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The Journal of Immunology, 2005, 175: 8060-8068.
Copyright © 2005 by The American Association of Immunologists

Elevated T Regulatory Cells in Long-Term Stable Transplant Tolerance in Rhesus Macaques Induced by Anti-CD3 Immunotoxin and Deoxyspergualin¹

Clement K. Asiedu^*, Karen J. Goodwin^*, Gansuvd Balgansuren^*, Stacie M. Jenkins^*, Stéphanie Le Bas-Bernardet^*, Uuganbayar Jargal^*, David M. Neville, Jr and Judith M. Thomas^2,*

^* Department of Surgery, Division of Transplant Immunology, University of Alabama, Birmingham, AL 35294; and National Institutes of Health, Laboratory of Molecular Biology, Bethesda, MD 20892

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Regulatory T cells (Tregs) are implicated in immune tolerance and are variably dependent on IL-10 for in vivo function. Brief peritransplant treatment of multiple nonhuman primates (NHP) with anti-CD3 immunotoxin and deoxyspergualin has induced stable (5–10 years) rejection-free tolerance to MHC-mismatched allografts, which associated with sustained elevations in serum IL-10. In this study, we demonstrate that resting and activated PBMC from long-term tolerant NHP recipients are biased to secrete high levels of IL-10, compared with normal NHP PBMC. Although IL-10-producing CD4⁺ Tregs (type 1 regulatory cells (TR1)/IL-10 Tregs) were undetectable (<0.5%) in normal rhesus monkeys, 7.5 ± 1.7% of circulating CD4⁺ T cells of tolerant rhesus recipients expressed IL-10. In addition to this >15-fold increase in Tr1/IL-10 Tregs, the tolerant monkeys exhibited a nearly 3-fold increase in CD4⁺CD25⁺ Tregs, 8.1 ± 3.0% of CD4 T cells vs 2.8 ± 1.4% in normal cohorts (p < 0.02). The frequency of CD4⁺CD25⁺IL-10⁺ cells was elevated 5-fold in tolerant vs normal NHP (1.8 ± 0.9% vs 0.4 ± 0.2%). Rhesus CD4⁺CD25⁺ Tregs exhibited a memory phenotype, and expressed high levels of Foxp3 and CTLA-4 compared with CD4⁺CD25^– T cells. Also, NHP CD4⁺CD25⁺ Tregs proliferated poorly after activation and suppressed proliferation of CD4⁺CD25^– effector T cells, exhibiting regulatory properties similar to rodent and human CD4⁺CD25⁺ Tregs. Of note, depletion of CD4⁺CD25⁺ Tregs restored indirect pathway antidonor responses in tolerant NHP. Our study demonstrates an expanded presence of Treg populations in tolerant NHP recipients, suggesting that these adaptations may be involved in maintenance of stable tolerance.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Recent studies have demonstrated that active T cell-mediated immune regulation is an important mechanism of peripheral immune tolerance. The most thoroughly characterized regulatory T cells are CD4⁺CD25⁺ regulatory T cells (Tregs),³ CD4⁺IL-10⁺ (type 1 regulatory cells (Tr1)/IL-10 Tregs), and CD4⁺TGF-1⁺ (Th3) (1, 2). Rodent Tregs mediate tolerance to MHC-mismatched allografts induced by various treatment modalities (3, 4, 5, 6). CD4⁺CD25⁺ Tregs are present in the peripheral blood of normal humans and are implicated in maintenance of tolerance to self Ags (7, 8, 9, 10, 11, 12). Rodent and human CD4⁺CD25⁺ Tregs exhibit an activated/memory phenotype, and express high levels of glucocorticoid-induced TNFR family-related gene and intracellular CTLA-4, distinct from CD4⁺CD25^– T cells (7, 13). Perhaps the most characteristic marker for CD4⁺CD25⁺ Tregs is the forkhead/winged helix transcription factor Foxp3, a deficiency of which causes a lack of CD4⁺CD25⁺ Tregs and a severe autoimmune syndrome in mice and humans (14, 15, 16, 17). Functionally, CD4⁺CD25⁺ Tregs are anergic in vitro and suppress TCR-induced proliferation of CD4⁺CD25^– T cells and CD8⁺ T cells (18, 19, 20). In vitro functional assays indicate that the suppressor activity of CD4⁺CD25⁺ Tregs is mediated by a contact-dependent, cytokine-independent mechanism (7, 21). In contrast, in vivo models suggest a role for IL-10 and TGF-1 in the regulatory activity of CD4⁺CD25⁺ Tregs (22).

