Supplementary Material for: Streptococcus pyogenes infection and CD4+ T cells crosstalk promote an antigen-presenting cell-like phenotype in neutrophils

Introduction: Neutrophils are the most abundant leukocytes in human blood and a key component of host defense against Streptococcus pyogenes (group A streptococci, GAS). They are rapidly recruited to the site of infection, where they mediate phagocytosis, degranulation, and release of neutrophil extracellular traps, thereby influencing both bacterial clearance and tissue damage. Traditionally considered short-lived effector cells, neutrophils are increasingly recognized for their immunomodulatory functions, including regulation of adaptive immunity. Methods: Human neutrophils were infected with GAS wild-type 5448 or isogenic mutants lacking streptolysin O (Δslo) or streptolysin S (ΔsagA) and analyzed either after infection alone or after infection followed by co-culture with autologous CD4⁺ T cells. Neutrophil activation and T cell responses were evaluated via flow cytometry. Results: GAS infection robustly activated neutrophils in a dose-dependent manner, driving expansion of the CD15bright/CD66bbright population and heparin-binding-protein release. Infection prompted neutrophils to acquire APC-like characteristics, including HLA-DR, CD40, and CD86 expression, particularly when exposed to autologous CD4⁺ T cells. In 5448ΔsagA infections, neutrophils showed lower expression of co-stimulatory markers and bacteria were more susceptible to intracellular killing. In co-cultures, CD4⁺ T cells were partially activated, as indicated by CD25 upregulation and Th1- and Th17-cell-associated cytokine release. Conclusion: GAS infected neutrophils acquire an APC-like phenotype and partially modulate CD4⁺ T cell activation, revealing a previously unrecognized role for infected neutrophils in shaping adaptive immunity in streptococcal infections.