Recombinant mouse hvem fc

Murine OT-1 BTLA KO cells overexpressing BTLA WT or mutants were re-stimulated with either 10 ng/ml anti-mouse CD3 (Clone 145-2C11, BD Pharmingen™) alone or with recombinant mouse HVEM-Fc (R&D systems) plate-bound for 24 h. Supernatants were collected to quantify the secreted cytokines using a MILLIPLEX MAP Mouse CD8⁺ T Cell Magnetic Bead Panel (Millipore).

Murine OT-1 BTLA KO cells overexpressing BTLA WT or mutants constructs were re-stimulated with either 10 ng/ml anti-mouse CD3 (Clone 145-2C11, BD Pharmingen™) alone or with recombinant mouse HVEM Fc (R&D systems) plate-bound for 8 h prior to harvest with the cell lysis buffer (kindly provided by RPPA core facility at The University of Texas M.D. Anderson Cancer Center). The cell lysates were centrifuged at 14,000 rpm for 10 minutes at 4°C. The protein supernatant was quantified using protein assay kit (Thermo scientific). RPPA was processed and normalized as previously described (20 (link)). Differential fold expression of protein was analyzed using Linear models and empirical Bayes methods(21 (link)). Volcano plots were generated using R system. For human TIL, four TIL lines were stained with anti-CD8 (clone RPA-T8, BD Pharmingen™), anti-BTLA (clone J168, BD Pharmingen™), and Sytox blue (Molecular Probe™) under aseptic condition. The cells were sorted based on expression of CD8⁺BTLA⁺ using FACSAria (BD Biosciences). On the next day, sorted TIL were re-stimulated with anti-human CD3 (clone OKT-3, eBioscience) with or without recombinant human HVEM-Fc (R&D systems) plate-bound for 8 h prior to harvest with the cell lysis buffer. The protein samples were processed, normalized, and analyzed as similar to the mouse experiment described above.