Brefeldin A

Colon LP cells were isolated as described previously and stained for flow cytometry (20 (link)). Briefly, the whole colon was washed, cut into 1-1.5 cm sections, incubated twice in Hanks' Balanced Salt Solution (HBSS, Sigma-Aldrich, St. Louis, MO) with 5mM EDTA at 37°C and then digested in RPMI-1640 containing 1 mg/ml collagenase type 1 (Worthington, Lakewood, NJ) and 10% FBS at 37°C for 1h in a shaking incubator. The cells were collected from the interface of 40/80% Percoll gradients (Sigma-Aldrich). The fluorescent dye conjugated-antibodies listed below were used for flow cytometry: Anti-CD3 (145-2C11), CD4 (GK1.5) (Biolegend, San Diego, CA), Thy1.2 (30-H12), CD45.2 (104) (BD Biosciences, San Jose, CA), FoxP3 (FJK-16s), RORγt (B2D), IL-22 (IL22JOP), IL-17A (TC11-18H10) (eBioscience, San Diego, CA). For intracellular cytokine staining, cells were stimulated with PMA (0.1 μg/ml, Sigma-Aldrich), ionomycin (0.5 μg/ml, Sigma-Aldrich) in the presence of Brefeldin A (10 μg/ml, Sigma-Aldrich) for 4 h. For IL-22 intracellular staining, mouse recombinant IL-23 (40 ng/ml, R&D systems, Minneapolis, MN) was added to the PMA, ionomycin and Brefeldin A cultures for 4 h. The cells were fixed and permeabilized using kits for intracellular staining (eBioscience) according to the manufacturer's instructions. All data were collected on a BD Fortessa LSRII (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, OR).

For intracellular staining, short-term reactivation of cryopreserved splenocytes and subsequent mass cytometry analysis were performed as described previously^{94 (link)}. In short, cells were kept at −80 °C for less than 2 months before thawing in a 37 °C water bath. Cells were then immediately resuspended in cell culture medium supplemented with 1:10,000 benzonase (Sigma-Aldrich), and centrifuged at 300 g for 7 min at 24 °C. Samples were then left overnight at 37 °C before restimulation with 50 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich) and 500 ng/mL ionomycin (Sigma-Aldrich) in the presence of 1× brefeldin A (BD Biosciences) and 1× monensin (Biolegend) for 4 h at 37 °C. For splenocytes, one anchor sample to correct batch effect among different acquisitions and one non-stimulated control sample were also included. For both PBMCs and reactivated cryopreserved splenocytes, surface staining was performed for 30 min at 4 °C. To identify dead cells, 2.5 μM cisplatin (Sigma-Aldrich) was added for 5 min on ice. To minimize technical variability, an equal number of cells from each sample were barcoded using Cell-ID 20-Plex (Fluidigm). Cells from all samples were then combined in one single tube. The combined sample was then processed for Live/Dead and surface staining. For intracellular cytokine staining of reactivated cryopreserved splenocytes, after surface staining, cells were fixed and permeabilized with FOXP3 staining kit (Invitrogen) and stained for intracellular markers and cytokines. The antibodies used are reported in Supplementary Table 2. Because CD4 molecules are internalized during phorbol 12-myristate 13-acetate/ionomycin stimulation^{95 (link)}, the anti-CD4 antibody was used in both the surface staining and the intracellular staining steps. After washing, the combined sample was incubated with 4% PFA overnight at 4 °C. Prior to acquisition in a Helios™ II CyTOF® system, samples were washed with cell staining buffer and mass cytometry grade water. Multidimensional datasets were analyzed using FlowJo and R (R Core Team, 2017).

