Multiparameter Flow Cytometry Profiling of Activated cTfh

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.