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The liver is generally regarded as the most “tolerogenic” of whole organ grafts (1, 2). Moreover, elective protocols have been developed that have allowed safe withdrawal of all immunosuppression in some liver transplant patients (3–5). Nevertheless, operational tolerance after clinical organ transplantation—defined as stable graft function in the absence of any immunosuppressive therapy, usually for >1 year—remains extremely rare.
Various mechanisms have been proposed to explain the induction and maintenance of liver transplant tolerance based largely on studies in experimental animals. These include a tolerogenic effect of soluble donor liver-derived major histocompatibility complex (MHC) class I antigens (Ags) (6), regulatory properties of donor-derived hematopoietic cells (7), especially dendritic cells (DC) (8), anergy or deletion of alloreactive T cells (9), and T regulatory cell (Treg) function (10). In recent years, evidence has accumulated that DC, regarded traditionally as instigators of rejection (11, 12) can regulate alloimmune responses (13), including the ability of donor or host DC to prolong graft survival or facilitate tolerance induction in rodents (13–16). In humans, precursors of classic myeloid (monocytoid) DC (mDC) and plasmacytoid DC (pDC) circulate as rare, lineage (lin)− HLA-DR+ cells, characterized by differential expression of cell surface antigens. Although both subsets play important roles in innate and adaptive immunity, there is recent evidence that pDC may be specialized for tolerance induction. Thus, freshly isolated precursor pDC of donor origin can markedly prolong murine organ allograft survival (17) and enhance donor hematopoietic cell engraftment and skin graft tolerance (18). pDC that present donor Ag in secondary lymphoid tissue have been implicated as critical promotors of organ transplant tolerance via the induction of alloAg-specific Treg (19). Human pDC are thought to different intrinsically from mDC in the expression of coregulatory molecules that drive distinct types of T-cell responses. Thus, maturing pDC prime interleukin (IL)-10-producing Treg (20) by inducible costimulatory ligand or induce the generation of CD4+CD25+ (21) or CD8+ Treg (22). Human pDC may also regulate graft-versus-host disease after allogeneic hematopoietic stem cell transplantation (23).
We have documented higher incidences of circulating pDC relative to mDC precursors in operationally tolerant (TOL) pediatric liver allograft recipients and in patients on low-dose immunosuppressive therapy undergoing prospective drug weaning (PW), compared with patients on maintenance immunosuppression (MI) (24). On the other hand, Li et al. (25) have reported elevated frequencies of circulating Treg (CD4+CD25hi) in clinically tolerant live donor pediatric liver allograft recipients compared with MI patients. In this study, we sought to ascertain whether, in operational liver transplant tolerance, there might be a relation between elevated precursor pDC and naturally-occurring Treg (CD4+CD25hiFoxp3+). Moreover, because DC function and the outcome of DC–T-cell interactions may depend on the net coregulatory signal delivered by DC-expressed B7 family molecules, we have examined the expression of coinhibitory B7-H1 (programmed death ligand-1 [PD-L1]; CD274) relative to costimulatory B7-2 (CD86) on tolerant patients’ precursor pDC and mDC relative to those of control groups. PD-L1/PD-1 signaling has been shown to negatively regulate lymphocyte activation (26) and to promote allograft survival (27) and fetomaternal tolerance (28). Moreover, blocking of PD-L1 on DC increases T-cell activation, suggesting that PD-L1 on DC plays an important role in the induction and maintenance of T-cell unresponsiveness (29). PD-1 pathway function also correlates with the presence of CD4+CD25+ Treg and regulation of alloimmune responses in vivo (30).
MATERIALS AND METHODS
Forty-three clinically stable liver transplant recipients (36 children and 7 adults) with normal graft function were eligible for study. All patients and normal healthy controls provided written informed consent in accordance with protocols approved by the local Institutional Review Board. No donor organs were obtained from executed prisoners or other institutionalized persons. The demographics of this population, subdivided into four study groups, are shown in Table 1. The mean age at transplantation was 8.9±14.4 years (range 0.2–49.8 years). Diagnoses at transplantation included biliary atresia (n=23), metabolic disease (n=2), cholestatic syndromes (n=3), viral induced cirrhosis (n=2), autoimmune liver disease (n=2), Wilson’s disease (n=2), cryptogenic cirrhosis (n=2), and other diagnoses in seven patients. No patients were on antiviral therapy at the time of analysis. There were no significant demographic differences between the patients off immunosuppression and the maintenance immunosuppression group.
