Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression of early memory and effector precursor CD8 T cells generated by differential TCR signaling strength

ABSTRACT: Asymmetric cell division (ACD) and the strength of initial T cell receptor (TCR) signaling are both implied to contribute to the establishment of cellular heterogeneity and CD8 T cell effector and memory differentiation. However, on a single cell level it remained unclear whether ACD generates progeny of different fates and whether the strength of TCR signaling affects this mechanism. Therefore, we developed experimental systems allowing monitoring the fate of single daughter cell progenies derived from an ACD either induced by weak or strong TCR stimulation by in vitro live imaging. We used the combinatorial expression of TCF1 and CD62L as markers indicating fate specification a few days after activation and analyzed the transcriptional profiles of in vitro generated CD62L+TCF1+ and CD62L-TCF1- cells derived from either antibody-induced (AB) activation or from gp33 (high affinity) or C6 (low affinity) peptide stimulation and compared them to in vivo generated effector and memory cells, respectively. Therefore, we sorted CD62L+TCF1+ and CD62L-TCF1- cells on day 6 post activation, followed by bulk RNA sequencing and found that CD62L+TCF1+ cells transcriptionally resembled in vivo generated memory cells, whereas CD62L-TCF1- cells were similar to in vivo generated effector cells.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Mus musculus

SUBMITTER: Fabienne Gräbnitz

PROVIDER: E-MTAB-12563 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Gene expression profiling of human CD8+ CD161hi and CD161lo central and effector memory and naïve T cell subsets. The mechanisms by which IL-17 secreting cells are regulated have not been completely elucidated. We previously identified a population of rhodamine-effluxing memory CD8+ T cells with high expression of CD161 that contributes to immune reconstitution after lymphopenia-inducing chemotherapy. Here we find that CD161hi CD8+ T cells share transcriptional programming with Th17 cells, but most do not secrete IL-17 or proliferate to stimulation through the T cell receptor (TCR). Transcriptional analysis of subsets identified by expression of CD161 and CD62L revealed a novel mechanism of TCR signaling pathway regulation in CD161hi CD8+ T cells that is distinct from that described in anergic or tolerant cells and renders them functionally dependent on costimulation through innate cytokine receptors or CD28. CD161hi CD8+ T cells, induced to proliferate by a TCR signal delivered with costimulation, demonstrated plasticity that was dependent on the nature of costimulation and resulted in expansion of IL-17 secreting cells that could not proliferate to a TCR signal alone or differentiation to Tc1-like cells that proliferated to TCR stimulation in the absence of costimulation. The data show an association between TCR signaling pathway downregulation and type 17 programming in CD161hi CD8+ T cells, whose dysregulation could mediate IL-17 dependent inflammatory diseases. T cell subsets were sort-purified from healthy adults and analyzed by gene expression profiling. Isolation: Effluxing CD161hi and non-effluxing CD161lo CD8+ TCM and TEM subsets were purified using magnetic bead separation and cell sorting to achieve >98% purity, as previously described (35). CD8+ T cells were positively selected using CD8 Microbeads (Miltenyi Biotec, Germany), loaded with Rh123 (Sigma, St. Louis, MO) and cultured for 30 min to allow Rh123 efflux, then labeled with fluorochrome-conjugated antibodies to CD4, CD16, TCRγδ, Vα24, CD8, CD95, CD62L and CD161. CD161hi and CD161lo TCM and TEM subsets were sort-purified on a FACS ARIA equipped with 405 nm, 488 nm and 633 nm lasers (BD Biosciences). CD161hi TCM and TEM subsets were identified as CD62L+/Rh123lo/CD161hi and CD62L-/Rh123lo/CD161hi events, respectively, in the CD4-/CD16-/TcRγδ-/Vα24-/CD8+/CD95+ population. CD161lo TCM and TEM subsets were identified as CD62L+/Rh123hi/CD161int/neg and CD62L-/Rh123hi/CD161int/neg events, respectively, in the CD4-/CD16-/TcRγδ-/Vα24-/CD8+/CD95+ population. Gene expression profiling: RNA was extracted from sort-purified subsets from 6 healthy individuals using the RNeasy Plus Minikit (Qiagen, Valencia, CA) and biotinylated, followed by generation of amplified cRNA for array hybridization using the Illumina TotalPrep RNA Amplification Kit (Applied Biosystems, Foster City, CA). Amplified biotinylated cRNA was then purified before quality control to ensure high quality cRNA was available for hybridization. Labeled cRNA was hybridized to Illumina WG-6 Expression BeadChips v3.0 (Illumina, San Diego, CA), which quantitate expression of 48,802 transcripts derived from the NCBI RefSeq (Build 36.2, Release 22) and UniGene databases (Build 199). BeadChips were washed before reading and image extraction, and then scanned on an Illumina BeadArray Reader. The resulting TIFF images were processed using GenomeStudio Gene Expression Module (GEM) software. Data quality was assessed using the Control Summary feature in GenomeStudio GEM. For a given analysis set, a GenomeStudio Probe-level Final Report was generated by combining the Sample Probe Profile and Control Probe Profile tables. The Final Report comprising the full dataset was initially processed using the Bioconductor package lumi by employing a background correction estimate and quantile normalization. A small adjustment (i.e. 20 counts) was made to the entire dataset to make all intensity signals non-negative and these values were log2-transformed. The dataset was initially filtered using the ‘shorth’ function of the Bioconductor package genefilter, resulting in retention of 31,084 of 48,802 original probes. Each pairwise comparison was further filtered by discarding probes whose signal intensity was less than a defined signal “noise floor” across all arrays in the data subset. This was achieved by calculating the median of the ‘negative control’ probe signals for each array, averaging these values, and setting the noise floor as 3 times this average. Differential gene expression was then determined using the Bioconductor package limma, and a false discovery rate (FDR) method applied to correct for multiple testing. Significant differential gene expression was defined by a log2 ratio > 0.585 (± 1.5-fold) and FDR (adjusted p value) < 0.05.

