Operational tolerance (graft acceptance in an immunosuppressive drug-free environment) is achieved more commonly in liver (up to 20% of patients) than in other types of organ transplantation (1–4), especially in children (5, 6). Underlying mechanisms have not been elucidated. Moreover, identification of patients that can discontinue immunosuppression, without risk of rejection, has proved difficult, due to the absence of reliable biomarkers/diagnostic tests that can discriminate the tolerant state (7). Recently, it has been reported (4) that transcriptional profiling of peripheral blood can identify adult liver graft recipients able to discontinue immune suppression, and that innate immune cells may play a major role in the maintenance of tolerance.
Dendritic cells (DCs) are innate immune system cells that are also important in the induction and regulation of adaptive immunity (8). They have inherent tolerogenic properties (9, 10), including the ability to induce regulatory T cells (Treg), that have been associated with organ transplant tolerance (11, 12), including the spontaneous acceptance of liver allografts in mice (13). Moreover, the adoptive transfer of tolerogenic DC to experimental organ graft recipients can promote indefinite transplant survival (14–17). Several subsets of DC have been identified, principally conventional monocytoid dendritic cell (mDC), and also type-1 interferon-producing plasmacytoid dendritic cell (pDC) (18, 19), that may be specialized for modifying the strength, quality, and duration of immune responses (20, 21). In previous reports, we have shown that the ratio of pDC:mDC is elevated significantly in operationally tolerant pediatric liver transplant recipients (22, 23), suggesting that pDC may play a functional role in the tolerant state. There is also evidence that circulating levels of Treg are elevated in tolerant liver transplant patients compared with nontolerant patients or healthy individuals (6, 24, 25).
Immune regulatory molecules expressed by DC subsets, including B7-H1 (B7 homologue-1=programmed death ligand-1) (26, 27), inducible costimulatory ligand (ICOSL) (28), glucocorticoid-induced tumor necrosis factor receptor-related ligand (GITRL) (29), and the nonclassical human leukocyte antigen (HLA) class I molecule HLA-G (30) and its receptor immunoglobulin-like transcript 4 (ILT4) (31), have been shown to regulate T-cell responses, including the induction of Treg. However, the expression of these molecules or the activation marker CMRF-44 (32) on DC has not been examined in relation to human organ transplant tolerance.
We examined the expression of these immune regulatory molecules on circulating DC subsets in operational pediatric liver transplant tolerance (TOL), patients undergoing prospective immunosuppressive drug weaning (PW), patients on maintenance immunosuppression (MI), and healthy controls (HC). Our data show that significantly elevated levels of HLA-G on mDC in TOL patients correlates with Foxp3 expression by CD4+CD25hiCD127− Treg, suggesting that HLA-G expression on mDC may be of functional significance in immune regulation in the tolerant state.
Expression of HLA-G, ILT4, CMRF44, GITRL, and ICOSL on Circulating DC Subsets
Flow cytometric analysis was first used to identify selected immune regulatory molecules on circulating mDC and pDC in 28 normal HC, as described in the Materials and Methods. Figure 1(A) shows the gating strategy used to identify lin−BDCA-2+ pDC and lin− BCDA-1+ mDC, and the expression of HLA-G and ILT4 on these DC subsets. As shown in Figure 1(B), the incidence of mDC expressing HLA-G (but not ILT4) was significantly higher than that of pDC (HLA-G%: 57.62±2.81 vs. 27.19±3.16, P<0.0001; ILT4%: 64.69±3.87 vs. 73.45±2.81, not significant). Mean fluorescence intensity (MFI), however, for both HLA-G (33.25±1.47 vs. 55.46±5.53, P<0.0002) and ILT4 (86.06±12.83 vs. 146.6±21.27, P<0.01) was lower on mDC compared with pDC. As shown in Figure 1(C), the frequency of circulating DC subsets positive for CMRF44 (pDC%: 1.62±0.39; mDC%: 1.87±0.54) and GITRL (pDC%: 3.88±0.93; mDC%: 4.48±0.80) was low, with no significant difference between pDC and mDC. However, the frequency of ICOSL+ pDC was higher than ICOSL+ mDC (28.33±3.82 vs. 13.47±2.77, P<0.0034). MFI, although low overall, was higher on pDC than on mDC for both CMRF44 (6.92±0.23 vs. 5.71±0.38, P<0.01) and GITRL (10.64±0.60 vs. 7.93±0.29, P<0.0003). Similarly, ICOSL MFI was higher on pDC than mDC (30.93±2.02 vs. 12.65±0.74, P<0.0001) in HC (Fig. 1C).
