a CD25⁺ phenotype. These data suggest that tolerated grafts harbor various types of infiltrating cells, including CD8⁺ T cells with a CD103⁺CD45RC^–CD62L^low phenotype, a population that has also been described as being potentially regulatory/activated T cells (22, 23).

View larger version (17K): [in this window] [in a new window]   FIGURE 2. Phenotype of cells infiltrating the grafts of tolerant and syngeneic recipients 100 days after transplantation. Graft-infiltrating cells from tolerant () or syngeneic () recipients were characterized by immunochemistry. The phenotype of graft-infiltrating cells is displayed as mean ± SD of positive cells per area of tissue section as previously described (19 ).

View larger version (50K): [in this window] [in a new window]   FIGURE 3. Phenotype of CD103+ graft-infiltrating cells from tolerant recipients 100 days after transplantation. Graft-infiltrating cells (see Materials and Methods for isolation process) of three tolerant recipients were stained with a mixture of TCR-PE, OX35-PE-Cy7, CD103-allophycocyanin, and MHC II-allophycocyanin-Cy7 mAbs along with different FITC-conjugated mAbs directed against TCR, TCR, CD25, CD161, CD11b, CD62L, CD172a, CD45RC, or CD8, and analyzed on a FACSAria. Cells were first separated, based on the expression of CD103 and TCR, into CD103+TCR–, CD103+TCR+, and CD103–TCR+ cells (top right panel). The phenotype of these subsets was then analyzed using different markers as indicated. Fig. 3 shows data obtained in graft-infiltrating cells of one tolerant recipient, representative of three. The expression of CD103 was tested without or with collagenase digestion and no significant differences were observed (data not shown).

Tolerated kidneys from anti-donor class II-treated tolerant rats displayed high levels of C, FOXP3, and IDO transcripts

Transcript levels of C, HO-1, foxP3, IDO, and the cytokines IFN-, IL-2, IL-4, IL-13, IL-10, TNF-, and TGF were measured in the grafts of tolerant rats and compared with those in syngeneic rats 100 days after transplantation and in normal kidneys. Kidney grafts from tolerant recipients displayed significantly higher levels of IDO transcripts (>8-fold, p < 0.05) and foxP3 (>100-fold, p < 0.05). The level of C was >30-fold (p < 0.05) when measured in syngeneic grafts and normal kidneys (Fig. 4). No difference was found for TGF1, HO-1, or for Th1 and Th2 cytokines in kidneys from the three groups (data not shown).

View larger version (15K): [in this window] [in a new window]   FIGURE 4. Kidneys from tolerant recipients display a higher level of foxP3, C, and IDO transcripts. IDO (A), C (B), and foxP3 (C) transcript levels were measured by RT-PCR in kidneys from tolerant recipients and compared with those in normal kidneys and syngeneic grafts 100 days after transplantation.

Splenocytes and purified spleen T cells from naive and tolerant (>100 days) LEW.1A rats were stimulated for 5 days with donor LEW.1W or with BN third-party APC-enriched populations. Splenocytes from tolerant recipients displayed a 5-fold decrease in their donor-specific proliferative response to LEW.1W APC (p < 0.05), whereas their response to third-party BN-enriched APC was unmodified (Fig. 5A). In contrast, purified T cells from the spleens of naive LEW.1A rats or tolerant recipients (>100 days) proliferated similarly when stimulated with donor LEW.1W APC (Fig. 5B).

View larger version (23K): [in this window] [in a new window]   FIGURE 5. Splenocytes but not spleen T cells from tolerant recipients exhibit donor-specific hyporesponse. Total spleen cells and purified spleen T cells from naive LEW.1A rats and tolerant recipients were stimulated for 5 days with donor Ag (LEW.1W) or third-party (BN)-enriched APC populations. A, Spleen cells from tolerant recipients displayed a decreased proliferative response to donor Ag APC in MLR (p < 0.05; a) but not to third-party APC (b). B, Purified T cells from tolerant recipients proliferated similarly to T cells isolated from naive recipients when stimulated with donor Ag.