Patients Off Immunosuppression or on Low Dose Antirejection Therapy Undergoing Prospective Weaning
Thirteen patients, off immunosuppression as described in Table 1 (group A), had been free from antirejection therapy for a mean of 9.3 years (range: 0.9–21.5 years). They were weaned off drugs by physician-directed protocol (n=8) as described (3), or emergently for life-threatening infectious disease indications (Epstein-Barr virus [EBV], n=1; posttransplant lymphoproliferative disease [PTLD], n=2) or had self-weaned by noncompliance (n=2). Initial immunosuppression had consisted of azathioprine and prednisone (n=3), cyclosporine and steroids (n=4), or tacrolimus and steroids (n=6).
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 cyclosporine and, when applicable, tacrolimus) were then withdrawn at 1- to 2-month intervals by 10–25% of the baseline amount. Azathioprine was the final drug withdrawn when present. 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 (group A).
Eighteen patients were undergoing prospective drug weaning (PW) and had tolerated uninterrupted drug withdrawal for a mean of 0.7±1.3 years (0–4.0 years). All were on minimal immunosuppression, defined as monotherapy with low or unmeasurable drug levels. Six months after blood sampling for the current study, the status of all of these patients remained unchanged.
Patients on Maintenance Immunosuppression
Twelve patients in this group had either failed drug withdrawal (n=5) 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=7).
Fourteen healthy volunteers of both sexes served as the normal control group.
Peripheral Blood Mononuclear Cells Isolation and Cryopreservation
Peripheral venous blood samples were collected in heparinized tubes (BD Vacutainer, Franklin Lakes, NJ) and rocked slowly overnight (18 hr) at room temperature. Peripheral blood mononuclear cells (PBMC) were then isolated by Ficoll-Paque (Amersham Pharmacia Biotech AB, Uppsala, Sweden) density gradient centrifugation and slowly suspended (dropwise) in cryopreservation freeze media (90% heat-inactivated fetal calf serum [FCS]; 10% dimethyl sulfoxide). The vials were placed in a Styrofoam container, stored at −70°C for 24 hr, and then transferred to liquid nitrogen for long-term storage. Cryopreserved PBMC were thawed in a 37°C water bath then washed and resuspended in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% FCS. Viability was confirmed by trypan blue exclusion. Prior to antibody (Ab) staining, cells were washed with phosphate-buffered saline (without Ca++ and Mg++) supplemented with 1% FCS. After centrifugation, the cells were suspended in supplemented phosphate-buffered saline containing 10% normal goat serum (Gibco-BRL) to block non-specific binding of Abs to Fc receptors. All Ab staining was performed on melting ice.
DC Subset Analyses
Rare Event, Four-Color Flow Cytometry
PBMC were stained with a lineage (lin) monoclonal antibody (mAb) cocktail (anti-CD3,-14,-19,-20), anti-HLA-DR, anti-CD123 (IL-3Rα), and anti-CD11c, as described (31). Precursors of mDC and pDC were identified as lin−, HLA-DR+, CD11c+, CD123−/lo and as lin−, HLA-DR+, CD11c−, CD123hi, respectively (Fig. 1A). The incidence of each subset was expressed as a percentage of lin− HLA-DR+ PBMC; in addition, the precursor pDC: precursor mDC1 ratio was determined for each subject at the time of sampling.
