Adoptively Transferred γδ T Cells Indirectly Regulate Murine Graft-Versus-Host Reactivity Following Donor Leukocyte Infusion Therapy in Mice

William R. Drobyski, Sanja Vodanovic-Jankovic and John Klein
J Immunol August 1, 2000, 165 (3) 1634-1640; DOI: https://doi.org/10.4049/jimmunol.165.3.1634

Abstract

The purpose of this study was to determine whether γδ T cells were able to regulate graft-vs-host (GVH) reactivity mediated by αβ T cells in murine recipients transplanted with MHC-mismatched marrow grafts. Studies were conducted using ex vivo-activated γδ T cells because this was a more clinically relevant strategy, and these cells have been shown to be capable of facilitating alloengraftment without causing GVH disease (GVHD). Coadministration of activated γδ T cells and naive αβ T cells at the time of bone marrow transplantation (BMT) significantly exacerbated GVHD when compared with naive αβ T cells alone. In contrast, when the administration of naive αβ T cells was delayed for 2 wk post-BMT, survival was significantly enhanced in mice transplanted with BM plus activated γδ T cells vs those given marrow cells alone. Mitigation of GVHD by activated γδ T cells occurred only at high doses (150 × 106) and was a unique property of γδ T cells, as activated αβ T cells were incapable of ameliorating the subsequent development of GVHD. Protection from GVHD was not due to the direct inhibition of naive αβ T cells by γδ T cells. Rather, γδ T cells mediated this effect indirectly through donor BM-derived αβ T cells that acted as the proximate regulatory population responsible for the decrease in GVH reactivity. Collectively, these data demonstrate that activated γδ T cells are capable of modulating the ability of MHC-incompatible nontolerant αβ T cells to cause GVHD after allogeneic BMT.

Donor T cells play a critical role in facilitating alloengraftment and in mediating the graft vs leukemia (GVL) effect after allogeneic bone marrow transplantation (BMT)3 in humans (1, 2, 3). These cells, however, also cause graft-vs-host disease (GVHD), which is the major complication after allogeneic marrow transplantation and has been the primary limitation to expanding the number of patients who might benefit from this therapy. T cells are comprised of two major subpopulations, identified by their expression of either the αβ or γδ TCR heterodimer. Cells that express the TCR-αβ are thought to be the primary T cell subpopulation responsible for mediating GVH/GVL reactivity and facilitating alloengraftment (4, 5, 6). The role of γδ T cells after marrow transplantation is less well defined.

Murine BMT studies have provided conflicting data as to whether naive γδ T cells play a role in the pathophysiology of GVHD. Although several studies have shown that γδ T cells can contribute to GVH reactivity (7, 8, 9), others have demonstrated that γδ T cells can inhibit GVHD that is induced by αβ T cells (10). A role for γδ T cells in the facilitation of alloengraftment in mice has also been proposed (11), suggesting that these cells can effect some of the same immune responses in allogeneic BMT as αβ T cells. Because γδ T cells comprise only a small percentage of total T cells, direct evaluation of the role of these cells in murine allogeneic marrow transplantation has been difficult. Moreover, the paucity of these cells in human peripheral blood makes the clinical translation of these results potentially problematic. For that reason, we opted to evaluate transplantation of ex vivo-activated and expanded γδ T cells as a more clinically relevant strategy. These studies showed that large doses of ex vivo-activated and expanded γδ T cells were capable of facilitating alloengraftment across the MHC barrier (12). Graft facilitating doses of these cells also failed to cause GVHD in MHC-mismatched donor/recipient strain combinations (13), demonstrating that transplantation of γδ T cells may be an approach to promote allogeneic engraftment without causing GVHD.

γδ T cells have been shown to have important immunological functions in mice. Studies have demonstrated a role for these cells in host defense against pathogens (14, 15). Additionally, a number of reports support a primary role for γδ T cells in the induction of tolerance to both inhaled and ingested Ags (16, 17, 18). In some, but not all, experimental settings, these cells also are capable of contributing to the resolution of inflammatory lesions (19, 20). One mechanism by which γδ T cells have been shown to induce tolerance and resolve inflammatory lesions is by down-regulating immune responses mediated by αβ T cells (17, 21, 22). These reports prompted us to explore whether γδ T cells were capable of regulating immune responses mediated by αβ T cells after allogeneic marrow transplantation. Because GVHD is the major complication of allogeneic BMT and is mediated primarily by αβ T cells, we elected to examine how these two cell populations interacted in the setting of a GVH reaction. To simulate human marrow transplantation, we assessed the effect of γδ T cells on the development of GVHD under two different scenarios: that occurring from the infusion of naive αβ T cells at the time of BMT, and that resulting from a delayed infusion of naive αβ T cells.

