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Single-cell RNA-seq of sorted CD8+ T cells from B16 melanoma tumors

ABSTRACT: The response of naive CD8 T cells to acute infections results in the generation of defined yet heterogeneous pools of effector and memory cells. In contrast, the complexity of tumor infiltrating CD8 T cell states is still not fully elucidated. To delineate the landscape of tumor infiltrating CD8 T cells in an unbiased manner, we performed single-cell (full-length) RNA-Seq of CD8 T cells infiltrating mouse B16 melanomas. Our analysis of cell heterogeneity revealed the presence of multiple distinct CD8 T cell subtypes including exhausted, terminal effector, naive and memory-like CD8 T cells, as well as novel intermediate states. TCR reconstruction and pseudotime ordering enabled us to follow differentiation of single clones and suggested distinct trajectories towards T-cell exhaustion.

ORGANISM(S): Mus musculus

PROVIDER: GSE113602 | GEO | 2021/04/24

REPOSITORIES: GEO

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Project description:During a T cell response, naÃ¯ve CD8 T cells differentiate into effector cells. Subsequently, a subset of effector cells termed memory precursor effector cells (MPECs) further differentiates into functionally mature memory CD8 T cells. The transcriptional network underlying this carefully scripted process is not well understood. Here, we report that the transcription factor FoxO1 plays an integral role in facilitating effector to memory transition and functional maturation of memory CD4 and CD8 T cells. We find that FoxO1 is not required for differentiation of effector cells, but in the absence of FoxO1, memory CD8 T cells displayed features of scenescence and progressive attrition in polyfunctionality, which in turn led to impared recall responses and poor protective immunity. These data suggest that FoxO1 is essential for active maintenance of functional CD8 T cell memory and protective immunity. Under competing conditions in bone marrow Single-cell suspensions from splenocytes of eight samples WT (control) and FoxO1-/- (experimental) LCMV-immune mice were prepared using standard procedures. CD8 T cells were then isoloated using Thy1.2 (CD90.2) (30-H12) microbeads (Miltenyi Biotec). Cells were then stained with anti-CD8, anti-CD44 and Db/NP396 MHC class I tetramer. Activated (CD8+CD44hi), naive (CD8+CD44lo), and virus-specific CD8 T cells were sorted using FACSAria II instrument (BD Biosciences). The purity of the cells was >95%. Total RNA was extracted from the sorted cells by Trizol Reagent. RNA samples were reverse transcribed and Cy3-labeled cDNAs were hyrbidized to Agilent whole Mouse Genome Oligo Microarrays. Fluorscence signals were detected using Agilent's Microarray Scanner system, data was analyzed using the Rosetta Resolver gene expression data analysis system and genes with a fold change < and p-values <0.01 were identified. Microarray data discussed in the paper is focused on virus-specific memory CD8 T cells from samples WT_Tet_2 vs KO_Tet_2.

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Single-cell RNA-seq of sorted CD8+ T cells from B16 melanoma tumors

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