mDC in TOL Patients Express Elevated Levels of HLA-G
We next compared the expression of HLA-G, ILT4, CMRF-44, GITRL, and ICOSL on mDC and pDC in the four study groups. As shown in Figure 2, expression of HLA-G (both % positive cells and MFI) was significantly higher on mDC in TOL patients compared with the MI (68.8%±2.49% vs. 58.89%±3.52%, P<0.05; MFI: 42.95±2.02 vs. 35.37±2.58, P<0.05) and HC groups (68.8%±2.49% vs. 57.62%±2.81%, P<0.05; MFI: 42.95±2.02 vs. 33.25±1.47, P<0.05) groups. No similar differences were observed for pDC. HLA-G expression on neither mDC nor pDC in the PW group differed from that in the other groups. ILT4 expression did not differ significantly between any of the four groups. Furthermore, we observed no differences in the low levels of expression of CMRF-44, GITRL, or ICOSL on either DC subset between the study groups (data not shown). HLA-G expression on mDC in the patient groups did not correlate with primary diagnosis, donor/recipient age at transplant, ABO typing, transplant type (whole or split liver), cold or warm ischemia time, induction therapy, or liver function. In the patients studied, more than 93% of PW and MI patients were under tacrolimus therapy (Table 1). To rule out a possible influence of tacrolimus in modulating the expression of HLA-G on circulating mDC, we analyzed the trough level and dose of tacrolimus in relation to HLA-G expression on mDC in individual PW and MI patients. As shown in Figure 2(C), no correlation between trough level/dose of tacrolimus and HLA-G expression on mDC (% positive cells or MFI) was observed.
Recently, it has been reported (33) that serum HLA-G levels are negatively correlated with the number of acute rejection (AR) episodes in liver transplantation. In our cohort of patients, we did not find any significant correlation between AR episodes (no AR episodes vs. one or more episodes, or 0 to 1 AR episodes vs. 2 or more episodes) and HLA-G expression on mDC (MFI or % positive cells) in each group.
Serum Levels of HLA-G Do Not Differ Significantly Among Tolerant, Prospective Weaning, and MI Patients
Given the elevated expression of HLA-G on circulating mDC in the TOL patients, we ascertained concomitant serum(s) HLA-G levels in each patient and control group by ELISA. Considerable variation was observed in serum human leukocyte antigen-G (sHLA-G) levels (TOL: 63.3±93.5 ng/mL; PW: 92.3±102.4 ng/mL; MI: 67.1±96.0 ng/mL), and no significant differences were detected between the groups. Recently, it has been reported that in heart transplantation, only 13% of patients with sHLA-G levels more than 100 ng/mL suffered clinically significant acute cellular rejection compared with 63% of patients with sHLA-G less than 100 ng/mL (34). Interestingly, in our study population, a higher percentage of patients with sHLA-G levels more than 100 ng/mL was observed in the TOL group (9 of 26, 34.6%) compared with PW patients (8 of 28, 28.5%) and MI patients (6 of 24, 25.0%) but these differences did not achieve statistical significance. As expected, in all HC, sHLA-G levels were low or undetectable (2.1±3.3 ng/mL). Although sHLA-G expression has been positively correlated with viral infection and neoplastic conditions (35), in our cohort of patients, the frequency of Epstein-Barr virus (EBV) infection and posttransplant lymphoproliferative disease (PTLD) was low or absent (Table 1). Therefore, we were not able to correlate the findings reported in our study with EBV infection or PTLD.
