Cd127 pe cf594

Cryopreserved PBMC were thawed at 37 °C, then washed in RPMI 1640 supplemented with 10% FCS and HEPES to remove DMSO. 5 × 10⁵ cells were stained for each patient at each time-point. Cells were stained with Fixable Viability Dye ef780 (ThermoFisher), plus antibodies against CD3-BV510 (BD, UCHT1, 563109), CD4-BV711 (BioLegend, OKT4, 317440), CD8a-eVolve605 (ThermoFisher, RPA-T8, 83–0088-42), CD14-APC-eF780 (ThermoFisher, 61D3, 47-0149-42), CD19-APC-eF780 (ThermoFisher, HIB19, 47-0199-), CD25-BV421 (BD, M-A251, 562442), CD45RA-BV785 (BioLegend, HI100, 304140), CD127-PE-CF594 (BD, HIL-7R-M21, 562397), PD-1-PECy7 (BioLegend, EH12.2H7, 329918), PD-L1-BUV395 (BD, HI30, 740320), ICOS-PerCP-eF710 (ThermoFisher, ISA-3, 46-9948-42). Cells were permeabilised with FoxP3 Fixation/Permeabilisation Kit (ThermoFisher), and stained for FoxP3-PE (ThermoFisher, 263A/E7, 12-4777-42) and Ki67-FITC (BD, B56, 51-36524X). Fixation was performed with Stabilising Fixative (BD) diluted in water. Data was acquired on a BD SORP Fortessa (BD) and analysed (Supplementary Fig. S1) with FlowJo software⁴⁰ (BD). Fluorescence minus one controls were used to determine gates.

Fluorochrome-conjugated monoclonal antibodies (mAbs) used in this study were CD45-PerCP and -Alexa Fluor 700, EpCam-FITC and -BV510, CD3-PerCP-Cy5.5, CD4-PE, -PE-Cy7, -BV786, and -AlexaFluor 700, CD8-APC-Cy7, CD45RA-BV605, HLA-DR-FITC, and -APC-Cy7, CD38-APC and -PE-Cy7, CD25-APC and -BB515, CD127-PE-CF594, CD103-PE, CXCR5-Alexa Fluor 647, CD16-BV421, and CD56-APC from BD Biosciences (San Jose, CA, USA); CD4⁺ 5RA-ECD (clone 2H4) from Beckman Coulter (Hialeah, FL, USA); CD127-eFluor450 and CD62L-APC-eFluor780 from eBioscience (San Diego, CA, USA); PD-1 (PE or APC, clone EH-12) and CD127-BV421 and -PE-Cy7 from BioLegend (San Diego, CA, USA); and IgA-PE and CD161-APC and -PE-Cy7 (Miltenyi Biotec, Germany). All antibodies were used according to the manufacturers’ directions.
For detection of surface markers, gut biopsy cells were stained with fluorochrome-conjugated antibodies for 15 min at room temperature, washed once with PBA [Dulbecco’s phosphate-buffered saline (DPBS) containing 0.5% BSA and 0.1% sodium azide], and resuspended in 0.5% paraformaldehyde (Electron Microscopy Sciences, PA, USA) in DPBS (PFA) for fixation, as previously described for human immunophenotyping (40 (link)). Intracellular staining for Ki-67 was performed as previously described (40 (link)), and intracellular staining for Bcl-6 was performed as previously described (31 (link)).

We analyzed the cell composition of the T cells specifically activated by SARS-CoV-2 in the IFN-γ assay by subtracting the basal cytokine response from the background control. We stained the cell surface for 20 min at 4°C using the following fluorochrome-conjugated antibodies titrated to their optimal concentrations: CD45RA FITC (BD Pharmingen), CD27 APC (BD Pharmingen), CD3 VioGreen (Miltenyi Biotec), CD4 PECy7 (BD Pharmingen), CD8 APC Cy7 (BD Pharmingen), and 7AAD (BD Horizon). For the Treg panel CD25 BV421 (BD Horizon) and CD127 PE-CF594 (BD Horizon), were used. For the activation panel, HLA-DR BV 421 (BD Pharmingen), CD69 BV421 (Biolegend), and CD25 BV421 (BD Horizon), were used. For the exhaustion panel PD1 AF700 (Biolegend) and NKG2A BV421 (Biolegend) were used. For the chemokine panel, CD103 BV421 (BD Horizon) and CCR7 PE-CF594 (BD Horizon) were used. Cell acquisition was performed using a Navios cytometer (Beckman Coulter), acquiring an average of 200,000 cells. The analysis was performed using FlowJo 10.7.1 (FlowJo LLC).