Gemcitabine and cisplatin were purchased from Pfizer and Fresenius Kabi, respectively. Mouse anti-PD1 antibody (clone RMP-014), anti-CTLA-4 (clone 9D9), anti-CD4 (clone GK1.5), and anti-CD8β (clone 53–5.8) were purchased from BioXcell (Lebanon, NH). Gemcitabine and cisplatin were administered intraperitoneally (i.p., 60 mg/kg for Gemcitabine and 0.3 mg/kg for cisplatin twice/week), anti-mouse PD1 antibody and anti-CTLA-4 antibody were administered by retrobulbar injection (10 mg/kg, every three days), and anti-CD4 and anti-CD8β depleting antibodies were administered by intraperitoneal injection (10 mg/kg, thrice a week).
For adoptive cell transfer studies, we enriched CD8+ T-cells from Cxcr3−/−/C57Bl/6 mice or non-transgenic C57Bl/6 mice to 80% purity using a negative selection method (Stemcell Technologies, Vancouver, Canada). Then, cells were sorted to 98% purity using AriaII FACS sorter in RPMI/with 10% FBS. Cells were washed with PBS twice and concentrated to 1×106 cells per 100μl. Cells were transferred via tail vein to Rag1−/−/C57Bl/6 mice two days before the ICC implantation into the liver.
For adoptive cell transfer studies, we enriched CD8+ T-cells from Cxcr3−/−/C57Bl/6 mice or non-transgenic C57Bl/6 mice to 80% purity using a negative selection method (Stemcell Technologies, Vancouver, Canada). Then, cells were sorted to 98% purity using AriaII FACS sorter in RPMI/with 10% FBS. Cells were washed with PBS twice and concentrated to 1×106 cells per 100μl. Cells were transferred via tail vein to Rag1−/−/C57Bl/6 mice two days before the ICC implantation into the liver.