|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 1 December 2007, Vol. 110, No. 12, pp. 3936-3948. Prepublished online as a Blood First Edition Paper on September 5, 2007; DOI 10.1182/blood-2007-04-083139.
IMMUNOBIOLOGY CD3+CD4low and CD3+CD8low are induced by HLA-G: novel human peripheral blood suppressor T-cell subsets involved in transplant acceptance1 Service de Recherches en Hemato-Immunologie, Commissariat à l'energie atomique-Direction des science, du vivant-Departement de la recherche medical (CEA-DSV-DRM), Hopital Saint-Louis, Institut Universitaire d'Hematologie (IUH), Paris; and 2 Departement de Nephrologie et Transplantation, Hopital du Kremlin-Bicetre, Le Kremlin-Bicetre, France
HLA-G is a tolerogenic molecule whose detection in sera and within allografted tissues is associated with better graft acceptance. HLA-G mediates T-cell differentiation into suppressor cells, which are thought to promote tolerance. Here, we investigated such T cells phenotypically and functionally and assessed their clinical relevance in the peripheral blood of patients who have undergone transplantation. Our results demonstrate that HLA-G expressed by antigen-presenting cells or present as soluble protein down-regulates the expression of CD4 and CD8 on allostimulated T cells at both transcriptional and posttranslational levels. These CD3+CD4low and CD3+CD8low T-cell subsets are characterized by an increased proportion of cells expressing CD45RA and HLA-DR, and a decreased number of cells expressing CD62L. In addition, these HLA-G–induced CD3+CD4low and CD3+CD8low subpopulations are Foxp3-negative suppressor T cells whose function involves IL-10. Biologic relevance came from analysis of patients who underwent transplantation, with high HLA-G plasma concentrations associated with better graft survival. Peripheral blood from these patients contains increased levels of IL-10 concomitantly to an enhanced representation of CD3+CD4low and CD3+CD8low T cells compared with HLA-G–negative patients who underwent transplantation and healthy individuals. These data define novel immunosuppressive subpopulations of peripheral blood T cells induced by HLA-G with potent implications in peripheral tolerance.
Although central tolerance mechanisms delete the majority of autoreactive T cells during a tightly controlled selection in the thymus, peripheral tolerance is required to avoid autoimmunity or aberrant immune responses.1 Furthermore, the induction of peripheral tolerance is a major goal in human allotransplantation where allograft rejection is mediated mainly by recipient alloreactive T cells.2 T-cell activation by antigen-presenting cells (APCs) needs at least 2 signals: the first is mediated by interaction of the T-cell receptor (TCR)–CD3 complex associated with CD4 or CD8 coreceptors with MHC class II or class I molecules on APCs, respectively. The second signal is costimulatory and originates from the engagement of CD28 with its ligands CD80/CD86 on APCs. Disrupting one of these signals may constitute a very efficient way to inhibit CD4+ and CD8+ T-cell activation, with therapeutic potential to prevent organ transplant rejection.3
HLA-G can be expressed as 7 different isoforms including 4 membrane-bound (HLA-G1 to -G4) and 3 soluble (HLA-G5 to -G7) proteins due to alternative splicing of the HLA-G primary transcript (see review in Carosella et al4). The HLA-G5 soluble isoform (37 kDa) differs from the membrane-anchored HLA-G1 protein (39 kDa) by the absence of both the transmembrane region and the cytoplasmic tail, which are substituted by a specific C-terminus sequence. The In opposition to classical HLA-class I molecules, HLA-G is of low polymorphism and its expression is found in a limited number of healthy tissues.4 HLA-G was first identified in cytotrophoblast where it is involved in the maternal-fetal tolerance.12 However, HLA-G expression can be up-regulated in various tissues under "pathological" conditions such as allotransplantation.13 Indeed, an increasing number of clinical studies reported HLA-G expression in recipients who did not reject their allograft following heart transplantation,14–16 kidney transplantation,17 liver transplantation,18 and liver-kidney transplantation.19,20 Concomitantly, in vitro functional studies showed that T cells stimulated by HLA-G1–expressing APCs9 or sensitized by HLA-G521 differentiate into suppressor T cells. These HLA-G–induced T cells were found to be different from the naturally occurring CD4+CD25+ regulatory T cells.21,22 Up to now, their precise phenotype, cytokine context, mechanism of action, and in vivo relevance remained to be characterized. The aim of this work was to address these points in vitro in mixed lymphocyte reactions and in vivo in human allotransplantation.
