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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2007 May 9;104(20):8415–8420. doi: 10.1073/pnas.0701031104

Low-intensity transplant regimens facilitate recruitment of donor-specific regulatory T cells that promote hematopoietic engraftment

Ling Weng *, Julian Dyson , Francesco Dazzi *,
PMCID: PMC1895964  PMID: 17494756

Abstract

Low- or reduced-intensity conditioning regimens for allogeneic hemopoietic stem cell transplantation are effective at establishing donor hematopoietic engraftment and host-vs.-graft (HvG) tolerance. We investigated the mechanisms of HvG tolerance induction and maintenance in an animal model in which transplantation of sublethally irradiated female recipients with bone marrow (BM) from syngeneic male donors produces mixed chimerism. Splenocytes from chimeric mice inhibited HY-specific CD8+ T cell responses both in vitro and in vivo, and their adoptive transfer facilitated donor hematopoietic engraftment. These properties were contained within the CD4+CD25+ population. The conditioning protocol alone led to a proportional expansion of regulatory T cells (Tregs), but the inhibitory activity was induced only if male BM was infused. The administration of anti-CD25-depleting antibodies to conditioned recipients at time of BM transplantation prevented donor-recipient chimerism but did not affect engraftment if performed after the establishment of chimerism, thus indicating that recipient Tregs are required for the generation but not the maintenance of HvG tolerance. We conclude that donor-specific Tregs of recipient origin are recruited when the donor antigens are present during reduced-intensity conditioning-induced Treg expansion.

Keywords: host vs. graft, reduced-intensity conditioning, stem cell transplantation, tolerance


Allogeneic hemopoietic stem cell transplantation (HSCT) is a well established treatment modality for malignant and nonmalignant diseases (1). The conventional approach to condition the patient with fully myeloablative regimens has recently been reconsidered in light of the therapeutic goals (2) and led to the development of low- or reduced-intensity conditioning (RIC) regimens whereby the role of conditioning is confined to the establishment of mixed hemopoietic chimerism and the consequent host-vs.-graft (HvG) tolerance (3, 4). Evidence exists that such approaches could also be used before solid organ transplantation (5) and to prevent the rejection of genetically modified cells (6).

The coexistence of donor and recipient hematopoiesis after RIC regimens creates durable HvG tolerance with no need for long-term immunosuppression. Despite the importance of central and peripheral deletional tolerance in the establishment of mixed chimerism (7), active regulation has emerged as being central (8, 9). Natural regulatory T (Treg) cells are a subpopulation of thymus-derived CD4+ T cells that constitutively express CD25 (10, 11) and the forkhead box P3 (FoxP3) gene product (12). They play a crucial role in peripheral tolerance, susceptibility to autoimmune disease (13), and tumor immunity (14), as well as in the induction of transplantation tolerance (1518).

Very little is known about the role of Tregs in allogeneic HSCT and to what extent they are affected by immune reconstitution (19) but, in animal models, their adoptive transfer controls graft-vs.-host disease (GvHD) (2024) and induce specific tolerance to bone marrow (BM) allografts (25, 26). How RIC determines HvG tolerance and whether this involves Treg function and specificity are unknown.

The detailed characterization of the HY minor antigen makes it an ideal model to dissect the basic mechanisms in transplantation tolerance. We have previously observed that the administration of nonmyeloablative doses of total body irradiation (TBI) to female recipient mice allows engraftment of male donor hematopoiesis and selectively prevents the generation of HY-specific CD8+ T cells (27). Here we demonstrate that recipient Tregs play a critical role in the establishment of HvG tolerance in this model. Further, expansion of Tregs is shaped by antigen producing a regulatory repertoire that suppresses with high efficiency antidonor immunity and facilitates donor hemopoietic engraftment.

Results

RIC Induces Treg Expansion in an Antigen-Independent Fashion.

