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Single cell analysis T cell activation

ABSTRACT: We isolated naÃƒÂ¯ve and memory T cells from healthy donors and cultured them in resting and stimulated conditions. We performed scRNA sequencing to assess T cell actiavtaion and perform sceQTL analysis. Major challenges for translating GWAS variants to function include identification of the disease relevant cell types in which the associated variants function. We have shown that immune-disease variants are functional in CD4+ T cell activation, and understanding how GWAS variants affect the activation efficiency is critical for development of new therapies. To perform sceQTL analysis of naÃƒÂ¯ve and memory T cell activation, here we stimulated T cells and profiled single cell transcriptome at different time points following stimulation. We mapped sceQTLs affecting cell activation. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

PROVIDER: EGAS00001003479 | EGA |

REPOSITORIES: EGA

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Project description:Memory T cells respond to stimulation with more rapid expression of effector cell functions than their naM-CM-/ve counterparts, yet the gene expression signature underlying this enhanced recall response is not known. Therefore, we performed comprehensive, whole-genome expression profiling of murine memory CD8 T cells before and shortly after ex vivo stimulation. We compared this differential expression profile to its counterpart from stimulated naive cells. Given that memory cells arise from naive cells, the quiescent state of both cell populations prior to stimulation, and the early time point analyzed (four hours post-stimulation), it was possible that the stimulation-induced changes in gene expression were identical between the two populations. While there was a high degree of overlap, we found that the majority of up-regulated genes were more highly induced following stimulation of memory cells. This more robust increase in transcript levels was observed for a functionally diverse set of genes, including cytokines, chemokines, amino acid metabolic enzymes and transporters, transcription factors and regulators of RNA processing. We also identified the unique, stimulation-induced signatures of naive and memory CD8 T cells and found that the former was enriched for factors involved in regulating chromatin modifications. Specifically, we found that Hdac 5,7 and 8 transcript levels were rapidly down-regulated following stimulation of naive cells, which correlated with an increase in their total level of acetylated histone H3 (AcH3). This was in contrast to stimulated memory cells, which had higher levels of total AcH3 ex vivo that did not change following short-term stimulation. Furthermore, the unique stimulation-induced expression profile of memory cells was enriched for factors involved in regulating transport of molecules between the nucleus and cytoplasm, including multiple members of the nuclear pore complex. Together, these results support a model whereby the chromatin modifications that occur during the differentiation of naM-CM-/ve cells into memory cells are preserved in resting memory populations, facilitating their more robust re-activation of a functionally diverse set of genes that contribute to rapid recall of effector functions. NaM-CM-/ve (CD44lo) and memory phenotype (CD44hi) CD8 T cells from 7-wk old female B6 mice were FACS-purified and cultured in vitro for four hours in the presence or absence of PMA and ionomycin. Total RNA was purified from un-stimulated (resting) naM-CM-/ve, resting memory, stimulated naive, and stimulated memory cells and hybridized to individual single-color arrays. This purification and stimulation protocol was performed four independent times.

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Single cell analysis T cell activation

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