APC-HLA-G1s and HLA-G5–secreting cells The HLA class II+ human B-lymphoblastoid cell line LCL 721.221 (ATCC, Manassas, VA) was transfected by electroporation with either pRc/RSV vector alone (Invitrogen, Frederick, MD) or the pRc/RSV vector containing the HLA-G1 cDNA, to obtain LCL-RSV and LCL-HLA-G1 cells, respectively.23 The LCL-HLA-G1 cells strongly expressed HLA-G1 at their cell surface (Figure 1A box). We used LCL-RSV and LCL-HLA-G1 as an APC-like model expressing or not HLA-G1.24 These cells will be referred in the text as APCs and APC-HLA-G1s, respectively. The HLA class I–positive, HLA-G–negative, M8 melanoma cell line was transfected with the pcDNA3.1 vector (Invitrogen) either alone to generate M8-pcDNA or containing the HLA-G5 cDNA to produce M8-HLA-G5 cells, as described previously.6
Patients who underwent transplantation and control individuals Blood samples from patients who received a kidney transplant (KT), liver transplant (LT), or combined liver-kidney transplant (LKT) were collected during their clinical follow-up. Thirty-four KT patients (Kremlin Bicetre Hospital, Le Kremlin-Bicetre, France), 21 LT patients (Paul Brousse Hospital, Villejuif, France), and 44 LKT patients (Paul Brousse Hospital, Villejuif, France) were investigated in the present study. Within a population that underwent transplantation, patients were divided into 3 groups according to the time interval between transplantation and blood collection. Patient characteristics are described in Table 1, Table 2, and Table 3 for KT, LT, and LKT populations, respectively. All patients had stable graft function at the time of blood collection. Histologic lesions of acute or chronic graft rejection were classified according to the Banff classification. Control individuals (n = 14) were healthy volunteer blood donors (HDs) from French Blood Establishment (EFS, Saint-Louis Hospital, Paris, France). The healthy donor population was age matched (49 ± 9 years old) with that of patients who underwent transplantation. Plasma and peripheral blood mononuclear cells (PBMCs) were isolated from blood samples to investigate plasma levels of soluble HLA-G, IL-10, and soluble CD4/CD8 and phenotype of peripheral T cells. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki, and the study was approved by the local ethics committee at the Hospital Saint-Louis.
Cell preparation, culture procedure, MLR, and suppression assays
PBMCs from heparinized whole blood of HDs, and KT, LT, or LKT patients were obtained by density-gradient centrifugation over Ficoll-histopaque 1077 (Sigma, St Louis, MO). For allostimulation assays, PBMCs were used as responder or CD4+ and CD8+ T-cell isolation After 6 days of MLR in presence or in absence of HLA-G, allostimulated CD4+ and CD8+ T cells were isolated by positive selection, respectively, using either CD4 or CD8 immunomagnetic beads according to the manufacturer's instructions (Dynal Biotech, Lake Success, NY). Positively selected CD4+ and CD8+ T-cell populations were collected after release from magnetic beads by overnight incubation at 37°C. Purity of each fraction was checked by flow cytometry showing that CD4+ and CD8+ fractions were more than 95%. In case of allostimulation using irradiated APC-HLA-G1s, we checked that none of these cells remained after 6 days of MLR, attesting that any contaminating HLA-G1+ cell was present in the CD4+ and CD8+ isolated T-cell fractions that were subsequently used in suppression assays. Antibodies and flow cytometric analysis The following Abs were used: MEM-G/9 anti–HLA-G1/HLA-G5 (Exbio, Vestec, Czech Republic); 5A6G7 anti–HLA-G5 (Exbio). Antibodies used for flow cytometric analyses were conjugated with either FITC, PE, ECD, or PC5 (Beckman Coulter, Hialeah, FL), except PD1-FITC (Pharmingen, San Diego, CA) and GITR-PE (R&D Systems, Minneapolis, MN). Briefly, cells were first incubated 30 minutes at 4°C in 20% human serum. Isotype control was systematically used to evaluate and compensate nonspecific signal. Cells were analyzed on EPICS XL4 cytometer using Expo32 software (Beckman Coulter).
