Critical role of post-transcriptional regulation for IFN-γ in tumor-infiltrating T cells

Abstract

Protective T cell responses against tumors require the production of Interferon gamma (IFN-γ). However, tumor-infiltrating T cells (TILs) gradually lose their capacity to produce IFN-γ and therefore fail to clear malignant cells. Dissecting the underlying mechanisms that block cytokine production is thus key for improving T cell products.Here we show that although TILs express substantial levels of Ifng mRNA, posttranscriptional mechanisms impede the production of IFN-γ protein due to loss of mRNA stability. CD28 triggering, but not PD1 blocking antibodies, effectively restores the stability of Ifng mRNA. Intriguingly, TILs devoid of AU-rich elements within the 3'untranslated region maintain stabilized Ifng mRNA and produce more IFN-γ protein than wild-type TILs. This sustained IFN-γ production translates into effective suppression of tumor outgrowth, which is almost exclusively mediated by direct effects on the tumor cells. We therefore conclude that post-transcriptional mechanisms could be modulated to potentiate effective T cell therapies in cancer.Recently, post-transcriptional mechanisms have become appreciated in modulating the production of cytokines. For instance, AU-rich elements (AREs) within the 3'untranslated region (3'UTR) determine the fate of mRNA by regulating mRNA stability, subcellular localization of the mRNA, and translation efficiency (17)(18)(19)(20). We recently found that these regulatory mechanisms differentially dictate the cytokine production of T cells (21). The immediate production of IFN- mainly depends on rapid translation of pre-formed mRNA, whereas prolonged cytokine production relies on de novo transcription and increased mRNA stability (21). Furthermore, limited de novo transcription and the lack of Ifng mRNA stabilization effectively restricts the magnitude and duration of IFN- production (22). Whether and how post-transcriptional mechanisms govern the production of IFN- in T cells during an acute infection, and how this compares to cytokine production during chronic antigen exposure in tumors is not well understood.Here, we discovered a hitherto unappreciated role of post-transcriptional regulation that restricts the production of IFN- T cells within the tumor environment. Importantly, removing AREs from the Ifng locus was sufficient for TILs to retain the production of IFN-, and thus their capacity to suppress the tumor outgrowth. We therefore propose that adoptive T cell therapy could be potentiated by relieving IFN- from post-transcriptional control mechanisms.

The relationship of mRNA with protein expression in CD8+T cells associates with gene class and gene characteristics

“…Also the presence of AU-rich elements was a strong predictor of mRNA and protein levels (Kontoyiannis et al, 1999;Salerno et al, 2018Salerno et al, , 2019a. Interestingly, ARE-mediated regulation in CD molecules and transcription factors did not alter mRNA and protein expression, irrespective of the number of ARE in cluster or the activation status.…”

“…Only AREcontaining transcripts encoding secreted proteins had high mRNA levels while having low to undetectable protein levels in memory T cells. Upon activation, however these transcripts showed high mRNA and high protein levels, reflecting the discrepancy of mRNA and protein levels of IFN-γ in murine memory and tumor-infiltrating T cells due to a ARE-dependent translational block (Salerno et al, 2018(Salerno et al, , 2019a. What makes transcripts susceptible to this 1 5…”

“…Furthermore, the production of IFN-γ, TNF-α and IL-2 in primary CD8 + T cells is subject to the type of signal and the signal strength that T cells receive (Nicolet et al, 2017;Salerno et al, 2016Salerno et al, , 2017. These signals define not only transcriptional regulation but also posttranscriptional events and alter the stability and translation efficiency of cytokine mRNA, which can result in discrepancies in mRNA and protein levels for these cytokines (Salerno et al, 2017(Salerno et al, , 2019b(Salerno et al, , 2019a. Untranslated regions (UTRs) are key regulatory elements in this process, as they are required for the modulation of mRNA transcript stability, translation initiation, and translation efficiency (Mayr, 2017).…”

Differential interferon-γ production by naive and memory-like CD8 T cells

“…This IFN‐γ production could have resulted from an increase in Ifng mRNA but also from post‐transcriptional mechanisms 19 . The influence of AU‐rich elements (AREs) within the 3ʹ untranslated region (3ʹUTR) on Ifng mRNA stability has already been shown 20 . To evaluate whether IFN‐γ production results from Ifng gene up‐regulation, we assessed Ifng mRNA levels from 0 to 8 h after in vitro TCR stimulation.…”

“…Bar graphs represent the mean of 3 independent experiments ± SE (n = 3). The ( * ) indicates P < 0.05, and ( *** ) indicates P < 0.0001 compared with CD44 low cells untranslated region (3ʹUTR) on Ifng mRNA stability has already been shown 20. To evaluate whether IFN-production results from Ifng gene up-regulation, we assessed Ifng mRNA levels from 0 to 8 h after in vitro TCR stimulation.…”