Clone cd28

CD3⁺ T cells were isolated from healthy donor PBMC by MACS (Milteny Biotec) and labeled with 10 µM cell proliferation dye eFluor 450 (eBioscience) at room temperature for 20 min. Then T cells were cocultured with in vitro generated MDSC in 100 µl L-lysine and L-arginine free RPMI medium with GlutaMAX (Thermo Fisher) supplemented with 150 µM L-arginine, 0.218 mM L-lysine hydrochloride (both Sigma Aldrich), 10% heat-inactivated FBS and 1% penicillin/streptomycin (both Thermo Fisher) for 4 days in 96-well round bottom plates (Sarstedt) precoated for 2 hours with anti-CD3 (1 µg/mL, clone OKT-3, eBioscience) and anti-CD28 antibodies (2 µg/mL, clone CD28.2, Beckman Coulter). The proliferation of T cells was assessed after 4 days of coculture by measuring proliferation dye eFluor 450 dilution using the BD FACSLyric flow cytometer.

The assay was performed according to the standardized Mye-EUNITER protocol as described.30 (link) Briefly, allogenic to monocytes CD3 T cells were isolated from healthy donor PBMC by MACS (Milteny Biotec) and labeled with 10 µM cell proliferation dye eFluor 450 (eBioscience) at RT for 20 min followed by the co-incubation for 4 days with monocytes prestimulated for 24 hours with rHSP90a (2 µg/mL). Cells were co-cultured in 96-well round-bottom plates (Sarstedt) precoated for 2 hours with anti-CD3 (1 µg/mL, clone OKT-3, eBioscience) and anti-CD28 antibodies (2 µg/mL, clone CD28.2, Beckman Coulter). T cell proliferation was measured by assessing proliferation dye eFluor 450 dilution by FACS-Lyric (BD Biosciences) flow cytometer.