Materials and Methods

Mice

C57BL/6 (H-2b), TCR β−/β− (αβ T cell-deficient, C57BL/6 background, H-2b), AKR/J (H-2k), and B10.BR (H-2k) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C.B-17 scid mice (BALB/c background, H-2d) were obtained from Taconic Farms (Germantown, NY). All animals were housed in the American Association for Lab Animal Care (AALAC)-accredited Animal Resource Center of the Medical College of Wisconsin. Mice received regular mouse chow and acidified tap water ad libitum. C.B-17 mice were housed in microisolator cages, while all other animals were housed in conventional cages.

Ex vivo activation and expansion of murine T cells

To expand γδ T cells, spleen cells were obtained from TCR β−/β− donor animals and passed through nylon wool columns to remove B cells. The resulting population was typically comprised of ∼50% cells expressing the γδ TCR. Cells were then resuspended in complete DMEM plus 5–10% FBS and cultured in flasks precoated with an immobilized γδ T cell-specific mAb (GL4, hamster IgG; PharMingen, San Diego, CA) at a concentration of 5–10 μg/ml. Twenty-four hours after the initiation of culture, human IL-2 (Proleukin, Chiron, Emeryville, CA) was added at a concentration of 200 IU/ml. All cultures were split into fresh flasks as needed to maintain a cell concentration of 0.5–1.5 × 106 cells/ml. Cells were exposed to immobilized mAb for the first 3–4 days of culture and thereafter grown only in medium plus 100 U/ml IL-2 to allow for reexpression of the γδ TCR. After a total of 7–8 days in culture, cells were counted, and the percentage of γδ T cells was analyzed by flow cytometry. Routinely, a total of 5–10 × 108 cells was obtained after expansion, with 95–99% of cells expressing the γδ TCR. Typically, 15–25% of activated γδ T cells coexpressed CD8. CD4 expression was not detected on these cells. A similar approach was employed for the activation and expansion of αβ T cells with the exception that spleen cells were obtained from C57BL/6 mice and cultured in flasks precoated with an immobilized anti-CD3 Ab (clone 145-2C11) (kindly provided by J. Bluestone, University of Chicago, Chicago, IL). Routinely, >95% of viable cells had reexpressed the CD3/TCR complex at the time that these cells were transplanted into recipients.

BM transplantation

BM was flushed from donor femurs and tibias with complete DMEM and passed through sterile mesh filters to obtain single cell suspensions. BM was T cell depleted (TCD) in vitro with anti-Thy1.2 mAb plus low toxicity rabbit complement (C6 Diagnostics, Mequon, WI). The hybridoma for 30-H12 (anti-Thy1.2, rat IgG2b) Ab was obtained from the American Tissue Culture Collection (Manassas, VA) and grown in complete DMEM plus 5% FBS. The culture supernatants was then harvested, precipitated in ammonium sulfate, and dialyzed against PBS before use in in vitro depletion experiments. BM cells were then washed and resuspended in DMEM before injection. To recover naive T cells, spleen cell suspensions were obtained by pressing spleens through wire mesh screens. Erythrocytes were removed from cell suspensions by hypotonic lysis with sterile distilled water. Naive T cells for admixture with TCD BM before transplantation were then obtained by passing spleen cells once or twice through nylon wool columns (Robbins Scientific, Sunnyvale, CA) to remove B cells. The percentage of αβ+ T cells from B6 donors was quantified by flow cytometry and defined as Thy1.2+ TCR-αβ+.