Tolerant Patients Exhibit Significantly Higher Levels of Circulating Treg Than MI Patients and Controls
HLA-G has been reported to impair DC maturation (36) and to induce the development of tolerogenic mDC that can induce the differentiation of anergic and Treg (37, 38). We therefore compared the incidence of CD4+CD25hiCD127− cells (Fig. 3A), a phenotype that correlates with human CD4+ Treg, and their function (39) between the four study groups. For this analysis, we used a smaller but not significantly different cohort of pediatric patients (18 patients for each group, see Table, Supplemental Digital Content 1,https://links.lww.com/TP/A404), for which sufficient peripheral blood mononuclear cell (PBMC) sample was available for analysis. As shown in Figure 3(B), the incidence of Treg was significantly higher in TOL than in MI patients (1.59%±0.19% vs. 0.86%±0.12%, P<0.05). Treg levels did not differ significantly between the TOL and PW groups (1.59%±0.19% vs. 1.56%±0.20%, NS) or between TOL and HC (1.59%±0.19% vs. 0.93%±0.09%, NS). Moreover, the incidence of Treg in the PW group did not differ significantly from that in HC (1.56%±0.20% vs. 0.93%±0.09%, NS). The intensity of Foxp3 expression by CD4+CD25hiCD127− Treg was elevated significantly in the TOL group compared with the MI group (162.7%±11.22% vs. 104.1%±13.68%, P<0.05) but not compared with other groups (Fig. 3C). Treg (% CD4+ cells and Foxp3 MFI) in the patient groups did not correlate with primary diagnosis, donor/recipient age at transplant, ABO typing, transplant type (whole or split liver), cold or warm ischemia time, induction therapy, or liver function. As shown in Figure 3(D), no correlation was found between Treg (% CD4+ cells or Foxp3 MFI) and trough/dose of tacrolimus in the PW and MI groups. There was also no correlation between rejection episodes and Treg in each patient group.
Elevated Expression of HLA-G by mDC in TOL Patients Correlates With Enhanced Levels of Treg Foxp3 Expression
We next examined the relationship between HLA-G expression on mDC and the extent of Foxp3 expression in Treg in the four study groups. As shown in Figure 4, there was a significant positive correlation between the intensity of HLA-G expression and that for Foxp3 in the TOL group (P=0.01, R=0.39) that was not observed in the other study groups (data not shown).
Both DC (12) and Treg (40) are believed to play important roles in the regulation of alloimmune responses and transplant outcome. Moreover, there is increasing evidence that these cells interact in the control of alloimmune reactivity (41). Thus, DC with tolerogenic potential can expand or induce Treg (17, 42–45), whereas Treg, in turn, can down-regulate the immunostimulatory properties of DC and enhance their inherent tolerogenic function (46), resulting in feedback inhibition of effector T-cell function and prolongation of graft survival (47). Our understanding of these mechanisms is based largely on work conducted in vitro, or in experimental animal models, and there is little information regarding the phenotype of DC and their relation to Treg in human transplant recipients (48). Studies of the expression by DC of molecules associated with immune regulation in clinical transplantation may provide new insights into the role of these cells in relation to graft outcome.
Human DC express a variety of surface molecules that correlate with their maturation or activation status, and with their ability to induce or regulate immune responses. These include MHC gene products, the maturation marker CD83, the activation/differentiation-associated molecule CMRF-44 (32, 49), T-cell co-stimulatory (e.g., CD80/86 and ICOSL) and co-inhibitory molecules (e.g., B7-H1), and molecules that may be associated with the induction of Treg (e.g., HLA-G and GITRL). In this study, we did not observe any difference in the expression of specific maturation or costimulatory molecules or activation markers or coregulatory molecules on DC between groups of liver transplant recipients. We did, however, observe for the first time, that conventional mDC, but not pDC in tolerant liver transplant recipients expressed significantly higher levels of the nonclassical MHC class I molecule HLA-G than those in patients on MI. We consider it unlikely that this difference can be ascribed to use of immunosuppressive drugs (in the MI group) because these agents have been associated with enhanced HLA-G transcript and protein levels, both in vitro and in vivo (50–52). Moreover, we found no correlation between HLA-G expression on mDC and tacrolimus dosage or trough blood levels. HLA-G has been ascribed tolerogenic functions, both in pregnancy and in transplantation (53, 54). In addition, we have found in this study that the enhanced expression of HLA-G by mDC correlates with concomitant, elevated expression of Foxp3 by blood-borne Treg, that, as reported herein and previously (6, 25), are elevated in tolerant pediatric liver graft recipients off all immunosuppression.
