T Cell enrichment

Inguinal and mesenteric lymph nodes and spleen were sterilely excised from non-treated C57BL/6 mice and placed on a 100 μm filter and dissociated using the plunger of a syringe followed by several washing steps with stain buffer (1X PBS + 2% FBS). Single cell suspension was treated with ACK lysis solution for 5 minutes at room temperature before being washed. Cells were then incubated with Fc block (anti-CD16/32, 10 μg/ml, Tonbo Biosciences, San Diego, CA) for 15-minute on ice. Antibody cocktail was added to surface stain cells for 30 minutes on ice, followed by washing in stain buffer. The following biotinylated antibodies were added to this cocktail at 1:50 dilution: anti-CD161 (clone# PK136, Ref# 30–5941), anti-CD11c (clone# N418, Ref# 30–0114), anti-Ly6G (Clone# RB6-8C5, Ref# 30–5931), anti-TER119 (Clone# TER-119, Ref# 30–5921), anti-CD11b (Clone# M1/70, Ref# 30–0112), and anti-B220 (Clone# RA3-6B2, Ref# 30–0452) from Tonbo Biosciences. Cells were then washed and resuspended in magnetic streptavidin beads (Cat# 557812, BD Biosciences, Franklin Lakes NJ according to the manufacturer’s protocol. Cells were incubated under mild rotation at 4°C for 30 minutes and then the solution volume was brought up to 1 mL total. Tube was placed in BD IMAG Cell Separation Magnet (Cat# 552311 BD Biosciences, Franklin Lakes NJ) for 8 minutes and the negative fraction was taken and placed into a fresh tube on the IMAG. This was again incubated for 6 minutes and repeated one additional time. All negative fractions were combined, and a fraction of the cells were stained for flow cytometry analysis to confirm T cell purity was above 90%.