B10.BR or AKR/J recipient mice were given varying doses of lethal total body irradiation (900 or 1100 cGy, respectively) as a single exposure at a dose rate of 70 cGy using a Shepherd Mark I Cesium Irradiator (J. L. Shepherd and Associates, San Fernando, CA). Irradiated recipients then received a single i.v. injection of TCD BM (10 × 106) with or without ex vivo-activated T cells. When a dose of 150 × 106 activated γδ T cells was administered to recipients, the total dose was split in half or in fourths and given over a period of 24 h. This was done to reduce immediate toxicity from the infusion of a large number of activated T cells.

Flow cytometric analysis

mAbs conjugated to either FITC or PE were used to assess chimerism in marrow transplant recipients. FITC-anti-Thy1.2 (clone 30-H12, rat IgG2b) was purchased from Collaborative Biomedical Products (Bedford, MA). FITC-anti-Ly5 (B220, rat IgG2a) and PE anti-CD8 (clone CT-CD8a, rat IgG2a) were obtained from Caltag (San Francisco, CA). PE anti-TCR αβ (clone H57-597, hamster IgG), PE anti-TCR γδ (clone GL3, hamster IgG), PE anti-CD3 (clone 145-2C11, hamster IgG), PE anti-CD4 (clone GK 1.5, rat IgG2b), FITC-anti-H-2Kb (clone AF6-885, mouse IgG2a) were all purchased from PharMingen. Spleen and thymus cells were obtained from chimeras at defined intervals posttransplant and stained for two-color analysis. Red cells were removed by lysis in distilled water. Cells were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Red cells and nonviable cells were excluded using forward and side scatter settings before analysis of spleen cell populations. Splenic T cell chimerism was assessed within a lymphocyte gated population, while thymic chimerism was evaluated using open gates. Ten thousand cells were analyzed for each determination whenever possible.

Statistics

Group comparisons of T cell chimerism in the spleen and thymus, and thymic size were performed using the unpaired Student t test. Data are presented as the mean ± SE. Survival curves were constructed using the Kaplan-Meier product limit estimator and compared using the log-rank rest. For experiments evaluating the effect of delaying the administration of naive αβ T cells to mice transplanted with and without γδ T cells, a Cox regression model was used to compare survival between the respective groups. In this model, no significant survival difference was found between groups of mice transplanted with TCD BM followed by naive T cells on either day 0, 7, or 14 post-BMT. For that reason, a Cox model was fit pooling the data from these three groups. Groups of mice transplanted with TCD BM and activated γδ T cells, and then given naive T cells on day 0, 7, or 14 post-BMT were compared with these pooled data to evaluate survival differences. In the γδ T cell dose titration experiments, survival was compared using the Mantel-Haenszel test. A two-tailed p value ≤0.05 was deemed to be significant in all experiments.

Results

Transplantation with activated γδ T cells results in protection from GVHD induced by a delayed infusion of naive donor T cells

Prior studies by others indicate that, in selected experimental settings, γδ T cells are able to down-regulate the ability of αβ T cells to mediate immune responses (17, 21). These data raised the question as to whether γδ T cells might be capable of mitigating or preventing GVHD induced by αβ T cells. To evaluate the immunoregulatory role of γδ T cells, we performed studies using a previously described donor leukocyte infusion (DLI) model (23, 24) which allowed us to vary the time at which mice received naive T cells posttransplant. Cohorts of lethally irradiated B10.BR mice were transplanted with TCD B6 BM alone or TCD BM plus 150 × 106 activated γδ T cells. Naive B6 T cells (comprised of >97% αβ T cells) were administered at the time of BMT, or at 7 or 14 days after transplant. For GVH control mice transplanted with TCD BM alone, the day on which naive αβ T cells were administered (day 0 vs 7 vs 14) did not significantly affect survival between any of the three groups (p = 0.096). In contrast, the timing of naive αβ T cell administration significantly affected survival in mice transplanted with TCD BM and activated γδ T cells. When mice were transplanted with activated γδ T cells and naive T cells at day 0, animals had significantly reduced survival relative to mice transplanted with naive T cells only (Fig. 1⇓A) (p = 0.019), indicating that activated γδ T cells exacerbated GVHD induced by naive αβ T cells. Delaying the administration of naive T cells for 7 days, however, resulted in equivalent survival for both groups (Fig. 1⇓B) (p = 0.75). Conversely, when the infusion of naive T cells was delayed to day 14 posttransplant, mice transplanted with activated γδ T cells were protected from lethal GVHD and had an enhanced survival rate (p = 0.019) (Fig. 1⇓C). These data indicated that γδ T cells were able to contribute to GVH reactivity when administered contemporaneously with naive αβ T cells. When the administration of naive T cells was delayed for 2 wk, however, transplantation with γδ T cells protected mice from GVHD and prolonged survival.