There is evidence that HLA-G plays an important role in regulating both the maturation and function of DC. Thus, interaction between HLA-G and its receptors leads to inhibition of DC maturation (55). Moreover, soluble HLA-G inhibits human DC-triggered allogeneic T-cell proliferation (56), whereas HLA-G-expressing antigen-presenting cells induce CD4+ T-cell anergy and differentiation of Treg (30). In HLA-G transgenic (tg) mice, DC maturation and T-cell responses are compromised and skin allograft survival is prolonged significantly (36). Moreover, tg mice expressing human ILT4 only on DC and triggered by HLA-G exhibit long-term skin graft survival. Furthermore, HLA-G-modified DC from these tg mice promote long-term graft survival by mechanisms that include T-cell anergy and induction of Treg (31). Taken together with our current observations, these findings suggest that elevated HLA-G on mDC in clinically tolerant liver transplant recipients may signify a functional role of HLA-G in achieving or maintaining the tolerant state. It would now be informative to determine the expression of HLA-G on mDC in sequential samples from liver graft recipients, to establish the relationship between these levels and progression towards the tolerant state. Such studies should include assessment of antidonor T-cell reactivity and its regulation that could not be performed in the present study due to the lack of donor cells.
The only previous study of HLA-G expression on both PBMCs and in serum of liver transplant patients led to the conclusion that the analyses performed could be beneficial in determining prognosis and response to treatment (51). However, in the present investigation, we detected a wide distribution of sHLA-G levels in the three patient groups, and there were no significant differences between these groups or between patients and HC. Nevertheless, in the TOL group, we found a higher incidence of patients (34.6%) with sHLA-G levels more than 100 ng/mL compared with the PW (28.5%) and MI (25.0%) groups, although the differences were not statistically significant. Recently, significantly higher sHLA-G levels were reported in tolerant pediatric liver transplant patients, compared with patients who had experienced AR episodes (33).
Transplant biopsies were not available in the present study, and therefore we were unable to determine intragraft expression of HLA-G. Previously however, HLA-G expression in biliary epithelial cells and high serum concentrations of HLA-G have been associated with allograft acceptance in combined human liver-kidney transplantation (57, 58), whereas elevated expression of HLA-G in bronchial epithelium of human lung allograft recipients has been associated with graft functional stability (59). In view of these and the current findings, it would be of interest to evaluate expression of HLA-G both on parenchymal cells and non-parenchymal cells (in particular interstitial DC and other liver antigen-presenting cells) in relation to intragraft and circulating Treg and human liver transplant outcome. In this regard, quantitative multiplex quantum dot immunostaining analysis of these variables in liver allograft biopsies sections (60) may be particularly informative.
In a previous cross-sectional analysis of liver allograft recipients (25), we observed that the ratio of costimulatory B7-H1:coinhibitory CD86 (B7-1) on circulating pDC, but not mDC, was higher in TOL compared with MI patients, and that this high ratio correlated with elevated Treg in operational liver transplant tolerance pDC undergo unique developmental programming and express a genetic profile that more closely resembles lymphoid than myeloid cell development (61), which may explain differences in regulation of surface molecule expression. The present findings concerning elevated expression of HLA-G on mDC (the predominant DC subset) in TOL patients provide further evidence of a possible functional relationship between the expression of immune regulatory molecules on DC subsets, the concomitant elevated frequency of Treg observed, and the state of operational liver transplant tolerance.
MATERIALS AND METHODS
Seventy-eight clinically stable, pediatric liver transplant recipients with normal graft function were eligible for study. All patients, and 28 normal HC, provided written informed consent in accordance with protocols approved by the local Institutional Review Board (protocol number: IRB010560). The demographics of this population, subdivided into four study groups, that is, TOL, tolerant patients; PW, prospective weaning patients; MI, MI patients; and HC are shown in Table 1. The mean age at transplantation for all patients was 5.3±5.5 years (range 0.4–20.2 years), the mean time from transplant to testing was 8.9±6.6 years (range 0.4–23.6 years), the mean age at testing was 14.2±8.0 (range 1.6–27.9 years), and the mean time off all immunosuppressive therapy was 10.6±4.5 years (range 3.5–16.9 years). Diagnoses at transplantation included cholestatic disease (n=40), metabolic disease (n=21), autoimmune disease (n=4), cryptogenic cirrhosis (n=2), and other diagnoses (n=10: comprising fulminant hepatic failure with unknown etiology, Budd-Chiari disease, total parental nutrition–induced liver failure, embryonal hepatoblastoma, and polycystic disease).
