Unknown,Transcriptomics,Genomics,Proteomics

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RNA sequencing of CD57+ and CD57- effector memory CD8 T cells in patients with type 1 diabetes

ABSTRACT: Type 1 diabetes is an autoimmune disease in which insulin-secreting β cells of the pancreatic islets are selectively destroyed. CD8 T cells are regarded as critical players in mediating β cell destruction and as a result, considerable effort has been expended to define CD8 T cell behaviour in this disease. The overarching aim of the experiment is to characterize a recently identified autoreactive CD57-positive CD8 T cell subset which is associated with loss of function of islet beta cells in type 1 diabetes, to compare the phenotype of the CD57-positive effector memory CD8 T cells versus the CD57-negative compartment, and provide an insight into the function of these cells in the disease process. To that aim, HLA-A*24 positive patients with T1D -within 2 years of diagnosis- were asked to provide a blood, and following consent, and PBMC were isolated. CD57-positive and CD57-negative CD8 T cell populations were sorted by FACS, and finally, RNA was extracted. Amplified cDNA was obtained and used for the library preparation.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Irma Pujol Autonell

PROVIDER: E-MTAB-6727 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:T cells contribute to host-tumor interactions in patients with monoclonal gammopathies. Expansions of CD8+CD57+TCRVÎ²+ restricted cytotoxic T cell (CTL) clones are found in 48% of patients with multiple myeloma and confer a favorable prognosis. We now report the presence of CTL clones with varying TCRVÎ² repertoire in 70% of patients with Waldenstromâs Macroglobulinaemia (WM) (n=20). Previous nucleoside analogue (NA) therapy, associated with an increased incidence of transformation to aggressive lymphoma, significantly influenced the presence of TCRVÎ² expansions (X2=11.6; P<0.001) as 83% of patients without (n=6) and only 7% with TCRVÎ² expansions (n=14) had received NA. Clonality of CD3+CD8+CD57+TCRVÎ²+ restricted CTLs were confirmed by TCRVÎ² CDR3 size analysis and direct sequencing. To characterize CTL clones, samples of CD3+CD8+CD57+TCRVÎ²+ cells were profiled using DNA microarrays and the results were validated on both gene and protein level. By gene set enrichment analysis, CTL clones not only expressed genes (GZMB, PRF1, FGFBP2) from cytotoxic pathways but also genes which suppress apoptosis, inhibit proliferation, arrest cell cycle G1/S transition and activate T cells (RAS, CSK and TOB pathways). Proliferation tracking confirmed their anergic state. Our studies demonstrate the incidence, NA sensitivity and anergic nature of clonal T cells in a B cell tumor. We used microarrays to detail the global programme of gene expression underlying T cell clonal expansion and identified distinct classes of up-regulated genes during this process. Experiment Overall Design: In this study we have sought T cell clones in the blood of patients with WM and related their presence with nucleoside analog therapy and transformation. We have also sorted CD3+CD4-TCRVÎ²+CD57+ from CD3+CD4-TCRVÎ²+CD57- cells and confirmed clonality by TCRVÎ² CDR3 size determination and sequencing and used carboxy-fluorescein diacetate succinmidyl ester (CFSE) tracking to monitor proliferation of CD3+CD4-TCRVÎ²+CD57+ clones. Finally we have performed microarray analysis to search for significant differentially expressed genes and pathways in the clonally expanded CD3+CD8+TCRVÎ²+CD57+ cells, focusing on cytotoxic pathways but also identifying other dysfunctional changes in these cells. Expression of a selection of the most differentially expressed genes detected by microarray was validated at both gene (qPCR) and protein level.

Project description:To understand better the nature of the poor response of human neonates to intracellular pathogens, we evaluated the transcriptome of neonates as compared to adult naÃ¯ve CD8-T cells. A specific transcription signature of the neonatal cells was found, characterized by a lower expression of signalling and CTL functional genes and a high expression of maturation, cell cycle and innate-immunity associated genes. Functional assays demonstrated that neonatal CD8-T cells undergo homeostatic proliferation, transcribe antimicrobial peptides and produce reactive oxygen species. Master transcription factors were found presumably responsible for this specific signature. Genome wide epigenetic studies showed a corresponding chromatin signature for a number of the differentially expressed genes. Altogether our results show that CD8 neonatal T cells have a particular genetic program with functions within the innate immune response, while still undergoing their maturation process. Neonates are highly susceptible to infections by intracellular pathogens, which are a major cause of infant morbidity and mortality. CD8-T cells control intracellular pathogens through cytotoxic mechanisms in an antigen-dependent manner. We found that human neonatal CD8-T cells, as compared to adult lymphocytes, had a distinctive pattern of gene transcription, characterized by the lower expression of genes involved in TCR signalling and cytotoxicity and a high expression of genes involved in cell cycle and innate immunity. Functional studies corroborated that neonatal CD8-T cells are less cytotoxic, transcribe antimicrobial peptides and produce reactive oxygen species. These properties could explain the high sensitivity of neonates to intracellular pathogen infections and outline novel functions of neonatal CD8-T cells. PBMC total RNAs from Adult and Neonate subjects were profiled after hybridization with Agilent SurePrint G3 Human GE 8x60K Microarray. CD8-T cells mRNA were extracted from 8 samples including: 4 Adults and 4 Neonates.

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RNA sequencing of CD57+ and CD57- effector memory CD8 T cells in patients with type 1 diabetes

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