Supplementary Material for: <b><i>Streptococcus pneumoniae</i></b> Impairs Maturation of Human Dendritic Cells and Consequent Activation of CD4⁺ T Cells via Pneumolysin

Influenza A Virus (IAV), <i>Staphylococcus aureus</i> (staphylococci), and <i>Streptococcus pneumoniae</i> (pneumococci) are leading viral and bacterial causes of pneumonia. Dendritic cells (DCs) are present in the lower respiratory tract. They are characterized by low expression of co-stimulatory molecules, including CD80 and CD86 and high capacity of antigen uptake. Subsequently, DCs upregulate co-stimulatory signals and cytokine secretion to effectively induce T-cell priming. Here, we investigated these processes in response to bacterial and viral single as well as coinfections using human monocyte-derived (mo)DCs. Irrespective of single or coinfections, moDCs matured in response to IAV and/or staphylococcal infections, secreted a wide range of cytokines, and activated CD4⁺, CD8⁺ as well as double-negative T cells. In contrast, pneumococcal single and coinfections impaired moDC maturation, which was characterized by low expression of CD80 and CD86, downregulated expression of CD40, and a mild cytokine release resulting in abrogated CD4⁺ T-cell activation. These actions were attributed to the cholesterol-dependent cytotoxin pneumolysin (Ply). Infections with a <i>ply</i>-deficient mutant resulted in restored moDC maturation and exclusive CD4⁺ T-cell activation. These findings show that Ply has important immunomodulatory functions, supporting further investigations in specific modalities of Ply-DC interplay.