View larger version (20K): [in this window] [in a new window]   FIGURE 6. CD103+ non-T cells, but not T cells, from tolerant recipients exhibit regulatory properties. A, The ability of purified T cells from tolerant recipients to inhibit proliferation of naive LEW.1A T cells against donor Ag APC was assessed in a coculture system by adding increasing numbers of T cells from tolerant recipients. B, Suppressive activity of TCR–CD103+ cells from tolerant recipients when added to cocultures of naive LEW.1A T cells stimulated with LEW.1W donor APC. C, Effect of the inhibition of IDO production, by addition of 1-MT, on the proliferative response of splenocytes from tolerant recipients and naive rats in MLR.

Because IDO, which was found to be overexpressed in tolerated grafts, has also been shown to be involved in several regulatory effects of non-T cells and particularly DC, we further investigated its potential role in vitro in controlling the tolerant T cell response to donor APC. Splenocytes from tolerant recipients were tested in MLR in the presence or absence of a specific IDO antagonist (1-MT). As earlier, splenocytes from tolerant recipients proliferated significantly less (5-fold) than naive splenocytes (p < 0.05; Figs. 5A and 6C). Addition of 1-MT significantly restored the proliferation of splenocytes from tolerant recipients stimulated with LEW.1W donor APC (p < 0.01; Fig. 6C), suggesting a role for this molecule in this model.

Transfer of splenocytes from anti-donor class II-treated recipients induces donor-specific tolerance of LEW.1W kidney grafts in secondary immunocompetent LEW.1A naive rats

To test the hypothesis of regulatory cell involvement in the maintenance phase of tolerance, we performed different series of cell transfers from tolerant recipients (>100 days) into secondary immunocompetent, nonirradiated, naive LEW.1A recipients of LEW.1W kidney grafts. Splenocytes harvested from tolerant recipients and administered i.v. to secondary LEW.1A recipients consistently induced long-term survival of LEW.1W kidneys (Table I and Fig. 7A) but not of third-party BN kidneys (data not shown). As a control, splenocytes from naive LEW.1A rats did not prolong survival of LEW.1W kidneys (mean survival time, 11 ± 1 days; Table I and Fig. 7A). Secondary hosts of a MHC-disparate LEW.1W kidney graft that had received an injection of splenocytes from anti-donor class II-treated recipients displayed a robust and dominant tolerance without histological or clinical signs of chronic rejection (Fig. 7B). The absence of chronic rejection in secondary hosts was also supported by normal protein:creatinine ratios in the urine and normal levels of blood urea and creatinine on day 100 after adoptive transfer (data not shown). Finally, this tolerant state was transferable over a second generation of naive recipients when splenocytes from secondary tolerant recipients were transferred into a third immunocompetent naive LEW.1A recipient of a LEW.1W kidney graft (data not shown).

View larger version (27K): [in this window] [in a new window]   FIGURE 7. Tolerance induced by anti-donor class II administration is infectious. A, Kidney graft survival in untreated LEW.1A recipients was 11 ± 1 days (n=6). Splenocytes (8 x 107) from tolerant recipients (n=12) induced long-term survival of secondary untreated and immunocompetent LEW.1W kidney grafts. T cell-depleted splenocytes (5–8 x 107) from tolerant recipients (n=6) only moderately prolonged graft survival. A total of 8 x 107 CD103+-depleted splenocytes from tolerant recipients had no effect on graft survival prolongation (n=5). B, The kidneys of secondary recipients that had received spleen cells from tolerant recipients did not show any pathological changes classically associated with chronic rejection.

TCR^– CD103⁺ spleen cells from tolerant rats are instrumental in transferring tolerance to a second LEW.1W kidney graft

Because splenocytes from tolerant recipients consistently induced tolerance in secondary nonirradiated LEW.1A hosts and because CD4⁺CD25⁺ regulatory T cells have been shown to play an important role in other models of tolerance in the same LEW.1W/LEW.1A strain combination (Ref.24 and D. Lair, in preparation), we first tested the capacity of purified T cells to transfer tolerance. However, even at high doses (8 x 10⁷), purified spleen T cells from tolerant recipients had no effect on the survival of subsequent LEW.1W kidney allografts placed into secondary LEW.1A hosts (n = 5; Table I and Fig. 7A). These data are in agreement with the in vitro findings (Fig. 6A) and demonstrate the inability of the T cell population from tolerant recipients to control the alloresponse of naive T cells.

