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Differential accumulation of splice variants and transcripts as a result of PI3K inhibition in T lymphocytes and the potential role of their gene products in T cell silencing

ABSTRACT: Using measles virus induced T cell suppression as a model, we established that T cell inhibitory protein isoforms can be produced from alternatively spliced pre-mRNAs as a result of virus-mediated ablation of T cell receptor dependent activation of the phosphatidylinositol-3-kinase (PI3K). To asses production of alternative splice variants in response to PI3K abrogation in T cells at a whole cell level, we performed a Human Exon 1.0 ST Array on RNAs isolated from T cells stimulated only or stimulated after PI3K inhibition. We developed a simple algorithm based on a splicing index to detect genes that undergo alternative splicing (AS) or are differentially regulated (RG) on T cell suppression. Applying our algorithm on this model 9% of the genes were assigned as AS, while only 3% were attributed to RG. Though there are overlaps, AS and RG genes differed with regard to functional regulated at the level of AS or RG were found enriched in different functional groups with AS targeting e. g. extra cellular matrix (ECM)-receptor interaction and focal adhesion, while cytokine-receptor interaction, Jak-STAT and p53 pathways were mainly RG. When combined, AS/RG dependent alterations targeted pathways essential for T cell receptor signaling, cytoskeletal dynamics and cell cycle entry strongly supporting the notion that PI3K abrogations interferes with key T cell activation processes at both levels, and that candidates represented within both categories bear the potential to actively contribute to T cell suppression

ORGANISM(S): Homo sapiens

PROVIDER: GSE38255 | GEO | 2013/05/22

SECONDARY ACCESSION(S): PRJNA167699

REPOSITORIES: GEO

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Project description:The immune system relies on the plasticity of its components to produce appropriate responses to frequent environmental challenges. Dendritic cells (DCs) are critical initiators of innate immunity and orchestrate the later and more specific adaptive immunity. The generation of diversity in transcriptional programs is central for effective immune responses. Alternative splicing is widely considered a key generator of transcriptional and proteomic complexity, but its role has been rarely addressed systematically in immune cells. Here we used splicing-sensitive arrays to assess genome-wide gene- and exon-level expression profiles in human DCs in response to a bacterial challenge. We find widespread alternative splicing events and splicing factor transcriptional signatures induced by an E. coli challenge to human DCs. Alternative splicing acts in concert with transcriptional modulation, but these two mechanisms of gene regulation affect primarily distinct functional gene groups. Alternative splicing is likely to have an important role in DC immunobiology because it affects genes known to be involved in DC development, endocytosis, antigen presentation and cell cycle arrest 6 samples were analysed. 3 were used as controls (non-stimulated replicates) and 3 were stimulated with E.coli for 3 distinct periods: (T1) 4 hours, to cover early changes that occur after the bacterial challenge; (T2) 8 hours, to assess intermediate changes; and (T3) 18 hours, to measure the late response stage. Microarray data pre-processing and summarization was performed using AltAnalyze application, which includes several rigorous statistical methods for data filtering, summarization and determination of differential splicing. Briefly, low quality probesets were removed according to detection above background (DABG) p-value and absolute expression values. Next, probesets were summarized with RMA algorithm and using the AltAnalyze annotations, derived from Ensembl [29] and UCSC databases [30]. Gene expression levels were determined using only probesets targeting constitutive exons. Determination of overall gene level variation was assessed using linear models and empirical Bayes methods s implemented in M-bM-^@M-^XlimmaM-bM-^@M-^Y package. The B-statistics gives the log odds of differential expression and it requires an M-bM-^@M-^Xa prioriM-bM-^@M-^Y value for the estimated proportion of differentially expressed genes. To determine this value, we visually inspected the volcano plot, which compares biological significance (represented by fold-changes) with statistical significance (B-values), finding the value which enabled genes to be distinguished from the majority (logFCM-bM-^IM-$~1.585). Additionally, we verified the P-values corresponding to moderated F-statistics. Using the Benjamini and Hochberg method, all genes selected as differentially expressed had adjusted P-values <0.05. . Detection of alternative splicing variations were determined using splicing-index and FIRMA approaches, calculating MiDAS and normalized intensity p-values (p-value <0.05).

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Differential accumulation of splice variants and transcripts as a result of PI3K inhibition in T lymphocytes and the potential role of their gene products in T cell silencing

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