Fig. 7: Effect of pharmacological inhibition of p38 phosphorylation of MDSC function and tumor growth.
a Left panel. PMN were isolated from the blood of healthy volunteers and cultured for 16–18 h with medium containing 20 ng/ml of GM-CSF with or without p38i (LY2228829 1 μM). IFNAR1 level was measured by flow cytometry. N = 4. p Value was calculated in two-sided unpaired Student’s t test. Right panel. Healthy donors PMNs were pre-treated with 1 µM LY2228820 (p38 inhibitor; p38i) followed by 16–18 h incubation with 30% TES. IFNAR1 levels were measured by flow cytometry (n = 3) and expressed as fold changes over untreated cells. p Values were calculated in one-way ANOVA test with correction for multiple comparisons. b PMN and Monocytes were isolated from the bone marrow of mice by cell sorting, pretreated with 1 μM p38i followed by incubation with 20% TES (n = 3) for 16–18 h. IFNAR1 was measured by flow cytometry. Geometric MFI is shown. Data are expressed as mean ± SEM. p Values were calculated in one-way ANOVA test with corrections for multiple comparisons. c HPC-derived PMN-MDSCs were treated with 1 µM p38i before assessing their suppressive activity in triplicates. Two experiments with the same results were performed. Data are expressed as mean ± SEM (n = 3). p Values were calculated in two-sided unpaired Student’s t test. d Suppressive function of PMN-MDSCs from the spleen of EL-4 TB mice after in vitro treatment for 3 h with 1 µM p38i before coculture with responder T cells. p Value was calculated in two-sided unpaired Student’s t test. e BM PMNs from tumor-free mice were treated with 1 µM p38i for 3 h prior to the suppression assay. f MC38 tumor growth in mice treated with Poly I:C and p38i (n = 4). p Values are calculated by two-way ANOVA test with correction for repeated measurements. g MC38 tumor growth in mice treated with anti-CD8 antibody, Poly I:C and p38i (n = 5). Control is mice treated with PBS. p Values are calculated by two-way ANOVA test with correction for repeated measurements. h Suppressive activity of spleen PMN-MDSCs from control or anti-CD8 antibody, Poly I:C and p38i treatment group (n = 4). p Values were calculated in unpaired, two-sided Student’s t test. *p < 0.05. i Totally, 106 MC38 cells was injected s.c. to the mice lacking IFNAR1 in myeloid cells (Ifnar1fl/fl Cre+) or control mice (Ifnar1fl/fl Cre−), and tumor growth was measured every 2–4 days (n = 5 in Ifnar1fl/fl Cre− group, n = 4 in all other groups). Treatment started at day 12 with poly: IC in PBS (10 µg/mouse) i.p. daily and p38 inhibitor (LY2228829 1 mg/kg) prepared in methylcellulose administered by oral gavage every other day. Control mice received an equal amount of vehicle (PBS i.p. and methylcellulose by oral gavage). p Values are calculated by two-way ANOVA test with correction for repeated measurements.
