Session: 703. Cellular Immunotherapies: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Biological therapies, GVHD, Diseases, Immune Disorders, immune mechanism, Therapies, Immunotherapy, Biological Processes, Transplantation
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.
Disclosures: No relevant conflicts of interest to declare.
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