Because unseparated splenocytes but not T cells induced tolerance, we then tested the effect of non-T cell splenocyte transfer. T cell-depleted splenocytes (5 x 10⁷–8 x 10⁷) prolonged survival of subsequent LEW.1W kidney allografts in three of six naive secondary recipients without inducing long-term acceptance or tolerance (Table I and Fig. 7A). The effect of this transfer was not dose dependent (three recipients injected with 5 x 10⁷ cells rejected their graft on days 14, 21, and 21 and three recipients injected with 8 x 10⁷ cells still rejected on days 12, 12, and 50). T cell-depleted splenocytes (8 x 10⁷ cells) from naive LEW.1A rats used as controls had no effect on graft survival (Table I).

Collectively, these data strongly suggest that whereas T cells alone are unable to transfer tolerance, they are instrumental for the full effect of non-T cells. We thus tried to further investigate the non-T cell compartment which could cooperate with T cells in transferring full tolerance in this model. Kidney grafts from tolerant recipients are mainly infiltrated by MHC II⁺ cells (Fig. 2). Since DC is the main population expressing MHC II molecules, we investigated their ability to cooperate with T cells to achieve tolerance transfer. Most of the DC present in the spleen are recognized by the OX62⁺ Ab specific for the ₁ integrin (CD103⁺) marker (25). Interestingly, 8 x 10⁷ CD103⁺-depleted splenocytes from tolerant recipients were no longer able to transfer prolongation of graft survival of subsequent LEW.1W kidney allografts placed in secondary LEW.1A hosts (n=5; Table I and Fig. 7A). These data, which are also in agreement with the in vitro findings (Fig. 6B), strongly suggest that CD103⁺ non-T cells from tolerant rats are not only able to educate naive host T cells when transferred in vivo but could also cooperate with T cells from the spleen to transfer full tolerance in this model.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The capacity of anti-donor MHC alloantibodies to prolong survival or induce tolerance to allografts is well documented. We previously showed that a single administration of anti-donor MHC II alloimmune serum at the time of transplantation induces significant prolongation of heart graft survival (10). In this study, using the same strain combination, we show that anti-donor class II Abs induced a full state of tolerance to a kidney graft, i.e., without functional or histological signs of chronic rejection, that could be transferred to immunocompetent secondary recipients over two generations. This contrasts with the tolerance induced by donor cell priming in which severe chronic rejection coexists with the capacity to transfer long-term survival to naive secondary recipients (Ref.26 and our similar observations in the LEW.1W/LEW.1A combination).

The ability to transfer tolerance to unmanipulated recipients by cell transfer over multiple generations is a well-known phenomenon referred to as "infectious tolerance" where the regulatory properties of cells from tolerant recipients can be transferred to a naive cell population, thus converting them into regulatory cells. Since regulatory CD4⁺CD25⁺ T cells have been shown to be capable of converting naive effector T cells into regulatory T cells (27), we initially set out to determine whether regulatory T cells were responsible for the infectious tolerance in this model.

The normal numbers of spleen CD4⁺CD25⁺ T cells in tolerant animals (data not shown), the lack of suppressive activity of T cells from tolerant animals when tested in vitro, along with the inability of purified T cells from tolerant recipients to transfer tolerance to naive secondary hosts suggested that CD4⁺CD25⁺ regulatory T cells alone could not explain the ability of splenocytes to transfer tolerance. Along the same lines, contrasting with the majority of tolerance transfer protocols including our own observations in the same strain combination (Ref.24 and Lair et al., submitted for publication), irradiation of the secondary recipient was not required. This suggests that the classical homeostatic expansion of CD4⁺CD25⁺ regulatory T cells in a secondary recipient where empty space has been made is not necessary here. The lack of effect of a large number of purified T cells in transferring long-term graft survival here contrasts with the reproducible effect of T cell transfer in the same strain combination in two other protocols of tolerance; donor-specific transfusion-induced tolerance (Ref.26 and Lair, in preparation) and deoxyspergualine derivative-induced tolerance (24), again indicating that T cells were not directly instrumental in transferring tolerance in this model.

We therefore addressed the possibility that non-T cells and, in particular, DC were responsible for tolerance transfer, as observed in other models (3, 10, 24, 28, 29). This was possible given our findings that proliferation of donor-reactive T cells from tolerant recipients in MLR was inhibited by the presence of non-T cells and that TCR^–CD103⁺ cells from tolerant recipients were able to suppress the proliferation of naive host T cells in vitro. However, the possibility of tolerance transfer being mediated solely by non-T cells or DC was also excluded by the finding that neither non-T cells nor splenocytes depleted of CD103 (a marker specific for spleen DC) were able to transfer tolerance.

