Fig. 3

Bone marrow MDSCs express a functional β2-adrenergic receptor and are affected by VTA activation. a Representative dot plot demonstrating staining for Gr-1 and CD11b in the bone marrow of LLC tumor-bearing mice. b qPCR analysis of β2-adrenergic receptor (ADRB2) mRNA expression levels by bone marrow M-MDSCs and PMN-MDSCs of tumor-bearing mice (P < 0.004; Mann–Whitney test; n = 5, 6 in each group). c Phospho-flow analysis of intracellular CREB phosphorylation levels in bone marrow MDSCs following incubation with isoproterenol (1 µM; 15 min). Values represent median florescence intensity (MFI) (M-MDSCs P < 0.023, PMN-MDSCs P < 0.0004; Student’s paired t-test; mean ± s.e.m; n = 8). Data represent two independent repeats. d qPCR analysis of ADRB2 mRNA expression levels by MDSCs sorted from the bone marrow or tumor of LLC tumor-bearing mice (P < 0.015; Mann–Whitney test; n = 5). Data represent two independent repeats. e MDSCs were isolated from the bone marrow of tumor-bearing mice and incubated with isoproterenol (1 µM). Changes in gene expression were analyzed by qPCR. f qPCR analysis of TNFα mRNA expression levels by bone marrow MDSCs of tumor-bearing mice, and incubated with isoproterenol (1 µM) (P < 0.016; Mann–Whitney test; n = 5). Data represent one experiment out of two independent repeats; *P < 0.1. g Intracellular TNFα levels in bone marrow MDSCs from VTA-activated mice and controls (injected with virus lacking the DREADD gene). Values represent MFI (P < 0.027; Student’s t-test; mean ± s.e.m; n = 4). h Intracellular TNFα levels in spleen MDSCs from VTA-activated mice and controls (injected with virus lacking the DREADD gene). Values represent MFI (P < 0.039; Student’s t-test; mean ± s.e.m; n = 5, 4). i Analysis of suppression assay (as described in the methods) using bone marrow MDSCs from tumor-bearing mice following treatment with isoproterenol or vehicle (P < 0.007; Student’s t-test; mean ± s.e.m; n = 7, 6). Data represent two independent repeats. j Schematic representation of the experiment measuring the effect of VTA activation on MDSCs immunosuppressive capacity. k Analysis of suppression assay using tumor MDSCs from VTA-activated mice and controls (injected with virus lacking the DREADD gene; P < 0.004; Student’s t-test; mean ± s.e.m; n = 5, 4). Data represent two independent repeats. l CD69 expression levels on tumor CD4⁺ T cells from VTA-activated mice and controls, indicated by MFI (P < 0.001; Student’s t-test; mean ± s.e.m; n = 7). Data represent two independent repeats