Fig 2: Example of gating strategy in flow analysisATOs from 2-week culture was used here. Cells were gated for scatter and singlets, DAPI- for viable cells, and mCD29- for gating human leukemic cells. Further gating on T cell (CD4, CD8, CD5, and CD7), NK (CD56) and CD19 (B cell) are shown to characterize T-ALL cells.

Fig 3: Gating strategy for the characterization of dendritic cell subsets in human gingiva. (i) Cells are pregated according to their size and granularity (FSC‐A/SSC‐A) to exclude debris and dead cells. (ii, iii) Doublets are excluded by gating on the FSC and SSC area (A) and height (H). (iv) Red blood cells are excluded by using SSC‐B‐H and SSC‐H. (v) Dead cells are excluded using Zombie staining. (vi) CD45+ hematopoietic cells are selected. (vii) CD3+ T cells and CD19+ B cells are excluded. (viii) CD66b+ neutrophils and CD56+ NK cells are excluded. (ix) HLA‐DR+ cells are selected. (x) CD123+, CD45RA+, and CD123‐CD45RA+∖− cells are further gated, which represent the pre‐DC, pDC, and DC subpopulations, respectively. (xi) CD5+AXL+ cells represent the pre‐DC subpopulation and CD5‐AXL‐ cells constitute the pDC subpopulation. (xii) LC cells are isolated from other DC cells by their expression of both CD207 and Epcam. (xiii) Conventional DC (cDC) are divided into cDC1 and cDC2 according to their CD141 and CD1c expression. While cDC1 are positive for CD141 and negative for CD1c, cDC2 are positive for CD1a and negative for CD141. Data acquisition was performed on a Cytek Aurora Flow Cytometry System and data was analyzed using FlowJo V10.8.1 software.

Fig 4: Dynamic changes of CT125A and lymphocyte subsets in patients(A and B) The copy number of the CAR transgene per microgram of genomic DNA (A) determined by ddPCR and the counts of CAR-T cells per microliter of peripheral blood (B) determined by flow cytometric analysis for each patient across various time points post CAR-T cell infusion.(C–G) The levels of the indicated immune cell populations (CD3+ T cells, CD19+ B cells, NK cells, and NKT cells) at different time points post CAR-T infusion, as well as the percentage of CD5+ T cells and the ratio of CD4+ to CD8+ T cells.See also Figure S3.

Fig 5: Effects of CD5 CAR-T cells in hCD5 knockin murine models(A) Schematic illustration of the generation of CD5 CAR-T cells and post-infusion monitoring in murine models.(B) Hematoxylin and eosin (H&E) staining results of various organs, including the heart, liver, spleen, lung, kidney, skin, and adrenal gland, from mice at the experimental endpoint. Black arrows indicate minor inflammatory cell infiltration; red arrows indicate hair follicles. Scale bars: 200 μm in heart and liver, 400 μm in spleen, lung, and kidney, and 100 μm in skin and adrenal gland.(C and D) Dynamic changes in the levels of human CD5+ T cells, murine CD3+ T cells, murine CD19+ B cells, and the ratio of CD4+ to CD8+ T cells in mice measured by spectral flow cytometry. Data are represented as mean ± SEM (n = 5 for CAR-T groups, and n = 2 for the mock T group).(E) Flow cytometric characterization of T cell subpopulations in vivo at different time points. Data are represented as mean ± SEM (n = 5 for CAR-T groups, and n = 2 for the mock T group).See also Figures S4–S6.