Profiling Single-Cell Cytokine Responses of CAR-T Cells

Ex vivo human T cells were isolated from IMR5-bearing mice spleens using Miltenyi Biotec tumor dissociation kit. Viable CAR-T cells were enriched using Ficoll. CD4⁺/CD8⁺ T cell subsets were separated using anti-CD4 or anti-CD8 microbeads (Miltenyi Biotec) and stimulated with B7-H3-positive IMR32 or IMR32 B7-H3 KO cells at a ratio of 1:2 for 20 h. Both CD4⁺ and CD8⁺ T populations were then recovered from the tumor cell-incubation systems. A single cell functional profile was determined as described previously^{32 (link),54 (link)}. Briefly, a total of 30,000 single cells were loaded onto an IsoCode chip (IsoPlexis) which contains ~12,000 microchambers. A 32-plex cytokine/chemokine antibody array was designed and embedded in each microchamber, including Effector: Granzyme B, IFN-γ, MIP-1α, Perforin, TNFα, TNFβ; Stimulatory: GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, IL-21; Chemoattractive: CCL11, IP-10, MIP-1β, RANTES; Regulatory: IL-4, IL-10, IL-13, IL-22, sCD137, sCD40L, TGFβ1; Inflammatory: IL-1β, IL-6, IL-17A, IL-17F, MCP-1, MCP-4. The single-cell functional subsets within the different CD4⁺ CAR-T cells groups were shown in a heatmap, and the combination of multiple cytokine/chemokine proteins in each sample was quantified as the percentage of polyfunctional cells.