2.4. Flow Cytometry

Violet ^TM510 from Tonbo-Biosciences was used as a viability dye. Cell-surface staining was done as previously described. The intracellular staining procedure was modified from our previously published paper [22]. Intracellular protein staining was combined with surface protein staining as a way to identify pTFH and pTFR cells. First, 4–5 × 10⁶ cells were washed with a staining buffer and then incubated with human Ab serum along with all surface staining antibodies. Surface staining antibodies included: CD3, CD4, CD8, CXCR5, CD25, CCR5, PD-1, ICOS, and TIGIT. Details of antibody fluorochromes and sources can be found in Table 1. Extra antibodies were washed off with the appropriate staining buffer [22]. The cells were then fixed and permeabilized with FOXP3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent (eBioscience, Santa Clara, CA, Cat. No. 00-5521-00) for 30 min at room temperature. Cells were washed twice with Permeabilization Buffer (eBioscience, Cat. No. 00-8333-56). Next, cells were stained intracellularly with FOXP3, BCL6, and appropriate IgG control (antibody sources in Table 1). Extra antibodies were washed off with the nanocrystal buffer. Finally, cells were re-suspended in a staining buffer before acquisition on a SORP HTLSRII Analytic Flow Cytometer (BD Immunocytometry Systems). Compensation tubes were not permeabilized, but were fixed with 1% paraformaldehyde.

Flow cytometry-Antibodies and their sources.

In all experiments gating was done on live cells followed by lymphocytes and single cells. TFH and TFR cells were distinguished by expression of BCL6 and FOX-P3 (Supplementary Materials Figure S1).