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Promotion of Allograft Survival by CD4⁺CD25⁺ Regulatory T Cells: Evidence for In Vivo Inhibition of Effector Cell Proliferation¹

Major K. Lee, IV^2,*, Daniel J. Moore^2,*, Beth P. Jarrett^*, Moh Moh Lian^*, Shaoping Deng^*, Xiaolun Huang^*, Joseph W. Markmann^*, Meredith Chiaccio^*, Clyde F. Barker^*, Andrew J. Caton and James F. Markmann^3,*

^* Harrison Department of Surgical Research, Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA 19104; and Wistar Institute, Philadelphia, PA 19104

	Abstract

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Regulatory T cells preserve tolerance to peripheral self-Ags and may control the response to allogeneic tissues to promote transplantation tolerance. Although prior studies have demonstrated prolonged allograft survival in the presence of regulatory T cells (T-reg), data documenting the capacity of these cells to promote tolerance in immunocompetent transplant models are lacking, and the mechanism of suppression in vivo remains unclear. We used a TCR transgenic model of allograft rejection to characterize the in vivo activity of CD4⁺CD25⁺ T-reg. We demonstrate that graft Ag-specific T-reg effectively intercede in the rejection response of naive T cells to established skin allografts. Furthermore, CFSE labeling demonstrates impaired proliferation of naive graft Ag-specific T cells in the draining lymph node in the presence of T-reg. These results confirm the efficacy of T-reg in promoting graft survival and suggest that their suppressive action is accomplished in part through inhibition of proliferation.

	Introduction

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Currently, organ transplantation represents the only curative therapy for a diverse range of devastating disease processes. However, the vigorous immune response to MHC-mismatched allografts necessitates adjunct immunosuppressive therapy to prevent acute rejection. Although modern immunosuppressive agents are efficacious in countering acute rejection, lifelong administration carries considerable risk of morbidity and mortality. Ideally, the use of chronic immunosuppression could be avoided altogether by induction of donor-specific tolerance.

Transplantation tolerance has been achieved in animal models through various strategies. The tolerant state has been attributed to a variety of mechanisms, including deletion, anergy, ignorance, and T cell-mediated suppression. Recently, characterization of suppressor/regulatory T cells (T-reg) has focused on the T cell population coexpressing the CD4 and CD25 surface Ags. CD4⁺CD25⁺ T cells are a naturally occurring, anergic, suppressive T cell population that promotes tolerance to self and foreign Ags (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). The capacity of these cells to induce tolerance to alloantigens suggested potential application in transplantation. A number of studies have demonstrated that CD4⁺CD25⁺ T cells are capable of prolonging allograft survival (1, 12, 13, 14, 15). However, interpretation of many of these data has been limited by 1) the use of immunodeficient hosts in which the properties of transferred regulatory and/or effector cells may be altered, 2) the uncharacterized specificity of the regulatory population, and 3) the inability to visualize the effects of the regulatory population on graft-reactive lymphocytes.

In this study we evaluated the capacity of graft Ag-specific CD4⁺CD25⁺ T cells to inhibit allograft rejection in immunocompetent hosts. We found that skin allograft rejection was associated with a robust in vivo proliferative response of graft-reactive T cells that was localized to the graft’s draining lymph nodes (LN).⁴ CD4⁺CD25⁺ T cells both prolonged graft survival and markedly contracted proliferation of the graft-reactive population. In light of findings correlating the extent of proliferation with the acquisition of effector function (16, 17), we suggest that inhibition of the proliferative response of graft-reactive T cells to donor Ags is in large part responsible for prolonged graft survival in vivo. Indefinite graft survival was reversed in some, but not all, cases by administration of neutralizing Abs to CTLA-4, IL-10, or TGF-, implicating these factors in regulatory activity in vivo. We conclude that CD4⁺CD25⁺ T cells inhibit the proliferation of graft-reactive cells in vivo, and this inhibition is associated with prolongation of graft survival.

