Fig. 7

CCL2 is involved in T-cell suppression via the Siglec-sialoglycan axis in suppressive myeloid cells. A MCP-1/CCL2 found in the supernatant of murine MDSC:T-cell cocultures at the endpoint of the experiment from Fig. 4B–D. MDSCs were untreated, or were pretreated with sialidase or a Siglec-E blocking antibody. n = 3 donors per group. B Correlation of MCP-1/CCL2 levels measured in supernatants of murine MDSC:T-cell cocultures at the endpoint and percentage of proliferating CD8⁺ T cells from the same conditions from (A). n = 3 donors per group. C Experimental setup: Neutralization of CCL2 using a neutralization antibody in SigE^ΔLysM mice and SigE^WT littermates bearing B16F10 tumors. Mice were injected with a CCL2 neutralization antibody up to 3 times a week (gray arrow) starting 1 day after subcutaneous B16F10 tumor injection (black arrow). Tumor growth and survival were monitored, and the suppressive capacity of MDSCs was analyzed in vitro. D Kaplan‒Meier survival curves from pooled data from 2 independent experiments. n = 5–8 mice per group. E B16F10 tumors at the endpoint (C) were digested, cocultured with pervanadate and analyzed by phospho-flow cytometry for phosphorylated STAT3 (pSTAT3). The percentage of pSTAT3 gated on total MDSCs is shown. Representative histograms for each condition are shown on the right. n = 3 mice per group. F Suppressive capacity of MDSCs against naïve T cells. Percentage of proliferating CD8⁺ T cells cocultured without MDSCs, (G) with MDSCs from SigE^WT mice or (H) MDSCs from SigE^ΔLysM mice with or without addition of CCL2 blocking antibody. N = 3–5 mice from N = 3 experiments. I Cytokine expression found in the supernatant of human primary CD33⁺:CD8⁺ cell cocultures at the endpoint of the experiment from Fig. 5H. CD33⁺ cells were left untreated or pretreated with sialidase. Z scores were calculated for each cytokine and are shown on a color scale from blue (low) to red (high). n = 3 donors per group. The data are presented as mean ± SD. Two-tailed paired t tests or unpaired t tests (E) were used. R shows the Pearson correlation coefficient. For survival analysis, the log-rank test was used, followed by the Šidák correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001