Intracellular Foxp3 staining was performed using Alexa 488–conjugated antihuman Foxp3 Ab (clone 259D; Ozyme, Saint Quentin, France). To assess cell permeabilization, anti– Anti–IL-10 blocking experiments To neutralize IL-10, anti–IL-10 mAb (clone B-S10; Diaclone, Besançon, France) was added at a concentration of 10 µg/mL on days 1 and 3 in allostimulation or suppression assay.27 An irrelevant isotype Ab was used as a control at the same concentration. Real-time reverse-transcription–polymerase chain reaction analysis
Total mRNA were extracted from healthy donor's peripheral blood leukocytes (PBLs; calibrator) and allostimulated T cells and reverse-transcribed to generate cDNA. Multiplex PCR amplifications were performed using commercially available primers and probe for CD4 and CD8 ELISA
Soluble HLA-G concentrations were measured in plasma from KT, LT, and LKT patients, and HDs, as well as in supernatants from M8 transfectants. Two distinct enzyme-linked immunosorbent assay (ELISA) were used that detect either both shed HLA-G1 and HLA-G5 (MEM-G/9 as capture antibody and anti-β2m as detection antibody) or HLA-G5 specifically (5A6G7 as capture antibody and the Pan HLA class I W6/32 as detection antibody), as described previously.28 Plasma samples were diluted
IL-2, IFN- Statistical analysis Statistical analysis was performed with Student t test; ANOVA test and linear regression analyses were used to assess significance and correlation between variables. The Kaplan-Meier method was used to estimate the survival time without acute and chronic rejection. Survival differences were analyzed using the log-rank test. Statistical test, P value, and significance were given with the results assuming P values less than .05 as significant (** and *** at the top of histogram indicate P values less than .001.
APC-HLA-G1s and soluble HLA-G5 induce immunosuppressive T cells We previously showed that T cells stimulated by HLA-G1–expressing APCs9 or sensitized by soluble HLA-G521 differentiate into suppressor T cells that were different from CD4+CD25+ regulatory T cells.21 To identify and characterize these HLA-G–induced T cells, we here used HLA-G as either a membrane form presented at the cell surface of APCs or a soluble form released in biologic fluids. Previously, the direct role of HLA-G in inhibiting T-cell alloproliferation was demonstrated in these 2 expression models.24 Both HLA-G forms are biologically relevant in transplantation since infiltrating mononuclear cells expressing HLA-G were detected in vivo within grafted tissues, and since high serum levels of soluble HLA-G were measured in patients who underwent transplantation who had better graft acceptance.18,19
First, we analyzed induction of immunosuppressive T cells by HLA-G. T cells were primed with either APCs or APC-HLA-G1s and then tested as IL-10 is required for the suppressive function of HLA-G–induced T cells
We then analyzed the direct contribution of IL-10 to the suppressive function of HLA-G–induced T cells. Results showed that neutralizing IL-10 with Ab reversed the suppressive activity of HLA-G–induced T cells (Figure 1H). We next investigated whether a particular cytokinic microenvironment could be related to their generation. Results showed that increased levels of IL-10 were observed at day 6 in MLR supernatants when APC-HLA-G1s (Figure 1I) or HLA-G5 (Figure 1J) was used to induce suppressor T cells. By contrast, IL-4, IFN- APC-HLA-G1s and soluble HLA-G5 induce CD3+ T-cell subpopulations expressing lowered levels of CD4 and CD8
To identify the phenotype of suppressor T cells induced by HLA-G, we then analyzed the expression of T-cell markers associated with suppressive function. Flow cytometric analysis showed that the percentage of positive cells as well as mean fluorescence intensity for PD-1 (programmed cell death-1 protein), CD25 (IL-2 receptor
