T cell infiltration in tumors derived from Ccr2^+/+ or Ccr2 ^−/− cancer cells. (A) There is no difference between Ccr2^+/+ and Ccr2^−/− tumors during the growth-restricted phase in the percentage of CD3⁺ T cells among CD45⁺ leukocytes, as determined by flow cytometry for CD45⁺CD3⁺ cells (n = 5). (B) FoxP3⁺CD4⁺ regulatory T cell infiltration is increased in Ccr2^+/+ tumors compared with Ccr2^−/− tumors during the growth-restricted phase, as determined by flow cytometry gated on CD45⁺CD3⁺CD4⁺ cells (n = 5). (C) Depletion of CD8⁺ T cells with anti-CD8a antibody significantly accelerated the growth of tumors from Ccr2^−/− cancer cells (arrows indicate treatment with antibody; n = 5 for all conditions). (D) Pharmacological inhibition of CCR2 increased OT-1 T cell cytotoxicity toward E0771 cancer cells expressing OVA peptide, as measured by chromium (Cr⁵¹) release (each dot is a triplicate from the same experiment, and similar results were obtained in an independently performed experiment). (E) Immune suppression induced by cancer cell CCR2 signaling is confined to the local tumor microenvironment. Ccr2^+/+ cancer cells transplanted into one mammary gland did not alter the growth of tumors from Ccr2^−/− cancer cells transplanted to the contralateral gland. Means ± SEM are indicated. P values were determined by Student’s t test (A, B, and D). Tumor burden was determined by weekly caliper measurements (n = 10 for all conditions). Tumor volume was analyzed at the end time point by one-way ANOVA, followed by Sidak's multiple comparisons test (C) or by Welch’s ANOVA followed by Dunnett’s T3 multiple comparisons test (due to unequal variances; E). *, P < 0.05; **, P < 0.01. N.S., nonsignificant.