Blockade of LIGHT/LTβ and CD40 signaling induces allospecific T cell anergy, preventing graft-versus-host disease (original) (raw)
Synergistic effects of LTβR-Ig and anti-CD40L mAb in the prevention of GVHD are accompanied by profound inhibition of anti-host CTLs. Upon transfer of B6 T cells, recipient BDF1 mice generate acute GVHD characterized by rapid weight loss, expansion of host-reactive donor T cells, shrinkage of the thymus, and eventual death (Figure 1, a and b, and data not shown). Infusion of LTβR-Ig, which blocks the LIGHT costimulatory pathway (9, 10), significantly reduced GVHD mortality as well as anti-host CTL activity. The treatment is not optimal because approximately 20% of mice still die of GVHD. However, combined treatment with anti-CD40L and LTβR-Ig in our study prevented death in 100% of recipient mice, whereas anti-CD40L treatment alone protected only about 50% of mice (Figure 1a). Combined therapy protected all recipient mice from death for more than 90 days (Figure 1a), and efficiently prevented weight loss in recipient mice (Figure 1b). This combined therapy also profoundly inhibited the generation of anti-host (H-2d) CTL activity, whereas treatment by anti-CD40L alone did not affect CTL activity, and LTβR-Ig alone resulted in a partial inhibition (Figure 1c).
Synergistic effect of LTβR-Ig and anti-CD40L mAb in amelioration of GVHD and inhibition of anti-host CTL activity. (a–c) B6 splenocytes (7 × 107 cells) were injected into sublethally irradiated BDF1 mice and treated with either anti-CD40L (open circles), LTβR-Ig (filled squares), or both (filled circles). Hamster IgG and human IgG1 were injected as control (open squares). (a) Survival of recipients was examined daily, and pooled data from four independent experiments are presented. Treatment with both anti-CD40L and LTβR-Ig significantly prolonged survival compared to other treatments (P < 0.05). (**b**) Average body weight. Symbols same as for **a**. (**c**) The CTL activity of recipient spleen cells against P815 (H-2d) and EL4 (H-2b) was examined on day 7 without in vitro restimulation. Results are expressed as the mean ± SD of triplicate wells. (**d**) Purified B6 CD8+ T cells (1 × 106 cells) were injected into sublethally irradiated bm1 mice followed by treatment with LTβR-Ig (filled squares) or control Ig (open squares) as described in Methods. Recipients of LTβR-Ig had a significantly (_P_ = 0.0002) higher survival rate than did control-treated recipients. The reduction in GVHD lethality by LTβR-Ig treatment was estimated to be approximately equivalent to that resulting in control-treated mice from a threefold lower number (3 × 105) of CD8+ cell transfer (open circles, _P_ > 0.1). (e) Purified B6 CD4+ T cells (1 × 105 cells) were injected into sublethally-irradiated bm12 mice followed by treatment with LTβR-Ig (filled squares) or control Ig (open squares) as described in Methods. No significant difference (P > 0.1) was noted between these two groups.
It has been suggested that the blockade of CD40-CD40L interaction prevents GVHD through a CD4+ but not CD8+ T cell–mediated mechanism (22). To investigate the differential effects of LTβR-Ig on CD4+ and CD8+ T cell–mediated GVHD, sublethally irradiated bm12 and bm1 recipients were injected intravenously with purified B6 CD4+ and CD8+ T cells, respectively. Infusion of LTβR-Ig significantly prolonged the survival of bm1 recipients of a minimum uniformly lethal dose of CD8+ T cells (Figure 1d). In contrast, survival was not significantly prolonged in bm12 recipients given a minimum uniformly lethal dose of CD4+ T cells (Figure 1e). Thus, LTβR-Ig treatment is more effective in inhibiting CD8+ T cell–mediated GVHD than CD4+ T cell–mediated GVHD under these conditions. Our results suggest that the synergistic effect of anti-CD40L mAb and LTβR-Ig in our model is mediated by inhibition of both CD4+ and CD8+ host-reactive T cells.