Recently, it was reported that 40% of human kidney allograft recipients without any history of acute rejection showed increased indirect pathway antidonor reactivity in vitro after depletion of CD4⁺CD25⁺ Tregs (23). Also, while lung transplant recipients with stable function had similar peripheral blood levels of CD4⁺CD25⁺ Tregs as healthy subjects, those recipients with chronic rejection had significantly reduced frequency of CD4⁺CD25⁺ Tregs (24). However, depletion of CD4⁺CD25⁺ T cells had no impact on direct pathway antidonor responses (25). In contrast, it was reported recently that infusion of ex vivo generated Tregs promoted long-term survival of renal allografts in nonhuman primates (NHP) by a mechanism that involves donor Ag-specific suppression of direct pathway reactivity (26).

Thus, Ag-specific Tregs may promote allograft survival by regulating indirect pathway antidonor immune responses in the periphery. Indeed, evidence is accumulating that supports the notion that the indirect pathway of allorecognition may underlie the generation and immune regulatory activity of Tregs (27, 28, 29, 30).

We have demonstrated previously that stable, drug-free tolerance induced by peritransplant anti-CD3 immunotoxin (IT) plus 15-deoxyspergualin (DSG) therapy is associated with donor-specific indirect pathway unresponsiveness (31) and sustained elevated levels of serum IL-10 (32, 33, 34). We found that when tolerant recipients accepted a second donor kidney allograft without additional immunosuppressive therapy, this was associated with an upsurge in the already elevated serum IL-10 levels (31), suggesting a role for active immune regulation in the maintenance of stable tolerance in this model. Therefore, we hypothesized that presentation of donor Ags by host APC in a milieu dominated by IL-10 may drive the generation and expansion of donor-specific Tregs that mediate stable tolerance induced by anti-CD3 IT plus DSG. In this study, we examined the frequency, phenotype, and function of Tr1/IL-10 and CD4⁺CD25⁺ Tregs in normal and long-term tolerant rhesus macaque recipients. To our knowledge, this is the first report demonstrating that CD4⁺CD25⁺ Tregs occur naturally in rhesus monkeys and that they are similar in phenotype and function to murine and human CD4⁺CD25⁺ Tregs. Notably, the frequency of Tr1/IL-10 Tregs and CD4⁺CD25⁺ Tregs is significantly increased in long-term tolerant rhesus recipients compared with normal cohorts. Furthermore, depletion of CD4⁺CD25⁺ Tregs uncovers the response of tolerant recipients’ T cells to cell-free donor Ags presented by the indirect pathway. In view of undetectable chimerism in these drug-free tolerant animals (32, 35, 36), we postulate that the elevated levels of Tregs may have a role in the genesis and/or maintenance of long-term, stable tolerance induced by anti-CD3 IT plus DSG.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Monoclonal Abs

Abs demonstrated to cross-react with rhesus Ags were used in these studies. Matching isotype control mAbs were included in all experiments. Anti-CD4 FITC (MT477), anti-CD4 PerCP (L200), anti-CD28 FITC (CD28.2), anti-CD69 FITC (FN52), and anti-CD95 (DX2) were purchased from BD Pharmingen. Anti-CD25 PE (4E3) and anti-CD45RA ECD (2H4) were bought from Miltenyi Biotec and Beckman Coulter, respectively.

Animals

Male rhesus macaques used in this study were purchased from Covance and LABS. The peritransplant tolerance induction protocol with anti-CD3 IT plus DSG has been described previously (31). The University of Alabama Animal Care and Use Committee approved all procedures.

Cell culture

PBMC were isolated from heparinized peripheral blood of normal and tolerant rhesus macaque by Ficoll-Hypaque density gradient centrifugation, as described previously (37). Equivalent numbers of cells (1 x 10⁶/well) were cultured in RPMI 1640/10% normal monkey serum. Polyclonal activation was achieved by seeding cells in RPMI 1640/10% normal monkey serum supplemented with 20 ng/ml PMA and 500 ng/ml ionomycin D or plate-bound anti-CD3 FNI8 (10 µg/ml) and soluble anti-CD28 (CD28.2, 10 µg/ml) mAbs. Supernatants were harvested on days 1, 2, 4, and 6 after initiation of culture for cytokine measurements.

