Single-cell cytokine profiling of BAFF-R CAR T-cell products by isolated subset or bulk stimulation. (A) Workflow of CAR T-cell product processing, comparing protocols in which cells were first separated into CD4⁺ and CD8⁺ subsets, stimulated with BAFF-R⁺ target cells, which were subsequently depleted (isolated stimulation), or cells were stimulated in bulk and then separated into subsets without the need for target cell depletion, before analysis (bulk stimulation). (B) Fluorescence-activated cell sorting (FACS) plots showing the frequency of EGFRt⁺ CAR T cells stimulated with either BAFF-R⁺ or BAFF-R⁻ targets (control) in cell suspensions prepared either by isolated or bulk stimulation protocols, before loading onto microchips. (C) Heat maps showing frequencies of single CD4⁺ or CD8⁺ T cells secreting selected cytokine combinations from isolated or bulk stimulation protocols. Brackets indicate differences observed in specific cytokine profiles between the 2 protocols. (D) Differences between isolated subset and bulk stimulation protocols as measured by PSI. Results are representative of 2 CAR T-cell products generated from healthy donors. Stim, stimulation.