FIGURE 1.
FIGURE 1.

Transplantation with activated γδ T cells protects mice from GVHD induced by the subsequent infusion of naive alloreactive donor T cells. Lethally irradiated (900 cGy) B10.BR mice were transplanted with TCD B6 BM alone (□) or TCD B6 BM plus 150 × 106 B6 activated γδ T cells (▪). In A, 2 × 106 naive B6 T cells were administered on the day of transplant to both groups (DAY 0)(n = 8–12 mice/group). In B, the same dose of naive B6 T cells was administered 7 days post-BMT (DAY 7)(n = 12–17 mice/group). In C, the same dose of B6 T cells was administered 14 days after transplant (DAY 14)(n = 23–27 mice/group). A control group received TCD BM only at BMT and no naive T cells post-BMT in each of the experiments (○). Actual survival is depicted. Data are cumulative results from two to five independent experiments.

To confirm that the protective effects we observed from γδ T cells were not strain dependent, we repeated these experiments with a different MHC-incompatible (B6→AKR) murine model. Administration of DLI 2 wk after BMT to AKR mice transplanted with activated γδ T cells again resulted in a significantly higher survival rate as compared with mice that received TCD BM only (p = 0.01) (Fig. 2⇓). Thus the effect of γδ T cells was not strain-specific.

FIGURE 2.
FIGURE 2.

Protection from DLI-induced GVHD by activated γδ T cells is not strain-specific. Lethally irradiated (1100 cGy) AKR mice were transplanted with TCD B6 BM alone (□, n = 21) or TCD B6 BM and 150 × 106 B6 activated γδ T cells (▪, n = 22). On day 14 both groups were administered 2 × 106 naive B6 T cells. A control group received TCD BM only at BMT and no naive T cells post-BMT (○, n = 20). Actual survival is depicted. Data are cumulative results from four independent experiments.

Protection from GVHD by γδ T cells is dose dependent

Experiments were performed to determine whether protection from GVHD by activated γδ T cells was dose dependent. AKR recipient mice were transplanted with TCD B6 BM and either 5 × 106, 25 × 106, 150 × 106, or no activated γδ T cells. Two weeks later, animals were given 2 × 106 naive B6 αβ T cells. No difference in survival was observed between mice that received no γδ T cells (20% survival at day 100, n = 56) and those that received either 5 × 106 (16%, n = 55) or 25 × 106 (25%, n = 53) activated γδ T cells at the time of BMT. In contrast, transplantation with 150 × 106 activated γδ T cells (36%, n = 47) mitigated the development of lethal GVHD and significantly prolonged survival when compared with the GVHD control population (p = 0.023 by Mantel-Haenzel test).

Protection from GVHD by activated T cells is a property unique to γδ T cells

We have shown previously that anti-CD3 Ab-activated αβ T cells cause significantly less GVHD than do an equivalent number of naive T cells when administered at the time of transplant (25). We therefore considered that protection from GVHD after a delayed infusion of αβ T cells might be a general property of activated T cells and not a unique characteristic of γδ T cells. To address this question, AKR mice were transplanted with TCD B6 BM alone or together with either a low dose (2 × 106) or a high dose (100 × 106) of anti-CD3 activated B6 αβ T cells. The mice were infused with 2 × 106 naive B6 T cells 2 wk later. The two doses of activated αβ T cells were selected to encompass a wide cell dose range and thereby account for any effect that large doses of activated T cells per se might have on GVHD protection. There was no difference in survival rates after DLI between mice transplanted with 2 × 106 activated T cells vs mice given TCD BM only (p = 0.14) (Fig. 3⇓A). In contrast, mice transplanted with 100 × 106 activated T cells had significantly worse survival after DLI than mice transplanted with TCD BM followed by DLI (p = 0.016) (Fig. 3⇓B). Death in the group of mice transplanted with 100 × 106 activated T cells was due to accelerated GVHD. These data indicated that protection from GVHD by activated T cells was due to a unique property of γδ T cells and not a property of T cells in general.