Cytokine measurements

Serum samples or culture supernatants were stored at –70°C until analyzed. Cytokine levels were measured by Luminex multiplex assay (BioSource International), according to the manufacturer’s instructions. IL-10 was measured using a monkey-specific IL-10 ELISA kit as stipulated by the manufacturer (BioSource International).

Isolation of PBMC subsets

PBMC were stained with anti-CD4 FITC and anti-CD25 PE mAbs, followed by positive selection of CD4⁺ cells with anti-FITC microbeads, according to the manufacturer’s instructions (Miltenyi Biotec). Anti-FITC microbeads were released from CD4⁺ cells (efficiency of release >95%), and then CD4⁺CD25⁺ cells were positively selected with anti-PE microbeads. Isolated CD4⁺CD25^– and CD4⁺CD25⁺ cells were typically >95% and >85% pure, respectively (data not shown). To generate T cell-negative accessory cells, the non-CD4 fraction was depleted of CD8⁺ cells using NHP CD8 microbeads, as recommended by the manufacturer (Miltenyi Biotec).

Flow cytometry

Rhesus whole blood, single cell suspensions of PBMC, or T cell subsets isolated from PBMC by magnetic bead sorting were stained for surface Ags and intracellular IL-10, according to standard methods. Stained samples were acquired on a Coulter Cytomics FC 500 flow cytometer (Beckman Coulter), and analyses of events in the lymphocyte gate were performed with WinList 3D 5.0 (Verity Software House) or Cytomics RXP software (Beckman Coulter).

Quantitative RT-PCR

Total RNA or mRNA was extracted from PBMC or magnetically sorted T cell subsets using RNeasy mini (Qiagen) or µMACS mRNA (Miltenyi Biotec) isolation kits, respectively, according to conditions stipulated by the manufacturer. First strand cDNA was generated using random hexamers in the Superscript first strand synthesis system, as per the manufacturer’s protocol (Invitrogen Life Technologies). mRNA or cDNA, gene-specific primers, fluorogenic probes, and Universal MasterMix (Applied Biosystems) were combined in a final volume of 20 µl. Rhesus pyruvate dehydrogenase (PDH) forward primer, 5'-GCTGCTGAAGAGGCGCTT-3'; rhesus PDH reverse primer, 5'-GCTCCTCATCCATACCCTGATTTT-3'; rhesus PDH probe, FAM-5'-CACGAACTGTCACCTGCAGCGCAG-3'-TAMRA (38). We cloned and sequenced rhesus macaque Foxp3 (G. Balgansuren, et al., manuscript in preparation) and submitted the sequence of a 400-base-long fragment to Applied Biosystems for design and synthesis of a 20x primers/probe mix for real-time PCR. The sequences of the primers used were as follows: rhesus Foxp3 forward primer, 5'-CACCTGGCTGGGAAAATGG-3'; rhesus Foxp3 reverse primer, 5'-CCTTGTCAGATGATGCCACAGAT-3'; rhesus Foxp3 probe, FAM-5'-CACTGACCAAGGCTTC-3'-nonfluorescent quencher. The primers and FAM probes for human CTLA-4 and actin, catalog numbers 4329510F and 4333762F, respectively, were purchased from Applied Biosystems. Reactions were set up in triplicates. One- or two-step real-time TaqMan PCR and analyses were performed using the ABI/PRISM 7900HT sequence detector system (Applied Biosystems). Target mRNA expression was normalized to mRNA levels of the housekeeping PDH or actin gene using comparative threshold cycle analysis. We verified that the efficiencies of the amplification reactions for housekeeping and target genes were similar (Applied Biosystems bulletin 2).

Proliferation assay

Equivalent numbers of CD4⁺CD25^– effector cells, CD4⁺CD25⁺ T cells, or both subsets (3 x 10³ to 1 x 10⁵/well) were stimulated with plate-bound anti-CD3 (FN18, 10 µg/ml) and soluble anti-CD28 (CD28.2, 10 µg/ml). After 5 days, [³H]thymidine (1 µCi/well) was added, and cells were harvested 16 h later. Scintillation counting was done to measure proliferation, as described previously (37).