To evaluate the phenotypes of T cells that responded to stimulation in IFN-γ ELISPOT assays, we conducted intracellular cytokine staining (ICS). The in vitro stimulation was similar to that described above in the IFN-γ ELLISPOT section. Briefly, after 12-14 days in vitro culture, PBMCs were restimulated with peptide. After 1 h, Brefeldin A was added to inhibit protein transport. After 4 additional hours of incubation, the cells were stained with T-cell surface markers CD4-PerCP (cat. 345770), CD8- FITC (cat. 345772), CD3-APC-H7 (cat. 560275) and a dead cell marker FVS510 (564406) (all from BD Biosciences). Samples were then fixed and permeabilized using eBioscience™ Fixation/Permeabilization buffers (eBioscience, cat. 00-5123-43, 00-5223-56) and stained with IFNg-APC (cat.341117, BD Biosciences), TNFa-BV421 (cat.562783, BD Biosciences) in eBioscience permeabilization buffer (eBioscience, cat. 00-8333-56) and analyzed on FACSCanto™ II (BD Biosciences) using BD FACSDiva software version 8.0.2 as described previously (32 (link)).

Frozen PBMCs and SFMCs were thawed and stained with a T cell focused panel of 37 heavy metal-conjugated antibodies (Supplementary file 2), as previously described (Chew et al., 2019 (link)), and analyzed by CyTOF-Helios (Fluidigm, San Francisco, CA). Briefly, PBMCs were stimulated with or without phorbol 12-myristate 13-acetate (150 ng/ml, Sigma-Aldrich) and ionomycin (750 ng/ml, Sigma-Aldrich) for 4 h, and blocked with secretory inhibitors, brefeldin A (1:1000, eBioscience) and monensin (1:1000, BioLegend) for the last 2 h. The cells were then washed and stained with cell viability dye cisplatin (200 μM, Sigma-Aldrich). Each individual sample was barcoded with a unique combination of anti-CD45 conjugated with either heavy metal 89, 115, 141, or 167, as previously described (Lai et al., 2015 (link)). Barcoded cells were washed and stained with the surface antibody cocktail for 30 min on ice, and subsequently washed and re-suspended in fixation/permeabilization buffer (permeabilization buffer, eBioscience) for 45 min on ice. Permeabilized cells were subsequently stained with an intra-cellular antibody cocktail for 45 min on ice, followed by staining with a DNA intercalator Ir-191/193 (1:2000 in 1.6% w/v paraformaldehyde, Fluidigm) overnight at 4°C or for 20 min on ice. Finally, the cells were washed and re-suspended with EQ Four Element Calibration beads (1:10, Fluidigm) at a concentration of 1×10⁶ cells/ml. The cell mixture was then loaded and acquired on a Helios mass cytometer (Fluidigm) calibrated with CyTOF Tunning solution (Fluidigm). The output FCS files were randomized and normalized with the EQ Four Element Calibration beads (Fluidigm) against the entire run, according to the manufacturer’s recommendations.
Normalized CyTOF output FCS files were de-barcoded manually into individual samples in FlowJo (v.10.2), and downsampled to equal cell events (5000 cells) for each sample. Batch run effects were assessed using an internal biological control (PBMC aliquots from the same healthy donor for every run). Normalized cells were then clustered with MarVis (Kaever et al., 2009 (link)), using Barnes Hut Stochastic Neighbor Embedding (SNE) nonlinear dimensionality reduction algorithm and k-means clustering algorithm, as previously described (Chew et al., 2019 (link)). The default clustering parameters were set at perplexity of 30, and p<1e−21. The cells were then mapped on a two-dimensional t-distributed SNE scale based on the similarity score of their respective combination of markers, and categorized into nodes (k-means). To ensure that the significant nodes obtained from clustering were relevant, we performed back-gating of the clustered CSV files and supervised gating of the original FCS files with FlowJo as validation. Visualizations (density maps, node frequency fingerprint, node phenotype, and radar plots) were performed through R scripts and/or Flow Jo (v.10.2). Correlation matrix and node heatmaps were generated using MarVis (Kaever et al., 2009 (link)) and PRISM (v.7.0).