show Abstracthide AbstractCytotoxic CD4 T lymphocytes (CD4-CTL) are important in anti-viral immunity.? For example, we have previously shown that in mice, CD4-CTL are important to control ectromelia virus (ECTV) infection.? How viral infections induce CD4-CTL responses remains incompletely understood. Here we demonstrate that not only ECTV but also vaccinia virus and Lymphocytic Choriomeningitis virus induce CD4-CTL, but that the response to ECTV is stronger.? Using ECTV, we also demonstrate that in contrast to CD8-CTL, CD4-CTL differentiation requires constant virus replication and ceases once the virus is controlled.? We also show that Major Histocompatibility Complex Class II molecules on CD11c+?cells are required for CD4-CTL differentiation and for mousepox resistance.? Transcriptional analysis indicated that anti-viral CD4-CTL and non-cytolytic T Helper 1 (Th1) CD4 T cells have similar transcriptional profiles, suggesting that CD4-CTL are terminally differentiated classical Th1 cells.? Interestingly, CD4-CTL and classical Th1 cells expressed similar mRNA levels of the transcription factors ThPOK and GATA-3, necessary for CD4 T cell linage commitment; and Runx3, required for CD8 T cell development and effector function.? However, at the protein level, CD4-CTL had higher levels of the three transcription factors suggesting that further post-transcriptional regulation is required for CD4-CTL differentiation.? Finally, using CRISPR-Cas9 deletion of Runx3 in CD4 T cells, we demonstrate that the development of CD4-CTL but not of classical Th1 CD4 T cells requires Runx3 following ECTV infection.? These results further our understanding of the mechanisms of CD4-CTL differentiation during viral infection and the role of post-transcriptionally regulated Runx3 in this process. Overall design: Murine T cell populations from the liver were sorted on a flow cytometer and total RNA was extracted at 7 days following ectromelia virus infection. Granzyme B (Gzmb) reporter mice (C57BL/6-Gzmbtm1Asmv/Orl) were used to identify cytotoxic T cells. CD44 low Gzmb-, CD44+ Gzmb-, and CD44+ Gzmb+ CD4 T cells as well as CD44+ Gzmb+ CD8 T cells were sorted from a total of 10 livers from ectromelia virus infected mice. As controls, CD4 and CD8 T cells that were CD44 low and CD44+ were also sorted from spleens of naive reporter mice. Total RNA was extracted for up to 10E6 cells for each population using RNA Clean and Concentrator-5' kit from Zymo Research into 25 microliters elution buffer, and 3 samples for each population were collected across independent experiments (total of 24 samples). RNA was sent to Novogene for RNA sequencing with 20 million, 150 PE reads and bioinformatic analysis.