Primary EBV Infection Induces an Acute Wave of Activated Antigen-Specific Cytotoxic CD4+ T Cells

CD4⁺ T cells are essential for immune protection against viruses, yet their multiple roles remain ill-defined at the single-cell level in humans. Using HLA class II tetramers, we studied the functional properties and clonotypic architecture of EBV-specific CD4⁺ T cells in patients with infectious mononucleosis, a symptomatic manifestation of primary EBV Infection, and in long-term healthy carriers of EBV. We found that primary Infection elicited oligoclonal expansions of T_H1-like EBV-specific CD4⁺ T cells armed with cytotoxic proteins that responded immediately ex vivo to challenge with EBV-infected B cells. Importantly, these acutely generated cytotoxic CD4⁺ T cells were highly activated and transcriptionally distinct from classically described cytotoxic CD4⁺ memory T cells that accumulate during other persistent viral infections, including CMV and HIV. In contrast, EBV-specific memory CD4⁺ T cells displayed increased cytokine polyfunctionality but lacked cytotoxic activity. These findings suggested an important effector role for acutely generated cytotoxic CD4⁺ T cells that could potentially be harnessed to improve the efficacy of vaccines against EBV.