6 research outputs found
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Investigating transcriptional regulators of memory helper T cells
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
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GTPBP1 resolves paused ribosomes to maintain neuronal homeostasis.
Ribosome-associated quality control pathways respond to defects in translational elongation to recycle arrested ribosomes and degrade aberrant polypeptides and mRNAs. Loss of a tRNA gene leads to ribosomal pausing that is resolved by the translational GTPase GTPBP2, and in its absence causes neuron death. Here, we show that loss of the homologous protein GTPBP1 during tRNA deficiency in the mouse brain also leads to codon-specific ribosome pausing and neurodegeneration, suggesting that these non-redundant GTPases function in the same pathway to mitigate ribosome pausing. As observed in Gtpbp2-/- mice (Ishimura et al., 2016), GCN2-mediated activation of the integrated stress response (ISR) was apparent in the Gtpbp1-/- brain. We observed decreased mTORC1 signaling which increased neuronal death, whereas ISR activation was neuroprotective. Our data demonstrate that GTPBP1 functions as an important quality control mechanism during translation elongation and suggest that translational signaling pathways intricately interact to regulate neuronal homeostasis during defective elongation
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Transcriptional programming of CD4+ TRM differentiation in viral infection balances effector- and memory-associated gene expression
After resolution of infection, T cells differentiate into long-lived memory cells that recirculate through secondary lymphoid organs or establish residence in tissues. In contrast to CD8+ tissue-resident memory T cells (TRM), the developmental origins and transcriptional regulation of CD4+ TRM remain largely undefined. Here, we investigated the phenotypic, functional, and transcriptional profiles of CD4+ TRM in the small intestine (SI) responding to acute viral infection, revealing a shared gene expression program and chromatin accessibility profile with circulating TH1 and the progressive acquisition of a mature TRM program. Single-cell RNA sequencing identified heterogeneity among established CD4+ TRM, which were predominantly located in the lamina propria, and revealed a population of cells that coexpressed both effector- and memory-associated genes, including the transcriptional regulators Blimp1, Id2, and Bcl6. TH1-associated Blimp1 and Id2 and TFH-associated Bcl6 were required for early TRM formation and development of a mature TRM population in the SI. These results demonstrate a developmental relationship between TH1 effector cells and the establishment of early TRM, as well as highlighted differences in CD4+ versus CD8+ TRM populations, providing insights into the mechanisms underlying the origins, differentiation, and persistence of CD4+ TRM in response to viral infection
Gut CD4+ T cell phenotypes are a continuum molded by microbes, not by TH archetypes
CD4 effector lymphocytes (T ) are traditionally classified by the cytokines they produce. To determine the states that T cells actually adopt in frontline tissues in vivo, we applied single-cell transcriptome and chromatin analyses to colonic T cells in germ-free or conventional mice or in mice after challenge with a range of phenotypically biasing microbes. Unexpected subsets were marked by the expression of the interferon (IFN) signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete clusters fitting classic helper T cell (T ) subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as T markers were distributed in a polarized continuum, which was functionally validated. Clones derived from single progenitors gave rise to both IFN-Îł- and interleukin (IL)-17-producing cells. Most of the transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activities of activator protein (AP)-1 and IFN-regulatory factor (IRF) transcription factor (TF) families, not the canonical subset master regulators T-bet, GATA3 or RORÎł. + eff eff eff H