Regardless of whether promotion info chemotherapeutic drugs or zoledronate were used to sensitize CICs, V��9V��2T cells killing of these targets was TCR- or NKG2D-mediated: consistent with our previous report [27] chemotherapy-sensitized colon CICs were killed following NKG2D-mediated recognition and TRAIL/DR5 interaction, while both mechanisms were dispensable to the cytotoxicity of zoledronate-sensitized colon CICs, which were almost exclusively killed by TCR-mediated interaction and the perforin/granzyme pathway. Previous studies have highlighted the importance of NKG2D-MICA/B interactions for tumour cell recognition and effective cytotoxic activity by V��9V��2T cells [35]�C[44].
The difference between NKG2D-mediated recognition of chemotherapy-sensitized colon CICs and TCR-mediated recognition of zoledronate-sensitized CIC targets cannot be explained differential expression of MICA/B or ULBPs since neither 5-FU nor DXR changed constitutive expression levels of these molecules. It is likely that phosphoantigens production/expression by colon CICs is very low, below the threshold required for efficient recognition by the reactive V��9V��2 TCR, hence target recognition only occurs through NKG2D: the finding that colon CICs become sensitive to V��9V��2T cell cytotoxicity upon exposure to zoledronate [27], which enhances phosphoantigen accumulation and production, supports this possibility. We conclude that in vivo activation of V��9V��2T cells or adoptive transfer of ex vivo-activated V��9V��2T cells, together with or soon after administration of certain chemotherapeutic drugs may substantially increase their anti-tumor effects.
Additional clinical studies are thus needed to assess the efficacy of this combinatory therapy, possibly including the novel �æ� T cell-based immunotherapeutic approach that ex-vivo expansion of polyclonal �æ� T cells followed by introduction of a CD19-specific chimeric antigen receptor render them bispecific and more efficient in killing of CD19+ tumor cell lines in vitro and in xenografts [45]. Materials and Methods Peripheral Blood and Colon Cancer Samples Human peripheral blood mononuclear cells (PBMC) and colon cancer tissues were obtained in accordance with the ethical standards of the institutional committee of human experimentation from patients undergoing a colon resection for colon adenocarcinoma.
Histological diagnosis was based on microscopic features of carcinoma cells determining the histological type and grade. PBMC were isolated from colon cancer patients by density gradient centrifugation using Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) Dacomitinib and were cryopreserved in 80% RPMI 1640 (Life Technologies, Monza, Italy), 10% DMSO (Sigma, St. Louis, MO) and 10% heat-inactivated fetal calf serum (FCS, Life Technologies). According to Italian rules (Article 13 of Legislative Decree no.