Granzyme B Contributes to the Optimal Graft-Versus-Tumor Effect Mediated by Conventional CD4+ T Cells

Abstract

Granzyme B (GzmB) is a key cytotoxic molecule utilized by T cells to kill pathogen-infected cells or transformed tumor cells. Previous studies using allogeneic hematopoietic cell transplantation (allo-HCT) murine models showed that GzmB is required for CD8⁺ T cells to cause graft-versus-host disease (GVHD). However, our recent study demonstrated that GzmB-mediated damage of CD8⁺ T cells diminished their graft-versus-tumor (GVT) activity. In this study, we examined the role of GzmB in GVT effect mediated by conventional CD4⁺CD25^- T cells (CD4⁺ Tcon). GzmB^-/-CD4⁺ Tcon cells exhibited decreased GVT activity compared to wild-type (WT) CD4⁺ Tcon cells, suggesting that GzmB is required for the optimal GVT activity of CD4⁺ Tcon cells. On the other hand, GzmB^-/- CD4⁺CD25⁺ regulatory T cells were as suppressive as WT regulatory T cells in suppressing GVT activity, which is consistent with our previous report showing that GzmB is not required for regulatory T cell-mediated suppression of GVHD. These results demonstrate that GzmB causes opposite impacts on GVT effect mediated by CD4⁺CD25^- versus CD8⁺ T cells. Interestingly, GzmB^-/- total T cells exhibited GVT activity equivalent to that of WT total T cells, suggesting that the opposite impacts of GzmB on the GVT effect of CD4⁺CD25^- versus CD8⁺ T cells may neutralize each other, which can only be observed when an individual T cell subset is examined. Importantly, these differential roles suggest that targeting GzmB in selective T cell subsets may have the potential to enhance the beneficial GVT effect.

Keywords: Allogeneic hematopoietic cell transplantation (allo-HCT); Graft-versus-host-disease (GVHD); Graft-versus-tumor (GVT) effect; Granzyme B (GzmB); T cells.

Figure 1. CD4⁺CD25⁻ T cell-mediated GVT effect is diminished in the absence of GzmB

(A) BALB/c (H-2d) host mice were irradiated with 800 rad at day −1. On day 0, host mice were injected with 2×106 TCD-BM cells alone or combined with 2×104 WT or GzmB−/− CD4+CD25− Tcon cells purified from C57BL/6 (H-2b) donor mice. On day 8, total cells harvested from the host spleens were analyzed with flow cytometry to examine GzmB expression in donor-derived T cells. Purified pre-HCT naive CD4+CD25− T cells were also examined as controls. Representative dot plots are gated on donor-derived T cells (H-2Kb+CD3+CD4+). (B) Summary data of the percentages of donor CD4+CD25− Tcon cells described in (A) that are GzmB+ are shown with each point representing an individual mouse. (C) BALB/c (H-2d) host mice were irradiated with 800 rad at day −1. On Day 0, mice were inoculated IV with 1×10⁵ luciferase-expressing A20 tumor cells and subsequently injected with 2×10⁶ TCD-BM cells alone or combined with 2×10⁴ WT or GzmB^−/− CD4⁺CD25⁻ Tcon cells isolated from C57BL/6 (H-2b) donor mice. (D) BALB/c (H-2d) host mice were irradiated with 800 rad at day −1. On Day 0, mice were inoculated IV with 1×10⁴ or luciferase-expressing A20 tumor cells and subsequently injected with 2×10⁶ TCD-BM cells alone or combined with 1×10⁴ WT or GzmB^−/− CD4⁺CD25⁻ Tcon cells isolated from C57BL/6 (H-2b) donor mice. Tumor burden was measured with bioluminescence imaging and shown as mean ± SD. 5–10 host mice were used in each group for each experiment. Two-way ANOVA was performed for statistical analyses.

Figure 2. GzmB deficiency did not alter the ability of CD4⁺CD25⁺ Treg cells to suppress GVT effect mediated by CD8⁺ and CD4⁺CD25⁻ T cells

(A) BALB/c (H-2d) mice were irradiated with 800 rad at day −1. At Day 0, mice were inoculated IV with 1×10⁶ luciferase-expressing A20 cells and subsequently given 2×10⁶ TCD-BM cells alone or combined with 1×10⁵ WT CD4⁺CD25⁻ T cells only or mixed with 8×10⁴ WT or GzmB^−/− CD4⁺CD25⁺ Treg cells isolated from C57BL/6 (H-2b) donor mice. (B) BALB/c (H-2d) mice were irradiated with 800 rad at day −1. At Day 0, mice were inoculated IV with 1×10⁶ luciferase-expressing A20 cells and subsequently given 2×10⁶ TCD-BM cells alone or combined with 4×105 GzmB^−/− CD8⁺ T cells only or mixed with 9×10⁴ WT or GzmB^−/− CD4⁺CD25⁺ Tregs isolated from C57BL/6 (H-2^b) donor mice. Tumor burden was measured with bioluminescence imaging and shown as mean ± SD. 5–10 host mice were used in each group for each experiment. Two-way ANOVA was performed for statistical analyses.

Figure 3. GzmB^−/− total T cells exhibited equivalent GVT activity to that of WT total T cells

(A) Before allo-HCT, Pan T cells isolated from C57BL/6 WT or GzmB^−/− donor mice were examined by flow cytometry for composition of CD8⁺, CD4⁺, CD4⁺Foxp3⁺ subpopulations and phenotypic differentiation via CD44 and CD62L expression. (B) BALB/c (H-2^d) mice were irradiated with 800 rad at day −1. At Day 0, mice were inoculated IV with 1×10⁵ luciferase-expressing A20 cells and subsequently given 2×10⁶ TCD-BM cells alone or combined with 5×10⁴ WT or GzmB^−/− Pan T cells isolated from 129/SvJ (H-2^b) donor mice. (C) BALB/c (H-2^d) mice were irradiated with 800 rad at day −1. At Day 0, mice were inoculated IV with 1×10⁵ luciferase-expressing A20 cells and subsequently given 2×10⁶ TCD-BM cells alone or combined with 10×10⁴ WT or GzmB^−/− Pan T cells isolated from 129/SvJ (H-2^b) donor mice. Tumor burden was measured with bioluminescence imaging and shown as mean ± SD. 5–10 host mice were used in each group for each experiment. Two-way ANOVA was performed for statistical analyses.