We enumerated the proportion of CD4+CD25+FoxP3+ in the peripheral blood of sublethally irradiated (400 cGy) C57BL/6 (B6) female mice transplanted with B6 male BM cells. At 8 weeks after transplant, we observed a significant increment in the percentage of Tregs (Fig. 1A). Serial monitoring of CD4+CD25+ cells revealed that Treg proportional expansion was more evident at 2 weeks (5.47% vs. 3.82%, P < 0.001) and persisted for at least 10 weeks after the transplant (Fig. 1B), thus confirming that low-dose TBI allows selective expansion and/or survival of Treg cells. CD4+FoxP3+ cells paralleled that of CD4+CD25+ at each time point. Treg changes similar to those in the peripheral blood were observed in BM and lymph nodes but not in the liver [see supporting information (SI) Table 1].

Fig. 1.

Fig. 1.

Tregs expand after RIC. Irradiated (400 cGy) female B6 mice received male or female B6Ly5.1 BM cells (5 × 106). (A) Peripheral blood (PB) CD4+CD25+FoxP3+ cells 8 weeks after transplant. PB was obtained from naïve female B6 controls (n = 7), female recipients of female BM (n = 7), or female recipients of male BM (n = 8). (B) CD4+ cells of donor origin (Ly5.1+) (□), total CD4+CD25+ (•), and CD4+FoxP3+ (▴) cells at different time points after transplant. Each point represents the average values in 14 mice. (C) CD4+CD25+ cells in donor or recipient CD4+ cells 5 (n = 8) or 7 (n = 8) weeks after female B6 mice were transplanted with male BM. ∗, P < 0.05; ∗∗∗, P < 0.01.

In peripheral blood, donor CD4+ cells could be detected only 4 weeks after BM transplantation (BMT) (Fig. 1B). Regardless of the magnitude of donor engraftment, the proportion of Tregs in recipient CD4+ cells was higher than the proportion of Tregs in donor CD4+ cells (Fig. 1C). These findings indicate that Treg expansion and/or survival are significantly higher in the recipient pool.

The observed proportional Treg increment could result from antigen-independent homeostatic expansion and/or low sensitivity to radiation (28) but might also involve stimulation by donor antigens. To address this issue, irradiated B6 female mice received female syngeneic BM and were likewise examined for Treg numbers. We observed a similar expansion in the Treg population (CD4+CD25+FoxP3+) between the female–female and the male–female chimeric mice (average of 5.7% and 6%, respectively) (Fig. 1A). Treg cells expanded also in mice that were irradiated but did not receive any BM (not shown). Therefore, nonmyeloablative doses of TBI are sufficient to produce the expansion of recipient Tregs. The transplantation of BM provides a further potential source of Tregs at later stages of hematopoietic recovery but is not required for the initial expansion.

Spleen Cells from Chimeric Mice Inhibit HY-Specific CD8+ T Cells via Tregs.

We asked whether RIC allografting was associated with the generation of suppressor activity. Spleen cells from RIC-BMT chimeric mice were tested in vitro by addition as third-party cells to anti-HY mixed lymphocyte reaction (MLR). Splenocytes from 5-week chimeric mice inhibited the expansion of HYDbUty tetramer+CD8+T cells in a dose-dependent manner. The same magnitude of inhibition was observed when using cells from mice transplanted 15 weeks earlier (Fig. 2A). The inhibitory effect could be detected also in BM and lymph nodes of chimeric mice and was still observable 20 weeks after the transplant (SI Table 2).

Fig. 2.

Fig. 2.

