Anti cd16 32 blocking antibody clone 93

All antibodies used for flow cytometry were purchased from either
ThermoFisher, BD Biosciences, or BioLegend and are listed in the Key resources table. Dead cells were
discriminated in all experiments using LIVE/DEAD fixable dead stain
(ThermoFisher). All stains were carried out in media containing anti-CD16/32
blocking antibody (clone 93, ThermoFisher). For intracellular staining,
cells were fixed and permeabilized using the FoxP3/Transcription factor
staining buffer set according to the manufacturer’s directions
(ThermoFisher). Intracellular staining for IL-10 production from Treg cells
was carried out on siLP cell suspensions treated with
PMA/ionomycin/brefeldin A for four hours and fixed using Fix/Perm (BD
Biosciences). All flow cytometry was acquired on an LSRFortessa FACS
analyzer(BD Biosciences) and cell sorting was carried out on a FACS Aria (BD
Biosciences). Cells were isolated from the small intestine, Peyer’s
Patches, spleen and lymph nodes for flow cytometry as described previously
(Hand et al., 2012 (link))

All antibodies used for flow cytometry were purchased from either ThermoFisher, BD Biosciences, or BioLegend. The following antibodies were used to discriminate cell surface or intracellular phenotype: TCRβ (H57-597), CD3 (500A2), CD90.2 (53-2.1), CD4 (RM4-5), CD8b (H35-17.2), CD45.1 (A20), CD45.2 (104) CD44 (IM7), IFN-γ (XMG1.2), TNFα (MP6-XT22), IL-17α (eBio17B7), IL-2Rα/CD25 (PC61), Vα2 (B20.1) and Foxp3 (FJK-16S). Dead cells were discriminated in all experiments using LIVE/DEAD fixable dead stain (ThermoFisher). All stains were carried out in media containing anti-CD16/32 blocking antibody (clone 93, ThermoFisher). For intracellular cytokine staining, cells were fixed in BD Cytofix buffer (BD Biosciences) and stained in BD PermWash buffer (BD Biosciences). For Foxp3 staining, cells were fixed and permabilized using the FoxP3/Transcription factor staining buffer set according to the manufacturer’s directions (ThermoFisher). APC-conjugated CBir1_464-72 tetramers (YSNANILSQ) and PE-conjugated HH1713_172-86 and HH1713_230-44 tetramers (QESPRIAAAYTIKGA and GNAYISVLAHYGKNG, respectively) were provided by the NIH Tetramer Core Facility (Atlanta, GA). All tetramer stains were performed at room temperature for 45–60 minutes. All flow cytometry was acquired on an LSRFortessa FACS analyzer, and cell sorting was carried out on a FACS Aria (BD Biosciences).