Thus, our data show that both T cells and CD103⁺ cells have to be present to transfer tolerance. Such a hypothesis could be validated by a "mixing approach" demonstrating the need for both T lymphocytes and CD103⁺ APC to transfer tolerance. However, to our knowledge, no study in the literature has reported this type of experiment. This may be explained by the difficulties encountered when attempting to reconstitute a mixed cell population in vivo after an in vitro cell purification. In this study, negative selection was chosen to avoid any possible alteration of the selected population resulting from positive selection through Ab binding. Using this approach, we were able to identify the different cell populations required for the transfer of tolerance and to minimize the procedures necessary to obtain a pure cell population.

With regard to the mechanisms of the maintenance of tolerance in this model, several lines of evidence point toward a role for regulatory T cells: high intragraft accumulation of IDO and foxP3 and large numbers of infiltrating TCR⁺CD103⁺ cells, a cell type previously described as having regulatory properties (22, 30). In fact, CD103⁺ has also been shown to be present on distinct regulatory T cell populations at different sites and its expression may be regulated locally (22, 31, 32). These cells were, however, not detected in the spleen (data not shown). The strong accumulation of C transcripts contrasting with the normal TCR⁺-infiltrating cells compared with syngeneic rats gives further credence to the hypothesis that T cells from the tolerant rats had acquired a regulatory/activated phenotype. Moreover, several models have described the existence of DC that function by inducing or enhancing the suppressive efficacy of different lineages of regulatory T cells (33, 34) as well as the induction of foxP3 expression by T cells that have acquired regulatory function following stimulation by autologous mature DC (35, 36).

The finding of IDO transcript accumulation in kidney grafts from tolerant recipients and that a specific IDO antagonist partially restored the proliferative anti-donor response of splenocytes from tolerant recipients in vitro suggests a role for this molecule in our model. IDO is expressed by non-T cells and particularly by DC (37, 38, 39) and can act directly on T cells or indirectly on non-T cells (38, 40) Its mechanism of action here remains to be explored further. The incomplete restoration of the proliferative response of splenocytes from tolerant recipients by the IDO inhibitor 1-MT may be related to the high sensitivity of activated T cells to inhibition by tryptophan metabolites (41). Moreover, the increased proliferation of naive responding cells in MLR during IDO blockade suggests that it may exert a "natural" immunosuppressive effect. This is in accordance with the fact that T cells have a regulatory checkpoint within their cell cycle that is sensitive to the level of free tryptophan (42). This effect may be intensified in tolerant recipients. However, the lack of IDO transcripts in the spleens of tolerant recipients suggests that this enzyme is mostly produced in the graft. Nevertheless, given that IDO was measured in whole splenocytes, it is possible that the DC from the spleen (1–2% of the total cells) may not be in sufficient numbers to allow differences with spleens from syngeneic grafts to be detected.

Altogether, these data show that following tolerance induction by anti-donor class II Ab administration, T cells alone are not sufficient to transfer a state of dominant tolerance but require the presence of TCR^–CD103⁺ DC. We suggest that these cells act in concert with T cells from tolerant recipients to educate naive host T cells. Finally, we suggest that the IDO pathway plays a role in this effect.

	Acknowledgments

 
We thank J. Ashton-Chess for help in editing the manuscript. We also thank Ignacio Anegon for technical advice and Dominique Baeten for reviewing the manuscript.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Address correspondence and reprint requests to Prof. Jean-Paul Soulillou, Institut National de la Santé et de la Recherche Médicale, Unité 643, Centre Hospitalier Universitaire-Hotel Dieu, 30 boulevard Jean Monnet, 44093 Nantes, Cedex 01, France. E-mail address: Jean-Paul.Soulillou{at}univ-nantes.fr

² J.P.S. and S.B. contributed equally to this project.

³ Abbreviations used in this paper: DC, dendritic cell; MHC II, MHC class II; 1-MT, 1-methyltryptophan; Cy7, cyanin 7.