Combined therapy inhibits the generation of anti-host CTL activity without peripheral deletion of T cells. To trace T cells in the recipients after treatment with costimulatory blockade, we used 2C TCR transgenic T cells as donor cells. 2C T cells react specifically against H-2Ld antigen and express a defined T cell receptor that can be specifically identified (27). 2C T cells that were transferred into BDF1 mice expanded vigorously and generated a high level of anti-host CTL activity, as early as 5 days after cell transfer (Table 1). Similar to the results obtained from polyclonal T cell transfer shown in Figure 1c, injections of LTβR-Ig, but not anti-CD40L mAb, significantly inhibited CTL activity. Nearly complete inhibition of 2C CTL activity was observed in recipients treated with a combination of LTβR-Ig and anti-CD40L mAb. Inhibition of antigen-specific CTL generation was noted through day 15 after transfer, although cell counting taken on both day 5 and day 15 showed that 2C T cells in treated recipients had expanded as vigorously as those in control recipients (Table 1). Our data thus suggest that combined treatment with LTβR-Ig and anti-CD40L mAb inhibits anti-host CTL generation without deletion of host-reactive T cells.
Modification of 2C T cell functions by blockade of LIGHT and CD40L costimulators
After transfer into syngeneic B6 recipients, the percentage of 2C T cells expressing a low level of CD62L (CD62Llow), which indicates an antigen-experienced phenotype (29), remained constant in the range of 10–20% (Table 1). In contrast, the majority of 2C T cells transferred into BDF1 recipients converted to CD62Llow cells as early as 5 days after transfer. The combined therapy delayed downregulation of CD62L expression on 2C T cells, an effect that was accompanied by suppressed CTL activity. However, a significant number of 2C T cells had converted to CD62Llow cells by day 15, even though CTL activity remained low. A similar pattern was observed using CD44 marker (data not shown). Taken together, our results suggest that combined treatment with LTβR-Ig and anti-CD40L mAb inhibits the effector function, but is less effective at inhibiting the priming and expansion of host-reactive CTLs.
Combined therapy leads to repopulation of donor-derived lymphocytes in long-term GVHD-surviving mice. In the recipients that received the combined treatment and survived GVHD more than 60 days after transfer of B6 splenocytes, all lymphohematopoietic cells were H-2Kb+ and H-2Kd–, indicating complete replacement of the recipient lymphoid system by donor cells. Repopulation of donor lymphocytes was observed in spleen, LNs (Figure 2a), thymus (Figure 2b), and BM (data not shown). In addition, proportions of T cells, B cells, and CD4+ and CD8+ T cell subsets in spleen, LN, and thymocyte subsets were similar to those in naive B6 mice. Myeloid cells expressing Mac-1 also converted to donor-derived cells (data not shown). The proportion of CD62Llow cells in recipient splenic and LN T cells, however, increased significantly compared with that in naive B6 mice (Figure 3a), suggesting constant exposure of T cells to host antigens. Similar results were also observed using 2C T cells as a donor source (Figure 3b). In this system, transferred 2C T cells were present more than 60 days in BDF1 recipients treated with the combined therapy and were comparable in number to those transferred into control B6 recipients. Importantly, a significant increase of CD62Llow cells was detected in 2C T cells in BDF1 recipients compared with those in B6 recipients (Figure 3b). Our results thus demonstrate that donor-derived hematopoietic cells can repopulate in treated recipient mice in which antigen-experienced, host-reactive T cells persist long-term, without inducing GVHD.
Repopulation of donor-derived lymphocytes in GVHD-surviving recipients. Sublethally irradiated BDF1 recipients were given B6 spleen cells (7 × 107 cells) and subsequently treated with a combination of anti-CD40L and LTβR-Ig. More than 60 days later, the recipient spleen cells, LN cells (a), and thymocytes (b) were stained with mAb’s against indicated antigens conjugated with FITC or phycoerythrin and subsequently analyzed by flow cytometry. Similar data was obtained from eight independent mice surviving GVHD. Numbers in the figure represent the percentage of lymphocytes located in the same quadrants.
Persistence of host-reactive T cells in long-term GVHD survivors. (a) In naive B6 and GVHD-surviving mice (more than 60 days), spleen and LNs were stained with anti-CD3 and anti-CD62L mAb’s and examined for CD62L expression of CD3-positive cells. (b) Sublethally irradiated B6 or BDF1 recipient mice received 4 × 107 LN cells from 2C TCR transgenic mice on day 0. BDF1 recipients were treated with anti-CD40L (100 μg, on day 0) and LTβR-Ig (100 μg on days 0, 3, and 6), whereas control Ig was injected into B6 recipients. More than 60 days later, recipient spleen cells were stained with anti-CD8, 1B2, and anti-CD62L mAb’s. CD62L expression of CD8+1B2+ double-positive cells was examined. Numbers in the figure represent the percentage of 2C T cells (b) and the percentage of CD62Llow cells (a and b).