Patients Off Immunosuppression (TOL) or on Low-Dose Antirejection Therapy Undergoing Prospective Weaning
Twenty-six patients, off all immunosuppression (TOL), as described in Table 1, had been weaned off drugs by physician-directed protocol (n=18) as described (3), or emergently, for life-threatening infectious disease indications (EBV, n=2; PTLD, n=2) or had self-weaned by noncompliance (n=3). Initial immunosuppression had consisted of azathioprine and prednisone (n=2), cyclosporine and steroids (n=4), or tacrolimus and steroids (n=20). Briefly, the protocol used to achieve drug withdrawal was as follows: prednisone was withdrawn in 50% decrements monthly with corticotrophin stimulation testing to detect adrenal insufficiency if indicated clinically. Calcineurin inhibitors (primarily tacrolimus and, when applicable, cyclosporine) were then withdrawn at 1- to 2-month intervals by 10% to 25% of the baseline amount. Azathioprine, when present, was the final drug withdrawn. For patients who presented with acute complications of immunosuppression, such as EBV infection or overt PTLD, all immunosuppression was stopped immediately. Antirejection therapy was resumed when the presenting infection had resolved and with documentation of rejection on biopsy. Patients who did not subsequently require reinstitution of immunosuppression because of normal liver function and/or a severe episode of PTLD, were then included in the study group of patients off immunosuppression (TOL). Twenty-eight patients were undergoing prospective drug weaning (PW). All were on minimal immunosuppression, defined as monotherapy (93% of patients in the PW group were on tacrolimus monotherapy), with low (<5 ng/mL) or undetectable drug levels. Six months after blood sampling for the current study, the status of all of these patients remained unchanged.
Patients on MI
Twenty-four patients in this group had failed drug withdrawal (n=10) or had never been weaned from immunosuppressive medications because of a concern for rejection and/or disease recurrence (i.e. history of autoimmune hepatitis) or previous rejection (n=14).
For ethical reasons, blood was not drawn from normal healthy children and, as documented in previous studies (22, 23, 25), healthy adult volunteers of both sexes (n=28) served as controls.
PBMC Isolation and Cryopreservation
Peripheral venous blood samples were collected, and PBMC isolated, cryopreserved and recovered, as described in detail (25). We have shown previously (23) that results obtained staining cryopreserved PBMC do not differ significantly from those obtained using freshly isolated cells.
DC Subset Analysis
As described previously (25), PBMC were stained on melting ice with a lineage (lin) monoclonal antibody (mAb) cocktail (anti-CD3, -CD14, -CD19, and -CD20) and anti-HLA-DR. The cells were also stained at the same time with the DC subset-specific mAbs blood DC Ag (BDCA)-1 (CD1; clone AD5–8E7) and BDCA-2 (CD303; clone AC144) (both Miltenyi Biotec, Auburn, CA), as well as for HLA-G (87G) and the human DC differentiation/activation antigen CMRF-44 (CMRF-44) (32) (all from BD PharMingen, San Diego, CA), CD85d (ILT4) (Beckman Coulter, Brea, CA), glucocorticoid-induced tumor necrosis factor-related protein ligand (GITRL; eBioAITR-L) and ICOSL (MIH12; eBioscience, San Diego, CA). DC subsets were identified as: mDC (lin−BDCA-1+BDCA-2−) and pDC (lin−BDCA-1−BDCA-2+; Fig. 1A). DC phenotype was further characterized by flow cytometric analysis (LSR II, BD Bioscience), gating on the mDC or pDC population. Data were analyzed by using Flow-Jo (Tree Star Inc., Ashland, OR) or WinMDI (freeware designed by J Trotter: http://facs.scripps.edu/software.htm).
PBMC were stained with fluorochrome-conjugated anti-CD4 (RPA-T4), and anti-CD25 (M-A251) from BD PharMingen, and with anti-CD3 (UCHT1), anti-CD127 (IL-3R; hIL-7R-M21), and anti-Foxp3 mAbs (PCH101) from eBioscience. Intracellular staining for Foxp3 was conducted after surface staining with anti-CD3, -CD4, -CD127 (IL-7R), and -CD25 mAbs, as recommended by the manufacturer (eBioscience). Treg were defined as CD4+CD127−CD25hiFoxp3+ by flow cytometry, and the results expressed as % total CD4+ cells. Foxp3 expression was also expressed as MFI.
