PROJECT SUMMARY Project 3 The existence of MHC class II-restricted CD4+ helper T cells with cytotoxic potential (CD4-CTLs) has been reported in humans with several viral infections. Importantly, the CD4-CTLs have been reported to play a protective role in several of these viral infections. However, little is known in humans about the biology of CD4- CTL generation and their functional properties. We recently performed single-cell RNA-seq in over 9000 cells to unravel CD4-CTL heterogeneity and functional properties. Our analysis led to the discovery of a distinct subset of long-lived CD4-CTL memory precursors. Understanding the biology of such long-lived CD4-CTL precursors will pave the way for developing strategies to boost durable CD4-CTL immune responses following vaccination against infections. In Project 3, specific aim 1, we will determine the phenotype and functional properties of pathogen-specific and tissue-specific CD4-CTLs in humans. (A) Pathogen-specific features of CD4-CTLs: We will compare the phenotype and molecular features of circulating DENV-, hCMV-, EBV-reactive CD4-CTL precursors and effector cells. (B) Tissue-specific features of CD4-CTLs: Here, we will compare hCMV-, EBV- and pertussis vaccine-reactive CD4-CTLs present in human lungs to those circulating in the blood. In addition, we will compare pathogen-specific CD4-CTLs present in tonsil (lymphoid) tissue vs. blood (collaboration with Crotty, Project 1). (C) Vaccine-induced CD4-CTLs: In collaboration with Crotty (Project 1), we will determine whether yellow fever vaccination induces the generation of CD4-CTL precursors in the blood and lymph nodes. In specific aim 2, (A) we will identify molecular transcription factors driving CD4-CTL differentiation and (B) test their function in in vivo models and in human cells. In summary, our work will fundamentally advance our understanding of the molecular basis of CD4-CTL immunity in humans and will benefit from the synergistic interactions with other Projects.