Phenotype and function of MDSC in tumor site. (A and B) 3 × 10⁵ EL-4 tumor cells were injected i.p. into C57BL/6 mice. After 3 wk, spleens and cells from tumor ascites were collected. Gr-1⁺CD11b⁺ MDSC were sorted (A) and their morphology was evaluated by staining with H&E (B; 200× magnification). Bars, 10 µm. (C, left) Surface markers in gated Gr-1⁺CD11b⁺ MDSC isolated from spleens and tumors of the same mice. (C, right) Ratio of granulocytic/monocytic MDSC. Five experiments were performed. Error bars show mean and SD. (D) Surface markers in gated Gr-1⁺CD11b⁺ MDSC isolated from spleens and solid tumors of the same mice. All tissues were subjected to the same enzymatic digestions. Three indicated tumor models were used. Each experiment included four mice. Mean ± SD is shown. (E and F) Gr-1⁺CD11b⁺ cells purified from spleens of tumor-free mice (naive) or spleens (SPL) and ascites (ASC) of EL-4 tumor-bearing mice were cultured at indicated ratios with 10⁵ splenocytes from transgenic 2C mice. (E) Splenocytes were stimulated with control and specific peptides. IFN-γ production was measured in quadruplicates in ELISPOT assay. Number of spots per 10⁵ 2C splenocytes is shown. Values in cells stimulated with control peptide were subtracted. *, statistically significant (P < 0.05) difference from naive mice. A typical example of four independent experiments is shown. (F) Splenocytes were stimulated with anti-CD3/CD28 antibodies. In addition to the measurement of IFN-γ production, evaluation of splenocyte proliferation was performed using ³H-thymidine uptake. CPM, counts per minute. Thymidine uptake in cells stimulated with control peptide was <1,000 cpm. All experiments were performed in triplicates. A typical result of three performed experiments is shown. Error bars show mean and SD. *, statistically significant (P < 0.05) differences from naive mice.