Cd3 pe cy5

The available number of PBMC allowed further analysis of eight samples by flow cytometry. In detail, 0.5 × 10⁶ cells were transferred to a 96-well round-bottom plate in 100 µL culture medium with fetal calf serum in the presence of the corresponding peptide pool or SEB or culture medium alone. Following 1-h incubation at 37 °C in a humidified 5% CO2, brefeldin A (Sigma-Aldrich) at a final concentration of 10 ug/mL was added. After overnight incubation, cells were washed in phosphate-buffered saline (PBS) ethylenediaminetetraacetic acid (EDTA) 2mM and incubated with the Live/Dead Fixable Far Red Dead Cell Stain Kit (ThermoFisher Scientific, Waltham, MA, USA) for 30 min at 4 °C. Cells were then washed with PBS, fixed and permeabilized with BD Cytofix/Cytoperm (BDBiosciences, San Jose, CA, USA) according to the manufacturer’s instructions for intracellular staining with the following monoclonal antibodies: IFNγ-FITC, CD4-ECD, CD8-PC7, CD3-Pe-Cy5 (all from BeckmanCoulter, Brea, CA, USA). Finally, the cells were resuspended in 4% paraformaldehyde and analyzed with a Navios flow cytometer (Beckman Coulter, Brea, CA, USA), obtaining the percentages of IFN-γ secreting lymphocyte cell responses and allowing phenotypical discrimination of individual cytokine-producing cells

To isolate mononuclear cells (MNCs) from PB and SF samples we diluted the samples 4 times with 1x PBS and proceeded with Ficoll density gradient centrifugation (PanEco). CD4+ and CD8+ T cells were isolated immediately from MNCs with anti-CD4 or anti-CD8 positive-selection magnetic beads (Dynabeads, Thermo Fisher Scientific). The PD-1+ T cells were sorted from fresh SF MNCs, and CD137+ T cells and CD103+CD69± T cells (tissue-resident memory) were sorted from cryopreserved SF MNCs using the following antibodies: CD3-PE/Cy5 (Beckman Coulter Cat# IM2635U, RRID : AB_10645166), CD8-PE/Cy5 (Beckman Coulter Cat# IM2638U, RRID : AB_131157), CD3-eFluor 450 (Thermo Fisher Scientific Cat# 48-0038-80, RRID : AB_1518801), PD-1-Brilliant Violet 421 (BioLegend Cat# 329920, RRID : AB_10960742), CD137-PE (Miltenyi Biotec Cat# 130-110-900, RRID: AB_2654985), CD103-FITC (Thermo Fisher Scientific Cat# 11-1031-81, RRID : AB_465175), CD69-PE/Cy5 (BioLegend Cat# 310907, RRID : AB_314842). The Helix NIR stain (Thermo Fisher Scientific) was used to exclude dead cells. Cell sorting was performed on FACSAria III (BD) directly into the RLT lysis buffer (Qiagen). Gating strategies are provided in Supplementary Figure 1.

Killer-cell immunoglobulin-like receptor repertoire staining, functional assays, and data analysis were performed as described in detail elsewhere (3 (link)). In brief, freshly thawed PBMCs of patient and controls were stained with the following monoclonal antibodies: CD3-PE.Cy5, CD14-PE.Cy5, CD19-PE.Cy5, CD56-ECD, ILT2-PE, CD161-PE, CD7-PE-Cy7, and NKG2A-APC.AF750, all from Beckman Coulter; NKG2C-PE or biotin from R&D Systems; PLZF-PE, NKp30-APC, and NKp46-PE from Becton Dickinson; FcϵRγ1-FITC from Millipore; and CD57-PB from Biolegend. Dead cells were excluded by using the aqua live/dead kit (Invitrogen).