By contrast, there were striking differences in T-cell morphology between APC- and APC-HLA-G1–primed T cells. APC-primed T cells were enlarged, while APC-HLA-G1–primed T cells were smaller with fewer granularities (Figure 3A). We showed that allostimulation by APC-HLA-G1s induced T-cell subsets with lower cell surface expression of CD4 or CD8 coreceptor compared with APC-primed T cells (Figure 3B,C). Indeed, flow cytometric analysis showed a 2-fold and 8-fold decrease of CD4 and CD8 surface expression, respectively, on CD3+ T cells primed with APC-HLA-G1s compared with APC-primed T cells (Figure 3C). Interestingly, these CD3+CD4low and CD3+CD8low T cells showed higher surface expression levels of CD3 (Figure 3B,C). Such CD3+CD4low and CD3+CD8low T cells were found after 6 days of allostimulation with APC-HLA-G1s. Similar results were observed with T cells primed in presence of HLA-G5 (data not shown). Since HLA-G–induced T cells emerge from IL-10 microenvironment (Figure 1I,J), we investigated whether this cytokine was directly involved in their differentiation into CD3+CD4low and CD3+CD8low T cells. In our experimental conditions, addition of neutralizing IL-10 mAb did not affect this differentiation. Indeed, CD4 MFI was 58 (± 4) versus 55 (± 1) and CD8 MFI was 1182 (± 202) versus 944 (± 105) with and without anti–IL-10 mAb, respectively (n = 3, P > .05, Student t test). In these experiments, CD4 and CD8 MFI was 84 (± 3) and 2192 (± 104), respectively, after T-cell priming in the absence of HLA-G.
To define the level at which HLA-G downmodulates CD4 and CD8 expression, we first analyzed CD4 and CD8 gene transcription. Results showed that both CD4 (Figure 4A) and CD8 (Figure 4B) transcriptional levels were significantly decreased in, respectively, HLA-G–induced CD3+CD4low and CD3+CD8low T cells compared with T cells primed in absence of HLA-G (P < .001, Student t test) (Figure 4A,B). Second, we investigated whether HLA-G–mediated CD4/CD8 downmodulation may also result from a release of both molecules from T-cell surface. Results showed that the amount of soluble CD4 (Figure 4C) and CD8 (Figure 4D) molecules was significantly higher in supernatants from HLA-G–induced T cells than in those of T cells primed in absence of HLA-G (P < .001, Student t test).
Extensive phenotypic analysis showed that the percentage of cells expressing CD45RA, HLA-DR, and CD62L differed among CD3+CD4low and CD3+CD8low T cells compared with CD3+CD4high and CD3+CD8high T cells primed without HLA-G (Figure 5). Indeed, CD4highCD45RA+ T cells represented 20% of CD3+CD4high T cells primed with APCs, whereas CD4lowCD45RA+ T cells represented 30% of CD3+CD4low T cells (Figure 5A). CD45RA+ cells reached 55% among CD3+CD8low T cells, while they represented 40% of the CD3+CD8high T cells primed with APCs (Figure 5A). These differences were significant with a mean of CD4highCD45RA+ versus CD4lowCD45RA+ T cells: 20 (± 1) versus 33 (± 2), and a mean of CD8highCD45RA+ versus CD8lowCD45RA+ T cells: 37 (± 1) versus 53 (± 4); (n = 3, P > .05, Student t test). Interestingly, the percentage of HLA-DR+ cells was increased in both CD3+CD4low and CD3+CD8low T cells by more than 1.5-fold (Figure 5C). This enhancement was significant with a mean of CD4highHLA-DR+ versus CD4lowHLA-DR+ T cells: 28 (± 4) versus 48 (± 4), and a mean of CD8highHLA-DR+ versus CD8lowHLA-DR+ T cells: 39 (± 4) versus 56 (± 3; n = 3, P < .05, Student t test). Concomitantly, CD62L+ T-cell subsets decreased among CD3+CD4low and CD3+CD8low T cells (Figure 5B). This diminution was significant with a mean of CD4highCD62L+ versus CD4lowCD62L+ T cells: 94 (± 3) versus 76 (± 3), and a mean of CD8highCD62L+ versus CD8lowCD62L+ T cells: 21 (± 2) versus 5 (± 2; n = 3, P < .05, Student t test). All these results were similarly observed with T cells primed in presence of HLA-G5 (data not shown).