FIGURE 3.
FIGURE 3.

Activated αβ T cells do not protect mice from a subsequent infusion of naive donor T cells. Lethally irradiated (1100 cGy) AKR mice were transplanted with TCD B6 BM alone (▪, n = 7–8/group) or TCD B6 BM plus B6 activated αβ T cells (○). Animals in each group were administered 2 × 106 naive B6 T cells 14 days after transplant. In A, the dose of activated T cells was 2 × 106 (n = 7), whereas in B, the dose of activated T cells was 100 × 106 (n = 6). A control group received TCD BM only at BMT and no naive T cells post-BMT (□, n = 3–5/group). Actual survival is depicted.

Activated γδ T cells do not protect against GVHD in scid mice

The protective effect of activated γδ T cells was likely due either to a direct effect of these cells or to an indirect effect that required the presence of another cell population. To address the question of whether another cell population was involved, we examined whether γδ T cells mediated protection against GVHD in a scid mouse model. This model was chosen because transplantation can be performed without the need for preconditioning or reconstitution with donor BM. scid mice can therefore be transplanted with activated γδ T cells alone and then given a delayed infusion of naive GVH reactive T cells. The validity of this model, however, is dependent upon activated γδ T cells not being rejected by host immune cells (e.g., NK cells) in the scid animals. To address this issue, scid mice (n = 8) were transplanted with 150 × 106 activated γδ T cells and then analyzed 2 wk posttransplant for donor chimerism. The mean percentage of γδ T cells in the spleen was 16.7% (range, 5.8–39.6), indicating that γδ T cells were not completely rejected by host NK cells. The percentage of γδ T cells was within the range observed for irradiated mice transplanted with TCD BM and the same dose of activated γδ T cells (W. R. Drobyski, unpublished observations). Thus, we concluded that the scid model was a valid one with which to address the role of other T cells. scid mice were transplanted with activated B6 γδ T cells and then administered naive B6 T cells (4 × 106) 2 wk later. GVHD control mice received the same naive B6 T cells without the antecedent infusion of γδ T cells. Mice transplanted with γδ T cells followed by naive T cells had significantly lower survival rates than animals which only received naive T cells (Fig. 4⇓) (p = 0.003). These data strongly suggested that the mitigation of DLI-induced GVHD by γδ T cells was not a direct effect of these cells but was dependent upon the presence of another cell population(s).

FIGURE 4.
FIGURE 4.

Protection from GVHD is not due to a direct effect of activated γδ T cells on αβ T cells. Unirradiated C. B-17 scid mice were transplanted with 150 × 106 B6 activated γδ T cells (▪, n = 13). An additional group of scid mice was given no cells (□, n = 11). On day 14 post-BMT, both groups were transplanted with 4 × 106 naive B6 T cells. Actual survival is depicted. Data are cumulative results from two independent experiments.

Protection from GVHD requires the presence of BM-derived donor αβ T cells

Because the scid mouse model did not employ donor BM, we considered that the additional cell population might be a BM-derived cell. Support for this hypothesis comes from a report by Johnson et al. (23) who demonstrated the presence of a BM-derived T cell that was able to down-regulate GVH reactivity after DLI. To address this question, lethally irradiated AKR recipients were transplanted with TCD BM from either normal B6 or αβ T cell deficient (β−/β−) B6 donors. Mice in both cohorts received 150 × 106 activated γδ T cells with their marrow grafts and 2 wk later were administered an equivalent dose of 2 × 106 naive αβ T cells. Animals transplanted with BM from β−/β− donors had significantly higher mortality from GVHD as compared with mice given normal B6 BM (25% vs 69% 100-day survival, p = 0.021) (Fig. 5⇓). These studies suggested that the protective effect of activated γδ T cells required that the donor marrow be capable of generating αβ T cells and that a BM-derived αβ T cell was the proximate cell responsible for mitigation of GVHD.

FIGURE 5.
FIGURE 5.

