Figure 8

Transcription factors promoting human MDSC suppressive function. A, HIF1α, STAT3, and C/EBPβ expression in tumor cell line-induced CD33⁺ or CD11b⁺ MDSC compared with medium only controls as measured by qRT-PCR. Mean shown (data from six unique donors, two independent experiments) +SEM; * indicates statistical significance, p <0.05, † indicates p = 0.06. B, Immunohistochemisty of C/EBPβ, p-STAT3, and HIF1α in CD33⁺ (left) and CD11b⁺ (right) MDSC and CD33⁺ medium controls (middle). Representative images shown from multiple samples stained (400x, original magnification) with arrows showing positive staining areas for p-STAT3 and HIF1α. C, Inhibition of CD33⁺ human MDSC subset by celecoxib and celecoxib analogs via a non-COX2 dependent mechanism. Studies in our laboratory have identified Celecoxib and analogs dimethyl celecoxib (DMC) and unmethylated celecoxib (UMC) as inhibitors of suppressive function in CD33⁺ MDSC in vitro. Of note, the reversal of MDSC effects by CXB and analogs DMX and UMC does not appear to rely upon cyclo-oxygenase (COX)2 enzyme inactivation, as demonstrated by the persistence of therapeutic effects in the presence of prostaglandin E₂ rescue, efficacy of analog DMC with low to absent COX inhibitory action, and the absence of effect seen with the structurally-unrelated COX2-seletcive inhibitor naproxen. For these studies, human CD33⁺ MDSC induced by cancer cell lines were co-cultured with fresh, autologous CFSE-labeled T cells at a ratio of 1:4 in the presence or absence of drugs (black bars) and prostaglandin E2 (PGE2, gray bars) as indicated. T cell stimulation was provided by anti CD3/CD28 microbeads. After three days in culture, T cell proliferation was measured as CFSE dilution by flow cytometry. Mean T cell proliferation shown where possible (n = 2 for no drug, 1 μM, and 10 μM; n = 1 for 20 μM and 20 μM + PGE2) + SD, two independent experiments. D, Transcriptional changes in MDSC subsets associated with inactivation of suppressive function. (left panel) Reversal of CD33⁺ MDSC suppressive function by ATRA, sunitinib, and CXB correlated with decreased STAT3 and HIF1α expression (green arrows). (right panel) Functional inhibition of human CD11b⁺ MDSC by ATRA and Sunitinib correlated with decreased C/EBPβ levels (green arrow), but no change in STAT3 and HIF1α mRNA levels. CXB was not found to have inhibitory actions on CD11b⁺ MDSC and it was not observed to decrease C/EBPβ levels in this population. Mean shown (data from three unique donors) + SEM, * indicates statistically significant decrease (p <0.05) in transcript level in drug-treated MDSC compared with untreated MDSC (ANOVA with Dunnett post-test).