	Materials and Methods

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Animals

TS1 transgenic mice possess a high frequency of CD4⁺ T cells specific for the immunodominant (site 1) epitope of the influenza hemagglutinin (HA) protein in the context of MHC class II I-E^d (18). HA104 mice provide a source of HA-expressing grafts, as they carry the HA transgene controlled by the SV40 early region promoter/enhancer (19), which results in ubiquitous transgene expression (20). (TS1 x HA28)F₁ mice were created and described by Caton et al. (6). TS1, HA28, and HA104 transgenic lines were maintained as hemizygotes backcrossed with BALB/c mice (The Jackson Laboratory, Bar Harbor, ME). All animals were maintained in a pathogen-free environment in the University of Pennsylvania animal facility under institutional animal care and use committee-approved protocols.

CFSE labeling

LNs were harvested, and single-cell suspensions were prepared by passage of tissue through a cell strainer (70 µm pore size; Falcon; BD Biosciences, Franklin Lakes, NJ). Cells were resuspended at a density of 1 x 10⁷ cells/ml in IMDM. An equal volume of 5 mM CFSE (Molecular Probes, Eugene, OR) in IMDM was added, and cells were incubated at 37°C for 5 min. The reaction was quenched through the addition of an equal volume of heat-inactivated FCS (Life Technologies). Labeled cells were washed twice and resuspended in PBS for i.v. injection or culture medium (IMDM and 10% FCS) for in vitro stimulations.

Flow cytometric analysis

LN single-cell suspensions were prepared in FACS buffer, and anti-CD4-allophycocyanin and anti-CD25-PE (BD PharMingen, San Diego, CA) Abs were used for analysis. In addition, 6.5-biotin (18) and strepavidin-Red670 (Life Technologies) were used to detect the transgenic TCR. Flow cytometric analysis was performed on a FACSCalibur cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA). Data processing was accomplished with CellQuest software (BD Biosciences).

FACS purification of cell populations

Cells were sorted on a BD FACSVantage SE (BD Biosciences) high speed cell sorter. The dual laser Vantage is equipped with a 5-W argon laser (Innova 305; Coherent, Santa Clara, CA) and mixed gas argon-krypton (Spectrum; Coherent) lasers. Cells were stained with anti-CD25-FITC, anti-CD45RB-PE, and anti-CD4-allophycocyanin. Sorted populations were gated on CD4⁺, CD25^–, and CD45RB^bright or CD4⁺, CD25⁺, and CD45RB^int. Forward scatter pulse width was used as an additional gated parameter to exclude cell aggregates. Purity checks on the sorted populations ranged from 95 to 98%.

Skin grafting

Skin grafts were transplanted to mice according to the technique described by Billingham and Medawar (21). Mice were anesthetized, and skin grafts were placed in a lateral thoracic position and stabilized with Vaseline gauze (Kendall, Mansfield, MA) and bandaids. The bandages were removed on the 10th day. Grafts were scored as rejected when >75% of the grafted tissue area had been lost.

	Results

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S1-specific T cells transferred to BALB/c mice mediate rejection of HA-expressing skin grafts

We previously reported that TS1 hosts expressing a transgene-encoded HA-specific TCR consistently reject HA-expressing skin and heart allografts (15, 22). BALB/c hosts do not reject these transplants, confirming that the HA-specific TCR is necessary for graft rejection. Because the transgenic environment consists of a supranormal frequency of Ag-reactive lymphocytes, we characterized the rejection of HA104 skin transplants after adoptive transfer of TS1 lymphocytes to BALB/c hosts. We found that BALB/c hosts receiving 5 x 10⁵ unfractionated TS1 LN cells consistently rejected established (grafted >30 days before transplant) HA104 skin grafts (Fig. 1; mean survival time (MST), 19.8 days). Prior experimentation excluded contribution of conventional minor antigenic disparities in rejection, and these experiments collectively confirm a requirement for both transgenic (6.5⁺) TS1 T cells and expression of the HA transgene in this rejection process (15).

View larger version (12K): [in this window] [in a new window]   FIGURE 1. Rejection of established HA104 skin transplants by adoptively transferred TS1 lymphocytes. BALB/c mice were transplanted with skin grafts harvested from HA104 mice, and these grafts were allowed at least 30 days to establish. For adoptive transfer, LNs were harvested from TS1 mice, single-cell suspensions were prepared, and 5 x 105 cells were transferred by tail vein injection into BALB/c mice bearing established HA104 skin grafts. Graft rejection by untreated BALB/c hosts (n = 5; MST, >200 days) and that by hosts receiving TS1 cells (n = 6; MST, among five rejectors, 19.8 days) are shown. Data are pooled from two separate experiments.

