Extended Data Fig. 3: Binding with PS is required for CD300ld-mediated tumor progression.
a, b: B16-F10 tumor growth curves (a) and the percentage of indicated immune cell types (b) in CD300ldfl/flS100a8cre mice (a, b, n = 6 mice). c: In vitro proliferation curves of B16-F10 cells treated with the indicated concentrations of Annexin V protein (c, n = 3 independent samples per group). d: Tumor weight measurements and flow cytometric analysis of CD8+ T cells and PMN-MDSCs on day 15 after B16-F10 inoculation in WT or CD300ld KO mice treated with Hexa-His or Annexin V-His protein (d, WT + His, n = 7 mice; WT + Annexin V, n = 7 mice; KO + His, n = 7 mice; KO + Annexin V, n = 8 mice). e: Tumor growth curves of WT or CD300ld KO mice treated with Isotype or PS Ab starting from day 7 post B16-F10 tumor inoculation (e, n = 7 mice per group). f: Tumor weights in mice treated with Fc, CD300ld-ECD (WT), or its point mutants (C62A or D116A) at day 7 post tumor inoculation (f, Fc, n = 6 mice; ECD (WT), n = 6 mice; ECD (C62A), n = 5 mice; ECD (D116A), n = 5 mice). g: The percentage of indicated immune cell types in the B16-F10 tumors from mice treated with Fc, ECD (WT), ECD (C62A) or ECD (D116A) (g, Fc, n = 6 mice; ECD (WT), n = 5 mice; ECD (C62A), n = 5 mice; ECD (D116A), n = 6 mice). h: Sanger sequencing validation of CD300ld-C62A and CD300ld-D116A mutant mouse strains. Heterozygotes genotypes are shown. i: Flow cytometric quantification of CD300ld expression on peripheral blood neutrophils from CD300ld WT, KO, C62A and D116A mice (n = 6 mice per group). j: PS liposome-binding percentages of PMNs isolated from CD300ld WT, KO, C62A and D116A mice (j, n = 6 mice per group). k: Indicated immune cell percentage in the B16-F10 tumors of indicated mice at day-15 post tumor inoculation as determined by flow cytometry (k, WT n = 9 mice, KO n = 9 mice, C62A n = 8 mice, D116A n = 8 mice). l: Representative images of livers harvested from CD300ld WT, KO, C62A and D116A mice at day 31 following hydrodynamic injection of Akt/Ras (n = 6 mice per group). m: T cell suppression by splenic PMN-MDSCs isolated from CD300ld WT, KO, C62A and D116A B16-F10 tumor-bearing mice (n = 3 independent samples per group). Representative flow cytometry plots are shown. 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, e, f, g, i, j, k) and Student’s two-sided unpaired t-test (a, b). Data are representative of two (a, b, c) or three (d, e, f, g, i, j, k, l, m) independent experiments.
