Long Non-Coding RNA Regulation of Allogeneic T-Cells

Abstract

The mechanisms governing human allogeneic T-cell responses remain incompletely understood which limits the wider clinical application of allogeneic BMT. Long non-coding RNAs (lncRNA) are an emerging class of non-coding RNAs that control gene expression with tissue and context specificity. However, their role in alloimmunity is unknown. To determine whether lncRNAs regulate T cell alloimmunity we performed RNA-seq of donor T-cells from clinical BMT patients that received increasing amounts of allogeneic stimulation. Hierarchical clustering and differential expression analysis showed that increasing allogeneic stimulation drove differential expression of lncRNAs. To validate the sequencing data, we confirmed the differential expression of 6 lncRNAs in an independent patient cohort by qRT-PCR and confirmed the altered expression of 4 of these candidate lncRNAs in a human mixed lymphocyte reaction (MLR). The database FANTOM-CAT showed strong enrichment of Linc00402 (88 fold) and RP11-348F1.3 (18 fold) in human T-cells, and CD4 and CD8 human T-cells demonstrated similar expression of Linc00402 and RP11-348F1.3. Linc00402 is predicted to be conserved, and its expression was detected by qRT-PCR primer tiling in mouse T-cells. The differential expression upon allo-stimulation of the Linc00402 mouse orthologue, GM34199, was confirmed both in vitro with an MLR and in vivo using both major and minor histocompatibility disparate murine models of GVHD. The functional validation of human RP11-348F1.3, human Linc00402, and Linc00402’s murine orthologue, GM34199, was tested by anti-sense oligonucleotide-mediated knockdown, which inhibited human Linc00402 (55% of control, p