Blockade of LIGHT/LTbeta and CD40 signaling induces allospecific T cell anergy, preventing graft-versus-host disease

Abstract

Previous studies have shown that blockade of LIGHT, a T cell costimulatory molecule belonging to the TNF superfamily, by soluble lymphotoxin beta receptor-Ig (LTbetaR-Ig) inhibits the cytotoxic T lymphocyte (CTL) response to host antigenic disparities and ameliorates lethal graft-versus-host disease (GVHD) in a B6 to BDF1 mouse model. Here, we demonstrate that infusion of an mAb against CD40 ligand (CD40L) further increases the efficacy of LTbetaR-Ig, leading to complete prevention of GVHD. We further demonstrate that alloantigen-specific CTLs become anergic upon rapid expansion, and persist in the tolerized mice as a result of costimulatory blockade. Transfer of anergic CTLs to secondary F1 mice fails to induce GVHD despite the fact that anergic CTLs can be stimulated to proliferate in vitro by antigens and cytokines. Our study provides a potential new approach for the prevention of lethal GVHD.

Figure 1

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-2^d) and EL4 (H-2^b) 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.

Figure 2

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.

Figure 3

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 CD62L^low cells (a and b).

Figure 4

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-2K^d 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.

Figure 5

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.

Figure 6

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.