Tolerance induced by combined therapy did not affect T cell responses to nominal antigens. To examine whether the tolerance is “infectious,” we used OT-I T cells, which express a transgenic TCR that uniformly reacts with OVA antigen in the context of H-2Kb (30), to facilitate isolation of antigen-reactive T cells. A mixture of OT-I T cells and B6.Ly5.1 splenocytes was transferred into BDF1 recipient mice, followed by combined treatment with LTβR-Ig and anti-CD40L mAb to induce tolerance. As a control, OT-I T cells mixed with BDF1 splenocytes were transferred into BDF1 recipients in which GVHD was not induced. Transferred OT-I T cells were recovered from recipient spleen by enrichment of a subpopulation that was negative for both H-2Kd and Ly5.1. After in vitro restimulation with antigenic OVA peptide, OT-I T cells recovered from the tolerant recipients generated significant CTL activity against OVA peptide–pulsed EL4 cells and E.G7 cells, but not nonpulsed EL4 cells. CTL activity of these OT-I T cells was identical to that of OT-I T cells purified from control recipients (Figure 4). CTL activity of OT-I T cells can be inhibited when antigenic OVA peptide is administered to mice receiving transferred OT-I T cells (data not shown). These data demonstrate that T cell tolerance to allogeneic antigens induced by combined therapy does not inhibit the responsiveness of nonalloreactive T cells to other antigens.
Intact T cell responses to nominal antigen in the combined therapy. Sublethally irradiated BDF1 mice received a mixture of OT-I LN cells (3 × 107 cells) and either 3 × 107 BDF1 spleen cells (open circles) or B6.Ly5.1 spleen cells (filled circles) on day 0. Recipients of transferred OT-I LN cells and B6.Ly5.1 cells received anti-CD40L (100 μg, on day 0) and LTβR-Ig (100 μg on days 0, 3, and 6). On day 8, cell populations negative for both Ly5.1 and H-2Kd were enriched from recipient spleen cells by magnetic cell sorting. The purified cells (1.5 × 106 cells/ml) were stimulated with 10 ng/ml OVA peptide in the presence of irradiated B6 spleen cells (1.5 × 106 cells/ml) for 4 days. The CTL activity against nonpulsed EL4 cells, EL4 cells pulsed with 10 μg/ml of antigenic OVA peptide (EL4/pep), and E.G7 cells was assessed by 51Cr release assay. Results are expressed as mean ± SD.
Combined therapy induces an early and persistent anergy of host-reactive CTLs. The hallmark of T cell tolerance is its unresponsiveness to antigen in the presence of appropriate antigen-presenting cells (31). We first examined whether T cells isolated from tolerant mice can be induced to resume their CTL activity. As shown in Figure 5a, B6.Ly5.1 T cells, which were isolated from BDF1 mice 9 days after combined therapy and restimulated in vitro by allogeneic DBA/2 spleen cells as a source of H-2d antigens, failed to lyse H-2d+ target cells. In contrast, B6.Ly5.1 donor cells transferred into syngeneic B6 recipients mounted considerable CTL activity in an H-2d–specific manner after in vitro restimulation. Our results indicate that combined treatment with LTβR-Ig and anti-CD40L mAb can induce donor T cell anergy to allogeneic antigens in an early phase of T cell activation.