Project description:CD8+ tumor infiltrating T cells (TIL) lack effector-phase functions due to defective proximal TCR-mediated signaling shown to result from inactivation of p56lck kinase. We identify a novel interacting partner for p56lck in nonlytic TIL, Protocadherin-18 (‘pcdh18’), and show that pcdh18 is transcribed upon in vitro or in vivo activation of CD8+ central memory T cells (CD44+CD62LhiCD127+) coincident with conversion into effector memory cells (CD44+CD62LloCD127+). Expression of pcdh18 in primary CD8+ effector cells induces the phenotype of nonlytic TIL: defective; proximal TCR signaling, cytokine secretion, and cytolysis; and enhanced AICD. pcdh18 contains a motif (centered at Y505) shared with src kinases (QGQYQP) which is required for the inhibitory phenotype. Thus, pcdh18 is a novel marker of CD8+ effector memory T cells expressed upon cell activation that can function as a negative regulator by restricting the effector phase. We used microarrays to detail the global programme of gene expression underlying CD8+ 'Central memory' T cells activation and identified distinct transcriptional pattern clusters. Ten spleens were pooled from 62 week old C57BL/6 male mice and enriched for CD8+ T cells by negative selection using a cocktail of biotinylated antibodies to deplete CD4, CD11c, CD11b, MHC-II, B220, and NK cells. Cells were then stained for CD8, CD44, CD62L, and CD127 and sorted (using a iCyt Reflection parallel cell sorter). Cells were collected and cultured (0.5 x106 cells/well) in 10% complete RPMI media supplemented with 0.005 ugr Con A and RNA prepared using TRIZOL and the 'RNeasy clean-up kit' (Invitrogen) after activation for different times. We established single transcriptional profiles for 6 time points after T cell activation (4, 6, 8, 16, 20, 24 h) including a control zero h time point (prior to activation). RNA was isolated by standard procedures and its quality was assessed by the NYU Center for Health Informatics and Bioinformatics. cDNAs were hybridized to GeneSpring arrays using the mouse genome MOE430 2.0 array (Affymetrix) which interrogates ~45,000 transcripts.

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Gene expression of early memory and effector precursor CD8 T cells generated by differential TCR signaling strength

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