Serum HLA-G Quantitation
Serum HLA-G levels in groups of TOL, PW, and MI patients and in HC were determined by ELISA, using commercial kits from Biovendor Research and Diagnostic Products (Modrice, Czech Republic) and following the manufacturer's instructions.
Results are expressed as arithmetic mean±95% confidence intervals. Statistical analysis was performed using Mann-Whitney U test or the Kruskal-Wallis test, where appropriate, followed by a posthoc test (Dunn's test). Correlation analyses were performed using Spearman's correlation. For biomarker analysis on pDC and mDC in HC, the significances of differences were determined using the unpaired Student's t test. P values less than 0.05 were considered significant.
The authors thank Miriam Freeman for administrative support.
1. Ramos HC, Reyes J, Abu-Elmagd K, et al. Weaning of immunosuppression in long-term liver transplant recipients. Transplantation
1995; 59: 212.
2. Mazariegos GV, Reyes J, Marino IR, et al. Weaning of immunosuppression in liver transplant recipients. Transplantation
1997; 63: 243.
3. Takatsuki M, Uemoto S, Inomata Y, et al. Weaning of immunosuppression in living donor liver transplant recipients. Transplantation
2001; 72: 449.
4. Martinez-Llordella M, Lozano JJ, Puig-Pey I, et al. Using transcriptional profiling to develop a diagnostic test of operational tolerance in liver transplant recipients. J Clin Invest
2008; 118: 2845.
5. Tzakis AG, Reyes J, Zeevi A, et al. Early tolerance in pediatric liver allograft recipients. J Pediatr Surg
1994; 29: 754.
6. Li Y, Koshiba T, Yoshizawa A, et al. Analyses of peripheral blood mononuclear cells in operational tolerance after pediatric living donor liver transplantation
. Am J Transplant
2004; 4: 2118.
7. Castellaneta A, Thomson AW, Nayyar N, et al. Monitoring the operationally tolerant liver allograft recipient. Curr Opin Organ Transplant
2010; 15: 28.
8. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature
1998; 392: 245.
9. Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells. Annu Rev Immunol
2003; 21: 685.
10. Thomson AW. Tolerogenic dendritic cells: All present and correct? Am J Transplant
2010; 10: 214.
11. Ochando JC, Homma C, Yang Y, et al. Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts. Nat Immunol
2006; 7: 652.
12. Morelli AE, Thomson AW. Tolerogenic dendritic cells and the quest for transplant tolerance. Nat Rev Immunol
2007; 7: 610.
13. Li W, Kuhr CS, Zheng XX, et al. New insights into mechanisms of spontaneous liver transplant tolerance: The role of Foxp3-expressing CD25+
regulatory T cells. Am J Transplant
2008; 8: 1639.
14. Lu L, Li W, Fu F, et al. Blockade of the CD40-CD40 ligand pathway potentiates the capacity of donor-derived dendritic cell progenitors to induce long-term cardiac allograft survival. Transplantation
1997; 64: 1808.
15. Lutz MB, Suri RM, Niimi M, et al. Immature dendritic cells generated with low doses of GM-CSF in the absence of IL-4 are maturation resistant and prolong allograft survival in vivo. Eur J Immunol
2000; 30: 1813.
16. Mirenda V, Berton I, Read J, et al. Modified dendritic cells coexpressing self and allogeneic major histocompatibility complex molecules: An efficient way to induce indirect pathway regulation. J Am Soc Nephrol
2004; 15: 987.
17. Turnquist H, Raimondi G, Zahorchak AF, et al. Rapamycin-conditioned dendritic cells are poor stimulators of allogeneic CD4+
T cells, but enrich for antigen-specific Foxp3+
T regulatory cells and promote organ transplant tolerance. J Immunol
2007; 178: 7018.
18. Shortman K, Liu YJ. Mouse and human dendritic cell subtypes. Nat Rev Immunol
2002; 2: 151.
19. Liu Y-J. IPC: Professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors. Annu Rev Immunol
2005; 23: 275.
