Differentiation of MLPGs. (A) Example of gating strategy and proportion of CD115⁺ cells among CD11b⁺Ly6C^hiLy6G⁻CD117⁺ MLPGs from BM of EL4-TB mice. Three mice were evaluated. The results of individual experiments, mean and SD, are shown. (B) CD115⁻ and CD115⁺ MLPGs were sorted from the BM of EL-4 TB mice and cultured for 3 d with GM-CSF with or without TES (20% vol/vol). The frequencies of CD115⁺Ly6G⁻ monocytic cells and of CD115⁻Ly6G⁺ granulocytic cells obtained upon differentiation of both subsets of MLPGs are shown. The results of individual experiments (n = 5), mean and SD, are shown. **, P < 0.01; ****, P < 0.0001 by two-tailed Student’s t test. (C) CX3CR1 expression was measured using naive reporter mice in the indicated cell populations. The results of individual experiments (n = 3), mean and SD, are shown. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 by two-tailed Student’s t test. MFI, mean fluorescence intensity. (D) Example of phenotype of cells recovered after 3 d of culture with GM-CSF of CD11b⁺Ly6C^hiLy6G⁻CD117⁻ monocytes and CD11b⁺Ly6C^hiLy6G⁻CD117⁺ MLPGs sorted from BM of naive mice. Numbers in the plots indicate the percentage of gated cells. (E) Numbers of cells generated from naive or EL-4 BM MLPGs after 3-d culture in complete RPMI with GM-CSF. Results of five experiments are shown. *, P < 0.05. (F) Antigen-specific proliferation of CD8⁺ T cells in the presence of Ly6G⁺ cells isolated after cultures of naive or EL-4 TB BM MLPGs. Cultures were done with GM-CSF and additional TES for EL-4 BM MLPGs. Proliferation was measured in triplicate by ³H thymidine uptake. PMEL T cells were used as responder cells in this antigen-specific suppression assay. Mean and SD are shown. Two experiments with similar results were performed. *, P < 0.05; ***, P < 0.001 by two-tailed Student’s t test. No stim, no stimulation; cpm, counts per minute; SSC, side scatter.