Both CD3+CD4low and CD3+CD8low T-cell subsets induced by HLA-G are suppressor cells We next investigated whether the HLA-G–induced suppressive function was associated with the CD3+CD4low/CD3+CD8low phenotype. We sorted the CD3+CD4low and CD3+CD8low T cells after priming with APC-HLA-G1s or HLA-G5, and the CD3+CD4high and CD3+CD8high T cells after priming in the absence of HLA-G after 6 days of MLR. These cells were then used as third-party cells in suppression assays. CD3+CD4low and CD3+CD8low T cells induced either by APC-HLA-G1s (Figure 5D) or HLA-G5 (Figure 5E) significantly inhibited alloproliferation compared with CD3+CD4high and CD3+CD8high. Indeed, CD3+CD4low and CD3+CD8low T cells induced by APC-HLA-G1s inhibited alloproliferation by 74% (± 2%) and 47% (± 2%; n = 3, mean ± SEM), respectively, compared with CD3+CD4high and CD3+CD8high T cells (P < .001, Student t test) (Figure 5D). Similar results were found using CD3+CD4low and CD3+CD8low T cells induced by HLA-G5 leading to alloproliferation inhibition of 74% (± 7%) and 65% (± 2%; n = 3, mean ± SEM, P < .001, Student t test), respectively (Figure 5E). These data show that HLA-G1 and HLA-G5 affect T-cell activation during in vitro allogeneic challenge inducing CD3+CD4low and CD3+CD8low suppressor T cells. To assess the clinical relevance of these HLA-G–induced suppressor T-cell subsets, we studied patients who received a combined liver-kidney transplant (LKT). Previous studies showed that LKT patients have high HLA-G plasma levels and peripheral blood T cells with suppressive function.21 They better accept their kidney graft compared with patients who received a single kidney transplant (KT), and this improved allograft acceptance could be associated with HLA-G expression.18,19 CD3+CD4low and CD3+CD8low suppressor T-cell subsets are overrepresented in the peripheral blood of patients who underwent transplantation, with high HLA-G plasma levels and better graft acceptance
Here, 44 newly recruited LKT patients were studied. We first checked HLA-G plasma levels in this novel panel of LKT patients in comparison with healthy donors (HDs, n = 14). For this purpose, 2 different ELISAs were performed that detect either shed HLA-G1 plus HLA-G5 (sHLA-G1 + HLA-G5) (Figure 6A) or only HLA-G5 (Figure 6B). Since HLA-G can be trapped within the clot when serum is collected,29 we worked with plasma that better reflects the in vivo concentration of circulating HLA-G. In agreement with our previous results,21 HLA-G plasma levels were significantly higher in LKT patients compared with HDs (Figure 6A,B). The respective roles played by the liver and the kidney graft were examined by measuring HLA-G in plasma from patients who received a single liver transplant (LT, n = 21) and patients who received a single kidney transplant (KT, n = 34). Considering the concentrations from HDs as basal levels of HLA-G in plasma (normal values), all LT and LKT patients had high HLA-G plasma levels (P < .001 comparing LT or LKT to HD, Student t test) (Figure 6A,B). By contrast, KT patients had HLA-G plasma concentrations close to normal values (P > .05 comparing KT to HD, Student t test) (Figure 6A,B). Significant differences were observed when comparing LT or LKT to KT (P < .001, Student t test). These results showed that high HLA-G plasma levels are always detected in patients who underwent a liver transplantation, strongly suggesting a specific role of the liver in up-regulating HLA-G expression. By delineating patients according to follow-up period posttransplantation, we found that HLA-G plasma levels were stable over time in the LKT and LT populations, while increased HLA-G levels were observed long term in KT patients (P < .05, Student t test) (Tables 1
