Effect of the tumor microenvironment on MDSC function and differentiation. MDSC isolated from spleens of congenic CD45.1⁺ mice bearing 3 wk s.c. EL-4 tumors were transferred into ascites of CD45.2⁺ EL-4 tumor-bearing recipients. CD45.1⁺ donor cells were recovered using magnetic beads 4 h after cell transfer. For controls, CD45.1⁺ MDSC were transferred i.v. into EL-4 tumor-bearing recipients or naive recipients and recovered from spleens 4 h after cell transfer. (A) After adoptive transfer, CD45.1⁺ MDSCs were cultured with 2C spleen responder cells (Resp.; 1:4 ratio) stimulated with control and specific peptides. IFN-γ production was measured by ELISPOT assay. The number of spots per 10⁵ splenocytes is shown. Values in cells stimulated with control peptide were subtracted. Each experiment was performed in triplicates and repeated twice. *, statistically significant differences (P < 0.05) from the values in splenocytes cultured alone. Error bars show mean and SD. (B) Similar experiments performed using stimulation with anti-CD3/CD28 antibodies. Error bars show mean and SD. (C) Evaluation of gene expression of argI, iNOS, and p47^phox in MDSC postadoptive transfer. Each experiment was performed in triplicates and repeated twice with the same results. Error bars show mean and SD. (D and E) Differentiation of MDSC at different time points after transfer. *, statistically significant (P < 0.05) differences between spleens and ascites. (D) The percentage of Gr-1⁺CD11b⁺ MDSC among all CD45.1⁺ donor cells. Before, the phenotype of MDSC before transfer to the mice. Error bars show mean and SD. (E) The percentage of mature macrophages (F4/80⁺CD11b⁺Gr-1⁻) and DCs (CD11c⁺CD11b⁺Gr-1⁻) among CD45.1⁺ donor cells. Mean ± SD from three experiments is shown. In D and E, * denotes statistically significant (P < 0.05) differences between the proportion of cells in spleens and tumors at the same time point. (F) Differentiation of MDSC in tumor-free peritoneum. Congenic CD45.1⁺ mice were injected i.p. with 1 ml thioglycollate. 3 d later, 5 × 10⁶ MDSCs isolated from spleen of EL-4 tumor-bearing C57BL/6 (CD45.2⁺) mice were injected i.p. Cells were collected 18 h after the injection and the proportion of different myeloid cells was measured within the gated population of CD45.2⁺ donor cells by flow cytometry. Two independent experiments were performed. Error bars show mean and SD.