Antigen-specific Tregs are recruited in chimeric mice. (A) Splenocytes from HY-immune female B6 mice were stimulated in vitro with irradiated male spleen cells in the presence or absence (positive control) of splenocytes from chimeric mice 5 (▴) or 15 (■) weeks after BMT or naïve control mice (○). Chimeric mice consisted of female B6 transplanted with BM from male B6. After 5 days, cells were harvested and restimulated with irradiated male cells and rIL-2 (10 units/ml) for 6 days, and DbUty-tetramer+ CD8+ cells were enumerated. Results are representative of seven experiments of identical design. (B) Splenocytes obtained from HY-immune female B6 mice were stimulated in vitro with irradiated male spleen cells in the presence of unfractionated (■), CD4− (•), or CD25-depleted (▴) splenocytes from chimeric mice (6–15 weeks after BMT) as a third party. Splenocytes from naïve mice were used as controls (○). After 6 days, DbUty-tetramer+ CD8+ cells were enumerated, and the percentage of inhibition was expressed as compared with MLR cultures in which no third-party splenocytes were added. Each point represents the average of four experiments of similar design. (C) Splenocytes obtained from HY-immune female B6 mice were stimulated in vitro with irradiated male spleen cells in the presence of chimeric splenocytes from female recipients of male (■) or female (▴) BM as a third party. Splenocytes from naïve mice were used as controls (○). After 6 days, DbUty-tetramer +CD8+ cells were enumerated, and the percentage of inhibition was expressed as compared with MLR cultures in which no third-party splenocytes were added. Each point represents the average of four experiments of similar design. (D) Purified CD4+CD25+ cells (>90%) from male–female or female–female chimeric splenocytes were added as a third party to anti-HY MLR at three different ratios. After 6 days, HY-specific T cells were enumerated by DbUty-tetramer. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.01.

When splenocytes from tolerant animals were depleted of CD4+ or CD25+ cells, neither CD4- nor CD25-depleted splenocytes exhibited a suppressive activity (Fig. 2B). Purified CD4+ T cells consistently exhibited a profound inhibitory effect that was >50% at 1:1 ratio as compared with an average of 35% when unfractionated cells were used at the same ratio (not shown).

The Immunosuppressive Effect Generated by RIC Allografting Is Antigen-Dependent.

We then assessed whether the increased proportion of Tregs generated in the absence of antigen exhibited similar suppressive activity. The effect of splenocytes from irradiated (400 cGy TBI) female mice transplanted with female BM cells on HY-specific MLR was comparable to naïve control cells (Fig. 2C). Therefore, despite similar expansion, only Tregs that develop in the presence of the antigen suppress the proliferation of effector T cells in response to the same antigen. Such a discrepancy could be attributable either to antigen-induced activation of Treg cells and/or an increased number of antigen-specific Tregs. CD4+CD25+ T cells were purified from the spleen of male–female and female–female chimeric mice and compared for their inhibitory activity on HY-specific CD8+ proliferation. The magnitude of inhibition at each of the ratios investigated was significantly superior when Tregs from male–female mice were used as compared with the activity exerted by the Tregs from female–female chimeric splenocytes (Fig. 2D). To exclude that the difference was the result of an impairment of the Tregs generated in the female–female chimeras, we compared the inhibitory effect of CD4+CD25+ T cells purified from the spleen of both types of chimeric mice on nonantigen specific polyclonal T cell stimulation. Tregs of both origins exhibited a similar inhibitory activity on CD3/CD28 induced proliferation (SI Fig. 6). These data confirm that antigen-specific Tregs are more active and/or numerous when the nominal antigen is present during Treg expansion.

The in Vivo Inhibitory Effect Is Mediated by Tregs and Is Antigen-Driven.

The immunosuppressive activity of cells from chimeric mice was also assessed by using an in vivo killing assay in which differentially carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled male and female spleen cells were injected into naïve untreated female mice with or without chimeric splenocytes. In the absence of chimeric splenocytes, female recipient mice rejected male but not female splenocytes. When splenocytes from chimeric mice were added to the inoculum, the rejection of male cells was greatly inhibited (14% male cell rejection vs. 48% in control, P < 0.001) (Fig. 3A). To exclude the possibility that this was due to a higher proportion of male cells in the recipients of chimeric splenocytes, we performed a control experiment in which, instead of the chimeric spleen cells, we used a cell population in which female and male cells obtained from naïve mice were mixed at the same female/male ratio as the chimeric spleen. Because no significant inhibition was detected in this control, we could rule out the contribution of the antigen dose.

Fig. 3.

Fig. 3.

