Cd8 apcy7

CD3-Alexa Fluor 700, CD4-BV605, CD8-APCy7, CD38-APC, CD27-PE-Cy7, CD19-PE-TxR, PD1-PE, ICOS-PcPCy5.5 and CXCR5-AF488 (BD Biosciences).

CD3-Alexa Fluor 700, CD4-BV605, CD8-APCy7, CD38-APC, TIGIT-PE-Cy7, CD45RO-PECF594, CCR7-BV421, PD1-PE, ICOS-PcPCy5.5 and CXCR5-AF488 (BD Biosciences).
Cells were washed twice and run/sorted on a BD FACS Aria III. Samples from 12 study participants were selected based on the magnitude of their cTfh response from d0 to d7 to undergo bulk sorting, with samples from four of these participants also undergoing single cell sorting. Sorted cells had 100U/mL of RNaseOUT added prior to sorting. Bulk sorted cells were centrifuged with the supernatant removed prior to freezing at -80°C. Data were analysed with FlowJo 10.1 (TreeStar). Activated and resting cTfh cells (CD4⁺CXCR5⁺PD-1⁺) were defined as CD38⁺ICOS⁺ and CD38^-ICOS^-, respectively (19 (link), 22 (link)). Activated plasmablasts were defined as CD19⁺CD27⁺CD38⁺. An example of flow gating for activation and sorting is depicted in Supplementary Figure 8.

For detailed analysis of T cell phenotype, 100 μl of blood samples were stained with a cocktail of the following monoclonal antibodies (mAbs): CD3 PE-Cy7, CD31 APC (eBioscience, San Diego, CA, USA), CD4 V450, CD8 APCy7, HLA-DR PerCP-Cy5.5, TCRα/β FITC, TCRγ/δ PE, and CD45RA V500 (all from BD Bioscience) for 20 minutes at room temperature. Erythrocytes were lysed with BD FACS Lysing solution (BD Bioscience) and washed twice with PBS containing 1% BSC. At least 500,000 cells per sample were collected using a BD Canto II flow cytometer (BD Biosciences, San Jose, CA, USA). T cell subsets were classified into i) cytotoxic T cells, ii) helper T cells, iii) recent thymus emigrants (CD4+ CD45RA CD31+) T cells, iv) naive (CD45RA+CD4+) helper T cells, (v) memory helper T cells (CD45RO+CD4+), (vi) T cells with T cell receptor (TCR) alpha/beta and gamma/delta, (vii) double negative T cells (DN T- CD4-CD8-): with alpha/beta TCR and gamma/delta TCR, and viii) activated (HLA-DR +) T cells. The volume of antibodies per test are shown in Supplementary Table S1, and the gating strategy is shown in Figure S2. Samples were analyzed using FlowJo 10.8 and BD FACSDiva v8.0.1 software (TreeStar Inc.) (Becton Dickinson, San Josè, CA).