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Gene exression in single T cells across division states.

ABSTRACT: Purpose: To compare diversity of primary human CD8+ T cells that have divided 0, 1, or 2 times on day 3 of ex vivo expansion from naïve resting state. Methods: Naïve T cells were enriched from human peripheral blood monoluclear cells (PBMCs), labeled with CFSE dye, and expanded for 3 days using rapid expansion protocol (Li, Y. & Kurlander, R.J. Journal of Translational Medicine, 2010). On day 3, 10,000 single live CFSE+ CD8+ T cells from each of divisions 0, 1, and 2 were sorted and immediately processed using 10X Genomics single-cell RNA-sequencing platform. Results: We found that undivided cells display the highest gene expression diversity. Using 1,000 most variably expressed genes, we created a force-directed layout, representing a phenotypic map of cellular differentiation across division states. To understand the basis of T-cell diversity, we defined and quantified regions of interest on this map based on diffusion pseudo-time (DPT), a metric of cell differentiation state. Finally, we examined gene expression in cells from each region and found that undivided cells acquire gene expression associated with effector cell function, while remaining cells go on to grow and differentiate. Conclusions: Our study provides insights into T-cell differentiation within an ex vivo expansion system for cancer immunotherapy applications.

ORGANISM(S): Homo sapiens

PROVIDER: GSE119139 | GEO | 2018/12/01

REPOSITORIES: GEO

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Project description:Peripheral tolerance induction is critical for the maintenance of self-tolerance and can be mediated by immunoregulatory T cells or by direct induction of T cell anergy or deletion. While the molecular processes underlying anergy have been extensively studied, little is known about the molecular basis for peripheral T cell deletion. Here, we determined the gene expression signature of peripheral CD8+ T cells undergoing deletional tolerance, relative to those undergoing immunogenic priming or lymphopenia-induced proliferation. From these data, we report the first detailed molecular signature of cells undergoing deletion. Consistent with defective cytolysis, these cells exhibited deficiencies in granzyme up-regulation. Furthermore, they showed antigen-driven Bcl-2 down-regulation and early up-regulation of the pro-apoptotic protein Bim, consistent with the requirement of this BH3-only protein for peripheral T cell deletion. Bim up-regulation was paralleled by defective IL-7Ra chain re-expression, suggesting that Bim-dependent death may be triggered by loss of IL-7/IL-7R signaling. Finally, we observed parallels in molecular signatures between deletion and anergy suggesting that these tolerance pathways may not be as molecularly distinct as previously surmised. NaÃ¯ve (CD44lo) OT-I T cells were CFSE labelled and transferred in a model of deletional tolerance (RIP-OVAhi mice), a model of immunity (mice primed with OVA coated splenocytes and LPS) or a model of lymphopenia induced proliferation (Rag-/- mice). 60 hrs (RIP-OVAhi and OVA coated splenocytes) or 5 days (Rag-/-) after transfer, OT-I cells that had undergone two or more divisions as determined by CFSE dilution were sorted, RNA extracted and samples were prepared for hybridisation to Affymetrix microarrays. As a control for naive cells, CFSE labelled OT-I cells were injected into antigen-free B6 mice and the undivided naive cells were sorted after 60 hrs and also used for microarray analysis. Two replicates were prepared for the naive cells, cells from RIP-OVAhi mice and cells from OVA coated splenocyte primed mice, while one replicate was prepared for the cells from Rag-/- mice.

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Gene exression in single T cells across division states.

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