Indirect MLR was done essentially as described previously (31), except that total CD4⁺ and CD4⁺CD25^– T cells were used as responders. Briefly, monocytes were isolated from PBMC by plastic adherence and cultured in recombinant human GM-CSF and IL-4 for 7 days. After 7 days, immature dendritic cells (DC) were harvested and pulsed with cell-free autologous or donor Ags. DC were then cultured for an additional 48 h with a cytokine mixture to induce maturation. Freshly isolated CD4⁺ or CD4⁺CD25^– T cells (i.e., depleted of CD4⁺CD25⁺ subset) were incubated with mature DC at a ratio of 5:1. Proliferation was assessed, as described above, after 5 days’ culture.

Statistical analyses

Comparisons were done using unpaired t tests (GraphPad). Statistical significance was assigned when p value was <0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Resting and activated PBMC from long-term tolerant recipients are high IL-10 secretors

We reported previously that long-term, rejection-free tolerance in NHP recipients treated briefly with anti-CD3 IT plus DSG associates strongly with elevated serum levels of IL-10 (33, 34). Tolerant rhesus recipients induced with anti-CD3 IT plus DSG are unable to respond to donor Ags presented by the indirect pathway, but are otherwise fully immunocompetent (31, 32). The sustained increase in serum IL-10, an immunosuppressive cytokine, in immunocompetent recipients suggests a role for immunoregulation in induction and/or maintenance of anti-CD3 IT plus DSG-induced tolerance. In this study, we determined serum IL-10 levels in tolerant NHP recipients >5 years after transplantation. As shown in Table I, serum IL-10 levels ranged from 71.4 to 872 pg/ml (321.0 pg/ml ± 99.6 SEM) in long-term tolerant recipients (n = 8). In contrast, IL-10 was below the assay detection limit (<15.6 pg/ml) in all normal monkeys (n = 12) examined, p < 0.02 for tolerant vs normal monkeys. Thus, serum IL-10 is significantly elevated in tolerant rhesus recipients >5 years after induction with anti-CD3 IT plus DSG therapy.

View larger version (22K): [in this window] [in a new window]   FIGURE 1. Anti-CD3 IT plus DSG-induced tolerance associates with enduring high IL-10 secretion. PBMC from normal and long-term tolerant monkeys were cultured without stimulation (A), or were polyclonally activated with plate-bound anti-CD3 FN18 and soluble anti-CD28 mAbs (B) or PMA/ionomycin (C). Culture supernatants were harvested at the indicated times, and IL-10 levels were measured by Luminex multiplex assay. Data are presented as mean ± SEM; n = 12 and n = 8 for normal and tolerant monkeys, respectively. Similar results were obtained in two other experiments.

View larger version (16K): [in this window] [in a new window]   FIGURE 2. Long-term tolerant NHP recipients are biased to secrete IL-10. Culture supernatants were harvested from unstimulated or PMA/ionomycin-activated PBMC 48 h after seeding. Supernatants from tolerant (A) or normal (B) monkeys’ PBMC were analyzed for IL-10 and proinflammatory cytokines using the Luminex multiplex assay. Cytokine levels are shown as mean ± SEM calculated from n = 3 animals in each group. Cytokines were essentially undetectable in supernatant from unstimulated tolerant (except IL-10) and normal monkeys’ PBMC.

To determine the peripheral blood source(s) of IL-10 in long-term tolerant NHP, we stained PBMC for surface Ags and intracellular IL-10 for FACS analysis. We found that peripheral blood CD4⁺, CD8⁺ T cells as well as non-T cells produce IL-10 (Fig. 3 and data not shown). The results demonstrate that a mean 7.5 ± 1.7% of CD4⁺ T cells in long-term tolerant NHP are Tr1/IL-10 Tregs (i.e., CD4⁺IL-10⁺), whereas these cells are undetectable (<0.5%) in the circulation of normal macaques (Fig. 3). Thus, Tr1/IL-10 Tregs are expanded in the peripheral blood of drug-free long-term tolerant NHP, years after peritransplant anti-CD3 IT plus DSG therapy.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. CD4+IL-10+ T cells are expanded in long-term tolerant NHP recipients. PBMC were stained for surface Ags and intracellular IL-10 before FACS analysis using an EPICS FC 500 flow cytometer. A, Representative data from one long-term tolerant recipient. B, Frequency of CD4+IL-10+ T cells expressed as a percentage of total CD4+ T cells obtained from 6 long-term tolerant NHP recipients and 12 normal animals.