Next, the peripheral blood CD3+CD4+ and CD3+CD8+ T-cell populations from HDs were compared with those of KT, LT, and LKT patients. We found an increase of CD3+CD4low and CD3+CD8low cells among circulating T cells from both LT and LKT patients compared with peripheral CD3+CD4+ and CD3+CD8+ T cells from HD and KT patients. One representative individual for each group is shown in Figure 6C. The low and high CD4- and CD8-expressing T cells were defined according to the basal expression level observed in healthy donors; all samples (ie, PBMCs from HD, KT, LT, and LKT) were processed together. Thus, our in vitro and in vivo data indicate that the presence of increased CD3+CD4low and CD3+CD8low T-cell subsets in both LT and LKT patients can be attributed to HLA-G. Supporting this conclusion and in agreement with in vitro results described in Figure 1I,J, we found (1) significantly higher amounts of IL-10 (Figure 6F) and of both soluble CD4 and CD8 molecules (Figure 6D,E) in plasma from HLA-G+ LT and LKT compared with those from HLA-G– KT patients (P < .001, Student t test), and (2) a significant correlation between sHLA-G plasma levels and the presently described HLA-G–related immunologic parameters (ie, IL-10 and suppressor CD3+CD4low and CD3+CD8low subpopulations) (Table 4; Figure 7C–G
Finally, regarding allograft acceptance status of these patients: one acute and no chronic graft rejection were observed in the 44 LKT patients studied (98% acceptance); 4 of 21 LT patients suffered from acute or chronic liver graft rejection (81% acceptance); and 16 of 34 KT patients suffered from acute or chronic kidney graft rejection (53% acceptance). Graft acceptance was significantly higher in HLA-G+ patients compared with HLA-G– patients. Indeed, LKT and LT patients had a significantly higher survival time without acute rejection (P < .001, log-rank test; Figure 7A) or without acute and chronic rejection (P < .001, log-rank test; Figure 7B) compared with KT patients. Thus, allograft acceptance is observed in patient groups with high HLA-G and IL-10 plasma levels and overrepresentation of peripheral blood CD3+CD4low and CD3+CD8low T-cell subsets (ie, LT and LKT groups).
HLA-G inhibits CD4+ T-cell proliferation, induces CD4+ T-cell long-term unresponsiveness, and causes the differentiation of T cells into immunosuppressive/regulatory T cells.9,21,25 First, attention has been focused on naturally occurring CD4+CD25+ regulatory T cells. Injection of HLA-G tetramer–coated beads to recipient mice just before skin allograft induced expansion of CD4+CD25+ regulatory T cells among splenocytes and allowed skin graft tolerance.10,11,30 However, current studies in humans have failed to demonstrate that HLA-G induces expansion of such CD4+CD25+ T cells.21,22 The aim of the present work was to characterize these HLA-G–induced T cells phenotypically and functionally, and to provide evidence of their clinical relevance in humans.
