Background The effect of transforming growth factor β (TGF-β) is cell type- and context-dependent. In the tumor microenvironment, TGF-β restricts T cells’ expansion, survival, and antitumor efficacy.1 2 Blocking of the TGF-β signaling enhanced the infiltration and antitumor responses of chimeric antigen receptor-T (CAR-T) cells and improved the cytotoxicity of tumor-infiltrating lymphocytes.3 4 However, TGF-β signaling is also required for IL-7Rα expression and the abrogation of TGF-β receptor expression led to the failed maintenance of peripheral CD4+ T cells,5 since IL-7 signaling is essential for homeostasis, persistence, and survival of naïve and memory T cells.6 7 Therefore we tested whether co-expression of IL-7 and dominant-negative TGFβRII (dnTGFβRII) could enhance the proliferation and antitumor efficacy of CAR-T cells and TILs isolated from primary hepatocellular carcinoma (HCC) tissues.
Methods T cells isolated from peripheral blood mononuclear cells or primary resected HCC tissues were activated by CD3/CD28 stimulation, lentivirally transduced with IL-7 and dnTGFβRII-expressing cassettes, and expanded ex vivo. For CAR-T cell preparation, anti-CD133 CAR was additionally transduced. Transgene expression was assessed by flow cytometry. In vitro, antitumor efficacy was analyzed by IFN-γ ELISA and real-time quantitative cytotoxicity assay with an IncuCyte instrument. The antitumor efficacy of the engineered T cells was further evaluated in a xenograft immunocompromised mouse model.
Results The expression of dnTGFβRII was analyzed with flow cytometry, and ELISA confirmed the secretion of IL-7 in TILs and anti-CD133 CAR-T cells (figure 1A-C). The genetically modified TILs revealed an increased activation level compared to unmodified TILs and the dnTGFβRII-expressing TILs as determined by IFN-γ ELISA (figure 1D). These engineered TILs also showed more potent and sustained killing efficacy against HCC cell line SK-Hep1 in the presence of TGF-β (figure 1E) in vitro and significantly higher tumor control efficacy in vivo (figure 1G). Similarly, IL-7 and dnTGFβRII co-expressing CAR-T cells also exhibited increased cytotoxicity against colorectal cancer cells HCT116 and HT-29 in the presence of TGF-β (figure 1F) in vitro and higher tumor control efficacy in vivo (figure 1H). Interestingly, a membrane-bound fusion protein, IL7:dnTGFβRII, linking IL-7 to the N terminus of dnTGFβRII further improved CAR-T cell proliferation when co-cultured with HT-29 cells. This finding prompts further investigation on this novel chimeric construct.
Conclusions A robust co-expression of IL-7 and dnTGFβRII on both TILs and CAR-T cells has been achieved. The combination of IL-7 signaling enhancement and TGF-β inhibition can synergistically improve T cells’ anti-tumor efficacy and their resistance to immunosuppression.
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