The adoptive transfer model is advantageous for the study of T-reg in transplantation because of the immunocompetent host and the specificity of the rejection process. However, investigating the effects of T-reg in the model requires large numbers of S1-specific CD4⁺CD25⁺ T cells. To obtain this regulatory population, we used LN cells from (TS1 x HA28)F₁ mice, which were previously characterized by Jordan et al. (6, 23). In this mouse, 50% of peripheral HA-specific (6.5⁺) CD4 T cells possess the cell surface phenotype CD25⁺CD45RB^int. In prior studies these cells were shown to inhibit the proliferation of cocultured CD4⁺CD25^– T cells in thymidine incorporation assays (6). These studies cannot, however, differentiate extensive division among a small fraction of naive cells from a small degree of proliferation across the entire population. To discriminate these alternatives, we used CFSE labeling to examine the effects of the regulatory population on individual T cells within the naive population. We found that CD25⁺CD45RB^int T cells proliferated poorly in culture in response to S1 peptide stimulation compared with CD25^–CD45RB^high cells (Fig. 2, A and B). Furthermore, the CD25⁺CD45RB^int population suppressed proliferation of CD25^–CD45RB^high cells in cocultures, with very few cells extending beyond 3–4 divisions (Fig. 2C). These results verify that CD4⁺CD25⁺ T cells from (TS1 x HA28)F₁ mice are anergic and suppressive and indicate that CD4⁺CD25⁺ cells limit proliferation across the entire naive cell population to three or four divisions. The regulatory population mimics previously characterized regulatory CD4⁺CD25⁺ T cells in this regard.

View larger version (55K): [in this window] [in a new window]   FIGURE 2. CD4+CD25+ T cells from (TS1 x HA28)F1 mice mimic previously characterized regulatory populations. A, CFSE-labeled CD4+CD25+ T cells (5 x 105) sorted from (TS1 x HA28)F1 mice were stimulated in the presence of 2.5 x 106 BALB/c APCs and 1 µM S1 peptide. Peptide-specific proliferation was detected using the anti-clonotypic Ab, 6.5. B, Proliferation of 5 x 105 CFSE-labeled CD4+CD25– T cells sorted from (TS1 x HA28)F1 mice in the presence of 2.5 x 106 BALB/c APCs and 1 µM S1 peptide. C, Proliferation of 2.5 x 105 CD4+CD25+ and 2.5 x 105 CD4+CD25– T cells sorted from (TS1 x HA28)F1 mice in the presence of 2.5 x 106 BALB/c APCs and 1 µM S1 peptide. D–F, Expression of CD103 (E7 integrin; D; n = 2), CTLA-4 (E; n = 3), and GITR (F; n = 2) on (TS1 x HA28)F1 LN cells.

Graft Ag-specific CD4⁺CD25⁺ T cells from (TS1 x HA28)F₁ mice suppress rejection of established HA-expressing skin allografts

Several reports have implicated CD4⁺CD25⁺ T cells in regulation of the immune response to skin allografts (1, 12, 13, 14, 15). To extend these studies we investigated the effects of Ag-specific CD4⁺CD25⁺ T cells on allograft survival in the immunocompetent adoptive transfer model. We compared rejection of established HA104 skin allografts by BALB/c hosts receiving TS1 vs (TS1 x HA28)F₁ LN cells to evaluate the rejection response of graft-reactive lymphocytes in the presence or the absence of a high frequency (50%) of Ag-specific T-reg. The use of established allografts in these experiments avoids inflammatory factors associated with acute skin grafts that may alter the activity of CD4⁺CD25⁺ T cells (28, 29, 30). As shown in Fig. 1, transfer of 5 x 10⁵ unfractionated TS1 LN cells resulted in consistent rejection of established HA104 skin grafts (MST, 19.8 days). In contrast, (TS1 x HA28)F₁ LN cells did not reject established HA104 allografts (Fig. 3). In fact, in 10/10 BALB/c hosts receiving (TS1 x HA28)F₁ LN cells, transplanted HA104 grafts survived indefinitely (p < 0.01 compared with TS1 cells).