Immune regulation mediated by CD4⁺CD25⁺ Tregs has emerged as a major mechanism of self and transplantation tolerance (6). Recent reports indicate that indirect recognition of donor alloantigens may be involved in the generation of donor-specific Tregs (39, 40). Our previous finding of specific indirect pathway hyporesponsiveness to donor Ags in long-term tolerant NHP recipients led us to hypothesize that Tregs might have a role in induction and/or maintenance of stable tolerance in our model. Because in vitro properties of CD4⁺CD25⁺ Tregs have not been described in NHP, we first characterized this population in normal animals. As shown in Fig. 4A, 2.8% ± 1.4 (range 1.3–5.7%, n = 9) of CD4⁺ T cells in the peripheral blood of normal rhesus monkeys coexpressed CD25. In normal monkey lymph node and spleen, 13.7% ± 2.1 and 18.8% ± 5.6 of CD4⁺ T cells were CD25⁺, respectively (n = 3; data not shown). Thus, like mice and humans, normal monkeys have naturally occurring CD4⁺CD25⁺ Tregs. Also, in normal monkeys, the frequency of these CD4⁺CD25⁺ Tregs is significantly higher in secondary lymphoid organs compared with peripheral blood.

View larger version (11K): [in this window] [in a new window]   FIGURE 4. The frequency of CD4+CD25+ T cells is elevated in long-term tolerant NHP induced with peritransplant anti-CD3 IT plus DSG therapy. PBMC from the peripheral blood of normal and long-term tolerant monkeys were stained with mAbs specific for CD4, CD25, and intracellular IL-10 or V24 and 6B11. Stained samples were analyzed as described in Fig. 3, and the frequency of CD4+CD25+ (A), IL-10+CD4+CD25+ (B), and V24+611+ (C) NKT cells in the lymphocyte gate was determined. These data are representative of 3 independent experiments performed.

Rhesus CD4⁺CD25⁺ T cells exhibit a memory phenotype

To further characterize the phenotype of rhesus peripheral blood CD4⁺CD25⁺ T cells, we used four-color flow cytometry. The representative data in Fig. 5, A and B, show that the majority of CD4⁺CD25⁺ T cells in the peripheral blood are negative for CD69, and therefore are not recently activated T cells. In contrast, most of the CD4⁺CD25⁺ T cells coexpressed CD95 (Fig. 5C), indicating a memory phenotype (41). Also, a majority of the CD4⁺CD25⁺ T cells exhibited a CD28⁺ phenotype (Fig. 5D), suggesting they are central memory cells. Consistent with this finding, real-time quantitative PCR analysis demonstrated 3-fold higher CCR7 expression in CD4⁺CD25⁺ T cells than CD4⁺CD25^– T cells (data not shown). Therefore, in line with murine and human CD4⁺CD25⁺ Tregs (42, 43), rhesus CD4⁺CD25⁺ T cells exhibit a heterogeneous phenotype in which central memory cells are prominent.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Rhesus CD4+CD25+ T cells display a memory phenotype. PBMC were stained for CD4 and CD25 in combination with CD69 or CD95 or CD28. Gated CD4+CD25+ T cells (A) were analyzed for coexpression of CD69 (B) or CD95 (C) or CD28 (D). Similar results were obtained when this experiment was performed in >8 tolerant animals and repeated several times in the same animal.

Foxp3 is expressed by CD4⁺CD25⁺ Tregs in mice and humans (15, 16, 44). Thus, we examined the relative expression of Foxp3 mRNA by rhesus CD4⁺CD25^– and CD4⁺CD25⁺ T cells. Our results demonstrate that freshly isolated CD4⁺CD25⁺ T cells from long-term tolerant rhesus recipients and normal cohorts expressed high levels of Foxp3 mRNA. In contrast, freshly isolated CD4⁺CD25^– T, CD8⁺, CD14⁺, and CD20⁺ cells expressed low levels of Foxp3 message that were barely detectable (Fig. 6, A and B). Although the frequency of CD4⁺CD25⁺ T cells was significantly elevated in tolerant rhesus recipients compared with normal monkeys, there was no significant difference in the relative Foxp3 mRNA levels within the CD4⁺CD25⁺ T cell population from tolerant and normal monkeys (Fig. 6A). Thus, Foxp3, per se, is not up-regulated in tolerant recipients.