Protection from GVHD is dependent upon the generation of αβ T cell precursors from the donor marrow graft. Lethally irradiated (1100 cGy) AKR mice were transplanted with TCD B6 BM and 150 × 106 B6 activated γδ T cells (▪, n = 16) or TCD β−/β− (β°) BM and 150 × 106 B6 activated γδ T cells (□, n = 16). On day 14 posttransplant, animals in each group were transplanted with 2 × 106 naive B6 T cells. Actual survival is depicted. Data are cumulative results from three independent experiments.

Activated γδ T cells promote engraftment of BM-derived donor Thy1.2+ T cells in the thymus

BM-derived T cell progenitors undergo selection and maturation in the thymus before emigrating into the periphery. Because these cells were necessary for mitigation of GVHD, we examined the thymi of mice transplanted with and without γδ T cells to determine whether γδ T cells enhanced thymic engraftment with BM-derived donor T cell precursors. Thymic engraftment was examined 2 wk after BMT which corresponded to the time that γδ T cells were the predominant donor T cell population in the spleen and the time that transplanted mice were given naive αβ T cells in the experiments above. Animals given activated γδ T cells had an increased percentage of donor T cells in the spleen (80 ± 2% vs 39 ± 4%) ascribable solely to the presence of donor γδ T cells. There was a commensurate reduction in the percentage of host T cells in these mice as well (3 ± 0% vs 7 ± 2%; p = 0.021). As shown in Table I⇓, the thymi of mice transplanted with activated γδ T cells contained a similar average percentage and absolute number of double-positive thymocytes as TCD BM control animals. This is consistent with a lack of GVHD in mice transplanted with γδ T cells. At 2 wk, animals transplanted with activated γδ T cells had a significantly greater percentage of donor Thy1.2+ T cells in the thymus when compared with control mice (Table I⇓). Both donor-derived CD4+ and CD8+ single-positive T cells were increased relative to mice transplanted with TCD BM alone. Because the average thymic size was the same in both groups, the absolute number of donor-derived CD4+ and CD8+ cells was increased. There was also an increased percentage of donor CD4+CD8+ double-positive cells in mice transplanted with activated γδ T cells. This provided evidence that γδ T cells increased the number of thymic T cell progenitors that were derived from the donor BM.

View this table:
Table I.

Activated γδ T cells facilitate thymic engraftment of BM-derived donor T cellsa

There was considerable variability in the degree of thymic engraftment of both Thy1.2+ CD4+ and Thy1.2+ CD8+ cells in animals transplanted with activated γδ T cells (ranges, 6–67% and 13–61%, respectively). Therefore we examined whether there was a correlation between the percentage of splenic donor γδ T cells and the degree of thymic engraftment by donor cells. A comparison of the percentage of splenic donor γδ T cells and the percentage of donor thymic Thy1.2+ CD4+ cells in individual mice 14 days posttransplant demonstrated a statistically significant correlation between these two variables (Fig. 6⇓) (Pearson’s coefficient, r = 0.79, p < 0.0001). A similar correlation was observed for CD8 cells (data not shown). These data indicated that mice with the highest percentage of γδ T cells in their spleens at day 14 exhibited the greatest degree of donor T cell engraftment in the thymus, suggesting a direct role for γδ T cells in promoting the posttransplant engraftment of BM-derived donor αβ T cells.

FIGURE 6.
FIGURE 6.

Correlation between the percentage of donor γδ T cells in the spleen and engraftment of BM-derived donor CD4+ T cells in the thymus 2 wk post-BMT. Lethally irradiated (1100 cGy) AKR mice were transplanted with TCD B6 BM plus 150 × 106 activated B6 γδ T cells (n = 26). On day 14 post-BMT, animals were sacrificed, and the percentage of donor γδ T cells in the spleen and the percentage of donor CD4+ T cells in the thymus were assessed. Data are presented as a dot plot for individual mice and were obtained from five independent experiments.

