Fig 2: Single-cell RNAseq analysis on CRC patients defines an effective prognostic signature based on CD8_Tem infiltration.A–F scRNAseq analysis on 8 CRC patients. A UMAP projection of CD8+ T cells. Colors represent different T cell subtypes. Key genes used for manual annotation are indicated. B Violin plots showing normalized expression of GZMB and GZMK in the different T CD8+ cell subtypes. C UMAP showing the expression of CD8_Tem and CD8_Tex signatures for each cell. Cells are color coded based on SNN-clustering, dot’s size reflects the expression of the signature for each cell. D Violin plots showing the expression of genes associated with T-cell exhaustion within the indicated cell subtype. Color code as in (B). E Monocle pseudotime analysis of the CD8+ T cell subtypes. The pseudotime line connects the indicated T cell subtypes identified in (A). F UMAP as in (C) showing the expression of Cytolysis and Allograft-rejection signatures. G Kaplan–Meyer analysis of the association of CD8_Tem cell subtype’ abundance with disease-free survival (DFS) on the TCGA-COAD cohort (n = 284; see “Methods” and Supplementary Table 1 for details). High infiltration in red, low infiltration in turquoise.

Fig 3: Contact-mediated interaction of neutrophils with CD8+ T cells induces GZMK expression.A Pearson correlation between CD15high neutrophils and GZMKhigh CD8+ TEM (n = 22; p = 0.0387) B Representative contour plots of GZMK expression in CD8+ TEM in LN and HN patients. C Frequency of GZMKhigh CD8+ TEM cells in LN (n = 7) and HN (n = 14, p = 0.009) patients. D GZMK Mean Fluorescence Intensity (MFI) in HN (n = 9) and LN (n = 8) tumors. Scale bar 10 µm. Pearson correlation between: CXCR2+ neutrophils and GZMKhigh CD8+ T cells (E, n = 22, p < 0.0001); CXCR2+ neutrophils and tumor size (F, n = 22, p = 0.0002), GZMKhigh CD8+ T cells and tumor size (G, n = 21, p = 0.0004) within tumor (T) of MC38 tumor-bearing mice. GZMKhigh CD8+ T frequency within T (H, p = 0.050) and tumor weight (I, p = 0.030) in mice upon aLy6G antibody-mediated neutrophil’s depletion (n = 6) or treatment with isotype control (n = 5). J Representative confocal images and quantification of GZMK MFI in CD8+ and CD8- (not-CD8) cells within a 10 µm distance from neutrophils (CD66b+). Scale bar 5 µm. K Representative confocal images and normalized quantification (see “Methods”) of GZMK MFI in CD8+ within 10 µm (Touching, n = 161) or between 10 and 20 µm (Near, n = 203) from neutrophils (CD66b+). Yellow dotted lines represent 20 µm (left) and 10 µm (right). Scale bar 10 µm. L. GZMK+ MFI within activated CD8+ T cells (CTR) co-cultured at 1:1 ratio with neutrophils isolated from Bone Marrow (BM, n = 9) or MC38 tumors (T, n = 3) (p = 0.015 for CTR vs 1:1 NEU T, p < 0.0001 for CTR vs 1:1 NEU BM. M. Frequency of GZMKhigh CD8+ T cells (CTR) after co-culture with HD neutrophils (n = 16), either in plate or in Transwell (TRANS). The CD8+ T cells-neutrophils ratios are indicated (p = 0.011 for CTR vs 1:1, p = 0.098 for CTR vs 1:10, p = 0.976 for CTR vs 1:1 TRANS). N. Co-culture of CD8+ isolated from patients’ PB with neutrophils isolated from tumor (NEU T) (n = 2), CD15high and CD15lowneutrophils. Bars represent mean ± SEM or box and whisker plots indicate Min to Max value, two-tailed paired t test (C, L, M, N), two-tailed Mann–Whitney test (H), two-tailed unpaired t test (I). Source data are provided as a Source Data file.

Fig 4: Flow cytometry data validate the transcriptional characterization of CD8+ CAR T cells. A, Box plots comparing the percentage of IFN?-producing CD8+ CAR T cells (top) and TOX-producing CD8+ CAR T cells (bottom) between effector precursor preinfusion cells (Tigit+, CD62Llo, CD27-; red) and preinfusion cells with the opposing surface phenotype (Tigit-, CD62Lhi, CD27+; blue) cocultured either with CD19 knockout (KO) tumor (left) or CD19+ tumor (right). Dashed lines link subsets from the same patient and sample. B, Representative flow cytometry data visualizing GzmB-BV421 and GzmK-FITC staining of preinfusion CD8+ CAR T cells (left) stimulated with either CD19+ tumor (red) or CD19 KO tumor (blue) and unstimulated postinfusion CD8+ CAR T cells (right).

Fig 5: Pseudotime trajectory analysis identifies a subset of dysfunctional postinfusion CAR T cells that arise directly from the GMP product rather than from prolonged antigen exposure. A–B, Monocle pseudotime map depicting trajectory analysis of 3,416 CAR T cells. Downsampling was necessary due to computational limitations. The analysis included 368 cells (the number of all cells at the month 3 time point) from each time point, as well as all 840 cells with TCRs matching known pre- and postinfusion lineages regardless of cluster designation. Pseudotime states were generated based on internal clustering by the pseudotime analysis. A, Cells are colored by pseudotime state. B, Cells are colored by either GMP or postinfusion sample types. C, Dot plot comparing relative expression of CASP8, LAG3, and TOX across pseudotime states, with the percentage of cells expressing a gene encoded by dot size. D, Dot plot comparing relative expression of effector genes (NKG7, GNLY, GZMB, and GZMK) across pseudotime states, with the percentage of cells expressing a gene encoded by dot size.