Bystander CD8⁺ conventional memory versus virtual memory T cells in the initial days post-Trypanosoma cruzi infection

Bystander activation has primarily focused on conventional antigen-specific T cells (T_MEM) and other innate immune cell types. However, the role of virtual memory T cells (T_VM) has been largely overlooked, despite their numerical superiority and highly cytotoxic phenotype. Bystander activation is particularly relevant in infections caused by intracellular pathogens. In this study, we aimed to compare the bystander activation potential of T_VM cells versus T_MEM cells during the early days following T. cruzi infection.

Our results demonstrate that T_VM and T_MEM cells, evaluated by flow cytometry, are present but do not undergo significant changes in frequency during the first four days post-infection (p.i.). In an in vitro co-culture system, T_VM or T_MEM cells pre-incubated with IL-12 and IL-18 (effector cells) were cultured with T. cruzi-infected enriched peritoneal macrophages (Tc-PM, target cells). Immunofluorescence assays revealed that both T_VM and T_MEM cells exhibit a highly efficient capacity to kill the parasite and induce degranulation, in contrast to naïve T cells (T_N), which showed almost no cytotoxic activity. Furthermore, intracellular flow cytometry assays confirmed that both T_VM and T_MEM cells produce substantial amounts of IFNγ up to 4 days p.i. when stimulated in vitro with IL-12 and IL-18, whereas T_N cells fail to produce this cytokine. Accordingly, T_VM and T_MEM cells exert their cytotoxic effects via IFNγ production, rather than NKG2D, which subsequently activates reactive oxygen species (ROS) and Nitric Oxide (NO) pathways in Tc-PM. Additionally, we demonstrate that in T_VM cells, IFNγ signaling occurs through STAT1 in Tc-PM. Finally, analysis of human T_VM cells within PBMCs, revealed increased expression of the functional marker granzymes in Chagas disease patients compared to healthy controls.

These results challenge the view that only T_MEM cells dominate early infection control. The equivalency of T_VM and T_MEM cells in parasite clearance suggests T_VM cells are valuable innate-like contributors, providing rapid protection. Their numerical prevalence in unprimed individuals indicates T_VM cells may be an underestimated component of early immunity.