Received for publication September 12, 2005. Accepted for publication January 10, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Soulillou, J. P., F. Blandin, E. Gunther, V. Lemoine. 1984. Genetics of the blood transfusion effect on heart allografts in rats. Transplantation 38: 63-67. [Medline]
2. Bugeon, L., M. C. Cuturi, M. M. Hallet, J. Paineau, D. Chabannes, J. P. Soulillou. 1992. Peripheral tolerance of an allograft in adult rats: characterization by low interleukin-2 and interferon- mRNA levels and by strong accumulation of major histocompatibility complex transcripts in the graft. Transplantation 54: 219-225. [Medline]
3. Josien, R., M. Heslan, S. Brouard, J. P. Soulillou, M. C. Cuturi. 1998. Critical requirement for graft passenger leukocytes in allograft tolerance induced by donor blood transfusion. Blood 92: 4539-4544. [Abstract/Free Full Text]
4. Madsen, J. C., R. A. Superina, K. J. Wood, P. J. Morris. 1988. Immunological unresponsiveness induced by recipient cells transfected with donor MHC genes. Nature 332: 161-164. [Medline]
5. Sayegh, M. H., A. M. Krensky. 1996. Novel immunotherapeutic strategies using MHC derived peptides. Kidney Int. Suppl. 53: S13-S20. [Medline]
6. Sonntag, K. C., D. W. Emery, A. Yasumoto, G. Haller, S. Germana, T. Sablinski, A. Shimizu, K. Yamada, H. Shimada, S. Arn, et al 2001. Tolerance to solid organ transplants through transfer of MHC class II genes. J. Clin. Invest. 107: 65-71. [Medline]
7. LeGuern, C.. 2004. Potential role of major histocompatibility complex class II peptides in regulatory tolerance to vascularized grafts. Transplantation 77: S35-S37. [Medline]
8. Pèche, H., B. van Denderen, J. C. Roussel, B. Trinité, J. P. Soulillou, M. C. Cuturi. 2004. Presentation of donor major histocompatibility complex class II antigens by DNA vaccination prolongs heart allograft survival. Transplantation 77: 733-740. [Medline]
9. Soulillou, J. P., C. B. Carpenter, A. J. d’Apice, T. B. Strom. 1976. The role of nonclassical Fc receptor-associated, Ag-B antigens (Ia) in rat allograft enhancement. J. Exp. Med. 143: 405-421. [Abstract/Free Full Text]
10. Gagne, K., S. Brouard, M. Guillet, M. C. Cuturi, J. P. Soulillou. 2001. TGF-1 and donor dendritic cells are common key components in donor-specific blood transfusion and anti-class II heart graft enhancement, whereas tolerance induction also required inflammatory cytokines down-regulation. Eur. J. Immunol. 31: 3111-3120. [Medline]
11. Bushell, A., M. Karim, C. I. Kingsley, K. J. Wood. 2003. Pretransplant blood transfusion without additional immunotherapy generates CD25⁺CD4⁺ regulatory T cells: a potential explanation for the blood-transfusion effect. Transplantation 76: 449-455. [Medline]
12. Spencer, S. C., J. W. Fabre. 1990. Characterization of the tissue macrophage and the interstitial dendritic cell as distinct leukocytes normally resident in the connective tissue of rat heart. J. Exp. Med. 171: 1841-1851. [Abstract/Free Full Text]
13. Hart, D. N., J. W. Fabre. 1981. Demonstration and characterization of Ia-positive dendritic cells in the interstitial connective tissues of rat heart and other tissues, but not brain. J. Exp. Med. 154: 347-361. [Abstract/Free Full Text]
14. Forbes, R. D., N. A. Parfrey, M. Gomersall, A. G. Darden, R. D. Guttmann. 1986. Dendritic cell-lymphoid cell aggregation and major histocompatibility antigen expression during rat cardiac allograft rejection. J. Exp. Med. 164: 1239-1258. [Abstract/Free Full Text]
15. Campos, L., A. Naji, B. C. Deli, J. H. Kern, J. I. Kim, C. F. Barker, J. F. Markmann. 1995. Survival of MHC-deficient mouse heterotopic cardiac allografts. Transplantation 59: 187-191. [Medline]
16. Roussey-Kesler, G., S. Brouard, C. Ballet, F. Moizant, A. Moreau, M. Guillet, H. Smit, C. Usal, P. Soulillou. 2005. Exhaustive depletion of graft resident dendritic cells: marginally delayed rejection but strong alteration of graft infiltration. Transplantation 80: 506-513. [Medline]
17. Mahnke, K., E. Schmitt, L. Bonifaz, A. H. Enk, H. Jonuleit. 2002. Immature, but not inactive: the tolerogenic function of immature dendritic cells. Immunol. Cell Biol. 80: 477-483. [Medline]
18. Hubert, F. X., C. Voisine, C. Louvet, M. Heslan, R. Josien. 2004. Rat plasmacytoid dendritic cells are an abundant subset of MHC class II⁺ CD4⁺CD11b^–OX62^– and type I IFN-producing cells that exhibit selective expression of Toll-like receptors 7 and 9 and strong responsiveness to CpG. J. Immunol. 172: 7485-7494. [Abstract/Free Full Text]
19. McWhinnie, D. L., J. F. Thompson, H. M. Taylor, J. R. Chapman, E. M. Bolton, N. P. Carter, R. F. Wood, P. J. Morris. 1986. Morphometric analysis of cellular infiltration assessed by monoclonal antibody labeling in sequential human renal allograft biopsies. Transplantation 42: 352-358. [Medline]