Here, we report that membrane-bound HLA-G1 on APCs, as well as soluble HLA-G5, induces T cells with suppressive function. The level of suppression is directly linked to the suppressor T-cell number added in the mixed lymphocyte reaction and remains significant with 4-fold fewer suppressors than responders. These HLA-G–induced suppressive T cells express lowered levels of CD4 and CD8 coreceptors and are phenotypically defined as CD3+CD4low and CD3+CD8low T cells. We found that the downmodulation of CD4 and CD8 surface expression on HLA-G–driven T cells results from both transcriptional and posttranslational regulation. Indeed, HLA-G induces a decrease of CD4 and CD8 mRNA transcription as well as an increase of release of both coreceptors from T-cell surface. The CD8 downmodulation may be due to direct interaction between HLA-G and CD8 coreceptor. Such hypothesis is supported by previous works demonstrating that HLA-G
These CD3+CD4low and CD3+CD8low T cells show an increased representation of CD45RA+ cells. It has been shown that primed CD4+ T cells expressing CD45RA secreted high amounts of IL-4, IL-10, and IL-13, whereas the production of IFN- Different types of regulatory/suppressor T cells have been described based on distinct expression patterns of markers and cytokines, suppressive mechanisms, and origin. Foxp3 is a transcription factor associated with the development of naturally occurring CD4+CD25+ regulatory T cells that express this factor at high levels constitutively.38 In the present study, HLA-G–induced suppressor T cells do not express high levels of CD25 or Foxp3. This expression pattern was previously observed for other regulatory T-cell types such as the IL-10–secreting T-regulatory type 1 (Tr1) cells.39 CD4+CD25+ Foxp3+ regulatory T cells exert their suppressive function via direct cell contact.40 The activity of Tr1 and T-helper 3 regulatory T cells is not dependent on cell contact and is based mainly on cytokines such as IL-10 and TGF-β.41 Cytokines are not only essential for function but also important for the generation of suppressor T cells.42 Here, IL-10 was identified as the cytokine directly involved in the suppressive function of HLA-G–induced T cells. This function did not occur through downmodulation of IL-2 secretion but rather through an up-regulation of IL-10 production counteracting the IL-2–mediated T-cell proliferation signal. However, although increased IL-10 levels correlated with the emergence of these HLA-G–induced suppressive T cells, IL-10 seems not necessary to their generation. Biologic relevance was provided by analyzing groups of patients who underwent transplantation who express HLA-G at high levels (ie, LT and LKT patients) in comparison with healthy controls or KT patients who have baseline levels of HLA-G. Suppressor T cells induced by HLA-G in vitro were overrepresented in patients who express HLA-G. Indeed, we consistently observed an increased representation of CD3+CD4low and CD3+CD8low within the peripheral blood from both LT and LKT patients. Notably, a correlation was clearly obtained between HLA-G, IL-10, and CD3+CD4low and CD3+CD8low T cells. Furthermore, the posttranslational mechanism by which HLA-G induces CD4 and CD8 down-regulation was supported by the increased amount of soluble CD4 and CD8 molecules in plasma from LT and LKT versus KT patients. These findings show that HLA-G but also its related immunologic parameters such as IL-10, soluble CD4/CD8, and CD3+CD4low/CD3+CD8low suppressor T cells all would contribute to induce tolerance in HLA-G+ patients. Incidentally, soluble CD4 and CD8 coreceptors are believed to be potent inhibitors of T-cell activation by competing with membrane-anchored coreceptors, thus preventing T-cell activation via coreceptor/CD3-TCR complex-peptide/MHC interaction.34 Better graft acceptance was observed in LT and LKT patient groups that all received a liver allograft. Therefore, HLA-G can be considered as a graft acceptance–associated marker whose expression may be up-regulated by liver factors. The liver has long been known to have a positive effect on the induction of peripheral tolerance.43 Previous studies showed that patients who received a combined liver-kidney transplant experienced significantly less kidney rejection compared with patients receiving a kidney transplant