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RNA-Seq analysis of P14 CD8+T cells in B16-GP tumors

ABSTRACT: RNA-Seq analysis of P14 CD8+T cells in B16-GP tumors

PROVIDER: PRJNA678289 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR13055323.fastq.gz	Fastqsanger.gz
	SRR13055324.fastq.gz	Fastqsanger.gz
	SRR13055325.fastq.gz	Fastqsanger.gz
	SRR13055326.fastq.gz	Fastqsanger.gz
	SRR13055327.fastq.gz	Fastqsanger.gz

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RNA-Seq analysis of P14 CD8+T cells in B16-GP tumors

Project description:CD8+ T cells play an important role in fighting against tumors. However, CD8+ T cells often become functionally impaired or “exhausted” in the tumor microenvironment. T cell exhaustion is a major hurdle that currently limits the efficacy of immunotherapeutic regimens. The exhausted T cell population is heterogeneous and contains T cells subsets at varying states of differentiation and with a range of effector functions and proliferative potential. Identification of exhausted T cell-specific markers will help to understand the heterogeneity of this population. Therefore, we have performed RNA-sequencing analysis of mouse tumor-infiltrating CD8+ T cells and splenic CD8+ T cells.

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Project description:The PI3K/Akt signaling pathway impacts various aspects of CD8 T cell homeostasis, such as effect versus memory cell differentiation, during viral infection. We used microarrays to determine which downstream molecules were affected and what other signaling pathways were interconnected with the Akt pathway by constitutive activation of Akt in LCMV-infected CD8 T cells. Splenocytes from naive P14/WT or P14/Akt mice were stained with anti-CD8 and anti-Ly5.1, and CD8 T cells were sorted using a FACSAria II instrument. Purified Ly5.1+ CD8 T cells from P14/WT or P14/Akt mice were transferred into B6 mice, which were subsequently infected with LCMV Armstrong. At day 8 post infection, splenocytes were stained with anti-CD8, anti-Ly5.1, anti-KLRG1, and anti-CD127. Following staining, short-lived effector cells (SLECs) and memory precursor effector cells (MPECs) were sorted using the FACSAria II instrument; the purity of the sorted cells was >95%. A total of 5 samples were analyzed, including WT naive, WT SLEC, WT MPEC, Akt naive and Akt SLEC.

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Project description:CD8 T cells are the main effectors of the adaptive immune response against intracellular pathogens and cancer. Chronic antigen stimulation and inflammation lead to CD8 T cell differentiation and function that differ from those generated during acute infections. A comprehensive definition of the heterogeneity existing within both tumor-specific and total CD8 tumor-infiltrating T cells remains to be clearly established. To investigate this heterogeneity at the transcriptomic level, we performed paired single-cell RNA and TCR sequencing of >3500 CD8 T cells infiltrating B16 murine melanoma tumors, including cells of known tumor-specificity.

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Project description:During acute viral infections, effector CD8+ T cells differentiate into memory precursors or short-lived terminal effectors. miR-17-92a over-expression skews CD8+ effector cells to the terminal differentiation. We used microarray to identify the genes that are differentially expressed caused by miR-17-92a over-expression. CD8+ T cells from P14 TCR transgenic mice were infected with miR-17-92a-MSCV-IRES-Thy1.1 vector and transfer to C57BL6 recipients. Chimeras were infected with LCMV Armstrong. Thy1.1+ miR-17-92a-MSCV-IRES-Thy1.1 transduced P14 cells and Thy1.1- non-transduced P14 cells were sorted by FACS. RNA was extracted from samples, labeled, and hybridized to Affymetrix microarrays.

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