A gradual decay in humoral and cellular immune responses over time upon SAR1S-CoV-2 vaccination may cause a lack of protective immunity. We conducted a longitudinal analysis of antibodies, T cells, and monocytes in 25 participants vaccinated with mRNA or ChAdOx1-S up to 12 weeks after the 3rd (booster) dose with mRNA vaccine. We observed a substantial increase in antibodies and CD8 T cells specific for the spike protein of SARS-CoV-2 after vaccination. Moreover, vaccination induced activated T cells expressing CD69, CD137 and producing IFN-γ and TNF-α. Virus-specific CD8 T cells showed predominantly memory phenotype. Although the level of antibodies and frequency of virus-specific T cells reduced 4-6 months after the 2nd dose, they were augmented after the 3rd dose followed by a decrease later. Importantly, T cells generated after the 3rd vaccination were also reactive against Omicron variant, indicated by a similar level of IFN-γ production after stimulation with Omicron peptides. Breakthrough infection in participants vaccinated with two doses induced more SARS-CoV-2-specific T cells than the booster vaccination. We found an upregulation of PD-L1 expression on monocytes but no accumulation of myeloid cells with MDSC-like immunosuppressive phenotype after the vaccination. Our results indicate that the 3rd vaccination fosters antibody and T cell immune response independently from vaccine type used for the first two injections. However, such immune response is attenuated over time, suggesting thereby the need for further vaccinations.

Background Tumor cells modulate host immunity by secreting extracellular vesicles (EV) and soluble factors. Their interactions with myeloid cells lead to the generation of myeloid-derived suppressor cells (MDSC), which inhibit the antitumor function of T and NK cells. We demonstrated previously that EV derived from mouse and human melanoma cells induced immunosuppressive activity via increased expression of programmed cell death ligand 1 (PD-L1) on myeloid cells that was dependent on the heat-shock protein 90α (HSP90α) in EV. Here, we investigated whether soluble HSP90α could convert monocytes into MDSC. Methods CD14 monocytes were isolated from the peripheral blood of healthy donors, incubated with human recombinant HSP90α (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 the immunosuppressive function of rHSP90α-treated monocytes. HSP90α levels were measured by ELISA in plasma of patients with advanced melanoma and correlated with clinical outcome. Results We found that the incubation of monocytes with rHSP90α resulted in a strong upregulation of PD-L1 expression, whereas reactive oxygen species (ROS) and nitric oxide (NO) production as well as the expression of arginase-1, ectoenzymes CD39 and CD73 remained unchanged. The PD-L1 upregulation was blocked by anti-TLR4 antibodies and a nuclear factor-κB inhibitor. rHSP90α-treated monocytes displayed the downregulation of HLA-DR expression and acquired the resistance to apoptosis. Moreover, these monocytes were converted into MDSC as indicated by their capacity to inhibit T cell proliferation, which was mediated by TLR4 signaling as well as PD-L1 and indoleamine 2,3-dioxygenase (IDO) 1 expression. Higher levels of HSP90α in plasma of patients with melanoma correlated with augmented PD-L1 expression on circulating monocytic (M)-MDSC. Patients with melanoma with high levels of HSP90α displayed shorter progression-free survival (PFS) on the treatment with immune checkpoint inhibitors (ICIs). Conclusion Our findings demonstrated that soluble rHSP90α increased the resistance of normal human monocytes to apoptosis and converted them into immunosuppressive MDSC via TLR4 signaling that stimulated PD-L1 and IDO-1 expression. Furthermore, patients with melanoma with high concentrations of HSP90α displayed increased PD-L1 expression on M-MDSC and reduced PFS after ICI therapy, suggesting HSP90α as a promising therapeutic target for overcoming immunosuppression in melanoma.