Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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ATAC-seq of isolated primary human CD8+ T cells treated with KDACi with polyclonal stimulation for 6 days

ABSTRACT: Lysine deacetylase inhibitors (KDACi) show immunomodulatory properties, promoting differntiation of an IL7RhiPD1lo memory precursor (MPEC) phenotype on polyclonal stimulation in vitro and enhancing functional memory responses during infection or immunisation in vivo. We isolated (MACS positive selection) primary human CD8+ T cells from healthy blood donors and treated them for 6 days with low dose sodium valproate (5mM, d0) with concurrent stimulation with anti-CD2/3/28 microbeads, then undertook bulk ATACseq of lysed cells.

INSTRUMENT(S): Miltenyi MACS isolation, Agilent Bioanalyser, NextSeq 500

ORGANISM(S): Homo sapiens

SUBMITTER: Eoin McKinney

PROVIDER: E-MTAB-13367 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Using killer cell lectin-like receptor G1 as a marker to distinguish terminal effector cells from memory precursors, we found that despite their diverse cell fates both subsets possessed remarkably similar gene expression profiles and functioned as equally potent killer cells. However, only the memory precursors were capable of making IL-2 thus defining a novel effector cell that was cytotoxic, expressed granzyme B, and produced inflammatory cytokines in addition to IL-2. This effector population then differentiated into long-lived protective memory T cells capable of self-renewal and rapid re-call responses. Mechanistic studies showed that cells that continued to receive antigenic stimulation during the later stages of infection were more likely to become terminal effectors. Importantly, curtailing antigenic stimulation towards the tail-end of the acute infection enhanced the generation of memory cells. These studies support the decreasing potential model of memory differentiation and show that the duration of antigenic stimulation is a critical regulator of memory formation Experiment Overall Design: An important question in memory development is understanding the differences between effector CD8 T cells that die versus effector cells that survive and give rise to memory cells. In this study we provide a comprehensive phenotypic, functional and genomic profiling of terminal effectors and memory precursors, towards better understanding the generation of these subsets. The two effector subsets were FACS purified during the early expansion phase (Days 4-5 post-infection) and extensively analyzed for their phenotypic (eg. CD127, CD62L, CD27, Bcl-2, Granzyme B, etc.) and functional properties (cytokine production, cytotoxicity, homeostatic proliferation, recall proliferation), and gene expression profiles (by genome-wide microarray analyses). Mechanistic studies involving the extent of proliferation and duration of antigen stimulation on memory differentiation potential of effectors were also performed using adoptive transfer techniques.

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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ATAC-seq of isolated primary human CD8+ T cells treated with KDACi with polyclonal stimulation for 6 days

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