The Staphylococcus aureus pore-forming toxin PVL is most likely causative for life-threatening necrotizing infections, which are characterized by massive tissue inflammation and necrosis. Whereas the cytotoxic action of PVL on human neutrophils is already well established, the PVL effects on other sensitive cell types, such as monocytes and macrophages, are less clear. In this study, we used different types of human leukocytes (neutrophils, monocytes, macrophages, lymphocytes) to investigate cell-specific binding of PVL subunits and subsequent proinflammatory and cytotoxic effects. In all PVL-sensitive cells, we identified the binding of the subunit LukS-PV as the critical factor for PVL-induced cytotoxicity, which was followed by binding of LukF-PV. LukS-PV binds to monocytes, macrophages, and neutrophils but not to lymphocytes. Additionally, we showed that PVL binding to monocytes and macrophages leads to release of caspase-1-dependent proinflammatory cytokines IL-1β and IL-18. PVL activates the NLRP3 inflammasome, a signaling complex of myeloid cells that is involved in caspase-1-dependent IL-1β processing in response to pathogens and endogenous danger signals. Specific inhibition of this pathway at several steps significantly reduced inflammasome activation and subsequent pyronecrosis. Furthermore, we found that PAMPs and DAMPs derived from dying neutrophils can dramatically enhance this response by up-regulating pro-IL-1β in monocytes/macrophages. This study analyzes a specific host signaling pathway that mediates PVL-induced inflammation and cytotoxicity, which has high relevance for CA-MRSA-associated and PVL-mediated pathogenic processes, such as necrotizing infections.

Classical dendritic cells (cDC) are professional antigen-presenting cells (APC) that regulate immunity and tolerance. Neutrophil-derived cells with properties of DCs (nAPC) are observed in human diseases and after culture of neutrophils with cytokines. Here we show that FcγR-mediated endocytosis of antibody-antigen complexes or an anti-FcγRIIIB-antigen conjugate converts neutrophils into nAPCs that, in contrast to those generated with cytokines alone, activate T cells to levels observed with cDCs and elicit CD8+ T cell-dependent anti-tumor immunity in mice. Single cell transcript analyses and validation studies implicate the transcription factor PU.1 in neutrophil to nAPC conversion. In humans, blood nAPC frequency in lupus patients correlates with disease. Moreover, anti-FcγRIIIB-antigen conjugate treatment induces nAPCs that can activate autologous T cells when using neutrophils from individuals with myeloid neoplasms that harbor neoantigens or those vaccinated against bacterial toxins. Thus, anti-FcγRIIIB-antigen conjugate-induced conversion of neutrophils to immunogenic nAPCs may represent a possible immunotherapy for cancer and infectious diseases.

BACKGROUND T cells control viral infection and promote vaccine durability and in COVID-19 associate with mild disease. We investigated whether prior Measles-Mumps-Rubella (MMR) or Tetanus-Diptheria-Pertussis (Tdap) vaccination elicit cross-reactive T cells that mitigate COVID-19. METHODS Antigen presenting cells (APC) loaded ex vivo with SARS-CoV-2, MMR or Tdap antigens and autologous T cells from COVID-19 convalescent and uninfected individuals, and COVID-19 mRNA vaccinated donors were co-cultured and T cell activation and phenotype were detected by IFN-γ ELISpot assays and flow cytometry. ELISA assays and validation studies identified the APC-derived cytokine(s) driving T cell activation. TCR clonotyping and scRNA-seq identified cross-reactive T cells and their transcriptional profile. A propensity-weighted analysis of COVID-19 patients estimated the effects of MMR and Tdap vaccination on COVID-19 outcomes. FINDINGS High correlation was observed between T cell responses to SARS-CoV-2 (Spike-S1 and Nucleocapsid) and MMR and Tdap proteins in COVID-19 convalescent and vaccinated individuals. The overlapping T cell population contained an effector memory T cell subset (TEMRA) implicated in protective, anti-viral immunity and their detection required APC-derived IL-15, known to sensitize T cells to activation. Cross-reactive TCR repertoires detected in antigen-experienced T cells recognizing SARS-CoV-2, MMR and Tdap epitopes had TEMRA features. Indices of disease severity were reduced in MMR or Tdap vaccinated individuals by 32-38% and 20-23% respectively, among COVID-19 patients. CONCLUSIONS Tdap and MMR memory T cells reactivated by SARS-CoV-2 may provide protection against severe COVID-19 disease. FUNDING National Institutes of Health (R01HL065095, R01AI152522, R01NS097719), donation from Barbara and Amos Hostetter and the Chleck Foundation.