Extended Data Fig. 6: CD300ld mediates PMN-MDSC engagement with PS^high CD8⁺ T cells.

a: Indicated T cell populations were isolated via FACS from B16-F10 tumors and Cell viability was detected by Flow cytometry (a, n = 3 independent samples per group). b: Percentage of PS^high CD8⁺ T cells in different organs from B16-F10 tumor-bearing mice, as determined by flow cytometry (b, n = 5 mice per group). c: PS^high and PS^low CD8⁺ T cells were isolated via FACS from B16-F10 tumors, and expression levels of indicated genes were detected by RT-qPCR (c, n = 3 independent samples per group). d: Cytotoxic activity of OT-1 CD8⁺ T cells sorted into PS^high and PS^low CD8⁺ T populations, measured as the percentage of lysed MC38 OVA-expressing tumor cells (d, n = 3 independent samples per group). e: Flow cytometry detection of CD107a positive CD8⁺ T cells from B16-F10 tumors (e, n = 6 mice per group). f: WT mice bearing B16-F10 tumors were treated with indicated antibodies post tumor inoculation. Tumor growth curves are shown (n = 5 mice). g: Splenocytes were incubated with CD300ld-ECD-Fc or Fc protein for 48 h, Proliferation of CD8⁺ T cells were assessed by flow cytometry. Representative histograms (left) and statistical analysis (right) are shown (g, n = 3 independent samples per group). h: Flow cytometric analysis of Biotin levels on 293t cells immobilized with or without SrtA protein. i: Flow cytometry analysis (left) and quantification (right) of the ratio of labeled bone marrow-derived DC (BMDC; Biotin⁺) after co-culture with SrtA⁺ CD8⁺ T cells, as determined by flow cytometry (i, n = 3 independent samples per group). j: Flow cytometry analysis and quantification of the ratio of labeled CD300ld WT, C62A, and D116A PMN-MDSCs (Biotin⁺) after incubation with control or SrtA⁺ CD8⁺ T cells (j, n = 4 mice per group). k: Flow cytometry analysis (left) and quantification (right) of the ratio of labeled WT PMN-MDSCs (Biotin⁺) after co-incubation with total or PS^high SrtA⁺ CD8⁺ T cells in the presence of CD300ld-ECD-Fc and Fc protein (k, n = 5 independent samples per group). l: Flow cytometry quantification of the ratio of labeled neutrophils (from tumor-free mice) or PMN-MDSCs (from tumor-bearing mice) after co-incubation with total or SrtA⁺ CD8⁺ T cells (l, n = 5 mice per group). Data are presented as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; NS, not significant. Statistical analysis was performed using ordinary one-way ANOVA (d, f, g, i, j), Student’s two-sided unpaired t-test (k, l) and Student’s two-sided paired t-test (e). Data are representative of two (a, b, f) or three (c, e, g, h, i, j, k, l) independent experiments.

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