Long noncoding RNAs (lncRNAs) are RNAs with lengths exceeding 200 nucleotides without encoding proteins. Whereas noncoding elements located in intergenic regions including lncRNA were in the past largely considered as junk sequences, more recently specific functions have been ascribed to them. We aimed to characterize lncRNAs which are expressed in human T lymphocytes and their regulation during ex vivo culture.

Human CD3+ lymphocytes were isolated using Fab-fragment based affinity column separation from peripheral blood of 8 male healthy donors. T cells were cultured in interleukin-2 and anti-CD3/CD28 for up to 7 days. RNA was extracted at time points T1 (freshly isolated), T2 (4h), T3 (12h), T4 (24h), T5 (3d) and T6 (7d), reverse transcribed and subjected to transcriptome analysis using Illumina TruSeq from cDNA libraries. Differential gene expression analysis was performed targeting lincRNA gene sets.

The number of differentially expressed LncRNAs at 4 hr, 12 hr, 24 hr, day 3 and day 7 compared to freshly isolated T cells was 168, 193, 239, 386 and 296, respectively. Of the main upregulated RNAs in cultured T cells, lncRNAs associated with suppressing cytokine production (e.g. IL-21-AS1; IL12A-AS1), with stimulating regulatory T cell differentiation (MIR155HG) and with T cell exhaustion (FOXP4-AS1) were found. Among the most downregulated were lncRNAs associated with stimulating T cell differentiation (GATA2-AS1), inhibiting interferon gamma production (IFNG-AS1) and inhibiting immune synapse (ITGB2-AS1), pointing at a role of lncRNAs towards activation of T cells during expansion culture.

Our results demonstrate regulation of lncRNA expression in T lymphocytes during ex vivo manipulation. First insights into the function of lncRNAs point to an association with initiating T cell activation during culture. A more profound knowledge on lncRNA functions and its application to T cell expansion culture might provide rationales to interfere with T cell fate during expansion culture, e.g. inhibiting senescence and promoting a more self-renewing phenotype.

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