Expression data from recombinant HSP90α (rHSP90α) treated human monocytes

Tumor cells modulate host immunity by secreting extracellular vesicles (EV) and soluble factors in circulation. Their interactions with myeloid cells could lead to the generation of myeloid-derived suppressor cells (MDSC), which strongly inhibit the anti-tumor function of T and NK cells. We demonstrated previously that EV derived from mouse and human melanoma cells induced such immunosuppressive activity via upregulating the expression of programmed cell death ligand 1 (PD-L1) on myeloid cells that was dependent on the heat-shock protein 90a (HSP90a) in EV and on the toll-like receptor (TLR) on myeloid cells. Here, we investigated whether soluble HSP90α could convert monocytes into immunosuppressive MDSC. CD14+ monocytes were isolated from the peripheral blood of healthy donors, incubated with human rHSP90α alone or in the presence of inhibitors of TLR4 signaling and analyzed by flow cytometry. Inhibition of T cell proliferation assay was applied to assess immunosuppressive function of rHSP90α-treated monocytes. The concentration of HSP90α was measured by ELISA in plasma of advanced melanoma patients and correlated with clinical outcome. We found that the incubation of monocytes with rHSP90α for 16 h resulted in a strong upregulation of PD-L1 expression, whereas ROS and NO production as well as the expression of arginase-1, adenosine producing ectoenzymes CD39 and CD73 remained unchanged. The PD-L1 upregulation can be blocked by anti-TLR4 antibodies and an NF-κB inhibitor. After longer incubation (for 24h), rHSP90α-treated monocytes downregulated HLA-DR expression and acquired an augmented viability and resistance to apoptosis. Moreover, these monocytes were converted into MDSC indicated by their capacity to inhibit T cell proliferation mediated by TLR4 signaling as well as PD-L1 and indolamin-2,3-Dioxygenase (IDO) 1 expression. Higher levels of HSP90α in plasma of melanoma patients correlated with augmented PD-L1 expression on circulating monocytic (M) MDSC. Furthermore, melanoma patients with high levels of HSP90α displayed shorter progression-free survival (PSF) upon the treatment with immune checkpoint inhibitors (ICI). Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy donors by density gradient centrifugation using Biocoll (Biochrom). CD14+ monocytes were isolated by MACS (Miltenyi Biotec) according to the manufacturer’s instructions. Monocytes were cultured at a concentration of 1x106/mL in RPMI-1640 medium supplemented with 10 mM HEPES, 1mM Sodium pyruvate, 1mM non-essential amino acids, 50µM β-mercaptoethanol, 10% FBS, 1% Penicillin/Streptomycin (all Gibco). Monocytes were treated with rHSP90a at a concentration of 2 μg/mL/106 cells or with PBS (control) for 24 hours. To obtain a more comprehensive view on the effect of rHSP90α on human monocytes, we performed a microarray analysis with Affymetrix Human Clariom S array, comparing rHSP90α-treated and control monocytes.