Granzyme B is not required for regulatory T cell-mediated suppression of graft-versus-host disease

Abstract

Regulatory T (T(reg)) cells can suppress a wide variety of immune responses, including antitumor and alloimmune responses. The mechanisms by which T(reg) cells mediate their suppressive effects depend on the context of their activation. We previously reported that granzyme B is important for T(reg) cell-mediated suppression of antitumor immune responses. We therefore hypothesized that granzyme B may likewise be important for suppression of graft-versus-host disease (GVHD). We found that allogeneic mismatch induces the expression of granzyme B in mixed lymphocyte reactions and in a model of graft-versus-host disease (GVHD). However, wild-type and granzyme B-deficient T(reg) cells were equally able to suppress effector T (T(eff)) cell proliferation driven by multiple stimuli, including allogeneicantigen-presenting cells. Surprisingly, adoptive transfer of granzyme B-deficient T(reg) cells prevented GVHD lethality, suppressed serum cytokine production in vivo, and prevented target organ damage. These data contrast strikingly with our previous study, which demonstrated that granzyme B plays a nonredundant role in T(reg) cell-mediated suppression of antitumor responses. Taken together, these findings suggest that targeting specific T(reg) cell-suppressive mechanisms, such as granzyme B, may be therapeutically beneficial for segregating GVHD and graft-versus-tumor immune responses.

Figure 1

Alloactivated T_reg cells express granzyme B during in vitro MLRs. The 129/SvJ WT (H-2K^b) splenocytes were cultured with irradiated (2000 cGy) Balb/c WT (H-2K^d) splenocytes in complete medium supplemented with 50 U/mL IL-2. At various time points, splenocytes were harvested and analyzed by flow cytometry for granzyme B expression. (A) Representative flow plots of granzyme B expression, gated on naive and day 6 MLR-stimulated CD8⁺ T cells. (B) Representative flow plots of granzyme B expression, gated on naive and day 6 MLR-stimulated CD4⁺Foxp3⁺ T_reg cells. (C) Summary graph of percentage granzyme B–expressing T_reg cells (n = 3 independent MLR cultures per time point).

Figure 2

Alloactivated T_reg cells express granzyme B in a mouse model of GVHD in vivo. Lethally irradiated Balb/c mice were reconstituted with 2 × 10⁶ bone marrow cells and 2 × 10⁶ T cells derived from 129/SvJ WT mice. Splenocytes were harvested from recipient mice on days 3 to 6 for flow cytometric analysis of granzyme B expression in donor-derived T_reg cells. (A) Representative flow plots, gated on H-2K^b+Foxp3⁺ T_reg cells, are shown. (B) Summary graph of percentage granzyme B–expressing T_reg cells is shown (n = 3 mice per time point).

Figure 3

Granzyme B is not required for T_reg cell–mediated suppression of ConA-activated and alloactivated T_eff-cell proliferation in vitro. (A) Suppression of CD4⁺CD25⁻ T_eff-cell proliferation stimulated by ConA in the presence of syngeneic T cell–depleted, irradiated APCs. (B) Representative flow plot of granzyme B expression, gated on wild-type T_reg cells cultured for 3 days with ConA-activated T_eff cells under maximal suppression conditions (1:1). (C) Dose-dependent suppression of 129/SvJ CD4⁺CD25⁻ T_eff-cell proliferation stimulated by fully mismatched Balb/c T cell–depleted APCs. (D) Representative flow plot of granzyme B expression, gated on wild-type T_reg cells cultured for 5 days with alloactivated T_eff cells under maximal suppression conditions (1:1). All data shown are representative of 3 independent experiments.

Figure 4

In vivo alloactivated T_reg cells do not require granzyme B to suppress T_eff-cell proliferation ex vivo. (A) Experimental protocol for generation of WT and Gzmb^−/− GVHD-activated T_reg cells: 2 × 10⁶ T cells from 129/SvJ WT or Gzmb^−/− FIG reporter mice were injected intravenously in lethally irradiated (900 cGy) Balb/c hosts, together with 2 × 10⁶ bone marrow cells from WT (non-FIG) mice. Four days after transplantation, splenic WT or Gzmb^−/− CD4⁺GFP⁺ T_reg cells were sort-purified and cultured ex vivo with DDAO-SE–stained CD4⁺CD25⁻ T_eff cells for 3 days. T cells were either unstimulated or stimulated with CD3/CD28 beads. (B) Representative flow plot and histogram of T_eff-cell proliferation (ie, loss of DDAO-SE staining) in the presence or absence of CD3/CD28 beads. (C) Dose-dependent inhibition of T_eff-cell proliferation mediated by wild-type or Gzmb^−/− GVHD-activated T_reg cells. (D) Summary graph of normalized data from 3 independent experiments.

Figure 5

T_reg cells do not require granzyme B to rescue hosts from GVHD lethality or to prevent GVHD target organ damage. Lethally irradiated (900 cGy) Balb/c mice received 2 × 10⁶ 129/SvJ TCD BM cells with or without 4 × 10⁵ 129/SvJ CD25⁻ T_eff cells (both CD4⁺ and CD8⁺) and either (A) 4 × 10⁵ or (B) 2 × 10⁵ wild-type or Gzmb^−/− CD4⁺CD25⁺ T_reg cells. Kaplan-Meier survival curves of recipient mice, pooled from 2 independent experiments (n = 10 mice per group), are shown. (C) Seven days after transplantation, 3 mice per experimental group (as outlined in panel A) were killed, and portions of lung, liver, and gut were prepared for histopathologic analysis. There was no statistically significant difference between groups receiving WT or Gzmb^−/− T_reg cells.

Figure 6

T_reg cells do not require granzyme B to suppress production of GVHD-associated cytokines in vivo. Lethally irradiated (900 cGy) Balb/c mice received 2 × 10⁶ 129/SvJ TCD BM cells with or without 4 × 10⁵ 129/SvJ CD25⁻ T_eff cells (both CD4⁺ and CD8⁺) and 4 × 10⁵ wild-type or Gzmb^−/− CD4⁺CD25⁺ T_reg cells. Seven days after transplantation, serum was harvested and analyzed via cytometric bead array for the production of (A) IL-2, (B) IL-4, (C) IL-5, (D) IL-10, (E) granulocyte-macrophage colony-stimulating factor, and (F) interferon-γ. There was no statistically significant difference between groups receiving WT or Gzmb^−/− T_reg cells.

Figure 7

Model-dependent role of granzyme B in T_reg cell–mediated suppressive function. Schematic illustrating the differential phenotypes observed with Gzmb^−/− Treg cells in suppressing antitumor responses and GVHD.