Staining of mouse cells for flow cytometry analysis was done after cells were collected and washed with PBS with 1% BSA (staining buffer), and then Fc-blocked with mouse IgG. Conjugated antibodies used were anti-PD-1 (clone 29F.1A12, Biolegend, San Diego, CA), anti-TIGIT (clone 1G9, Biolegend), anti-CD4 (clone RM4-5, Biolegend), anti-CCR6 (clone 29-2L17), anti-CCR7 (clone 4B12), anti-CXCR6 (clone SA051D1). Mouse spleen cells were washed with staining buffer (PBS + 1% BSA), blocked with mouse IgG in staining buffer, then stained with conjugated antibodies.
Human PBMCs were stained with anti-CD4 (clone OKT4, Biolegend), human CD25 (clone BC96, Biolegend), human FoxP3 (clone 236A/E7, Biolegend), human PD-1 (clone EH12.2H7, Biolegend), CCR6 (clone GO34E3, Biolegend), anti-CCR7 (clone GO43H7, Biolegend), anti-TIGIT (clone A15153G, Biolegend). Prior to anti-FoxP3 staining, the cells were fixed for 18 hours and permeabilized.
Stained cells were analyzed in the Oklahoma Medical Research Facility (OMRF) Flow Cytometry Core Facility on a BD LSRII (BD Biosciences, Franklin Lakes, NJ) or in the Ocular Immunobiology P30 Core Module at Dean McGee Eye Institute on a Cytek Aurora (Cytek, Fremont, CA). Spectral unmixing of full spectrum flow cytometry data was done with SpectroFlo software (Cytek) and data was analyzed using FlowJo Software (Tree Star, Inc., Ashland, OR).
Human PBMCs were stained with anti-CD4 (clone OKT4, Biolegend), human CD25 (clone BC96, Biolegend), human FoxP3 (clone 236A/E7, Biolegend), human PD-1 (clone EH12.2H7, Biolegend), CCR6 (clone GO34E3, Biolegend), anti-CCR7 (clone GO43H7, Biolegend), anti-TIGIT (clone A15153G, Biolegend). Prior to anti-FoxP3 staining, the cells were fixed for 18 hours and permeabilized.
Stained cells were analyzed in the Oklahoma Medical Research Facility (OMRF) Flow Cytometry Core Facility on a BD LSRII (BD Biosciences, Franklin Lakes, NJ) or in the Ocular Immunobiology P30 Core Module at Dean McGee Eye Institute on a Cytek Aurora (Cytek, Fremont, CA). Spectral unmixing of full spectrum flow cytometry data was done with SpectroFlo software (Cytek) and data was analyzed using FlowJo Software (Tree Star, Inc., Ashland, OR).