View larger version (12K): [in this window] [in a new window]   FIGURE 3. HA-specific CD4+CD25+ T cells prolong the survival of established HA104 skin grafts. (TS1 x HA28)F1 cells (n = 10), CD4+CD25+CD45RBint cells sorted from (TS1 x HA28)F1 mice (n = 7; survival times, 40 and 59 days for rejectors), or CD4+CD25–CD45RBhigh cells sorted from (TS1 x HA28)F1 mice (n = 6; MST, 37.4 days among rejectors) were transferred to BALB/c mice bearing established HA104 skin transplants. Recipients were examined regularly for graft rejection. Rejection by 5 x 105 TS1 cells from Fig. 1 above is also shown for comparison (n = 6; MST among five rejectors, 19.8 days).

CD4⁺CD25⁺ T cells inhibit in vivo proliferation of HA-specific T cells to established grafts

The fact that CD4⁺CD25⁺ T cells within the (TS1 x HA28)F₁ population prolong the survival of established HA104 allografts led us to characterize the activity of the regulatory population in vivo. As shown in Fig. 2, the CD4⁺CD25⁺ T cell population from (TS1 x HA28)F₁ mice arrested proliferation of the CD25^– fraction in vitro. We therefore theorized that these cells might similarly inhibit HA-specific proliferation in vivo. In prior studies, we observed extensive proliferation of HA-specific (6.5⁺) T cells within the draining LNs to acute HA-expressing skin and heart transplants after adoptive transfer (22). Because established HA104 grafts were consistently rejected by transferred TS1 lymphocytes, we expected a similarly robust proliferative response to these transplants. As shown, transfer of 5 x 10⁵ to 1 x 10⁶ CFSE-labeled TS1 LN cells resulted in an extensive proliferative response to established HA104 skin allografts (Fig. 4A). This response remained predominantly localized to the draining LNs (data not shown).

View larger version (15K): [in this window] [in a new window]   FIGURE 4. HA-specific CD4+CD25+ T cells from (TS1 x HA28)F1 mice inhibit proliferation to established HA104 skin grafts. Unfractionated, CFSE-labeled TS1 LN cells (5 x 105 to 1 x 106; A; n = 6), (TS1 x HA28)F1 LN cells (B; n = 10), CD4+CD25+CD45RBint T cells sorted from (TS1 x HA28)F1 mice (C; n = 3), or CD4+CD25–CD45RBhigh T cells sorted from (TS1 x HA28)F1 mice (D; n = 4) were transferred to BALB/c hosts bearing established HA104 skin transplants. To assess the suppression of naive TS1 cells in vivo, 1 x 106 unlabeled CD4+CD25+CD45RBint T cells sorted from (TS1 x HA28)F1 mice were combined with 1 x 106 CFSE-labeled TS1 LN cells, and the mixed population was transferred to BALB/c hosts bearing established HA104 skin transplants (E; n = 6; data were pooled from two separate experiments). The proliferative response within the draining LNs was in each instance assessed 14 days after cell transfer. All histograms shown are gated on CFSE+, CD4+ T cells expressing the transgenic TCR (6.5+).

CD4⁺CD25⁺ T cells exhibit an aborted proliferative response in vivo and contract proliferation of graft-reactive CD25^– cells

Given the anergic and suppressive nature of the CD4⁺CD25⁺ population, we surmised that the contracted proliferative response of the unfractionated (TS1 x HA28)F₁ population reflected suppression of CD4⁺CD25^– T cells by a nondividing CD4⁺CD25⁺ population. We therefore hypothesized that isolated CD4⁺CD25⁺ T cells would exhibit a limited proliferative response in vivo, and that the proliferative response of CD4⁺CD25^– cells would be restored in the absence of the regulatory population. To test this premise, we transferred 5 x 10⁵ sorted, CFSE-labeled CD4⁺CD25⁺ or CD4⁺CD25^– populations from (TS1 x HA28)F₁ mice into transgene-negative BALB/c mice with established HA104 skin grafts and evaluated the proliferative responses of the two groups.

