Rainbow beads

Stimulated cells were washed with PBS, and stained with Zombie Aqua (Biolegend) dye for 15 min at 4°C to discriminate viable cells. Subsequently, samples were washed with FACS buffer (PBS supplemented with 2% FCS, 2 mM EDTA, and 0.05% NaN₃), fixed, and permeabilized in CytoFix/CytoPerm Solution (BD Bioscience) for 15 min at 4°C. Cells were then washed with 1x Perm/Wash Buffer (BD Bioscience), and stained for intracellular markers for 15 min at 4°C using the following antibody conjugates; anti-CD3-APC-Cy7 (clone UCHT1; Biolegend), anti-CD4-BV786 (clone SK3; BD Bioscience), anti-IFNγ-PE (clone B27; Biolegend), anti-TNFα-BV421 (clone Mab11; Biolegend), and anti-IL2-AF488 (clone MQ1-17H12; Biolegend). Labeled cells were then washed with PBS and acquired on FACS Celesta (BD Bioscience). Frequencies of antigen-specific CD4+ T cells were calculated as negative-control-subtracted data. Possible longitudinal fluctuations in laser intensity were monitored and adjusted before each experiment using fluorescent beads (Rainbow beads, Biolegend). The data were analyzed with the FlowJo Software version 10.0.7 (TreeStar).

Cryopreserved PBMCs were thawed and rested for 2 h at 37°C in R‐20 media prior to staining. Rested cells were harvested and resuspended in PBS before being transferred to a 96‐well plate for staining. PBMCs were first stained with antibodies against CXCR5 (BB515, clone RF8B2, BD Biosciences), CCR7 (APC/Cy7, clone G043H7, Biolegend) and CXCR3 (PE/Cy5, clone 1C6/CXCR3, BD Biosciences) at 37°C, followed by a second panel of antibodies at 4°C targeting CD3 (PerCP/eFluor710, clone SK7, Thermo Fisher), CD4 (PE/Dazzle594, clone RPA‐T4, Biolegend), CD8 (BV711, clone RPA‐T8, Biolegend), CD14 (BV510, clone M5E2, Biolegend), CD19 (APC, clone HIB19, Biolegend), CD25 (PE/Cy7, clone 2A3, BD Biosciences), CD38 (BV785, clone HIT2, Biolegend), CD45RA (AF700, clone HI100, Biolegend), CD56 (BV605, clone NCAM16.2, BD Biosciences), CD127 (BV650, clone A019D5, Biolegend) and PD‐1 (BV421, clone EH12.2H7, Biolegend), as well as a viability dye (Live/Dead Fixable Aqua, Thermo Fisher). Cells were fixed prior to acquisition using 2% paraformaldehyde (Biolegend). Samples were acquired on a BD Biosciences LSRFortessa calibrated using Rainbow beads (Biolegend).

At indicated time points, blood samples or CAR-T cell products were processed for flow cytometry. All anti-human antibodies were used at the manufacturer’s recommended concentration. Flow cytometry using a BD LSRFortessa instrument. Gates were set using fluorescence minus one (FMO) controls. Laser intensities were standardized using Rainbow beads (Biolegend) to minimize variability between runs. Data was analyzed using FlowJo v10 (TreeStar). A gating strategy for T-cell blood samples is shown in Supplementary Fig. 1a and the gating strategy for CAR-T cell products is shown in Supplementary Fig. 1b. All CAR-T cell products were phenotyped to determine the frequency of CAR⁺ cells prior to the following analyses.