Figure 2

Induction and functional characterization of canonical CD33⁺ MDSC by human tumor cell lines. A, HNSCC-induced MDSC inhibit autologous T cell proliferation and IFNγ production. A subset of HNSCC cell lines induces a CD33⁺ population with suppressive function characteristic of MDSC, including inhibition of autologous T cell proliferation (left) and IFNγ secretion (right). Tumor cell lines are grouped by strength of MDSC induction: strong (black), weak (gray), and non-inducing (white). For both graphs, mean shown (n ≥ 2 donors) +SEM. * indicates statistical significance by ANOVA followed by Dunnett post-test for comparison to T cells alone, p <0.05. B, Human MDSC mediate suppression through up-regulation of ARG-1, NOX2, iNOS, VEGF, and TGFβ. A, Expression of putative suppressive genes ARG-1, iNOS, NOX2-component NCF1, VEGF, and TGFβ in a subset of tumor cell line-induced CD33⁺ MDSC. Mean fold change (n ≥ 2 donors per tumor cell line) +SEM, relative to CD33⁺ cells cultured in medium alone, are shown. * indicates statistical significance, p <0.05, by ANOVA followed by Dunnett test for pairwise comparisons to medium only CD33⁺ controls. C, Heatmap showing expression of immune modulatory cytokines by HNSCC cell lines in relation to their ability to induce CD33⁺ MDSC. MDSC-inducing cell lines produce increased IL-1β, IL-6, TNFα, VEGF, and GM-CSF. Expression of ten putative MDSC-inducing factors was measured in MDSC-inducing (bold) and non-inducing HNSCC cell lines by qRT-PCR. Increased CD33⁺ MDSC-induction capacity was associated with greater expression of IL-1β, IL-6, TNFα, and VEGF (p <0.05). Mean fold change (n = 2) relative to human reference RNA (gray shading = increased, white = decreased expression), p value shown is for linear regression analysis for factors having significantly higher gene expression in MDSC-inducing compared with non-inducing human HNSCC cell lines by one-way ANOVA followed by Tukey's post-test. D, Removal of GM-CSF, IL-6, or IL-1β from co-culture impairs CD33⁺ MDSC induction by tumor cell lines. T cell proliferation when co-cultured with CD33⁺ MDSC from tumor cell line (SCCL-MT1 or USC-HN2) co-cultures with neutralizing antibodies to GM-CSF, IL-6, IL-1β, TNFα, or VEGF. Mean shown (n = 5, four independent experiments), +SEM. * indicates statistical significance, p <0.05.