DC Subset-Specific Staining
PBMC were stained with the lin mAb cocktail and anti-HLA-DR. They were also stained with the DC subset-specific mAbs blood DC Ag (BDCA)-1 (CD1; clone AD5-8E7) and BDCA-2 (CD303; clone AC144) (Miltenyi Biotec, Auburn, CA), as well as for CD40 (5C3), CD80 (L307.4), CD86 (2331), and PD-L1 (CD274; MIH1) (BD PharMingen, San Diego, CA). DC precursor subsets were identified as: mDC1 (lin−; BDCA-1+, BDCA-2−; equivalent to 90% of total mDC) and pDC (lin−; BDCA-1−, BDCA-2+; Fig. 1B). DC phenotype was further analyzed by flow cytometric analysis (LSR II, BD Bioscience) gating on the mDC1 or pDC population.
Regulatory T-Cell Analysis
PBMC were stained with fluorochrome-conjugated anti-CD4 (RPA-T4), -CD25 (M-A251; BD PharMingen), anti-CD3 (UCHT1), and anti-Foxp3 (PCH101; eBioscience, San Diego, CA). Intracellular staining for Foxp3 was conducted after surface staining with anti-CD3, -CD4, and -CD25 Abs, as recommended by the manufacturer (eBioscience). Using flow cytometry, Treg were defined as CD4+CD25high and expressed higher CD25 fluorescence intensity when compared to CD4−CD25+ cells (Fig. 3B).
PBMC Culture and Exposure to Immunosuppressive Drugs
PBMC were isolated from buffy coats (leukopacks) of healthy human donors (Institute for Transfusion Medicine, Pittsburgh, PA) by Ficoll density centrifugation. The volume of each bag was approximately 70–80 ml, yielding 500−800×106 PBMC. PBMC were incubated in supplemented RPMI 1640 media containing 5% heat-inactivated normal human serum (Gemini Bio-Products, Woodland, CA), with or without water-soluble dexamethasone (Dex; Sigma, Chemical Co., St. Louis, MO) or tacrolimus (FK-506 monohydrate; Sigma). After 6 hr, the percentage of Treg and the intensity of surface expression of PD-L1, CD86 and HLA-DR on pDC were determined by mAb staining, as described above.
Measurement of Tacrolimus Trough Blood Levels
Tacrolimus levels in whole blood were determined by conventional enzyme-linked immunosorbent assay at the time of PBMC sampling.
Results are expressed as arithmetic means ±95% confidence interval. Statistical analysis was performed using the Mann-Whitney U test or the Kruskal-Wallis test, where appropriate. A Bonferroni correction was used to take account of multiple comparisons between patient groups. Correlation analyses were performed using Spearman’s correlation. For the cell culture studies, data are expressed as means±SD and the significances of differences determined by unpaired Student’s t test. P values <0.05 were considered significant.
Patients off Immunosuppression (tolerant; TOL) and Those on Low-Dose Antirejection Therapy Undergoing Prospective Weaning (PW) Exhibit Higher Relative Incidences of Precursor pDC Compared With Those on Maintenance Immunosuppression (MI): Confirmation by DC Subset-Specific mAb Staining
Circulating precursor pDC and mDC subsets in the same peripheral blood samples from TOL, PW, and MI patients and healthy controls were determined simultaneously on the basis of our previously-described, four-color flow cytometric analysis of lin− HLA-DR+ cells and by three-color staining using DC subset-specific mAbs (Fig. 2A and B). As reported previously, the incidence of CD11c− CD123hi (IL-3Rαhi) lin− HLA-DR+ cells (precursor pDC) compared with precursor mDC (CD11c+ CD123−/lo lin− HLA-DR+), such that %pDC/%mDC was significantly elevated in the TOL and PW groups compared to the MI group (using the Bonferroni correction for multiple comparisons; Fig. 2A). Using the three-color method, the incidence of precursor pDC (lin− BDCA2+) compared with precursor mDC1 (lin− BDCA1+) was also significantly higher in the TOL and PW groups compared with the MI group and between the TOL and PW groups and controls (Fig. 2B). Moreover, a positive correlation was observed between the two methods (Fig. 2C).