Chimeric splenocytes inhibit the in vivo rejection of donor cells. Equal numbers of male and female spleen cells from naïve mice labeled with different concentrations of CFSE were injected i.v. into female B6 recipients with or without (Control) the following third-party cells. (A) Splenocytes from male–female chimeric mice, a mixture of splenocytes from male and female naïve animals comparable to the proportion of male and female in the chimeric mice (M/F). ∗∗∗, P < 0.01. (B) Splenocytes from female–female (F-chim) or male–female (M-chim) chimeric mice. Results are expressed as the percentage of rejection of male splenocytes (see Materials and Methods). Five to six mice were used per group, and rejection was evaluated 7–10 days after inoculum. ∗∗∗, P < 0.01.

The removal of CD4+ or CD25+ cells from the chimeric splenocytes dramatically reduced the inhibitory effect also in the in vivo killing assay (SI Fig. 7 A and B). In fact, the proportion of male cell rejection was restored to the same levels as the controls when using CD4-depleted chimeric cells (84% vs. 85%, respectively) (SI Fig. 7A). As a confirmation, the use of purified CD4+ cells greatly increased the inhibitory effect as compared with the suppression exerted by the unfractionated splenocytes (29% vs. 57%, P < 0.05). The inhibitory activity of chimeric splenocytes was equally diminished when CD25+ cells were depleted; in fact, the level of rejection of male cells was similar to controls (54% vs. 48%, respectively) (SI Fig. 7B).

To further determine whether the generation of such inhibition was driven by the antigen, female–female chimeric mice were used as donor of immunosuppressive cells in the in vivo assay. Consistent with the in vitro findings, the infusion of splenocytes from these mice did not prevent the rejection of male cells (Fig. 3B). Therefore, the expansion of Treg cells after transplantation generated in the presence of HY is associated with the presence in the spleen of cells inhibiting HY-specific T cells.

Chimeric Splenocytes Promote Donor Cell Engraftment.

We tested whether the immunosuppressive effect of chimeric splenocytes would also facilitate donor BM engraftment. Recipient female mice were irradiated with the suboptimal dose of 300 cGy TBI for hematopoietic “space” (27). After this dose, male donor cells either do not engraft or do so at a very low level (1/11 of recipients engraft with >1.5% of donor cells). When these mice received chimeric spleen cells together with the donor BM, there was evidence of engraftment (>1.5% of donor cells) in 6/10 mice (2.21–8.83% of donor cells). In accord with the previous data, the depletion of CD4+ cells abolished the facilitating effect on donor engraftment, because 0/5 mice showed evidence of donor engraftment (Fig. 4).

Fig. 4.

Fig. 4.

The adoptive transfer of chimeric splenocytes improves donor BM cell engraftment. Female B6 mice, irradiated with 300 cGy, were injected i.v. with male B6Ly5.1 BM cells alone (BM) or with unfractionated (Chim) or CD4-depleted (Chim-CD4) chimeric splenocytes. Blood samples were collected after 4 weeks, and male donor cells were enumerated by Ly5.1 staining. ∗∗∗, P < 0.01.

Treg Cells Are Required for Donor Cell Engraftment.

To assess whether preexisting recipient Treg cells are required for engraftment, recipient mice received two doses of CD25-depleting antibodies (29); the first dose was given 2 days before the transplant (day −2), the second 7 days later (day +5). Two weeks after the transplant, BM engraftment, as measured by the percentage of donor cells in the peripheral blood, was significantly reduced in mice that had received the CD25-depleting antibody as compared with controls (13% vs. 23%, respectively; P < 0.001) (Fig. 5A). Transplanted mice treated with anti-CD25 antibodies showed lower numbers of CD4+CD25+ than untreated transplanted mice (SI Fig. 8).

Fig. 5.

Fig. 5.

Treg cells are required for donor cell engraftment. Sublethally irradiated (400 cGy) female B6 mice were transplanted with male B6Ly5.1 BM cells. Recipient mice also received two doses of PC61 CD25-depleting antibodies 2 days before (day −2) and 5 days after (day +5) BM infusion. Two weeks after transplant, the number of Ly5.1+ donor cells (A) were assessed in the peripheral blood. Six to nine mice were used per group. (B) A dose of anti-CD25 Ab was given to chimeric mice 6 weeks after BMT and followed by a second dose 1 week later. The number of Ly5.1+ donor cells was assessed 2 weeks after injection. Three mice per group were analyzed. ∗∗∗, P < 0.01.