20. Colonna M, Trinchieri G, Liu YJ. Plasmacytoid dendritic cells in immunity. Nat Immunol
2004; 5: 1219.
21. Matta BM, Castellaneta A, Thomson AW. Tolerogenic plasmacytoid DC. Eur J Immunol
2010; 40: 2667.
22. Mazariegos GV, Zahorchak AF, Reyes J, et al. Dendritic cell subset ratio in peripheral blood correlates with successful withdrawal of immunosuppression in liver transplant patients. Am J Transplant
2003; 3: 689.
23. Mazariegos GV, Zahorchak AF, Reyes J, et al. Dendritic cell subset ratio in tolerant, weaning and non-tolerant liver recipients is not affected by extent of immunosuppression. Am J Transplant
2005; 5: 314.
24. Martinez-Llordella M, Puig-Pey I, Orlando G, et al. Multiparameter immune profiling of operational tolerance in liver transplantation
. Am J Transplant
2007; 7: 309.
25. Tokita D, Mazariegos GV, Zahorchak AF, et al. High PD-L1/CD86 ratio on plasmacytoid dendritic cells correlates with elevated T-regulatory cells in liver transplant tolerance. Transplantation
2008; 85: 369.
26. Selenko-Gebauer N, Majdic O, Szekeres A, et al. B7–H1 (programmed death-1 ligand) on dendritic cells is involved in the induction and maintenance of T cell anergy. J Immunol
2003; 170: 3637.
27. Latchman YE, Liang SC, Wu Y, et al. PD-L1-deficient mice show that PD-L1 on T cells, antigen-presenting cells, and host tissues negatively regulates T cells. Proc Natl Acad Sci USA
2004; 101: 10691.
28. Akbari O, Freeman GJ, Meyer EH, et al. Antigen-specific regulatory T cells develop via the ICOS-ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity. Nat Med
2002; 8: 1024.
29. Tuyaerts S, Van Meirvenne S, Bonehill A, et al. Expression of human GITRL on myeloid dendritic cells enhances their immunostimulatory function but does not abrogate the suppressive effect of CD4+
regulatory T cells. J Leukoc Biol
2007; 82: 93.
30. LeMaoult J, Krawice-Radanne I, Dausset J, et al. HLA-G1-expressing antigen-presenting cells induce immunosuppressive CD4+
T cells. Proc Natl Acad Sci USA
2004; 101: 7064.
31. Ristich V, Zhang W, Liang S, et al. Mechanisms of prolongation of allograft survival by HLA-G/ILT4-modified dendritic cells. Hum Immunol
2007; 68: 264.
32. Hock BD, Starling GC, Daniel PB, et al. Characterization of CMRF-44, a novel monoclonal antibody to an activation antigen expressed by the allostimulatory cells within peripheral blood, including dendritic cells. Immunology
1994; 83: 573.
33. Zarkhin V, Talisetti A, Li L, et al. Expression of soluble HLA-G identifies favorable outcomes in liver transplant recipients. Transplantation
2010; 90: 1000.
34. Sheshgiri R, Rao V, Mociornita A, et al. Association between HLA-G expression and C4d staining in cardiac transplantation. Transplantation
2010; 89: 480.
35. Menier C, Rouas-Freiss N, Favier B, et al. Recent advances on the non-classical major histocompatibility complex class I HLA-G molecule. Tissue Antigens
2010; 75: 201.
36. Horuzsko A, Lenfant F, Munn DH, et al. Maturation of antigen-presenting cells is compromised in HLA-G transgenic mice. Int Immunol
2001; 13: 385.
37. Ristich V, Liang S, Zhang W, et al. Tolerization of dendritic cells by HLA-G. Eur J Immunol
2005; 35: 1133.
38. Gregori S, Magnani CF, Roncarolo MG. Role of human leukocyte antigen-G in the induction of adaptive type 1 regulatory T cells. Hum Immunol
2009; 70: 966.
39. Liu W, Putnam AL, Xu-Yu Z, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+
T reg cells. J Exp Med
2006; 203: 1701.
40. Wood KJ, Sakaguchi S. Regulatory T cells in transplantation tolerance. Nat Rev Immunol
2003; 3: 199.