Our findings parallel the in vitro findings reported in this study and by Caton et al. (6). CD25⁺ T cells exhibited an aborted, yet demonstrable, proliferative response resembling that of the unsorted population (Fig. 4C). In contrast, CD25^– T cells separated from the regulatory population demonstrated a more advanced proliferative response, with a significant fraction of cells advancing into late division peaks (Fig. 4D). The extensive proliferative activity of the sorted CD4⁺CD25^– population correlates with consistent rejection of established HA104 skin grafts (Fig. 3). These data provide further evidence that CD4⁺CD25⁺ T cells are anergic and suppressive in vivo, and in combination with our graft survival data suggest that the proliferative arrest effected by these cells prevents acquisition of effector functions necessary to mediate allograft rejection.

CD4⁺CD25⁺ T cells from (TS1 x HA28)F₁ mice inhibit the proliferation and rejection response of naive T cells

Although our results suggest that CD4⁺CD25⁺ T cells from (TS1 x HA28)F₁ mice inhibit the proliferative and rejection responses of the CD4⁺CD25^– population, it could be argued instead that the prolonged graft survival and contracted T cell proliferation profiles observed upon transfer of (TS1 x HA28)F₁ cells are attributable to poor effector function in the CD4⁺CD25^– T cell fraction that developed in an ubiquitously HA-positive environment (23). In this scenario, both CD25^– and CD25⁺ T cells from (TS1 x HA28)F₁ mice are hyporesponsive to graft Ag due to maturation in the presence of HA. In vitro data indicate that CD4⁺CD25^– cells sorted from (TS1 x HA28)F₁ mice are highly responsive to HA (Fig. 2), but this cannot rule out a hyporesponsive state in vivo. In vivo, this argument is contradicted in part by the fact that the rejection of established grafts is mediated by CD4⁺CD25^– T cells sorted from (TS1 x HA28)F₁ mice (Fig. 3), and that these cells mount an advanced proliferative response in the absence of the regulatory population (Fig. 4D). However, CD4⁺CD25^– T cells from (TS1 x HA28)F₁ mice rejected established HA104 skin less quickly than TS1 cells (Fig. 3; MST, 37.4 for CD4⁺CD25^– from (TS1 x HA28)F₁ mice vs 19.8 for naive TS1 cells), and the proliferative response of these cells was not as profound as that of TS1 cells alone (Fig. 4, A and D). To address this issue more directly, we assessed the capacity of CD25⁺ T-reg to suppress the activity of HA-specific lymphocytes from naive TS1 mice.

For these studies, we combined 1 x 10⁶ purified, unlabeled CD25⁺CD45RB^int cells from (TS1 x HA28)F₁ mice with 1 x 10⁶ CFSE-labeled naive TS1 LN cells. The mixed population was transferred to BALB/c mice with established HA104 skin grafts, and the division profile of draining LN T cells was examined 2 wk later. CFSE-labeled naive TS1 lymphocytes exhibited a contracted proliferative response to established HA104 skin grafts in the presence of the cotransferred CD25⁺ population. Although the majority of TS1 cells were localized to peaks 5–8 of the proliferative profile against established HA104 skin grafts in the absence of the CD25⁺ population (Fig. 4A), in their presence, division was arrested, with the majority of cells remaining undivided, and divided cells remaining in peaks 2–5 (Fig. 4E). This division profile was similar to that observed in transfer of unfractionated (TS1 x HA28)F₁ LN cells (Fig. 4B).

To correlate these findings with graft survival, we combined 5 x 10⁵ CD4⁺CD25⁺ T cells sorted from F₁ mice with 5 x 10⁵ TS1 LN cells and transferred this mixed population to BALB/c mice bearing established HA104 skin grafts. In contrast to hosts receiving TS1 cells alone (Fig. 1), all eight mice receiving cotransferred TS1 and CD4⁺CD25⁺ T cells maintained their transplants indefinitely (all eight grafts survived >120 days). Collectively, these data confirm that CD4⁺CD25⁺ T cells inhibit the proliferation and effector function of graft-reactive T cells.