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Freshly isolated rhesus peripheral blood CD4+CD25+ T cells differentially express high levels of Foxp3 and CTLA-4 mRNA. Real-time quantitative PCR was used to analyze Foxp3 and CTLA-4 mRNA expression in CD4+CD25– and CD4+CD25+ T cells isolated from normal (n = 3) and long-term tolerant (n = 3) NHP. Foxp3 (A and B) and CTLA-4 (C) mRNA levels were normalized to pyruvate dehydrogenase. The data shown here are representative of >10 experiments that were performed.

Rhesus CD4⁺CD25⁺ T cells exhibit regulatory activity

Following TCR-mediated stimulation in vitro, rodent and human CD4⁺CD25⁺ Tregs are anergic and suppress proliferation of CD4⁺CD25^– T cells (42). We examined rhesus CD4⁺CD25⁺ T cells isolated from normal and tolerant monkeys for evidence of regulatory activity in vitro. In contrast to the strong proliferation of CD4⁺CD25^– T cells observed after TCR-mediated polyclonal stimulation, expansion of rhesus CD4⁺CD25⁺ T cells was reduced, a mean 52% of that of the CD4⁺CD25^– T cells (range 21–79% in 6 of 6 experiments). The representative experiments in Fig. 7, A and B, show this trend and also demonstrate that coculturing CD4⁺CD25⁺ T cells with autologous CD4⁺CD25^– T cells suppressed proliferation of the latter (range 43–65% suppression in 6 of 6 experiments). Overall, there was no significant difference in the regulatory activity of normal and tolerant monkeys’ CD4⁺CD25⁺ Tregs. These data confirm that rhesus macaque CD4⁺CD25⁺ T cells exhibit regulatory activity in vitro.

View larger version (15K): [in this window] [in a new window]   FIGURE 7. Rhesus CD4+CD25+ T cells display regulatory activity in vitro. CD4 subsets isolated from a normal monkey (A) or a long-term tolerant recipient (B) were stimulated with plate-bound anti-CD3 FN18 and soluble anti-CD28 mAbs. Proliferation was quantified by [3H]thymidine incorporation. A, * and **, Denote p = 0.05 and p = 0.01, respectively. B, * and **, Denote p = 0.002. C, Monocyte-derived DC were pulsed with cell-free autologous or donor Ags before combining with self CD4+ or CD4+CD25– (depleted of CD4+CD25+ cells) at a DC:T cells ratio of 1:5. Proliferation was assessed by thymidine uptake after 5 days.

We demonstrated previously that long-term NHP tolerance induced by peritransplant anti-CD3 IT plus DSG therapy is associated with unresponsiveness to donor Ags presented by the indirect pathway. In contrast, T cells from tolerant recipients exhibited robust responses to third party Ags presented by the indirect pathway, as well as to donor Ags presented by the direct pathway (31). In this study, we compared the ability of tolerant recipients’ total CD4⁺ or CD4⁺CD25^– T cells to respond to cell-free donor Ags presented by autologous monocyte-derived DC. The representative data in Fig. 7C demonstrate that the response of total CD4⁺ T cells to autologous and donor cell-free Ags is similar. In contrast, depletion of CD4⁺CD25⁺ Tregs resulted in significantly greater proliferation of CD4⁺CD25^– effector cells to cell-free donor Ags relative to autologous Ags (p = 0.005). These results suggest that CD4⁺CD25⁺ Tregs may contribute to the suppression of indirect pathway responses to cell-free donor Ags.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Our previous studies demonstrated that brief peritransplant treatment of rhesus macaque recipients with two doses of anti-CD3 IT and a 14-day course of DSG resulted in stable, chronic rejection-free tolerance to MHC-mismatched kidney and islet allografts persisting for >5 years in many animals. Long-term tolerance in this clinically relevant NHP model associates strongly with early and sustained increases in serum IL-10 levels (33, 34). The specificity of tolerance has been demonstrated, with tolerant recipients accepting a second kidney allograft from the original donor without additional immunosuppressive therapy, while rejecting third party grafts in normal first set time. Of note, an upsurge in the elevated serum IL-10 levels occurred following transplantation of a second graft from the original donor, putatively enhancing an active IL-10-mediated immunoregulatory mechanism (24, 25, 31). These in vivo findings implicate active immunoregulation in the maintenance of long-term rejection-free tolerance in this NHP model.