We then evaluated whether CD4+CD25+ were still required after engraftment was established. Six weeks after engraftment, mice received a dose of anti-CD25-depleting antibody and a second dose 1 week later. The treatment did not produce any effect on the engraftment (Fig. 5B), although the level of CD4+CD25+ cells in these chimeric mice was similar to that in naïve mice (data not shown), thus suggesting depletion of Tregs does not break tolerance once chimerism has been established.

To examine the contribution of the donor T cell compartment in the generation of Treg cells, donor BM was depleted in vitro of T cells before being transplanted. We did not observe any difference in the levels of CD4+CD25+ cells at 2 weeks after transplant (SI Fig. 9A). Furthermore, hematopoietic engraftment in the recipient using T cell-depleted donor BM was similar to the engraftment achieved when using unfractionated donor BM (SI Fig. 9B).

Discussion

We have shown that Tregs play a critical role in controlling HvG after allogeneic HSCT after a RIC regimen. By using an animal model in which HvG tolerance is established by low-dose TBI, we observed that this active tolerance can be detected by cell mixing experiments in vitro (Fig. 2A) and adoptively transferred in vivo (Fig. 3A). The cells affecting tolerogenic activity were contained in the CD4+ and CD25+ population (Fig. 2B and SI Fig. 7 A and B).

After the transplant, recipient mice exhibited a proportional expansion of T cells with a regulatory phenotype (CD4+CD25+FoxP3+) in peripheral blood, BM, and lymph nodes (Fig. 1 A and B and SI Table 1). Because conditioning produces very low blood counts at early time points, we do not know whether this increment in Treg numbers is absolute. However, at later time points (13 weeks after BMT), the absolute number of Treg cells in transplanted mice is almost double (40 cells/μ vs. 21 cells/μ) that of naïve controls (not shown). It has been documented both in mice (30) and humans (31, 32) that Tregs undergo homeostatic proliferation when transferred into lymphopenic recipients (30). Furthermore, early studies have shown that irradiation does not interfere with the function of T cells with suppressive activity (33). Interestingly, the Treg expansion generated in our RIC model was not necessarily associated with increased suppressive activity. Although similar Treg levels were detected in female recipient mice that had received female or male BM, as well as in those that did not receive any BM, only splenocytes from the group receiving male BM exhibited a suppressive activity toward HY-specific T cells (Fig. 2 C and D). When Tregs were purified from the spleen of female–female and male–female chimeric mice, they showed similar suppressive activity in the conventional assay (SI Fig. 6), but the inhibitory effect on HY-specific responses was significantly greater when using CD4+CD25+ cells selected from the gender mismatch combination as compared with the Tregs from the same gender-transplant recipients. Here we demonstrate that transplantation of BM expressing antigenically different molecules facilitates the generation of Tregs with the selective ability to suppress conventional T cells specific for the transplantation antigen. Therefore, the presence of the antigen at the time of the homeostatic expansion produces a competitive advantage for Treg cells expressing donor-specific T cell receptors. It will be of great interest to establish which cell population(s) is responsible for mediating this effect.

Although Wu et al. (34) showed that antibody-mediated T cell depletion prevented tolerance to a subsequent cardiac allograft, our depleting conditioning was associated with the administration of BM, which is particularly effective at generating transplantation tolerance (35). Our findings might explain why HSCT is a successful approach to tolerance induction (5). Accordingly, we have observed that, after low-dose TBI, whereas the infusion of BM induces HvG tolerance to hemopoietic cells as well as to skin graft, a skin graft does not facilitate the engraftment of subsequently infused hemopoietic cells (R. Laylor et al., personal communication).

Our findings support the notion that Treg expansion is required for HvG tolerance, because the in vivo depletion of CD25+ cells before the transplant impaired donor cell engraftment (Fig. 5A). Furthermore, these data rule out the possibility that a change in the repertoire of the conventional T cell pool of the recipient as a result of the homeostatic expansion (36) is the main factor responsible for a reduction or deletion in the number of HY-specific T cell precursors. As a confirmation of this, we have recently observed that in patients after HSCT, the TCR repertoire of Tregs, as analyzed by spectratyping, is much more diverse as compared with the frequent oligoclonalities detected in conventional CD4+ cells from the same patients (37).