Involvement of CTLA-4, IL-10, and TGF- in CD4⁺CD25⁺ T cell function in vivo

Previous studies have implicated three factors in the function of CD4⁺CD25⁺ T cells in vivo: CTLA-4, IL-10, and TGF- (reviewed in Ref. 31). However, the involvement of these molecules in suppression has been inconsistent, with a variety of studies showing suppression by the CD25⁺ T cell population in the absence of one or more (31). We thus attempted to determine the involvement of these factors in suppression of the allograft response using this immunocompetent model. We transferred 5 x 10⁵ unfractionated (TS1 x HA28)F₁ lymphocytes into BALB/c hosts with established skin grafts and treated recipients with neutralizing Abs to CTLA-4, IL-10, or TGF- (0.5 mg on days 0, 4, and 10, then once weekly after cell transfer). As shown previously, hosts receiving (TS1 x HA28)F₁ lymphocytes alone do not reject established HA104 skin grafts (Fig. 3). In contrast, hosts receiving neutralizing Abs to CTLA-4, IL-10, or TGF- in addition to (TS1 x HA28)F₁ lymphocytes rejected their established skin allografts in a significant proportion of instances in each group (Fig. 5). In all treatment groups, however, a significant number of allografts survived indefinitely. This may signify incomplete blocking by the regimen used or that suppression can remain operative in the absence of these factors. In addition, as effector cells and regulatory cells in this model compete for the same ligand, it may be impossible to completely overcome the effect of the regulatory cells by the administration of these Abs. That is, although these blocking Abs may prevent CD4⁺CD25⁺ cells from regulating potential effectors, these same effectors may still not have access to their complementary ligand. That our results are reflective of a suboptimal dose of Ab administered is unlikely, because these results have been observed at higher Ab doses as well (data not shown).

View larger version (10K): [in this window] [in a new window]   FIGURE 5. Abs to CTLA-4, IL-10, and TGF- restore rejection of established skin grafts by (TS1 x HA28)F1 lymphocytes in some instances. LN cells (5 x 105) were harvested from (TS1 x HA28)F1 mice and adoptively transferred to BALB/c mice bearing established HA104 skin grafts. Recipient mice received 0.5 mg of anti-CTLA-4 (n = 7), anti-IL-10 (n = 7), or anti-TGF- (n = 5) by i.p. injection on days 0, 4, and 10, then once weekly after cell transfer. Grafts were examined regularly for rejection. Four untreated mice are included for comparison and represent a subset of the 10 (TS1 x HA28)F1 cell recipients from Fig. 3 that received cells concurrent with these treatment groups. Data are pooled from two separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

That tolerant lymphocytes contract the proliferation of graft-reactive T cells to some extent in vivo has been suggested previously in an islet allograft model (32). The use of an immunocompetent host and an Ag-specific population in our study may explain the clearer effects of the regulatory population on the proliferation of naive cells and the fact that the survival benefit observed was indefinite rather than only slightly prolonged.

Although the data presented in this study suggest that CD4⁺CD25⁺ T cells function to contract the proliferative response of Ag-reactive cells in vivo, this population may have additional effects. Data in several studies indicate that CD4⁺ regulatory cells exert their suppressive capacity in the absence of any demonstrable effect on proliferation (33, 34). McHugh et al. (33) showed that CD4⁺CD25⁺ T cells can inhibit development of gastritis in BALB/c nu/nu mice receiving CD4⁺CD25^– T cells without affecting the proliferation of these cells. Because the specificity of the CD25^– population was not certain, it is possible that the proliferation of that subset of CD25^– cells that actually promotes gastritis in the host was diminished, but extensive proliferation among nonreactive cells obscured this effect. Perhaps more relevant are the findings of Lin et al. (34) in a transplant system. This study showed that CD8⁺ T cells transferred into a tolerant host proliferated significantly, but that effector functions among the population (graft rejection, IFN- production, and CTL activity) were reduced. Regulatory T cells had previously been shown to sponsor tolerance in this setting (reviewed in Ref. 35). Although this study best examines the effects of CD4 T-reg (not specifically CD4⁺CD25⁺ T cells) on CD8⁺ T cells, collectively these data and ours suggest that T-reg may act to inhibit the proliferation and effector function of graft-reactive T cells independently. In our study as both proliferation and effector function were inhibited, we cannot draw specific conclusions regarding the direct effect of regulatory cells on effector function.