41. Thomson AW, Turnquist HR, Zahorchak AF, et al. Tolerogenic dendritic cell-regulatory T-cell interaction and the promotion of transplant tolerance. Transplantation
2009; 87(9 suppl): S86.
42. Lan A, Wang Z, Raimondi G, et al. ‘Alternatively-activated' dendritic cells preferentially secrete IL-10, expand Foxp3+
T cells and induce long-term organ allograft survival in combination with CTLA4-Ig. J Immunol
2006; 177: 5868.
43. Buckland M, Jago CB, Fazekasova H, et al. Aspirin-treated human DCs up-regulate ILT-3 and induce hyporesponsiveness and regulatory activity in responder T cells. Am J Transplant
2006; 6: 2046.
44. Luo X, Tarbell KV, Yang H, et al. Dendritic cells with TGF-beta1 differentiate naive CD4+
T cells into islet-protective Foxp3+
regulatory T cells. Proc Natl Acad Sci USA
2007; 104: 2821.
45. Yates SF, Paterson AM, Nolan KF, et al. Induction of regulatory T cells and dominant tolerance by dendritic cells incapable of full activation. J Immunol
2007; 179: 967.
46. Fallarino F, Grohmann U, Hwang KW, et al. Modulation of tryptophan catabolism by regulatory T cells. Nat Immunol
2003; 4: 1206.
47. Min WP, Zhou D, Ichim TE, et al. Inhibitory feedback loop between tolerogenic dendritic cells and regulatory T cells in transplant tolerance. J Immunol
2003; 170: 1304.
48. Solari MG, Thomson AW. Human dendritic cells and transplant outcome. Transplantation
2008; 85: 1513.
49. Lau J, Sartor M, Bradstock KF, et al. Activated circulating dendritic cells after hematopoietic stem cell transplantation predict acute graft-versus-host disease. Transplantation
2007; 83: 839.
50. Moreau P, Faure O, Lefebvre S, et al. Glucocorticoid hormones upregulate levels of HLA-G transcripts in trophoblasts. Transplant Proc
2001; 33: 2277.
51. Basturk B, Karakayali F, Emiroglu R, et al. Human leukocyte antigen-G, a new parameter in the follow-up of liver transplantation
. Transplant Proc
2006; 38: 571.
52. Luque J, Torres MI, Aumente MD, et al. Soluble HLA-G in heart transplantation: Their relationship to rejection episodes and immunosuppressive therapy. Hum Immunol
2006; 67: 257.
53. Carosella ED, Moreau P, Le Maoult J, et al. HLA-G molecules: From maternal-fetal tolerance to tissue acceptance. Adv Immunol
2003; 81: 199.
54. Rouas-Freiss N, Naji A, Durrbach A, et al. Tolerogenic functions of human leukocyte antigen G: From pregnancy to organ and cell transplantation. Transplantation
2007; 84(1 suppl): S21.
55. Liang S, Baibakov B, Horuzsko A. HLA-G inhibits the functions of murine dendritic cells via the PIR-B immune inhibitory receptor. Eur J Immunol
2002; 32: 2418.
56. Le Friec G, Laupeze B, Fardel O, et al. Soluble HLA-G inhibits human dendritic cell-triggered allogeneic T-cell proliferation without altering dendritic differentiation and maturation processes. Hum Immunol
2003; 64: 752.
57. Creput C, Durrbach A, Menier C, et al. Human leukocyte antigen-G (HLA-G) expression in biliary epithelial cells is associated with allograft acceptance in liver-kidney transplantation. J Hepatol
2003; 39: 587.
58. Creput C, Le Friec G, Bahri R, et al. Detection of HLA-G in serum and graft biopsy associated with fewer acute rejections following combined liver-kidney transplantation: Possible implications for monitoring patients. Hum Immunol
2003; 64: 1033.
59. Brugiere O, Thabut G, Pretolani M, et al. Immunohistochemical study of HLA-G expression in lung transplant recipients. Am J Transplant
2009; 9: 1427.
60. Isse K, Grama K, Abbott IM, et al. Adding value to liver (and allograft) biopsy evaluation using a combination of multiplex quantum dot immunostaining, high-resolution whole-slide digital imaging, and automated image analysis. Clin Liver Dis
2010; 14: 669.
61. Reizis B. Regulation of plasmacytoid dendritic cell development. Curr Opin Immunol
2010; 22: 206.