Human T cells employ conserved AU-rich elements to fine-tune IFN-γ production

Abstract

Long-lasting CD8 + T cell responses are critical in combatting infections and tumors. The proinflammatory cytokine IFN-γ is a key effector molecule herein. We recently showed that in murine T cells, the production of IFN-γ is tightly regulated through AU-rich elements (AREs) that are located in the 3' Untranslated Region (UTR). Loss of AREs resulted in prolonged cytokine production in activated T cells and boosted anti-tumoral T cell responses. Here, we investigated whether these findings can be translated to primary human T cells. Utilizing CRISPR-Cas9 technology, we deleted the ARE region from the IFNG 3'UTR in peripheral blood-derived human T cells. Loss of AREs stabilized the IFNG mRNA in T cells and supported a higher proportion of sustained IFN-γ protein-producing T cells. Importantly, this was also true for tumor antigen-specific T cells. MART-1 TCR engineered T cells that were gene-edited for ARE-deletion showed increased percentages of IFN-γ producing MART-1specific ARE-Del T cells in response to MART-1 expressing tumor cells. Combined, our study reveals that ARE-mediated post-transcriptional regulation is highly conserved between murine and human T cells. Furthermore, generating antigen-specific ARE-Del T cells is feasible, a feature that could potentially be exploited for therapeutical purposes. elements. revealed that IFN- sensing is instrumental to protect the host from infections by Mycobacteria species [14]-[16]. IFN- also prevents the development of cancers. In fact, mice lacking the Ifng gene, or the signaling protein downstream of Ifngr1/2, Stat1, spontaneously develop tumors [17],[18]. Furthermore, a high IFNG-mediated gene signature correlates with clinical response rates to immunotherapy in humans [19],[20]. Conversely, copy number alterations of IFN- pathway genes correlates with poor response to immunotherapy [21]. The regulation of IFN- production is multi-layered. The IFNG locus is only demethylated in effector and memory T cells [22], allowing for locus accessibility and transcription upon T cell activation. While the production of T cell effector molecules has been mainly attributed to changes in transcription and the presence of transcription factors [23]-[27], recently, the role of post-transcriptional regulation in T cells has also become appreciated [28]-[33]. Posttranscriptional regulation is mediated by sequence elements and structures present in both the 5' and 3' untranslated regions (UTRs) of mRNA molecules [34]-[37] and nucleoside modifications, such as adenine methylation [38]. By facilitating the binding of RNA binding proteins (RBPs), microRNAs and long non-coding RNAs, these regulators combined determine the actual protein output of a cell [37]. One of these sequence elements are adenylate uridylate-rich elements (AREs). AREs are AUUUA pentamers present in multimers in the 3'UTR of mRNA molecules [39],[40]. Interestingly, many cytokine transcripts contain AREs [37],[39]. They function as binding hubs for RBPs and microRNAs [39]-[41]. Binding to AREs by these factors me...

Toll‐like receptor‐2/7‐mediated T cell activation: An innate potential to augment CD8+ T cell cytokine production

“…The production of inflammatory cytokines such as IFN‐γ is regulated on multiple levels 57‐62 . Besides the well‐characterized transcriptional and epigenetic regulation that is enforced by transcription factor availability and activation status, as well as histone modification, 57,60,63‐65 post‐transcriptional regulatory events play a crucial role to fine‐tune T cell responses 6,16,62,66‐73 . Indeed, also the timing and magnitude of IFN‐γ production is, at least in part, dictated by post‐transcriptional regulation 16,62,69,73‐75 .…”

“…For the sustained production of IFN‐γ, mRNA stabilization is required 6 . This is, at least in part, regulated via the AREs present in the 3’UTR of the IFNG gene, 69 a feature that is conserved between mouse and men 73,75,89 . Whether AREs play a role in modulating IFN‐γ production in TLR2/7 ligand‐induced T cell responses remains to be determined.…”

Exploiting HIV‐1 tropism to target CD4+ T cells for CRISPR

“…Usually, Cas9 is precoupled to the guide RNA and trans‐activating CRISPR RNA that are used to target the Cas9 protein to and activate the Cas9 protein at the genomic locus of interest. The combined Cas9–RNA complex is referred to as a Cas9 ribonuclear protein (RNP), and Cas9 RNP electroporation was shown to be an effective method for the genetic modification of human T cells 6,7 . However, for large amounts of cells and/or targets, electroporation is impractical.…”

“…Of note, the HIV‐1‐pseudotyped version of the Cas9‐VLP resulted in lower genome editing frequencies (approximately 14–28%) in primary human T cells compared with conventional methods such as electroporation 6,7 . This can potentially be increased by making use of recently described more efficient Cas9 molecules, 15 but this remains to be determined.…”

“…The combined Cas9-RNA complex is referred to as a Cas9 ribonuclear protein (RNP), and Cas9 RNP electroporation was shown to be an effective method for the genetic modification of human T cells. 6,7 However, for large amounts of cells and/or targets, electroporation is impractical. Hamilton et al 5 report on a system that shuttles Streptococcus pyogenes Cas9 RNPs into cells utilizing retrovirus-like particles (VLPs; Figure 1a).…”