alone.44 Here, we detected increased levels of soluble HLA-G in plasma from all LT and LKT patients compared with those observed in HD and KT patients. Thus, we postulate that the liver allograft plays a role in HLA-G expression. The liver may be directly involved in secretion of HLA-G since its expression was detected on biliary epithelial cells, as we previously described.19 However, only some patients were displaying HLA-G+ biliary epithelial cells in the grafted liver. Another nonexclusive hypothesis is that liver cells secrete soluble factors that up-regulate HLA-G expression in an endocrine manner. Thereby, the hepatocyte growth factor (HGF) could be one candidate because its roles in vivo in graft acceptance45,46 and in vitro on monocyte differentiation into dendritic regulatory cells47 were recently reported. Besides, dendritic cells are described as efficient HLA-G secretors under adequate stimuli.48 Another candidate is the anti-inflammatory cytokine IL-10, which is produced by the liver49 and can up-regulate HLA-G expression in dendritic cells and monocytes.50 HLA-G+ dendritic cells may be preferentially localized in draining lymph nodes, from which soluble HLA-G molecules are delivered in the blood circulation. Otherwise, CD3+CD4low and CD3+CD8low cells have been previously identified in 3 biologic contexts: in the thymus, in HIV-infected individuals, and in patients with chronic lymphocytic leukemia.51–53 Interestingly, HLA-G has also been described in thymic epithelial cells and in the course of the same pathologies,36,54,55 supporting our observation that HLA-G generates CD3+CD4low and CD3+CD8low suppressive T cells. Such peripheral suppressive T cells may contribute to maintain an immune tolerant environment improving graft acceptance in LT and LKT patients. Here, we propose HLA-G as a biomarker of graft acceptance in the clinical follow-up of patients who underwent transplantation. HLA-G measurement in blood may be useful to predict graft outcome and provide physicians with a new approach to better manage patients who underwent transplantation. Furthermore, this study reinforces the potential use of HLA-G as an immunosuppressive agent to prevent allograft rejection. Finally, our present findings can be extended to other contexts in which HLA-G expression is up-regulated such as pregnancy,4 cancer,56 autoimmune disorders,57 and viral infections.36,58
Contribution: A.N., J.L., E.D.C., and N.R.-F. designed and analyzed all experiments; A.N. and N.R.-F. wrote the paper; A.N., S.L.R., I.K.-R., M.D., and J.C. performed experiments; A.D. and C.C. provided samples from patients who underwent transplantation; A.D. analyzed all clinical data. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Nathalie Rouas-Freiss, Service de Recherches en Hemato-Immunologie, CEA-DSV-DRM, Hopital Saint-Louis, Institut Universitaire d'Hematologie, 1, avenue Claude Vellefaux, 75010 Paris, France; e-mail: nathalie.rouas-freiss{at}cea.fr.
This work was supported by the Commissariat à l'Energie Atomique and the Agence de la Biomedecine.
Submitted April 2, 2007; accepted July 29, 2007.
Prepublished online as Blood First Edition Paper, September 5, 2007
DOI: 10.1182/blood-2007-04-083139
  CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2007 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
1 and 
-irradiated third-party cells with HLA-mismatched PBMCs as 
cpm). (Box) Both APCs and APC-HLA-G1s were analyzed for cell surface expression of HLA-G1 by flow cytometry using the MEM-G/9 antibody. Dashed lines correspond to APCs and solid lines to APC-HLA-G1s. Numbers on the right correspond to the mean fluorescence intensity (MFI) observed with APCs (top) and APC-HLA-G1s (bottom). Cells were stained with an isotype-matched antibody as negative control (filled histograms). (B) Concomitantly, IL-10 and IL-2 concentrations were measured by ELISA at day 6 in suppression assay supernatants. Results from 3 independent experiments are shown. (C) Similar experiment was performed with T cells primed with supernatant from either M8-HLA-G5 (HLA-G5+ SNs) or M8-pcDNA (HLA-G5– SNs). Results are expressed as the mean of thymidine incorporation (cpm) in triplicate wells plus or minus SEM from 3 allogeneic combinations and corrected for background values (
or 
prior to measuring HLA-G. 