TOL Patients Express Significantly Higher PD-L1/CD86 Ratios on Precursor pDC Compared With MI Patients
We have shown that, compared with mDC, stimulated murine pDC express a higher ratio of cell surface PD-L1 to CD86 (17) and that this correlates with reduced levels of T-cell allostimulatory ability. To address the level of expression of PD-L1 relative to CD86 on circulating precursor mDC and -pDC in the 4 study groups, lin− BDCA1+ (mDC1) and lin− BDCA2+ (pDC) were stained concomitantly for PD-L1 and CD86. As shown in Figure 3A, no significant differences in the PD-L1/CD86 ratio on precursor mDC1 were observed between the groups. By contrast, precursor pDC from TOL patients exhibited significantly higher PD-L1/CD86 ratios (similar to those values observed in normal controls) compared with the MI patients.
TOL Patients Exhibit Significantly Higher CD4+CD25hi Treg Compared With Patients on MI
Li et al. (25) have reported a significant increase in the incidence of circulating CD4+CD25hi T cells in tolerant pediatric liver allograft recipients compared with patients on MI and healthy controls, while Sandner et al. (30) have observed that, in mice, PD-L1 function in vivo correlates with the presence of CD4+CD25+ Treg. We therefore examined the frequency of CD4+CD25hi cells, which we confirmed expressed intracellular Foxp3 (consistently >90%; Fig. 3B) in each group. As shown in Figure 3C, a higher frequency of circulating CD4+CD25hi cells was observed in TOL compared with MI patients (P<0.01). Interestingly, the incidence of circulating Treg in the TOL and PW groups did not differ significantly from that in controls (normal individuals).
High PD-L1/CD86 Ratio on Precursor pDC Correlates With Elevated Frequency of CD4+CD25hi Treg in TOL Patients
We next ascertained whether there was a relationship between the surface PD-L1/CD86 ratio on precursor pDC identified by direct mAb staining and the frequency of CD4+CD25hi Treg in the four study groups. As shown in Figure 4, a significant positive correlation was demonstrated in the TOL group (P<0.01), but not in the other groups, either within their respective group or pooled together (data not shown). No correlation was observed between CD4+CD25+ (Foxp3+) cells and the PD-L1/CD86 ratio on precursor mDC. Further, there was no correlation between the PD-L1/CD86 ratio on precursor pDC or the frequency of CD4+CD25hi T cells and length of time off immunosuppression (from 0.9 to 21.5 years; Spearman r=0.4 and 0.62, respectively; data not shown).
No Relation Between Tacrolimus and Prednisone Dosage or Tacrolimus Blood Level and the PD-L1/CD86 Ratio on Precursor pDC in PW and MI Patients
No correlation was observed between tacrolimus and prednisone dosage, or tacrolimus whole blood trough level at the time of blood sampling, and the PD-L1/CD86 ratio on circulating precursor pDC in PW and MI patients. Furthermore, there was no significant difference in PD-L1/CD86 ratio between patients with comparatively low (<2.0 ng/ml) and high (>2.0 ng/ml) trough blood tacrolimus levels (Supplementary Fig. 1, available for viewing online only).
No Relation Between Tacrolimus and Prednisone Dosage or Tacrolimus Blood Levels and the Incidence of CD4+CD25hi Treg in PW and MI Patients
There was no correlation between tacrolimus and prednisone dosage and the frequency of CD4+CD25hi T cells, nor was there a correlation between tacrolimus whole blood trough level and the incidence of Treg (Supplementary Fig. 2, available for viewing online only). Thus, it can be concluded from these observations that neither the PD-L1/CD86 ratio on precursor pDC nor the frequency of Treg in the patients studied was determined by the presence or extent of immunosuppressive drug therapy.
