In brief, ICOS and Eomes cDNAs were inserted into the polylinker site of the empty retroviral vector (a generous gift from Dr. Retroviruses (RV) were prepared as described ( 16) by using constructs linked with a GFP marker. Harvested splenocytes and total lymph node cells from PMEL-1 or PMEL-1ICOS −/− mice were briefly activated with 1 μg/mL hgp100 overnight in Click's medium supplemented with 10% (vol/vol) FBS and 1% (vol/vol) penicillin–streptomycin. Transferred Thy1.1 + CTLs collected from spleen and lymph nodes were then analyzed for intracellular cytokine production after brief stimulation with 50 ng/mL phorbol-12-myristate 13-acetate (PMA Sigma) plus 1 μmol/L ionomycin (Sigma) in the presence of monensin or were directly examined for expression of transcriptional factors, as described below. Then, CD8 + bead–purified cytotoxic T lymphocytes (CTL 6 million) were transferred into recipient Rag-1 −/− mice or into sublethally irradiated CD45.1 and ICOSL −/− mice (400 rad/mouse using a sealed irradiator with a Cs 137 source, MARK I irradiator from J.L Shepherd & Associates) that had been allowed to rest for more than 4 hours before the intravenous infusion of CTLs, followed by immunization with 50 μg of hgp100 25-33 emulsified in CFA (Complete Freund's Adjuvant) per mouse on the next day. Harvested splenocytes and total lymph node cells from PMEL-1 or PMEL-1ICOS −/− mice were briefly activated with 1 μg/mL hgp100 overnight. In conclusion, our preclinical studies provide a strong rationale for combining anti–CTLA-4, anti–PD-1, and ACT to treat solid malignancies. Collectively, our study showed the critical role of the ICOS signaling in sustaining effector function of tumor antigen–specific PMEL-1 Tg + CD8 T cells. Deletion of ICOS largely abolished the therapeutic benefits of ACT combined with immune-checkpoint blockade (ICB), which coupled with the selective downregulation of Eomesodermin (Eomes), IFNγ, and perforin in PMEL-1 Tg + CD8 T cells. To determine whether ICOS played an important role in PMEL-1 Tg + CD8 T cells, we generated ICOS −/−PMEL-1 mice. We demonstrated that the combination of anti–CTLA-4 plus anti–PD-1 therapy with ACT consisting of ICOS +PMEL-1 CD8 + T cells, compared with monotherapy using either anti–CTLA–4 or anti–PD-1 or ACT alone, led to improved antitumor responses and durable survival benefit. In this model, we noted that the gp100 peptide induces expression of CTLA-4, PD-1, and ICOS on transgenic CD8 T cells. To test our hypothesis, we used the PMEL-1 transgenic murine model, which consists of CD8 + T cells bearing a specific T-cell receptor for the gp100 antigen ( 12). In our study, we hypothesized that the combination of anti–CTLA-4 plus anti–PD-1 would improve the efficacy of ACT and provide a survival benefit. Our study provides a foundation of testing combinatorial therapy of ACT of CD8 T cells and dual blocking of CTLA-4 and PD-1 in patients with melanoma. Together, our data provide direct evidence that ACT combined with immune-checkpoint therapy confers durable antitumor responses, which largely depended on CD8 + T-cell–intrinsic expression of ICOS. Higher expression of IFNγ and Eomes was noted in human ICOS hi CD8 + T cells compared with ICOS low counterparts. Using PMEL-1ICOS −/− mice, we showed that deletion of the inducible T-cell costimulator (ICOS) receptor abolished the therapeutic benefits, with selective downregulation of Eomesodermin (Eomes), interferon gamma (IFNγ), and perforin. Treatment with anti–CTLA-4 plus anti–PD-1 compared with monotherapy resulted in durable antitumor responses, enhanced effector function of ACT, utilizing PMEL-1 transgenic (Tg +) CD8 + T cells, and improved survival. To improve the efficacy of ACT, we combined ACT with dual blockade of CTLA-4 and PD-1. Failures with this therapy are primarily due to inadequate infiltration and poor function of adoptively transferred cells in the tumor microenvironment. Adoptive transfer of tumor-reactive T cells (ACT) has led to modest clinical benefit in the treatment of solid tumors.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |