Ifnγ af647

Cells used for in vitro or in vivo cytokine analysis were stimulated with PMA (20 ng/ml; Sigma-Aldrich), Ionomycin (1 µg/ml; Sigma-Aldrich), and Brefeldin A (2 µg/ml; Sigma-Aldrich) for 4 hr prior to flow staining (Lu et al., 2015 (link)). For intracellular staining, cells were fixed and permeabilized using the Mouse FOXP3 Buffer Set (BD) per the manufacturer’s protocol. The fluorophore-conjugated antibodies used in this study were as follows: Live/Dead Fix Blue (Invitrogen), CD3 PerCPCy5.5 (Biolegend), TCRb PE/Cy7 (Biolegend), CD4 PB (Biolegend), CD4 AF700 (Biolegend), CD8 BV650 (Biolegend), CD25 BV421 (Biolegend), FOXP3 AF488 (Biolegend), ROR gamma T PE (Invitrogen), TCF1 AF647 (Cell Signaling Technologies), TCF1 PE (Biolegend), IFNγ AF647 (Biolegend), IL17A FITC (Biolegend), and IL17A PE (ebioscience). Samples were analyzed using BD LSR II flow cytometer (BD Biosciences) and FlowJo software (TreeStar).

The I-A^b+ B cell lymphoma line LB27.4 was purchased from the American Type Culture Collection. The following FACS antibodies were used in this study: Fc Block, CD8a BV 421, CD45.2 BV 510, B220 BV 605, CD127 AF 488, CD45.1 PE, KLRG1 PE-Cy7, CD25 AF 647, TNF-α AF 488, and IFN-γ AF 647 (all from Biolegend). Anti-alpha-adducin (ab40760) from Abcam was used for western blots. Anti-PKC-θ (C-18), anti-alpha-adducin (H-100; used for microscopy), and anti-phospho-alpha-adducin (Ser 726) antibodies were from Santa Cruz Biotechnology. CellTrace Violet, CellTrace CFSE, and AF 568-labeled phalloidin were from ThermoFisher. Class I (257-264) and Class II (323-339) ovalbumin peptides were from AnaSpec. GolgiPlug was from BD Biosciences. Anti-CD3 (145-2C11) and anti-CD28 (37.51) were from Bio-X-Cell.

Cells used for in vitro or in vivo cytokine analysis were stimulated with PMA (20ng/mL; Sigma Aldrich), Ionomycin (1μg/mL; Sigma Aldrich), and Brefeldin A (2μg/mL; Sigma Aldrich) for 4 hours prior to flow staining (W. Lu et al., 2015 (link)). For intracellular staining, cells were fixed and permeabilized using the Mouse FOXP3 Buffer Set (BD) per the manufacturer’s protocol. The fluorophore-conjugated antibodies used in this study were as follows: Live/Dead Fix Blue (Invitrogen), CD3 PerCPCy5.5 (Biolegend), TCRb PE/Cy7 (Biolegend), CD4 PB (Biolegend), CD4 AF700 (Biolegend), CD8 BV650 (Biolegend), CD25 BV421 (Biolegend), FOXP3 AF488 (Biolegend), ROR gamma T PE (Invitrogen), TCF1 AF647 (Cell Signaling Technologies), TCF1 PE (Biolegend), IFNγ AF647 (Biolegend), IL17A FITC (Biolegend), IL17A PE (ebioscience). Representative flow cytometric gating and quantification strategy for detection of lung Th1/Th17 and Tc1/Tc17 cell populations is shown in Supplementary Figure 3. Samples were analyzed using BD LSR II flow cytometer (BD Biosciences) and FlowJo software (TreeStar).