20. Brouard, S., A. Dupont, M. Giral, S. Louis, D. Lair, C. Braudeau, N. Degauque, F. Moizant, A. Pallier, C. Ruiz, M. Guillet, D. Laplaud, J. P. Soulillou. 2005. Operationally tolerant and minimally immunosuppressed kidney recipients display strongly altered blood T-cell clonal regulation. Am. J. Transplant. 5: 330-340. [Medline]
21. Voisine, C., F. X. Hubert, B. Trinité, M. Heslan, R. Josien. 2002. Two phenotypically distinct subsets of spleen dendritic cells in rats exhibit different cytokine production and T cell stimulatory activity. J. Immunol. 169: 2284-2291. [Abstract/Free Full Text]
22. Shao, L., A. R. Jacobs, V. V. Johnson, L. Mayer. 2005. Activation of CD8⁺ regulatory T cells by human placental trophoblasts. J. Immunol. 174: 7539-7547. [Abstract/Free Full Text]
23. Haddeland, U., A. B. Karstensen, L. Farkas, K. O. Bo, J. Pirhonen, M. Karlsson, W. Kvavik, P. Brandtzaeg, B. Nakstad. 2005. Putative regulatory T cells are impaired in cord blood from neonates with hereditary allergy risk. Pediatr. Allergy Immunol. 16: 104-112. [Medline]
24. Chiffoleau, E., G. Bériou, P. Dutartre, C. Usal, J. P. Soulillou, M. C. Cuturi. 2002. Role for thymic and splenic regulatory CD4⁺ T cells induced by donor dendritic cells in allograft tolerance by LF15-0195 treatment. J. Immunol. 168: 5058-5069. [Abstract/Free Full Text]
25. Trinité, B., C. Voisine, H. Yagita, R. Josien. 2000. A subset of cytolytic dendritic cells in rat. J. Immunol. 165: 4202-4208. [Abstract/Free Full Text]
26. Koshiba, T., H. Kitade, B. Van Damme, A. Giulietti, L. Overbergh, C. Mathieu, M. Waer, J. Pirenne. 2003. Regulatory cell-mediated tolerance does not protect against chronic rejection. Transplantation 76: 588-596. [Medline]
27. Jonuleit, H., E. Schmitt, H. Kakirman, M. Stassen, J. Knop, A. H. Enk. 2002. Infectious tolerance: human CD25⁺ regulatory T cells convey suppressor activity to conventional CD4⁺ T helper cells. J. Exp. Med. 196: 255-260. [Abstract/Free Full Text]
28. Lechler, R., W. F. Ng, R. M. Steinman. 2001. Dendritic cells in transplantation–friend or foe?. Immunity 14: 357-368. [Medline]
29. Gregori, S., M. Casorati, S. Amuchastegui, S. Smiroldo, A. M. Davalli, L. Adorini. 2001. Regulatory T cells induced by 1,25-dihydroxyvitamin D₃ and mycophenolate mofetil treatment mediate transplantation tolerance. J. Immunol. 167: 1945-1953. [Abstract/Free Full Text]
30. Myers, L., M. Croft, B. S. Kwon, R. S. Mittler, A. T. Vella. 2005. Peptide-specific CD8 T regulatory cells use IFN- to elaborate TGF--based suppression. J. Immunol. 174: 7625-7632. [Abstract/Free Full Text]
31. El-Asady, R., R. Yuan, K. Liu, D. Wang, R. E. Gress, P. J. Lucas, C. B. Drachenberg, G. A. Hadley. 2005. TGF--dependent CD103 expression by CD8⁺ T cells promotes selective destruction of the host intestinal epithelium during graft-versus-host disease. J. Exp. Med. 201: 1647-1657. [Abstract/Free Full Text]
32. Suffia, I., S. K. Reckling, G. Salay, Y. Belkaid. 2005. A role for CD103 in the retention of CD4⁺CD25⁺ Treg and control of Leishmania major infection. J. Immunol. 174: 5444-5455. [Abstract/Free Full Text]
33. Bilsborough, J., J. L. Viney. 2004. In vivo enhancement of dendritic cell function. Ann. NY Acad. Sci. 1029: 83-87. [Abstract/Free Full Text]
34. Juedes, A. E., M. G. Von Herrath. 2003. Using regulatory APCs to induce/maintain tolerance. Ann. NY Acad. Sci. 1005: 128-137. [Abstract/Free Full Text]
35. Verhasselt, V., O. Vosters, C. Beuneu, C. Nicaise, P. Stordeur, M. Goldman. 2004. Induction of FOXP3-expressing regulatory CD4^pos T cells by human mature autologous dendritic cells. Eur. J. Immunol. 34: 762-772. [Medline]
36. Rao, P. E., A. L. Petrone, P. D. Ponath. 2005. Differentiation and expansion of T cells with regulatory function from human peripheral lymphocytes by stimulation in the presence of TGF-}. J. Immunol. 174: 1446-1455. [Abstract/Free Full Text]
37. Kai, S., S. Goto, K. Tahara, A. Sasaki, S. Tone, S. Kitano. 2004. Indoleamine 2,3-dioxygenase is necessary for cytolytic activity of natural killer cells. Scand. J. Immunol. 59: 177-182. [Medline]
38. Fallarino, F., U. Grohmann, K. W. Hwang, C. Orabona, C. Vacca, R. Bianchi, M. L. Belladonna, M. C. Fioretti, M. L. Alegre, P. Puccetti. 2003. Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol. 4: 1206-1212. [Medline]
39. Munn, D. H., M. D. Sharma, A. L. Mellor. 2004. Ligation of B7-1/B7-2 by human CD4⁺ T cells triggers indoleamine 2,3-dioxygenase activity in dendritic cells. J. Immunol. 172: 4100-4110. [Abstract/Free Full Text]
40. Finger, E. B., J. A. Bluestone. 2002. When ligand becomes receptor-tolerance via B7 signaling on DCs. Nat. Immunol. 3: 1056-1057. [Medline]
41. Terness, P., T. M. Bauer, L. Rose, C. Dufter, A. Watzlik, H. Simon, G. Opelz. 2002. Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. J. Exp. Med. 196: 447-457. [Abstract/Free Full Text]
42. Munn, D. H., E. Shafizadeh, J. T. Attwood, I. Bondarev, A. Pashine, A. L. Mellor. 1999. Inhibition of T cell proliferation by macrophage tryptophan catabolism. J. Exp. Med. 189: 1363-1372. [Abstract/Free Full Text]

