Aurora full spectrum flow cytometer

Flow cytometry analysis was performed using a Cytek^® Aurora full spectrum flow cytometer (Cytek Biosciences, California, USA). Cryopreserved PBMCs were gently thawed with a mean recovery of 87.50% viable cells, assessed by the Zombie UV™ Fixable Viability Kit (BioLegend, BioLegend, San Diego, CA, USA). 0.5 × 10⁶ PBMCs were stained in duplicate by using anti-CD3 BV570 (UCHT1), anti-CD4 VioBright R720 (REA623), anti-CD25 PE-Vio615 (REA570), anti-CD45RA VioGreen (REA1047), anti-CD197 (CCR7) PE-Vio770 (REA108), anti-CD279 (PD-1) VioBright 515 (REA1165), and anti-FoxP3 PE (REA1253) (Miltenyi Biotec, Bergisch Gladbach, Germany). The Transcription Factor Staining Buffer Set (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for nuclear staining. Isotype controls were used to rule out unspecific binding of antibodies. Gating was performed using FlowJo^™ 10.8.1 (see Fig. 2 for gating strategy). T_regs were gated as CD4⁺CD25^highFoxP3⁺, PD-1⁺ effector T_regs (eT_regs) as CD4⁺CD25^highFoxP3⁺CD45RA⁻PD-1⁺, and naïve T_regs (nT_regs) as CD4⁺CD25^highFoxP3⁺CD45RA⁺CCR7⁺. Median fluorescence intensity (MFI) was calculated in FlowJo™ and used as a relative surrogate marker of protein level.Fig. 2

Gating strategy

Validation of the CyTOF data was conducted by detection of similar markers with a fluorescence-conjugated antibody cocktail using a CyTEK Aurora full-spectrum flow cytometer (Cytek Biosciences) as described previously (56 (link)) through the Immunotherapy Assessment Core at Northwestern University. These fluorescence-conjugated antibodies were purchased from BioLegend and BD Biosciences and are listed in Supplemental Table 10.