Tumor infiltrating lymphocytes (TILs) express LAG-3 detected using ab252271.

Method: Freshly dissociated single cell suspensions of renal cell carcinoma TILs are incubated with ab252271 at 5 µg/ml and anti-MHC Class II molecules (PE) for 30 mins and washed twice in saline buffer.

Additional staining with anti-CD3 allowed a gate analysis of total T cells.

The LAG-3 and MHC II profiles of CD3⁺-gated cells for 3 patients are shown.

Specific inhibition of 17B4 staining.

Method: ab252271 at 10 µg/ml was preincubated with a specific peptide epitope (208b) or a control tetanus toxoid (TT) peptide at different molarities prior to staining of TILs. Stained cells are then analyzed by FACS.

LAG-3 expression on activated human peripheral blood mononuclear cells (PBMC) detected with ab252271.

Method: T lymphocytes from human PBMC are stimulated with 1 µg/ml of PHA for three days. Then, after seven days of culture, 3x10⁶ three-days PHA-activated human PBMC are treated with ab252271 or FITC coupled isotype-matched (IgG1) control MAb (used at a saturating dilution of 1/800 and 1/150 respectively) for 30 mins at 4°C in RPMI 1640 and washed twice with 1x PBS. Stained cells are then analysed by FACS.

Expression of LAG-3 on CD4⁺ and CD8⁺ subpopulations of tumour infiltrating lymphocytes (TILs) detected with ab252271.

Method: TILs from a dissociated renal cell carcinoma sample, stained with 5 µg/ml ab252271 and FITC-coupled anti-CD4 or -CD8, are analyzed by a two-colour FACS analysis. Additional staining with anti-CD3 allowed a gate analysis of total T cells. Values indicate percentages in each quadrant.