Tetramer staining and sorting

PBMC were thawed in warm thaw media (R10 with 2 μl/ml Benzonase (Millipore, Billerica, MA) sterile-filtered) and centrifuged at 700 × g for 5 min. The supernatant was decanted, and the cells were resuspended in R10 and counted by Trypan Blue (Millipore, Billerica, MA) exclusion. The cells were centrifuged at 700 × g for 5 min and plated at a density of 1 million cells per well in a 96-well U-bottom plate. A portion of the PBMC were resuspended in R10 at a density of 2 million cells per ml in 50 ml conicals with the caps lightly in place and rested overnight at 37 °C in humidified incubators supplemented with 5% CO₂. The PBMC in the 96-well plate were washed with FACS buffer (1× phosphate-buffered saline (PBS) (Gibco, Waltham, MA) supplemented with 0.2% bovine serum albumin (BSA) (Sigma, St. Louis, MO) and centrifuged at 700 × g for 3 min. Next, the cells were washed twice with PBS and stained with Aqua Live/Dead stain (cat. {"type":"entrez-nucleotide","attrs":{"text":"L34966","term_id":"522209","term_text":"L34966"}}L34966; Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Following a 15 min incubation at room temperature, the cells were washed twice in PBS. They were then blocked with human serum (Valley Biomedical, Winchester, VA) and FACS buffer mixed 1:1 for 10 min at 4 °C. The wells were washed twice with FACS buffer and then resuspended in 50 µl FACS buffer with 1 µl of unloaded CD1b tetramer and 1 µl of each SGL-loaded CD1b tetramer labeled with APC or ECD and incubated at room temperature for 40 min in the dark. After this incubation period, the cells were washed twice with FACS buffer and then labeled with anti-CD3 ECD (1:50 dilution) (cat. IM2705U; Beckman Coulter, Brea, CA), anti-CD4 APC Ax750 (1:50 dilution) (cat. A94685; Beckman Coulter, Brea, CA), and anti-CD8β BB700 (1:10 dilution) (cat. 745761; BD Biosciences, San Jose, CA) antibodies for 30 min at 4 °C. After two final washes in FACS buffer, the cells were fixed in 1% paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA) and acquired on a BD LSRFortessa running BD FACSDiva (BD Biosciences, San Jose, CA) equipped with blue (488 nm), green (532 nm), red (628 nm), violet (405 nm), and ultraviolet (355 nm) lasers.

T cell lines or transductants were plated at one million cells per well in a 96-well U-bottom plate. Cells were washed twice with PBS and resuspended with Live/Dead Fixable Aqua or with Live/Dead Fixable Green Dead Cell Stain Kit (cat. {"type":"entrez-nucleotide","attrs":{"text":"L34966","term_id":"522209","term_text":"L34966"}}L34966 or cat. {"type":"entrez-nucleotide","attrs":{"text":"L23101","term_id":"632938","term_text":"L23101"}}L23101, respectively; Life Technologies, Carlsbad, CA) per the manufacturer’s instructions. For this step and all subsequent steps, the cells were kept in the dark. Following a 15 min incubation, cells were washed twice with PBS and blocked with human serum (Valley Biomedical, Winchester, VA) prepared in FACS buffer (1× PBS (Gibco, Waltham, MA) supplemented with 0.2% BSA (Sigma, St. Louis, MO)) mixed 1:1 for 10 min at 4 °C. Cells were then resuspended in 50 µl of FACS buffer containing 1 µl of SGL-loaded CD1b and 1 µl of mock-loaded control CD1b tetramers, then incubated at room temperature for 60 min. The tetramer titers were determined prior to use in the present study. Following a 15 min incubation at room temperature, the cells were washed twice in PBS and then stained with anti-CD3 ECD (1:50 dilution) (cat. IM2705U; Beckman Coulter, Brea, CA), CD4 APC-Ax750 (1:50 dilution) (cat. A94685; Beckman Coulter, Brea, CA) and anti-CD8α PerCP Cy5.5 (1:10 dilution) (cat. 341051; BD Biosciences, San Jose, CA). When T cells transduced with exogenous TCR were used, anti-mouse TCR β chain APC (1:50 dilution) (cat. 553174; BD Biosciences, San Jose, CA) was also included in the antibody cocktail. The optimal titers of all antibodies were determined prior to use. After two final washes in FACS buffer, the cells were fixed in 1% paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA) and acquired on a BD LSRFortessa as above.

The transduced T cells were sorted to purity using a modified version of the tetramer staining method described above. After the antibody stain, the transduced T cells were resuspended in 200 μl of FACS buffer and tetramer-positive T cells were sorted at the UW Department of Immunology Flow Cytometry Core using a FACS Aria II (BD Biosciences, San Jose, CA) cell sorter equipped with blue (488 nm), red (641 nm), and violet (407 nm), lasers. Cells were sorted into 3 ml of TCM in 4 ml FACS tubes.