PROJECT SUMMARY Infants are especially susceptible to infectious agents, in particular intracellular pathogens, which require mounting a Th1 response for immune protection. The fact that CD4 T cells before birth and in early life are inherently skewed in favor of T regulatory or Th2 responses contributes to this clinical outcome and helps explain why infections are a leading mortality cause among children under the age of 5 years. Contrary to CD4 T cells, innate-like Vγ9Vδ2 (Vδ2) T cells are poised to secrete Th1 cytokines even before birth and acquire potent cytotoxic function shortly after birth. They mount rapid responses against a broad array of pathogens, including protozoa, bacteria and viruses, by sensing microbial intermediates of cholesterol biosynthesis in an MHC-unrestricted manner. Additionally, they can be activated by innate cytokines such as IL-18 and IL-15, secreted by myeloid cells during infections. Despite these key antimicrobial features, Vδ2 cell role against pathogens in early life is understudied and our ability to harness their potential is very limited. Our long- term goal is to fill this gap in knowledge and exploit Vδ2 cells to improve vaccine responses in infants. We recently observed that the inhibitory receptor PD1, which we know serves as a key regulator of Vδ2 cells at birth, appears to mark cells that are unlikely to have developed cytotoxic potential. Specifically, both ex vivo and after expansion, cord blood PD1+CD56- Vδ2 cells contain the lowest proportion of perforin+ cells among the four subsets identified by concomitant assessment of the two markers, while the PD1-CD56+ subset contains the highest. Bulk RNA sequencing performed on sorted Vδ2 cell subsets after in vitro expansion validated the flow cytometry findings. We hypothesize that expression of PD1 in neonatal Vδ2 lymphocytes, especially in absence of CD56, marks cells with specific functional features, possibly representing a unique differentiation state. With the help of key collaborators, we aim to test our hypothesis with existing cord blood samples and assess feasibility and effectiveness of state-of-the-art single cell approaches to study ex vivo sorted Vδ2 cells. We will rely on Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) and a variation of the Assay for Transposase-Accessible Chromatin with sequencing (ATAC-Seq), called ATAC with Select Antigen Profiling by sequencing (ASAP-seq), to investigate the significance of PD1 for Vδ2 cell differentiation and function. We will perform paired CITE-seq and ASAP-seq analyses to address two aims: Aim 1) Compare the transcriptional profile of ex vivo sorted Vδ2 cells at a single cell level using a CITE-seq approach; and Aim 2) Compare the chromatin accessibility profile of sorted PD1+ and PD1- Vδ2 cells by ASAC-seq. This study will advance our understanding of Vδ2 cell function in early life. The results of the proposed research will inform future studies, including a propo...