This article has been cited by other articles:

				C. H. Cook, A. A. Bickerstaff, J.-J. Wang, T. Nadasdy, P. Della Pelle, R. B. Colvin, and C. G. Orosz Spontaneous Renal Allograft Acceptance Associated with "Regulatory" Dendritic Cells and IDO J. Immunol., March 1, 2008; 180(5): 3103 - 3112. [Abstract] [Full Text] [PDF]

				V. Jovanovic, A.-S. Dugast, J.-M. Heslan, J. Ashton-Chess, M. Giral, N. Degauque, A. Moreau, A. Pallier, E. Chiffoleau, D. Lair, et al. Implication of Matrix Metalloproteinase 7 and the Noncanonical Wingless-Type Signaling Pathway in a Model of Kidney Allograft Tolerance Induced by the Administration of Anti-Donor Class II Antibodies J. Immunol., February 1, 2008; 180(3): 1317 - 1325. [Abstract] [Full Text] [PDF]

				C. J. Callaghan, F. J. Rouhani, M. C. Negus, A. J. Curry, E. M. Bolton, J. A. Bradley, and G. J. Pettigrew Abrogation of Antibody-Mediated Allograft Rejection by Regulatory CD4 T Cells with Indirect Allospecificity J. Immunol., February 15, 2007; 178(4): 2221 - 2228. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar