Unknown,Transcriptomics,Genomics,Proteomics

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Expression data from CD8+ T cells undergoing deletional tolerance

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Ian Parish

PROVIDER: E-GEOD-14699 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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The molecular signature of CD8+ T cells undergoing deletional tolerance.

Parish Ian A IA Rao Sudha S Smyth Gordon K GK Juelich Torsten T Denyer Gareth S GS Davey Gayle M GM Strasser Andreas A Heath William R WR

Blood 20090209 19

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. Although 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 o ...[more]

PMID: 19204323

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Project description:T lymphocytes responding to microbial infection give rise to effector cells that mediate acute host defense and memory cells that provide long-lived immunity, but the fundamental question of when and how these cells arise remains unresolved. Here we combine single-cell gene expression analyses with machine-learning approaches to trace the transcriptional roadmap of individual CD8+ T lymphocytes throughout the course of an immune response in vivo. Gene expression signatures predictive of eventual fates could be discerned as early as the first T lymphocyte division and may be influenced by asymmetric partitioning of the interleukin-2 receptor during mitosis. These findings underscore the importance of single-cell analyses in understanding fate determination and provide new insights into the specification of divergent lymphocyte fates early during an immune response to microbial infection. The goal of the study was to profile the gene expression in single CD8+ T cells responding in vivo to a microbial infection over multiple timepoints. 5 x 103 CD8+ CD45.1+ T cells transgenic for the OT-1 T cell receptor (which recognizes Listeria monocytogenes expressing ovalbumin (Lm-ova)) were adoptively transferred into congenic wild-type CD45.2 C57/B6J recipients, followed by infection intravenously one day later with 5 x 103 colony-forming units (CFU) of Lm-OVA. Splenocytes were isolated from recipient mice at 5, 7, or 45 days post-infection. To isolate cells at 3 days post-infection, 2 x 104 OT-1 CD8+ T cells were adoptively transferred. To isolate cells that had undergone their first division, 2 x 106 OT-1 CD8+ T cells were first labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) prior to adoptive transfer and recipient mice were sacrificed at 48 hours post-infection. Unactivated naM-CM-/ve OT-1 CD45.1+ CD8+ T cells were also included. Cells were stained with fluorochrome-labeled antibodies against CD8, CD44, CD4, CD11b, CD11c, and F4/80, and sorted on a MoFlo (Beckman Coulter) or FACS Aria II (BD Biosciences) flow cytometer. The Ct value for each gene analyzed in each individual cell at each time point is reported in the Matrix non-normalized. The 94 ABI TaqMan Assay IDs are listed on the left (A2-A96), along with the accession number (B2-B96) and corresponding gene name (C2-C96) of each gene studied. The Il2 gene expression assay was duplicated. Each heading (D1-BCV)) represents a single cell from each of 13 timepoints or T cell subset that were profiled. The timepoints/T cell subsets and number of each cells profiled from each timepoint/T cell subset were: unactivated naM-CM-/ve (149 cells), distal daughter (48), proximal daughter (83), division 1 (144), day 3 post-infection (143), day 5 post-infection memory precursor (Tmp) (90), day 5 post-infection short-lived effector (Tsle) (79), day 5 post-infection (154), day 7 post-infection memory precursor Tmp (62), day 7 post-infection short-lived effector Tsle (89), day 7 post-infection (134), central memory Tcm (138), and effector memory Tem (136).

Dataset Information

Expression data from CD8+ T cells undergoing deletional tolerance

Publications

The molecular signature of CD8+ T cells undergoing deletional tolerance.

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