Modulating Donor T Cell Graft-Versus-Host and Graft-Versus-Tumor Reactivity By Stat6

Stimulation of T helper-2 (Th2) pathway regulates acute graft-versus-host disease (GVHD) and preserves the graft-versus-tumor (GVT) effect after bone marrow transplantation (BMT). Cellular proteins associated with Th2 maturation include those induced by the transcription factor STAT6, which promotes immune regulation and alleviates GVHD in different models of BMT. In this study, we investigated whether augmentation of the Th2 pathway in donor T cells by genetic overexpression of STAT6 regulates GVHD and preserves GVT activity.

We applied a BMT and acute GVHD model with major MHC I/II mismatch (H2b into H2d) after myeloablative preparation with total body irradiation (850 cGy). We used donor T cells from STAT6VT+ transgenic mice on the H2b background whose T cells overexpress a constitutively active form of STAT6, driven by the CD2 promoter. We also used STAT6VT- nontransgenic mice (H2b) as donor T cell source and C57BL/6 wildtype (WT)(H2b) mice to provide donor T cell-depleted bone marrow (TCD-BM) cells as well as splenic donor T lymphocytes. WT BALB/c mice (H2d) constituted BMT recipients. Luciferase expressing A20 leukemia/lymphoma cell line syngeneic with BALB/c recipients (H2d) was used for graft-versus-tumor (GVT) studies. Tumor load assessment in BMT recipients was performed using bioluminescence imaging.

T cells from STAT6VT+ transgenic mice generated high quantities of Th2 and immune regulatory cytokines, such as interleukin-4 (VT+: 2.8 ± 0.2 ng/ml vs. VT-: 0.1 ± 0.03 ng/ml; p<0.0001) or interleukin-10 (VT+: 700 ± 89 pg/ml vs. VT-: 107 ± 10 pg/ml; p<0.0001)(Data are representative examples from at least 4 independent experiments for each cytokine and calculated as mean± SD from multiple samples). Cellular expression of immune regulatory TGF-beta propeptide (latency-associated peptide) was also increased on Foxp3+ CD4 regulatory T cells (Tregs) of STAT6VT+ mice, as shown by flow cytometry (MFI VT+: 517 ± 200 vs. MFI VT-: 119 ± 100; p<0.05). As also demonstrated by flow cytometry, Foxp3+ CD4 Tregs constituted a higher proportion of T cells from STAT6VT+ T mice (20 ± 5%) compared to T cells from STAT6VT- mice (8 ± 3%) in the mesenteric lymph nodes (p<0.001), and unlike splenic T cells, which displayed similar Foxp3+ CD4 Treg percentages (VT+: 12 ± 2 % vs. VT-: 12 ± 3; p:NS).

Most CD3+ T cells from STAT6VT+ mice exhibited an effector memory phenotype, with CD62L-CD44+ cells constituting ~75% of T lymphocyte pool, compared to ~25% of wildtype C57BL/6 or STAT6VT- T cells (p<0.05). Strikingly, STAT6VT+ transgenic splenic T lymphocytes functioned as effector memory cells after BMT in that they did not cause GVHD and promoted survival of BMT recipients (Figure 1A), but still preserved GVT reactivity (Figure 1B): Tumor-related mortality in BALB/c recipients (H2d) of donor wildtype (WT) C57BL/6 T cell-depleted bone marrow (TCD-BM) cells was 100% compared to 0% tumor-related mortality of BALB/c recipients of donor WT C57BL/6 TCD-BM and STAT6VT+ splenic T cells (p<0.05). Furthermore, and unlike memory T cells, STAT6VT+ T lymphocytes regulated GVHD when co-transferred with GVHD-causing WT splenic C57BL6 T cells and yet maintained the GVT activity: Mice receiving STAT6VT+ T cells alongside WT splenic T cells displayed a phenotype of regulated GVHD with no GVHD- or tumor-related mortality (100% survival), compared to 100% GVHD-related mortality for mice receiving only WT C57BL/6 T cells with WT C57BL/6 TCD-BM cells (p<0.05). This latter group did not show any sign of A20 tumor load by bioluminescence imaging. BALB/c recipients of C57BL/6 WT TCD-BM donor cells were used as another control group, which - as expected - did not display signs of GVHD but all of them died with high A20 tumor load, as shown by bioluminescence imaging (0% GVHD-related mortality but 100% tumor-related mortality). These data suggest that donor T cells expressing a constitutively active STAT6 protein do not only preserve GVT without causing GVHD (similar to donor memory T cells), but STAT6VT+ donor T cells also regulate GVHD and preserve GVT.

Taken together, our data indicate a crucial regulatory role of donor T cell STAT6 expression in the context of memory T cell- and Th2-dependent regulation of GVHD/GVT and suggest that engineering donor T cells to express active STAT6 can have a similar effect, improving the outcome of BMT.