Our data documenting a clear and reproducible pattern of three to five divisions executed by the CD4⁺CD25⁺ population raises several possibilities, including that 1) in vivo suppression mediated by CD4⁺CD25⁺ T cells requires a limited, but measurable, proliferative response; and 2) the CD4⁺CD25⁺ population is heterogeneous, with some cells more capable of division than others. The data presented in this study cannot discriminate between these possibilities, but other studies have indicated that the suppressive properties of these cells may extend with division (36, 37, 38). More extensive proliferation of CD4⁺CD25⁺ T cells than that observed here has been demonstrated in recent studies. Annacker et al. (39) found that at least a fraction of CD4⁺CD25⁺ T cells undergo five or more divisions after transfer into immunodeficient mice, but that these cells maintain a small pool size at their homeostatic equilibrium. The proliferative response observed in our study was less extensive; this may result because the immunocompetent host contains a pool of pre-existing CD4⁺CD25⁺ T cells. Robust proliferation of CD4⁺CD25⁺ T cells was also demonstrated by Walker et al. (40) in a transplantation model. This group found that the proliferative response of Ag-specific CD4⁺CD25⁺ T cells to cognate Ag and IFA was nearly identical with that of CD25^– cells, but observed a far less extensive proliferative response to cognate Ag transgenically expressed on pancreatic cells in the absence of adjuvant. These data in combination with our own raise the possibility that the proliferation of regulatory CD4⁺CD25⁺ T cells may be linked to the context in which their Ag is presented; the combination of Ag and adjuvant may engender maximal proliferation because of the use of adjuvant. Transplantation of skin grafts expressing cognate Ag also generates a nonspecific inflammatory response, perhaps explaining the proliferative response observed among CD4⁺CD25⁺ T cells in our study. This activation of the immune response probably does not occur in the case of transgene-encoded Ag expression on pancreatic cells, which may explain why even fewer division cycles occur in this instance. These results may suggest that the proliferative response of regulatory cells is tied to the strength of the immune response/inflammation in which their Ag is detected.

The transfer model used in this study can also be adapted to investigate the function of T-reg cells in a memory response, which may be more relevant to the clinical situation in which Ag experienced T cells are likely to participate in the rejection response. We have previously demonstrated immunologic memory in rejection of HA104 transplants by TS1 mice (22). In preliminary studies we have found that CD4⁺CD25⁺ T cells are able to inhibit the proliferation of graft-reactive memory cells in vivo, but that suppression may be less extensive than with the primary response (data not shown).

Our results are also of interest in considering the anatomic location at which suppression of the allograft response occurs by T-reg. Recent data have indicated that T-reg accumulate within long term accepted grafts (41, 42). However, similar studies seeking the presence of T-reg in the graft early post-transplant have not been reported. In contrast, a convincing body of literature indicates that the draining LN is the critical site of immune sensitization in initiation of an anti-skin graft response (43, 44, 45, 46). In this context, it is not surprising then that the draining LN would provide an effective site for early intercession in the anti-graft response by CD4⁺CD25⁺ T-reg. Although our data cannot exclude the possibility that T-reg act simultaneously within the graft, the contraction of the proliferative response of naive graft reactive T cells in the LN suggests that this location is an important site of T-reg activity.

In summary, we demonstrate that regulatory CD4⁺CD25⁺ T cells contract the proliferative response of graft-reactive cells in vivo in a manner similar to that previously reported for in vitro studies. We suggest that this contracted proliferative response precludes the development of the T cell effector function required for graft rejection. Although the consistent involvement of particular factors in the function of the regulatory population remains unclear, these findings provide a mechanistic basis for the in vivo function of T-reg.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant RO1AI048820.

² M.K.L. and D.J.M. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. James F. Markmann, Department of Surgery, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104. E-mail address: james.markmann{at}uphs.upenn.edu

⁴ Abbreviations used in this paper: LN, lymph node; GITR, glucocorticoid inhibitory TNF receptor; HA, hemagglutinin; MST, mean survival time; T-reg, regulatory T cell.

Received for publication October 10, 2003. Accepted for publication March 16, 2004.

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