Extended Data Fig. 8: Representative flow cytometry gating strategies used in this study.

a, Gating strategy for analysis of CAR-T cell differentiation subsets and PD-1 expression in participant peripheral blood samples. Lymphocytes were gated, followed by singlets, CD3⁺ T cells and CAR-T cells; differentiation subsets were then defined on the basis of CD45RO and CD62L expression, and PD-1 was assessed. Applicable to Fig. 1b and Fig. 4a–c. b, Gating strategy for analysis of lipid ROS and labile iron in CAR-T cells from participant peripheral blood samples. Lymphocytes, singlets, CD3⁺ T cells and CAR-T cells were sequentially gated, followed by quantification of lipid ROS and labile iron signals. Applicable to Fig. 1h–k and Extended Data Fig. 1i, j. c, Gating strategy for functional analysis of CAR-T cells in mouse bone marrow and spleen samples; the same strategy was also used for in vitro CAR-T functional assays. After gating lymphocytes, singlets and live cells, CAR-T cells were analysed for differentiation status, lipid ROS, labile iron and functional or exhaustion-associated markers, including CD25, CD69, LAG3, PD-1 and TIM3. Applicable to Fig. 2i–r, Fig. 3j–o, Extended Data Fig. 3d, g, j, m, Extended Data Fig. 7b–h, Fig. 7d, Fig. 6d, e, k–o, and Fig. 4d, e, i, j, l, n, o. d, Multicolour flow cytometry gating strategy used primarily to assess phenotypic and functional changes in cultured CAR-T cells in vitro. Lymphocytes, singlets and CAR-T cells were sequentially gated, followed by CD4/CD8 subset separation, differentiation subset analysis based on CD45RO and CD62L, and detection of exhaustion markers including PD-1, LAG3 and TIM3. Applicable to Extended Data Fig. 2d–m, Extended Data Fig. 6p–u, and Fig. 4f–h, q–s, y.