The CD25 antibody-depleting treatment at time of the transplant allows us to attribute with certainty that Tregs of recipient rather than donor origin are responsible for facilitating donor hemopoietic engraftment. Furthermore, donor BM preparation was depleted of T cells, and there was no sign of donor CD4+ cells before 4 weeks after transplant (Fig. 1B) and at 6 weeks (SI Fig. 9B), the proportion of donor CD4+ cells was only ≈10%. We do not know what proportion of the Tregs was recruited from the preexisting peripheral pool or whether they were new thymic emigrants. The only-partial myeloablation and the speed of the expansion would argue in favor of the first hypothesis (28). Furthermore, at least in the case of the antigen-driven expansion, Treg cells might have been recruited from the FoxP3 T cell pool, as recently reported (38).

Finally, when CD25 in vivo depletion was performed after establishment of the chimerism, there was no effect on tolerance (Fig. 5B). Although performed in a different system, early studies by Doyle et al. (39) could confirm our findings. In fact, they observed that the suppressive activity associated with the induction of tolerance was not obligatory for its maintenance (39). However, we cannot exclude an impaired efficacy of the CD25-depleting antibody related to the higher number of Tregs after transplantation, and it should also be considered that other types of cells with suppressive activity may play a role in this scenario (40). This is consistent with the fact that the treatment at time of the transplant did not completely abolish engraftment. Therefore, it is sensible to conclude that recipient Tregs are certainly the most important initial players for the induction of HvG tolerance in this system, whereas both recipient and donor cells with immune regulatory activity, not necessarily expressing CD25, play a role in the maintenance of the tolerance status.

Although this model is based on the use of a single antigen mismatch, the principles established here can be taken forward into more complex models. In fact, even in the presence of multiple minor mismatches, T cell responses tend to develop against a single or very few immunodominant epitopes (41). Our results show that a better understanding of the effects of current preparatory regimens for HSCT can contribute to the development of uncomplicated strategies capable of effectively manipulating immunosuppressive networks for the establishment of transplantation tolerance.

Materials and Methods

Mice.

B6 mice were purchased from Olac (Bicester, U.K.). B6.PL-Thya/Cy mice (Thy1.1 congenic), originally obtained from The Jackson Laboratory (Bar Harbor, ME), were subsequently bred in the Central Biomedical Service (CBS), Imperial College, Hammersmith Campus. B6 CD45.1 (Ly5.1) mice were obtained from Charles River Breeding Laboratory (Brussels, Belgium), and then bred in the Central Biomedical Service. Mice were used between 6 and 12 weeks of age. All procedures were conducted in accordance with the Home Office Animals Act of 1986.

BMT and HY Immunization.

BM cells (5 × 106) donor were injected i.v. into 6- to 8-week-old irradiated recipients with 300 or 400 cGy from a 137Cs radiation source (0.57 Gy/min). In vivo depletion of CD25+ cells in recipient mice was obtained by giving two doses (0.5 mg/mouse) of anti-CD25 antibody (29) (PC61, LGC Promochem, Middlesex, U.K.) at days −2 and +5 after transplant.

Female B6 mice were immunized with 5 × 106 male splenocytes by i.p. injection on two to four occasions several weeks apart.

Flow Cytometry Analysis.

Cells were stained as described (27) and analyzed by using a FACScaliber and CellQuest software (Becton-Dickinson, San Jose, CA). Appropriate isotype controls were also included. All Abs were from PharMingen (Oxford, U.K.).

Tetramers for HY peptide WMHHNMDLI (encoded by Uty) were produced as described (42) and conjugated with phycoerythrin (PE)-labeled ExtrAvidin-R-PE complex (Sigma, St. Louis, MO).

Cell Separations.

CD4+ cells were positively selected by using PE-conjugated anti-CD4 antibodies (PharMingen) and MiniMACS anti-PE MicroBeads and passed through selection columns in a magnetic field (Miltenyi Biotec, Camberley, U.K.). Purity was >95%.

