Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Expression data from CD8+ Central memory T cells after different time periods of Concanavalin A in vitro activation.

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Roy Blum

PROVIDER: E-GEOD-34618 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Immune memory cells are poised to rapidly expand and elaborate effector functions upon reinfection. However, despite heightened readiness to respond, memory cells exist in a functionally quiescent state. The paradigm is that memory cells remain inactive due to lack of TCR stimuli. Here we report a unique role of Tregs in orchestrating memory quiescence by inhibiting effector and proliferation programs through CTLA-4. Loss of Tregs resulted in activation of genome-wide transcriptional programs characteristic of potent effectors, and both developing and established memory quickly reverted to a terminally differentiated (KLRG-1hi/IL-7R±lo/GzmBhi) phenotype, with compromised metabolic fitness, longevity, polyfunctionality and protective efficacy. CTLA-4, an inhibitory receptor overexpressed on Tregs, functionally replaced Tregs in trans to rescue Treg-less memory defects and restore homeostasis of secondary mediators as well. These studies present CD28-CTLA-4-CD80/CD86 axis as a novel target to potentially accelerate vaccine-induced immunity and improve T-cell memory quality in current cancer immunotherapies proposing transient Treg-depletion. We used microarray analysis to detail the global programming of gene expression in LCMV GP33-specific CD8 T cells differentiated in the presence or absence of regulatory T cells Differentiation of memory CD8 T cells entails a progressive transition of highly activated effector program to a quiescent memory program. A key question in the field is to understand the factors that aid in the differentiation of memory cells from effector cells. It is a generally accepted paradigm that effector cells transition to a memory state by default after antigen clearance, since TCR stimuli is the key driver of effector programs in CD8 T cells. We hypothesized that the effector to memory transition of CD8 T cells involves active immunological brakes through regulatory T cells (Tregs) that allow the highly activated effector cells to convert into quiescent memory cells. To address this hypothesis, we used FoxP3-DTR mice to deplete Tregs during the window following antigen clearance, during which the effector CD8 T cells convert to long-lived memory cells. To get a deeper understanding of the global transcriptome of CD8 T cells as they transition from an effector to a memory state, we isolated and arrayed the antigen-specific CD8 T cells at day 16 post-infection that have experienced the transitional environment with and without the presence of Tregs.

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Expression data from CD8+ Central memory T cells after different time periods of Concanavalin A in vitro activation.

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