UC San Diego

Upon infection, naive T cells proliferate and differentiate into highly specialized effector cells to combat the invading pathogen. Naive CD4+ T cells have the potential to differentiate into multiple functionally distinct T helper (TH) subsets based on the type of infection. Differing pathogens elicit distinct infection milieux that help direct the differentiation of naive CD4+ T cells to ensure that each class of pathogen is countered with the appropriate immune response. The majority of effector T cells will die as the infection wanes, while a small proportion of cells will survive to established a long-lived memory population. This memory population is essential for improved antibody responses and also enables a rapid and robust secondary response against recurring pathogens, thus conferring lasting cellular immunity. However due to the functional breadth of CD4+ T cell lineages, the identification of a conserved memory CD4+ T cell precursor and memory population across TH lineages has proved challenging. Lack of such knowledge impedes the ability to investigate conserved mechanisms of memory CD4+ T cell formation and regulation. To better understand the biology of CD4+ memory T cells, we sought to identify a conserved marker of memory CD4+ T cells across different TH subsets. Utilizing fluorescent reporter mice, we found that expression of Id3, an inhibitor of E protein transcription factors, identified a population of cells within both the CD4+ TFH and TH1 helper lineages that exhibited memory potential in response to secondary infection. Notably, a subset of TH1 memory cells expressing Id3 exhibited enhanced expansion upon response to pathogen, generating both TH1 and TFH secondary effector cell populations, and displayed enrichment of key molecules associated with memory potential when compared to Id3lo TH1 cells. Relative to Id3lo TH1 memory cells, Id3hi TH1 cells exhibited a transcriptomic profile more akin to that of memory T lymphocytes. Thus, we found that Id3 expression serves as an important marker of multipotent memory CD4+ T cells.To investigate novel regulators of CD4+ memory T cells, we took a computational ap- proach by using Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and bulk RNA sequencing (RNA-seq) of effector and memory CD4+ T cell popula- tions. We leveraged the PageRank algorithm to first predict putative regulators based on changes in transcriptomic expression as well as chromatin accessibility between effector and memory CD4+ T cells. Validation of predicted targets utilized the electroporation of CRISPR/Cas9 RNP complex to achieve loss-of-function disruptions of target genes in CD4+ T cell prior to LCMV- Armstrong infection. Although initial testing of predicted targets Srebf2 and Rorb did not reveal significant effects in CD4+ memory T cell formation, the optimization of the CRISPR/Cas9 RNP system has provided an efficient and reliable method for gene-disruption in T cells that undoubtedly expands our ability to investigate T cell biology. Future experiments utilizing this workflow have the potential to identify conserved regulators of CD4+ precursor and memory T cell populations across TH lineages, shedding light on possible mechanism for CD4+ T cell memory formation.