Anti cd44 buv395

In Vitro Memory Differentiation Memory OT-I T cells were generated as described previously (Balmer et al., 2016; van der Windt et al., 2013) . Briefly, the lymph nodes from MHC class I-restricted OVA-specific T cell receptor (OT-I) transgenic mice and the spleen of C57BL/6 mice were aseptically removed and incubated in liberase TL (Roche) for 30 min. After mashing through a 70 mm cell strainer (BD Biosciences), red blood cells were lysed with RBC Lysis Buffer Solution (eBioscience). The isolated cell suspensions were washed in RPMI medium (RPMI 1640 containing 10% FCS, 100 U/mL penicillin, 100 mg streptomycin, 0.29 mg/mL L-glutamine, 50 mM 2-Mercaptoethanol (Life Technologies)) and re-suspended to 10 6 cells/mL. The splenocytes and OT-I cells were pooled in a ratio of 1:1 and activated with OVA peptide (Eurogentec) at 10 À9 M at 37 C for 3 days. The cells were then washed and re-suspended to 2 3 10 6 cells/mL and cultured in the presence of IL-15 (10 ng/mL) at 37 C for another 3 days to generate OVA-specific memory CD8 + T cells. Phenotyping was performed using BUV395-anti CD44 (BD), APC-anti CD8, BV421-anti KLRG1, PE-anti-CD62L, APC-Cy7-anti CD43, BV421-anti PDL1, PE-anti-CD25, BV510-anti CD27 (all Biolegend) and PE-Cy5-anti CD127 (eBioscience).

The mouse model of foreign-body infection (John et al., 2011; Nowakowska et al., 2014) was used in the present study. Briefly, a sterile tissue cage (Angst + Pfister AG, Zurich, Switzerland) was implanted subcutaneously in the back of female C57BL/6 mice, 13 weeks old (janvierlab, France). After complete wound healing (2 weeks), cages were tested for sterility by culturing the aspirated tissue cage fluid (TCF). Teflon cages were infected with 785 CFU of MSSA ATCC 29213. The infection was confirmed at day 1 directly before treatment start by plating. Mice were i.p. treated twice a day with 5% glucose for 11 days. Tissue cage fluid (TCF) was aspirated at different time points (day 3, 6, 9, 11 and 14) and apropriate dilutions were plated to determine the amount of planktonic MSSA. Tissue cage fluids were then analyzed for acetate, calcium and phosphate levels and CD8 + T cells isolated by MACS-purification and frozen in Trizol Reagent (Thermo Fisher Scientific). For phenotyping, cells were stained with APC-anti CD8 (Biolegend), BUV395-anti CD44 (BD), PE-anti CD62L (Biolegend) antibodies and Zombie-Red Viability staining (Biolegend).