Exposure of PBMC to Clinically-Relevant Concentrations of Glucocorticoid or Tacrolimus In Vitro Did Not Affect the PD-L1/CD86 Ratio on Precursor pDC or the Frequency of CD4+CD25hi T Cells
To further assess the possibility that immunosuppressive drugs might affect expression of the molecules of interest on precursor pDC and naturally-occurring Treg, PBMC freshly-isolated from leukopacks were incubated with various concentrations of dexamethasone (Dex) or tacrolimus for different periods, as described in the Materials and Methods. The peak concentration (Cmax) levels of tacrolimus in kidney or liver transplant recipients vary between 20 and 60 ng/ml (32, 33). Whole blood trough levels between 15 and 20 ng/ml correspond to a Cmax level of 60 ng/ml (32), whereas tacrolimus trough levels in our patients (at sampling) were all <10 ng/ml. The highest Dex concentration used (10−7 M; equivalent to 400 ng/ml prednisone) was equivalent to peak plasma levels reached during daily treatment with 10 mg prednisone (34). As shown in Figure 5, while very high concentrations (above therapeutic levels) of Dex or tacrolimus downregulated the frequency of CD4+CD25hi Treg and CD4+CD25+Foxp3+ cells, neither Dex nor tacrolimus affected the frequency of naturally occurring Treg at concentrations relevant to the patients’ dose or drug level. Neither Dex nor tacrolimus affected the PD-L1/CD86 ratio on precursor pDC, even at high concentrations, whereas both agents reduced the intensity of HLA-DR expression.
In this study, we have confirmed using rare event, flow cytometric analysis, that the frequency of circulating precursor pDC relative to precursor mDC is elevated significantly in clinically tolerant (TOL) pediatric primary liver allograft recipients and in stable patients successfully undergoing physician-controlled, prospective immunosuppressive drug weaning (PW), compared to subjects with a history of rejection on maintenance immunosuppression (MI). Confirmation was attained using two, multicolor staining procedures—our historical four-color approach (24) and, for the first time, a three-color, DC subset-specific staining protocol, which implements recently developed human pDC- and mDC1-specific mAbs to identify these DC subsets in lin− HLA-DR− PBMC populations. Importantly, in the present study, a strong positive correlation was observed between the precursor pDC/mDC ratios determined using the two staining protocols on the same patient PBMC samples. Thus, our initial findings (24, 31) have been corroborated using two different/complementary procedures in a new cohort of liver graft recipients. Previously, we have shown that the precursor pDC/precursor mDC ratio is not influenced significantly by time posttransplant, time off immunosuppression, or a history of PTLD (31). Moreover, we have ascertained that there is no correlation between patient age and either the precursor pDC:precursor mDC ratio, the incidence of CD4+ CD25hi or CD4+ Foxp3+ T cells, or the PD-L1/CD86 ratio on precursor pDC.
Accompanying the relatively high precursor pDC/mDC ratio in the TOL patients was a significantly higher cell surface PD-L1(B7-H1)/CD86(B7-2) ratio on precursor pDC (but not precursor mDC) compared to MI patients. Of further significance, was the higher incidence of circulating CD4+CD25hiFoxp3+ Treg in TOL patients compared with those in the MI group. This latter observation is consistent with the elevated frequency and absolute number of naturally-occurring CD4+CD25hi Treg observed in tolerant pediatric recipients of live donor liver grafts compared with patients on immunosuppression (25). Others have reported that clinically tolerant renal transplant recipients display normal levels of CD4+CD25hi Treg and Foxp3 transcripts, whereas chronic rejection is associated with a decrease in these parameters (35). These naturally occurring and other types of Treg have been implicated in the establishment of tolerance to alloAgs (36, 37), including liver allografts in rodents (38, 39). In the present study, elevated Treg correlated significantly with the PD-L1/CD86 ratio on the TOL patients’ precursor pDC. There was no correlation between either the dose or trough blood level of tacrolimus, the prednisone dose, or the aggregate level of immunosuppression at the time of blood sampling and either the PD-L1/CD86 ratio on precursor pDC or the incidence of CD4+CD25hi Treg in PW and MI patients. Thus, the differences in net cosignaling molecule expression on precursor pDC and the frequency of Treg observed between the study groups could not be ascribed to the influence of systemic immunosuppressive agents. This was confirmed by analysis of the PD-L1/CD86 ratio on precursor pDC and the incidence of CD4+CD25hiFoxp3+ cells in PBMC cultures exposed to therapeutic levels of tacrolimus or glucocorticoid (dexamethasone). Interestingly, Segundo et al. (40) have reported that calcineurin inhibitors (but not rapamycin) reduce the incidence of CD4+CD25+ Foxp3+ Treg in blood of stable renal transplant recipients and have suggested that increased numbers of circulating Treg may help identify candidates for reduced immunosuppression.