Discussion

The purpose of these studies was to investigate whether γδ T cells were capable of regulating the ability of αβ T cells to cause GVHD. Initial studies showed that when γδ T cells and αβ T cells were co-administered to lethally irradiated recipients, there was an exacerbation of GVHD when compared with mice that received αβ T cells alone. Because transplantation of activated γδ T cells alone does not cause significant GVHD (13), these results indicated that γδ T cells could contribute to GVH reactivity but required the presence of αβ T cells. This is consistent with data by Tsuji et al. (8) who showed that γδ T cells were incapable of inducing GVHD by themselves but could be induced to proliferate by donor αβ T cells. A possible explanation for the exacerbation of GVHD is the release of soluble factors from αβ T cells that can stimulate the proliferation of γδ T cells (26, 27). Activated γδ T cells also secrete Th1-type cytokines, such as IL-2 and IFN-γ (28, 29) and may thereby contribute to the amplification of the GVH reaction in a paracrine fashion. When the administration of naive αβ T cells was delayed until 2 wk posttransplant we observed that animals given γδ T cells at the time of BMT had a significant decrease in GVHD and improvement in survival. Even though γδ T cells were still detectable in the spleen of recipients at 2 wk posttransplant, the infusion of naive donor T cells did not exacerbate GVHD (Figs. 1⇑C and 2). This effect was dose dependent and required transplantation of large numbers of activated γδ T cells (i.e., 150 × 106). At this dose there was a consistent 2- to 3-fold improvement in the survival rate of these mice (Figs. 1⇑C and 2). Protection from GVHD was not complete, however, as some mice did develop GVHD and recent data suggest that this effect can be overwhelmed by a high dose of naive T cells (i.e., 4 × 106) (our unpublished observations). Notably, mitigation of GVHD was specific to activated γδ T cells because activated αβ T cells had no inhibitory role when examined at a low dose (2 × 106) (Fig. 3⇑A) and exacerbated GVHD at a high dose (100 × 106) (Fig. 3⇑B). Collectively, these data suggested that γδ T cells altered the immune environment of these mice rendering them less susceptible to GVHD when exposed 2 wk later to alloreactive donor T cells.

Studies were conducted to examine the mechanism by which transplantation with activated γδ T cells protected mice from a subsequent challenge of αβ T cells. To determine whether γδ T cells had a direct regulatory effect on the alloreactivity of αβ T cells infused into host mice, transplants were done in scid recipients which obviated the need for preconditioning therapy and infusion of donor BM. Animals previously transplanted with γδ T cells developed significantly more severe GVHD after a delayed infusion of naive αβ T cells than mice that received only naive T cells (Fig. 4⇑). These results were similar to those observed when both T cell populations were coadministered at the time of BMT (Fig. 1⇑) and indicated that γδ T cells did not directly prevent αβ T cells from causing GVHD. This suggested that the protective effects induced by γδ T cells were indirect (i.e., dependent upon interaction with another regulatory cell population), although these results did not exclude the possibility that irradiation played a contributory role. Because the scid model did not employ donor BM, we reasoned that a putative regulatory cell population might be derived from the marrow. Subsequent studies utilizing αβ T cell-deficient donor BM in an irradiation model resulted in a loss in the protective effect induced by γδ T cells (Fig. 5⇑), providing confirmatory evidence that the regulatory cell was a donor BM-derived αβ+ T cell. These results are supportive of studies by Johnson et al. (23) who demonstrated the existence of a donor, BM-derived, TCR-αβ+ T cell that is normally present in reconstituting chimeras and capable of down-regulating GVH reactivity after DLI. They noted that this cell was present only in marrow recipients that possessed an intact thymus, indicating that these regulatory T cells were derived from the donor-reconstituted host thymus. Elimination of this regulatory population posttransplant significantly exacerbated GVHD induced by a delayed infusion of donor T cells, demonstrating a critical role for these cells in establishing peripheral tolerance.

The key question of how transplantation with activated γδ T cells promoted the ability of BM-derived αβ T cells to mitigate GVHD has not been fully answered. We have shown previously that activated γδ T cells can facilitate alloengraftment, but that high doses are required (i.e., ∼150 × 106) (12). In the current study, a protective effect was observed only when a similarly high dose of γδ T cells was utilized. At this dose, there was a significant reduction in the percentage of residual host T cells in the spleen after conditioning therapy, substantiating a role for these cells in alloengraftment. Based on these collective data, our hypothesis is that transplantation of activated γδ T cells enhances the emergence of a regulatory population of donor BM-derived αβ T cells from the thymus posttransplant. The strong correlation between the percentage of γδ T cells in the spleen and percentage of donor-derived thymic cells 2 wk after BMT supports this premise and suggests a causal role for γδ T cells in thymic engraftment of donor BM-derived αβ T cell precursors. How these cells augment donor thymic engraftment is unknown. One possibility is that the graft-promoting effects of donor activated γδ T cells are responsible for protection from DLI-induced GVHD. That is to say, by contributing to the elimination or inactivation of host T cells that mediate graft resistance, γδ T cells may accelerate the emergence of a BM-derived regulatory population.

