Anti pe magnetic beads

Tumors were harvested and prepared for cell isolation as previously described by us ^{8 (link),57 (link)}. In brief, tumors were minced in cold DMEM with 1 g/L glucose. Tumors were further digested using a mechanical tissue homogenizer (Miltenyi). Samples were incubated at 37°C with 5 ml Collagenase T2 (2 mg/ml, Worthington), 1 mL neutral buffered protease (2.5 U/ml, Worthington), and 75 μL deoxyribonuclease (1 mg/mL, Worthington) for 75 minutes. Red blood cells were lysed with 1X Pharmlyse B (BD PharMingen). Cells were suspended in FACS Buffer (degassed phosphate-buffered saline containing 2 mM EDTA and 0.5% BSA), Fc receptors were blocked with Fc Block (Miltenyi), and 10 μg PE-conjugated anti-PECAM1 antibodies were added for 20 minutes. Cells were washed and resuspended with anti-PE magnetic beads (Miltenyi) for 15 minutes. Cells were then washed and passed over a magnetic column, washed, and then eluted. Eluted cells were washed and plated in EC-Media. Cells were grown for several weeks, and the isolation was repeated until cultures reached ~ 99% PECAM1 positivity, at which point single cell clones were made by limiting dilutions.

Twenty million PBMCs in culture medium at a concentration of 150 million cells/ml were treated with dasatinib (49 (link)) for 10 min at 37°C, followed by staining with of PE-labeled pMHCII tetramers (20 μg/ml) at room temperature for 100 min. After tetramer staining, cells were then washed twice and incubated with anti-PE magnetic beads (Miltenyi Biotec) at 4°C for another 20 min. The cells were washed again and enriched using a magnetic column according to the manufacturer’s instructions (Miltenyi Biotec). Frequency was calculated as previously described (50 (link)). For unbiased FACS screen analysis, CRT_H2-labeled PBMCs and cells in the tetramer-bound fractions were both stained with antibodies against markers of interest (table S1) or corresponding isotype-matched monoclonal antibodies. A combination of the vital dye Via-Probe (BD Pharmingen) as a viability marker, CD19 (eBioscience), and CD14 (eBioscience) was used to exclude dead cells, B cells, and monocytes from the analysis, respectively. A FACSAria II was used for multiparameter analysis, and data were analyzed with FlowJo software (Tree Star, Inc.).

Freshly isolated PBMC were aliquotted for ex vivo analysis, with random assignment to HA control tests and citrulline peptide tests, where the total number of ex vivo tetramers tested was dependent on the number of PBMC available. All samples for ex vivo analysis were labeled, enriched (20 (link)) and subjected to phenotypic analysis as previously described (21 (link)). Briefly, 40 million PBMC in 200 µl of T cell culture medium were treated with dasatinib for 10 minutes at 37°C and then stained with 20 µg/ml PE-labeled tetramers at room temperature for 90 min. Cells were washed and incubated with anti-PE magnetic beads (Miltenyi Biotec) at 4°C for 20 minutes, washed again, and a 1/100th fraction saved for analysis. The other fraction was passed through a magnetic column (Miltenyi). Bound, PE-labeled cells were flushed and collected. Both enriched and non-enriched fractions were labeled with CD4 V500 (BD), CXCR3 Pacific Blue (BioLegend), CD45RO eFlour 650 NC (eBioscience), CD28 PE-Cy7 (BioLegend), CCR7 Alexa-700 (BD), CD25 APC (BD), CCR6 PerCP-Cy5.5 (BD), and CD14 (BioLegend), CD19 (BioLegend), & Annexin V (BD) conjugated in FITC. Samples were then run on a BD LSRII flow cytometer, and data was analyzed using FlowJo software version 9.6.2.