Induction of T cell anergy by combined treatment with LTβR-Ig and anti-CD40L mAb. (a) Sublethally irradiated BDF1 (filled circles) or B6 (open squares) mice were infused with B6.Ly5.1 splenocytes together with anti-CD40L and LTβR-Ig on day 0. On day 9, B6.Ly5.1+ cells were purified and stimulated with irradiated DBA/2 splenocytes for 5 days. CTL activity against P815 and EL4 was assessed. The same recipient mice were injected with splenocytes from BDF1 mice as the controls (open circles). (b) CTL activity of splenocytes from recipients that survived GVHDmore than60 days was assessed against indicated targets without in vitro culture (filled circles). Splenocytes from BDF1 recipients that had received B6 (open circles) or BDF1 splenocytes (open squares) for 10 days were used as controls. (c) Splenocytes from recipients survived more than 60 days (filled circles) were stimulated for 5 days as described above, and subsequently examined for CTL activity against indicated targets. As controls, splenocytes from naive B6 mice (open squares) or B6 BM–reconstituted BDF1 mice (filled squares) were used as responder cells. (d) Splenocytes from recipients that survived (more than 60 days) were stimulated as described above in the absence (dark gray bars) or presence (black bars) of IL-2. Naive B6 splenocytes were similarly stimulated in the absence (white bars) or presence (light gray bars) of IL-2. After 5 days, CTL activity against P815 cells was examined. Results are expressed as the mean ± SD of triplicate wells. E/T ratio: ratio of effector cells to target cells in CTL assay.
Host-reactive CTL activity was also assessed in tolerant mice more than 60 days after combined treatment was completed. No CTL activity against P815 target cells (H-2d cells) was detected, using spleen cells from tolerant mice, when the cells were directly used as effectors without further restimulation (Figure 5b). Upon restimulation with irradiated splenocytes from DBA/2 mice as a source of antigen-presenting cells expressing H-2d antigens, CTL activity specific to P815 target cells could be detected in spleen cells from tolerant mice. The level of CTL activity, however, was significantly lower than that in cells from the positive control, in which CTLs were generated from splenocytes of naive B6 mice 5 days after coculture with allogeneic DBA/2 spleen cells (Figure 5c). In addition, the decreased CTL activity in tolerant mice could be restored in vitro to a level comparable to that induced in naive B6 mice by addition of IL-2 (Figure 5d). As a negative control, spleen cells from BDF1 mice that had been reconstituted with T cell–depleted B6 BM cells did not induce any CTL activity (Figure 5c), probably because of negative selection of host-reactive T cells in the thymus.
Although cytolytic activity of host-reactive T cells in tolerant mice can be recovered in vitro by exposure to appropriate antigens, antigen-presenting cells, and cytokines, it is not known whether the same environment exists in vivo. To address this point, spleen cells from recipients tolerized by combined therapy were transferred into naive BDF1 recipient mice. Transfer of naive B6 splenocytes mediated profound H-2d–specific CTL activity in the recipients. In contrast, BDF1 mice receiving splenocytes transferred from the tolerized mice or from B6 BM–reconstituted BDF1 mice did not generate CTLs (Figure 6a), in spite of a comparable number of T cells in transferred cells (Figure 2a). Administration of 50,000 IU/day IL-2 (Figure 6a) or 50 μg of LPS (data not shown) did not reverse the tolerant state of allogeneic T cells. Furthermore, vigorous expansion of donor T cells (CD3+H-2Kd–), along with the elimination of host B cells (CD3–H-2Kd+) — typical consequences of acute GVHD — were observed after transfer of naive B6 spleen cells but not after transfer of B6-derived cells present in BDF1 hosts tolerized by combined therapy or by B6 BM reconstitution (Figure 6b). Therefore, despite recovery of CTL activity by in vitro manipulation, anergic T cells in the mice given combined therapy remained tolerant in vivo even in the presence of corresponding antigens and exogenous IL-2.
In vivo tolerance of host-reactive T cells in GVHD-surviving recipients. Spleen cells (5 × 107 cells) from recipient mice that survived GVHD for more than 60 days due to the combined treatment were injected intravenously into secondary BDF1 recipient mice on day 0 (open squares). In some mice, 50,000 IU of IL-2 was injected daily intraperitoneally from day 0 to day 10 (filled squares). As control, either naive B6 spleen cells (open circles) or spleen cells from B6 BM–reconstituted BDF1 mice (filled circles) were injected into BDF1 recipients. (a) After 10 days of cell transfer, recipient spleen cells were examined for CTL activity against P815 and EL4 cells without in vitro culture. Results are expressed as the mean ± SD of triplicate wells. (b) After 10 days of transfer of spleen cells from either naive B6 mice (left panel), GVHD-surviving mice (center panel), or B6 BM–reconstituted BDF1 mice (right panel), spleen cells of recipient mice were stained with mAb’s against indicated antigens. Numbers in figure represent the percentage of lymphocytes located in that quadrant.