Anti nk1.1 pk136

YAC-1 cells were labelled with 0.5 μM carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen) for 20 min at room temperature. Subsequently, the labelled YAC-1 cells were incubated with enriched NK cells at effector-to-target ratios as indicated for a total time of 4 h 30 min. Anti-CD107a antibody (1D4B; BD Biosciences) was present from the beginning of the coculture. After the assay, the cells were stained with anti-NK1.1 (PK136; BioLegend), anti-CD3 (145-2C11), and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen). The percentages of live target cells were calculated based on the numbers of cells negative for the live/dead marker in each sample over total CFSE positive cells in the samples without NK cells. All experiments were run in triplicates.

For enrichment of CD4⁺ T cells, total splenocyte samples from WT and Rictor^−/− mice at day 8 after infection with LCMV were subjected to depletion of cells that were positive for lineage markers (Lin⁺ cells) using biotin-conjugated antibodies [anti-CD8 (53–6.7), anti-B220 (RA3-6B2), anti-CD11c (N418), anti-Gr-1 (RB6-8C5), anti-TER119 (TER-119), and anti-NK1.1 (PK136), all from Biolegend] coupled to the BeaverBeads Mag500 Streptavidin Matrix (22302, Beaver). The surfaces of the Lin⁻ cells were then stained with anti-CD4, anti-CD44, anti-GITR, anti-CD25, and anti-CXCR5 to identify T_FH cells. Next, 4 × 10⁵ T_FH cells from WT or Rictor^−/− mice were loaded into the upper chamber of a 24-well transwell plate (5-µm pore, Corning), and 600 µl of chemotaxis medium supplemented with or without the CXCL13 (4 µg/ml, 4583906, Biolegend) was added to the lower chamber. The cells were allowed to migrate for 3 h in a 5% CO₂ incubator at 37°C. Then, all the migrated cells were collected from the lower chamber, and the numbers of migrated T_FH cells were determined by flow cytometry (FACS Canto II). Based on the absolute number of T_FH cells, the “net migration (% of input)” was calculated as follows: Net migration (% of input) = (# of migrated T_FH cells to CXCL13 − # of migrated T_FH cells in the absence of CXCL13)/(# of T_FH cells in the input sample).