The data presented in this work extend our previous findings. We have shown that serum IL-10 levels remain elevated in long-term tolerant recipients >5 years posttransplantation. In addition, at the cellular level, resting and activated PBMC from tolerant monkeys are high IL-10 secretors and are biased to secrete higher levels of IL-10 than TNF-, compared with cells from normal animals. Thus, our in vitro data are in agreement with the in vivo finding that stable, drug-free tolerance associates with high levels of IL-10 and low/undetectable proinflammatory cytokine levels in the serum (33, 34). Also, we reported previously that durable tolerance is associated with donor-specific indirect pathway unresponsiveness and absence of donor microchimerism (31, 36). Without chimerism or chronic immunosuppressive therapy, other mechanisms must account for the enduring lack of rejection of highly incompatible grafts. We have postulated that stable tolerance develops in these outbred NHP when, following anti-CD3 IT therapy, T cell repopulation proceeds in a peritransplant environment in which innate immunity is dampened (34) and donor Ags are processed by host myeloid DC, whose maturation and survival are compromised by DSG (Ref.35 ; J. Wu, J. He, F. T. Thomas, X. L. Jiang, and J. M. Thomas, submitted for publication). This scenario, wherein systemic levels of proinflammatory cytokines are negligible, while TGF- (46) and IL-10 are heightened (33, 34), mimics the milieu favoring expansion of donor Ag-specific regulatory T cell populations (31, 47).

The data show that CD4⁺CD25⁺CD69^– T cells with in vitro regulatory function are present in the peripheral blood, and to a greater extent in the secondary lymphoid organs, of normal and tolerant rhesus macaques. The fact that these cells do not express the early activation marker CD69 suggests that they are bona fide CD4⁺CD25⁺ Tregs (48). Similar to corresponding cells in rodents and humans, freshly obtained rhesus CD4⁺CD25⁺ Tregs have a memory phenotype and express high levels of Foxp3 and CTLA-4 compared with CD4⁺CD25^– T cells (1). To our knowledge, this is the first report to document phenotypic and functional characteristics of NHP CD4⁺CD25⁺ Tregs.

We have demonstrated that Tr1/IL-10 and CD4⁺CD25⁺ Tregs are expanded in the blood of drug-free, long-term tolerant NHP. It is possible that the IL-10⁺CD4⁺CD25⁺ subset are Tr1/IL-10 Tregs because others have noted phenotypic and functional overlap between Tr1/IL-10 and CD4⁺CD25⁺ Tregs (49, 50, 51). Conceivably, tolerogenic DC induced by this brief peritransplant treatment may in turn induce the generation of Tregs in tolerant recipients. Experiments are underway to examine the frequency of plasmacytoid DC and expression of ILT3/ILT4 by APC in normal and tolerant rhesus macaques.

The most notable finding in this study is that peripheral blood of long-term tolerant rhesus recipients contains an elevated frequency of Tr1/IL-10 and CD4⁺CD25⁺ Tregs relative to normal animals. Examination of our most recent tolerant islet recipients (up to 12 mo posttransplantation) indicates that the elevation in CD4⁺CD25⁺ Treg frequency is observed as early as 2 mo posttransplantation and is sustained thereafter. In contrast, animals that reject their allografts exhibit a short-lived increase in CD4⁺CD25⁺ T cells within the first month, followed by rapid decline toward pretransplant levels (data not shown). Thus, the data suggest that adaptations leading to expansion of Tregs may be involved in the genesis and maintenance of the durable tolerance induced by anti-CD3 IT plus DSG therapy.

