Live dead fixable aqua dead cell dye

Cells were harvested as described above for nucleofection, then stained with LIVE/DEAD Fixable Aqua Dead Cell dye (Thermo Fisher Scientific) in 50 µl of 1× PBS for 10 min at room temperature. Cells were washed by the addition of 150 µl of 1× PBS to each well, and the cells were centrifuged for 3 min at 400 ×g. The pelleted cells were resuspended and incubated for ≥30 min with fluorophore-conjugated antibodies in 50 µl of FACS buffer at 4°C. Cells were washed as before and fixed with 2% PFA for 10 min at room temperature. Cells were then washed with 1× FACS buffer and resuspended in 180 µl of FACS buffer for analysis by flow cytometry. Monocyte-derived macrophages and DCs were identified by expression of CD64 or DC-SIGN, respectively. KO was determined by gating for negatively stained cells using the NTC. Heat-killed cells and unstained cells were used as controls for live-dead and positive antibody staining. All samples were analyzed by the FACSymphony system (BD).

Four polychromatic flow cytometry panels were used to measure MAIT cell function, phenotype, and for cell sorting for transcriptomics^{59 (link)}. Briefly, thawed samples were washed, stained with LIVE/DEAD Fixable Aqua Dead Cell dye (ThermoFisher), blocked for Fc receptors using Normal mouse serum (ThermoFisher), and surface stained with antibody cocktail. Samples were surface stained at room temperature for 30 min, and some were intracellularly stained at room temperature for 30 min. Some samples were fixed in 2% paraformaldehyde or BD FIX/PERM Buffer (BD Biosciences) before acquisition on a 5 laser, 16-parameter BD LSRII SORP flow cytometer (BD Biosciences) within 12 h of staining. Other samples used for sorting for downstream transcriptomics were resuspended in sorting buffer (PBS containing 1% BSA) and sorted for bulk MAIT cells for either RNA-Seq or targeted transcriptomics with Fluidigm Biomark. Data were analyzed with FlowJo v.9.9.4 (TreeStar). See Supplemental Experimental Procedures and Supplementary Table 8 for specific antibodies, MR1 tetramer, and reagents used in the study.

Frequency and phenotype of peripheral blood and mucosal CD161⁺ CD4⁺ T cells were determined as previously described [17 (link)]. Briefly, thawed samples were washed, stained with LIVE/DEAD Fixable Aqua Dead Cell dye (ThermoFisher, Waltham, MA, USA), blocked for Fc receptors using normal mouse serum (ThermoFisher), and surface-stained with an antibody cocktail. Samples were surface-stained at room temperature for 30 min. Surface staining was performed at 37 °C for panels, including CCR5 antibodies. Cells were then washed and fixed in 2% paraformaldehyde. Cells were fixed in Cytofix/Cytoperm or in Transcription Factor Fixation/Permeabilization buffer (both from BD Biosciences, San Jose, CA, USA) as appropriate for transcription factor analysis. Intracellular staining was performed using the relevant mAbs in Perm/Wash or Transcription Factor Perm/Wash buffer as appropriate (both from BD Biosciences). The LEGENDScreen was performed as previously described [18 (link)]. Samples were acquired on a five-laser, 16-parameter BD LSRII SORP; an 18-parameter LSR Fortessa; or a four-laser, custom-built LSR Fortessa (all from BD Biosciences). Data were analyzed with FlowJo v.9.9.4 or higher (BD Biosciences). See Supplemental Experimental Procedures for specific antibodies used throughout the study.

The frequency and phenotype of peripheral blood and mucosal iNKT cells were determined as previously described (64 (link)). Briefly, thawed samples were washed, stained with LIVE/DEAD Fixable Aqua Dead Cell dye (Thermo Fisher Scientific), blocked for Fc receptors using normal mouse serum (Thermo Fisher Scientific), and surface stained with antibody mixture. Samples were surface stained at room temperature for 30 min. For panels including CCR5 antibodies, surface staining was performed at 37 °C. Samples were fixed in 2% paraformaldehyde before acquisition on a five-laser, 16-parameter BD LSRII SORP flow cytometer or a four-laser custom-built LSR Fortessa (BD Biosciences). Total evens acquired in the iNKT cell gate ranged from 147 to < 7,000. Other samples used for sorting for downstream transcriptomics were resuspended in sorting buffer (phosphate buffered saline [PBS] containing 1% bovine serum albumin [BSA]) and sorted for bulk iNKT cells for targeted transcriptomics with Fluidigm Biomark. Data were analyzed with FlowJo version 9.9.4 or higher (TreeStar). See SI Appendix, Methods for specific antibodies used throughout the study. Anti-human α4β7 [clone Act-1, NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH (catalog No. 11718) from Dr. A. A. Ansari (65 (link))] was labeled using Alexa Fluor 647 antibody–labeling kit from Invitrogen.