A number of studies have supported a role for γδ T cells in the induction of tolerance in the nontransplant setting. Studies by Ke et al. (16) and Mengel et al. (30) have demonstrated that γδ T cells are the critical cell population necessary for the induction of oral tolerance when animals are administered an antigenic challenge with OVA. Moreover, γδ T cells can prevent the development of insulin-dependent diabetes when adoptively transferred into unaffected recipients (31) and prolong skin allograft survival (32). Collectively, these studies have shown that γδ T cells can induce tolerance in both humoral and cell-mediated responses. In some (17, 21, 22) but not all instances (33, 34), γδ T cells have also been shown to be able to down-regulate immune responses mediated by αβ T cells. Kaufmann et al. (21) have shown that αβ T cells from mice treated in vivo with a γδ T cell-specific Ab had increased proliferation, cytokine secretion, and cytotoxicity in vitro, suggesting that these cells had an inhibitory effect on αβ T cells. Supportive data for this premise also have come from work showing that γδ T cells down-regulate αβ T cell responses in murine listeriosis (35), mycobacterial tuberculosis (36), and experimental Trypanosoma cruzi infection (37). A common finding in the majority of these studies is that the tolerogenic effects of γδ T cells are mediated by a direct interaction between these cells and αβ T cells, or that γδ T cells elaborate cytokines that down-regulate the immune response (35). The current study suggests another mechanism by showing that γδ T cells can mediate a tolerogenic effect indirectly through a secondary regulatory αβ T cell population. Additional studies will be required to determine whether this is unique to the BMT setting.

From a clinical perspective, these data may prove relevant to the emerging use of adoptive immunotherapy as a means to prevent relapse after allogeneic BMT. Due to the increased incidence and severity of GVHD when donor T cells are administered at the time of transplant, recent approaches have focused on the delayed infusion of T cells at defined time points post-BMT (38, 39, 40, 41, 42). Although the administration of DLI appears to cause less GVHD than when a comparable number of donor T cells are given at the time of marrow grafting, GVHD remains a formidable problem, particularly in recipients of mismatched or unrelated grafts. The ability of activated γδ T cells to protect recipients from GVHD induced by a subsequent DLI infusion is an observation that may allow for this strategy to be more clinically efficacious. This approach has the added advantage that activated γδ T cells appear to be able to prevent graft rejection (12). Therefore, alloengraftment might not be compromised, but actually enhanced with this strategy. A barrier to transplanting naive γδ T cells is the limited number of these cells in the peripheral blood. However, ex vivo expansion technologies for the large-scale production of γδ T cells are currently being developed to make this a clinically feasible approach (43, 44). These studies demonstrate that large numbers of relatively pure populations of activated γδ T cells can be obtained from the peripheral blood. The continued refinement of this approach offers the potential to translate these preclinical studies into clinical marrow transplantation settings.

Acknowledgments

We thank Tina Agostini and David Majewski for technical assistance. We also thank Robert Truitt for critically reviewing the manuscript.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Grant HL55388 and the Mallinckrodt Foundation.

  • ↵2 Address correspondence and reprint requests to Dr. William R. Drobyski, Bone Marrow Transplant Program, Froedtert Memorial Lutheran Hospital, 9200 West Wisconsin Avenue, Milwaukee, WI 53226. E-mail address: bill{at}bmt.mcw.edu

  • ↵3 Abbreviations used in this paper: BMT, bone marrow transplantation; GVH, graft-vs-host; GVHD, GVH disease; DLI, donor lymphocyte infusions; TCD, T cell depleted.

  • Received February 17, 2000.
  • Accepted May 16, 2000.

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The Journal of Immunology: 165 (3)
The Journal of Immunology
Vol. 165, Issue 3
1 Aug 2000
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