Previously (31), we speculated that the comparatively high frequency of precursor pDC in clinically tolerant liver graft recipients and in patients on minimal calcineurin inhibitor monotherapy might signify a role of these APC in the regulation of antidonor reactivity. Indeed, evidence has accumulated that, in rodents, freshly isolated precursor pDC mediate tolerogenic effects on CD4+ T cells in vitro (41, 42). Moreover, freshly isolated, purified precursor pDC enhance donor hematopoietic cell engraftment and promote donor-specific, MHC-mismatched skin graft tolerance in radiation chimeras (18) or the survival of organ allografts in unmodified recipients (17). Further, host pDC appear to play a critical role in the promotion of tolerance and the induction of alloAg-specific Treg in organ allograft recipients (19). In humans, precursor pDC isolated from peripheral blood can elicit Ag-specific anergy in CD4+ T cell lines (43). Moreover, coculture of human CD8+ T cells with allogeneic pDC gives rise to IL10-producing CD8+ Treg (44).
Our interest in the differential expression of PD-L1 and CD86 on the surface of the liver recipients’ precursor pDC (compared with precursor mDC) was stimulated by evidence that the balance between inhibitory PD-L1 and costimulatory B7-1 (CD80)/B7-2(CD86) ligands may regulate their interactions with T cells (26, 45). Indeed, we have observed previously a much higher (5- to 6-fold) ratio of PD-L1 to CD80 or CD86 on CpG-stimulated murine pDC compared with mDC, and that blockade of PD-L1 on pDC increases their T-cell allostimulatory ability (17). Interestingly, Sandner et al. (30) found that the enhancing effect of PD-L1 blockade on murine alloreactive T-cell responses was dependent on the presence of CD4+CD25+ Treg, suggesting an important role for the PD-1 pathway in modulating the function of these cells and in regulation of alloimmunity. The significant positive correlation we have observed between increased net coinhibitory molecule expression on TOL patients’ precursor pDC and the incidence of circulating CD4+CD25hiFoxp3+ cells may reflect a causal relationship, the outcome of which may depend on the immunological context of their encounter and location (46). Unfortunately, we could not test the ability of patients’ precursor pDC to modulate antidonor T-cell responses because no stored donor cells were available.
Very few clinical studies have assessed the functional significance of circulating Treg in stable organ allograft recipients. Recently however, Velthuis et al. (47) observed that in some (28%)—but not all—stable, long-term (>5 years), calcineurin inhibitor-free renal allograft recipients on low-dose immunosuppressive therapy, antidonor T-cell proliferative activity could be restored in mixed lymphocyte reaction after depletion of CD4+CD25bright cells in the responder (host) T-cell population. Moreover, reconstitution of these cells suppressed the donor reactivity in a dose-dependent manner. Conceivably, in other stable patients, different regulatory mechanisms (including possibly, other types of Treg) operate to maintain the absence of responsiveness to donor. Thus, in a small number of kidney transplant patients free of immunosuppression, allograft acceptance has been associated with immune regulation (48), which may be mediated by Treg (49). Unfortunately, transplant biopsies were not available for the TOL patients to allow us ascertain whether, as in rodents (50), Treg may be recruited to the tolerated grafts. Animal studies show that such Treg can prevent nontolerant lymphocytes from mediating rejection in a donor-specific manner (50).
We thank Drs. Diana Metes, Iulia Popescu, Camilla Macedo, and Hidetaka Hara for valuable technologic advice and discussion, Dr. Hongmei Shen for expertise with flow cytometry, Dr. Igor Dvorchak for statistical advice, and Jennifer Dobberstein and Miriam Freeman for administrative support.
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