Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8<sup>+</sup>T cells

Salerno, Fiamma; Paolini, Nahuel A.; Stark, Regina; Lindern, Marieke von; Wolkers, Monika C.

doi:10.1073/pnas.1704227114

Cited by 60 publications

(136 citation statements)

References 43 publications

(52 reference statements)

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“…We highlight that although the antigen threshold for producing different cytokines may be comparable, or indeed identical, there are multiple mechanisms that enable differential regulation. For example, it is clear that there are differences in bulk cytokine production kinetics (32,33), which may be a result of different mRNA expression and stability (35)(36)(37) and sequential production programs (32). Although differences in individual cytokine levels are clearly observed in individual experiments (see representative curves in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…A technically more demanding assay based on single-cell cytokine secretion has shown that a single pMHC can induce both TNFα and IL-2 implying that their antigen threshold is comparable (31). In addition, it is now clear that different cytokines exhibit different production kinetics (32,33) and production depends on continuous TCR/pMHC engagement (34). Therefore, the pMHC degradation rate may introduce apparent differences in thresholds with cytokines having faster production kinetics appearing to have a lower threshold.…”

Section: Discussionmentioning

confidence: 99%

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Human CD8⁺T cells exhibit a shared antigen threshold for different effector responses

Abu-Shah

Trendel

Krüger

et al. 2020

Preprint

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T cells recognising cognate pMHC antigens become activated to elicit a myriad of cellular responses, such as target cell killing and the secretion of different cytokines, that collectively contribute to adaptive immunity. These effector responses have been hypothesised to exhibit different antigen dose and affinity thresholds, suggesting that pathogen-specific information may be encoded within the nature of the antigen. Here, using systematic experiments in a reductionist system, where primary human CD8 + T cell blasts are stimulated by recombinant pMHC antigen alone, we show that different inflammatory cytokines have comparable antigen dose thresholds across a 25,000-fold variation in affinity. Although co-stimulation by CD28, CD2, and CD27 increased cytokine production in this system, the antigen threshold remained comparable across different cytokines. When using primary human memory CD8 + T cells responding to autologous antigen presenting cells equivalent thresholds were also observed for cytokine production and killing. These findings imply a simple phenotypic model of TCR signalling where multiple T cell responses share a common rate-limiting threshold and a conceptually simple model of antigen recognition, where the chance factor of antigen dose and affinity do not provide any additional response-specific information.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Human CD8⁺T cells exhibit a shared antigen threshold for different effector responses

Abu-Shah

Trendel

Krüger

et al. 2020

Preprint

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“…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).…”

Section: Introductionmentioning

confidence: 99%

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

Nicolet

Wolkers

2020

Preprint

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T cell differentiation and activation induces substantial alterations in gene expression. While RNA sequencing and single cell RNA sequencing analysis provided important insights in the gene expression dynamics of T cells, it is not well understood how the mRNA expression translates into the protein landscape. By combining paired RNA-sequencing and mass spectrometry data of primary human CD8 + T cells, we found that mRNA expression is a poor proxy for the overall protein output. Irrespective of the differentiation or activation status, the correlation coefficient of human CD8 + T cells reached a mere 0.41-0.43. Only gene classes that mediate conserved cellular processes such as protein translation or cellular metabolism showed a strong correlation of mRNA with protein expression. In contrast, the mRNA expression and protein output of transcription factors, cell surface molecules, and secreted proteins -including cytokines -only mildly correlated. Conversely, highly conserved genes correlated well with the protein output. This was also true for the presence of AU-rich elements in the 3'untranslated region, in particular for mRNAs that encode secreted proteins.In conclusion, the in-depth characterization of the transcriptome and proteome in human CD8 + T cells emphasizes the need of combined mRNA and protein analysis for our understanding of T cell biology and function.

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“…Whereas murine T cells show a t1/2 >2h upon antigen stimulation [30], [33], human control T cells activated with -CD3/-CD28 only reach a t1/2 ~ 70min. This also translates into lower overall IFN- protein output in human T cells, both in terms of responding T cells (60-80% in murine wild type T cells versus 30-50% in human T cells upon PMA-Ionomycin stimulation [47], [54]), and in terms of production kinetics. In fact, even though human and murine T cells initiate the IFN- production in the same time frame, human T cells cease to produce IFN- faster.…”

Section: Discussionmentioning

confidence: 99%

“…To determine whether ARE-deletion also modulated the IFN- protein production and/or kinetics, we activated ARE-Del and control T cells with -CD3/-CD28. To visualize the production kinetics, we added Brefeldin A for a maximum of the final 2h of stimulation [54], and measured the production of IFN- protein via intracellular cytokine staining. In nonactivated T cells, we did not observe any protein production by any T cell type (Figure 2C), indicating that like control T cells, ARE-Del T cells require T cell activation for cytokine production.…”

Section: Are-deletion Stabilizes Ifng Mrna and Augments Protein Produmentioning

confidence: 99%

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

Heeren

Popović

Guislain

et al. 2019

Preprint

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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...

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Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8⁺T cells

Cited by 60 publications

References 43 publications

Human CD8⁺T cells exhibit a shared antigen threshold for different effector responses

Human CD8⁺T cells exhibit a shared antigen threshold for different effector responses

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

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

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Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8+T cells

Cited by 60 publications

References 43 publications

Human CD8+T cells exhibit a shared antigen threshold for different effector responses

Human CD8+T cells exhibit a shared antigen threshold for different effector responses

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

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

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Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8⁺T cells

Human CD8⁺T cells exhibit a shared antigen threshold for different effector responses

Human CD8⁺T cells exhibit a shared antigen threshold for different effector responses

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