The depletion of CD4+ cells was carried out by magnetic separation with rat anti-mouse CD4 (PharMingen), followed by sheep anti-rat IgG Dynabeads (M450) (Dynal, Wirral, U.K.). CD25 depletion was achieved by PE-anti-mouse CD25 (PharMingen) followed by anti-PE microbeads (Miltenyi) and passage through selection columns. Depletion resulted in <1% of CD4+ or CD25+ cells at FACS analysis.

CD4+CD25+ and CD4+CD25 cells were purified in two steps by using MACS CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi). CD4+CD25+ cell purity was >90%, and CD4+CD25 cell purity was >95% with <1% of CD25+ cells.

MLR.

Spleen cells (2 × 106 per well) from HY immune female mice were stimulated with irradiated (60 Gy) male splenocytes (5 × 106 per well) in 24-well plates (Costar, Cambridge, MA) in a final volume of 2 ml of medium (RPMI 1640 with Glutamax; Life Technologies, Rockville, MD), supplemented with 10% FCS (Labtech International, Sussex, U.K.), 2.5 × 10−5 M 2-mercaptoethanol (Sigma–Aldrich, Poole, U.K.), 100 units/ml penicillin, and 100 μg/ml streptomycin (GibcoBRL, Paisley, U.K.). Cultures were incubated at 37°C in a 5% CO2 humidified atmosphere. In the inhibition assays, unfractionated or fractionated CD4+, CD4, or CD25 cells from tolerant or naïve splenocytes were added to cultures at different ratios as specified. After 5 days, cultures were restimulated with irradiated male spleen cells (5 × 106 cells per well) and rIL-2 (10 units/ml) for an additional 6 days and assessed for Uty-specific CD8 cells at FACS. When purified CD4+CD25+ cells were tested as suppressor cells, they were added to a primary MLR in 96-well plates in which 5 × 104 cells per well of the responder cells were stimulated with equal numbers of irradiated male splenocytes in a 0.2-ml volume. CD8+Uty-Tetremar+ cells were enumerated as above.

CD3/CD28 Stimulation.

Splenocytes from naïve female mice (5 × 105 per well) were stimulated with CD3/CD28 antibody-coated Dynabeads (5 × 104 beads per well) (Dynal) in a 96-well plate for 3 days and pulsed with 1 μCi per well of 3H-Thymidine (Amersham, Buckinghamshire, U.K.) for an additional 16 h. 3H-Thymidine incorporation was measured on an LKB Betaplate counter (Wallac Oy, Turku, Finland). The results are expressed as mean cpm for triplicate cultures (standard errors were routinely <10%).

CFSE Labeling and in Vivo Killing Assay.

Spleen cells from naïve male or female mice were labeled with 0.5 or 0.05 mM CFSE (Invitrogen, Paisley, U.K.), respectively. After 10 min at room temperature, the reaction was stopped by the addition of equal value of FCS (Labtech International), followed by washing in PBS. The cells were mixed at 1:1 ratio, and 2 × 107 cells of the mixture with or without 1.5 × 107 cells from chimeric or naïve mice were injected into B6 female recipients intravenously. Blood samples were taken after 1 or 2 weeks, and the proportion of male and female CFSE-labeled cells was determined by FACS analysis. The percentage of rejection of male splenocytes was calculated as follows: 100 × (preinjection male/female ratio − postinjection male/female ratio)/preinjection male/female ratio.

Acknowledgments

We are grateful to E. Simpson and G. Stockinger for critical review of the manuscript and to J. Chai for helpful discussions. This work was supported by Cancer Research U.K.

Abbreviations

RIC

reduced-intensity conditioning

HSCT

hematopoietic stem cell transplantation

HvG

host vs. graft

Treg

regulatory T cell

TBI

total body irradiation

BM

bone marrow

BMT

BM transplantation

FoxP3

forkhead box P3

B6

C57BL/6

CFSE

carboxyfluorescein diacetate succinimidyl ester

MLR

mixed lymphocyte reaction

PE

phycoerythrin.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0701031104/DC1.

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