Tregs have been demonstrated to prevent allograft rejection and to mediate transplantation tolerance in experimental models (6, 47, 52). Information on the role of NHP Tregs in transplantation is scant, although there is growing information on the role of Tregs in human transplantation. Meloni et al. (24) reported recently that the frequency of peripheral blood CD4⁺CD25⁺ Tregs in lung transplant recipients in stable condition was similar to those in normal subjects. In contrast, CD4⁺CD25⁺ Treg frequency was significantly lower in lung transplant recipients experiencing chronic rejection compared with normal individuals, suggesting that maintenance of this population in recipients with chronic immunosuppressive therapy is an important variable for successful graft outcome. In our studies, the frequency of CD4⁺CD25⁺ and Tr1/IL-10 Tregs was 3- and 15-fold higher in tolerant recipients than normal monkeys, respectively. The difference between our results in NHP and those obtained in the clinical study may be due to the fact that the lung transplant recipients were on immunosuppressive drugs, while tolerant monkeys did not receive immunosuppressive therapy after the initial 2-wk induction period. This viewpoint is bolstered by the report of Li et al. (53), showing an increased frequency of CD4⁺CD25^high cells in the peripheral blood of liver transplant patients who maintained graft function after discontinuance of immunosuppressive therapy. Unlike the tolerant human recipients, our tolerant monkeys showed a significant elevation in CD4⁺CD25⁺ Tregs regardless of the intensity of CD25⁺ expression. Additionally, the monkeys showed a greater difference in Treg frequency between tolerant recipients and normal cohorts than did those in the human study. The relatively greater increase in frequency in our tolerant monkeys might be related to the longer duration of immunosuppressive drug therapy (1–6 years in humans vs 2 wk in NHP). Also, the rhesus MHC is more complicated than the human MHC, the former having a far greater number of MHC genes per locus (54). The recipient/donor pairs used in our study had multiple MHC mismatches, as defined by rhesus-specific PCR sequence-specific primers for class II DR loci (31, 55), class IA (56), and the class IB locus (our unpublished data). Consequently, the extent of mismatches in our recipient/donor pairs generally exceed those in clinical transplantation and may be part of the reason Tregs frequencies are elevated in tolerant rhesus recipients.

Relevant to our findings, 50% of human kidney allograft recipients with indirect pathway donor-specific hyporesponsiveness exhibited increased responses against donor immunogenic peptides after depletion of CD4⁺CD25⁺ T cells (23). Stable tolerance in our preclinical model is associated with donor-specific indirect pathway unresponsiveness (31). In this study, we have demonstrated that long-term tolerance also associates with elevated frequencies of Tr1/IL-10 and CD4⁺CD25⁺ Tregs. Furthermore, depletion of CD4⁺CD25⁺ Tregs alleviated suppression of indirect pathway antidonor responses. Frozen normal, pretransplant cells from the long-term tolerant rhesus macaques are no longer available, so we are unable to directly test for suppression of indirect pathway antidonor responses with isolated subsets. Nevertheless, our data suggest that donor-specific CD4 regulatory cells promote allograft survival and tolerance by regulating antidonor indirect pathway responses.

Intragraft CD4⁺TGF-1⁺ regulatory T cells were demonstrated in NHP kidney allograft recipients with metastable tolerance following withdrawal of prolonged immunosuppressive therapy (57). Breakdown of tolerance was preceded by disappearance of these from graft biopsies. In contrast, stable, rejection-free tolerance induced in NHP following brief therapy with anti-CD3 IT plus DSG is associated with the absence of TGF-1 from graft biopsies (32). Thus, the mechanisms underpinning long-term durable NHP tolerance (32, 34, 46) appear to be distinct from NHP metastable tolerance with approaching chronic rejection (57, 58, 59).

	Acknowledgments

 
We are indebted to Jennifer McCarn and Celina Rodriguez for sample collection and excellent animal care. Albert J. Savage provided valuable technical assistance.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
J. M. Thomas has a CRADA (cooperative research and development agreement) with Novartis/National Institutes of Health/University of Alabama.

	Footnotes

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

¹ This work was supported by National Institutes of Health Grants 2 U19 DK057958 and U19 AI056542-02 and Juvenile Diabetes Research Foundation Grant 4-2004-364.

² Address correspondence and reprint requests to Dr. Judith M. Thomas, Department of Surgery, Division of Transplant Immunobiology, University of Alabama, Birmingham, AL 35294.

³ Abbreviations used in this paper: Treg, regulatory T cell; Tr1, type 1 regulatory cells; DC, dendritic cell; DSG, 15-deoxyspergualin; IT, immunotoxin; NHP, nonhuman primate; PDH, pyruvate dehydrogenase.

Received for publication December 28, 2004. Accepted for publication October 10, 2005.

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