Cancer immunoediting is the dynamic process whereby cancer cells modify the immune microenvironment to their advantage. In a typical cancer scenario, altered protein products caused by genetic mutations function as neoantigens, eliciting an immune response against cancer cells (Schumacher and Schreiber, 2015). In parallel, the inflammatory, hypoxic, and often necrotic microenvironment of tumors modifies the immune response (Kalluri, 2016). Tumor-infiltrating T lymphocytes (TILs) are the major players in the immune response (Quail and Joyce, 2013). Conventional classification of T lymphocytes divides them in CD8⁺ cytotoxic T cells, and various subtypes of CD4⁺ cells, each with a precise range of action. In general, CD8⁺ cytotoxic T cells, CD4⁺ T-helper 1 cells producing interferon-γ, and natural killer cells are associated with favorable anti-tumor immune responses (Tosolini et al., 2011). In contrast, myeloid-derived suppressor cells, and CD4⁺ FOXP3⁺ regulatory T (Treg) cells producing IL-10 and TGF-β have immunosuppressive and protumoral effects (Shang et al., 2015). Other T cell subsets, present at lower frequency, have been linked to patient survival in specific tumors, such as CD4⁺ Tr1 (Bonnal et al., 2021), or CD4^- CD8^- unconventional αβ T cells (Ponzetta et al., 2019). Recently, single-cell RNAseq studies have extended the phenotypic description of TILs (De Simone et al., 2016; Guo et al., 2018; Miller et al., 2019; Stuart and Satija, 2019; Yao et al., 2019; Zheng et al., 2017) showing distinct patterns of immune activation and exhaustion, and increasing the number of T cell subtypes. Although many of these subtypes are present across different tumor types, not all cancer types with similar TIL landscapes respond similarly to immunotherapy (Paijens et al., 2021). These observations highlight the complexity of the tumor-immune interactions and suggest the presence of environmental modifiers of the T cell transcriptional repertoire. Notably, in spite of the elevated number of T cell players found in the tumor microenvironment, the most consistent survival predictors are (i) the number of TILs and (ii) the ratio between CD8⁺ cytotoxic T cells and CD4⁺ FOXP3⁺ Tregs (Gooden et al., 2011).

The high complexity of the T cell transcriptional repertoire is only one side of the coin. Several studies show that the presence of a messenger RNA (mRNA) may not reflect the active synthesis of the encoded protein (Schwanhäusser et al., 2011). It is known that some mRNAs are translated poorly and only in response to specific stimuli (Pelletier and Sonenberg, 2019). Regulation of protein synthesis, called translational control, occurs both at the global and at the specific mRNAs level. Protein synthesis is one of the most energy-consuming pathways in cell metabolism (Buttgereit and Brand, 1995), and microenvironment conditions and nutrients are major controllers of the global rate of synthesis (Biffo et al., 2018). For instance, hypoxia leads to the downregulation of high-energy processes, including protein synthesis (Horman et al., 2002; Liu et al., 2006). Local conditions of amino acid concentration affect translation, and global translation decreases in response to amino acid deprivation. Both hypoxia and amino acid deprivation activate also specific translation. When an essential amino acid is limiting, the concentration(s) of the non-aminoacylated (‘uncharged’) tRNA increase(s). Uncharged tRNAs bind to the regulatory domain of GCN2 kinase causing its activation, the phosphorylation of eIF2α, a shutdown of global protein synthesis, and the specific derepression of the translation of uORF-controlled mRNAs (Wek, 2018). Four kinases (eIF2AK1–4) are able to phosphorylate eIF2α (Loreni et al., 2014) and are all expressed in T cells (Mitchell et al., 2015). Hypoxia is a stimulator of eIF2AK3 known also as PERK (Koumenis et al., 2002). Consequently, the hypoxic tumor microenvironment may inhibit protein synthesis through specific cascades, leading to a mismatch between the presence of mRNAs and their encoded proteins. It is therefore evident that translational inhibition may be particularly relevant for T cells that respond to the conditions of the microenvironment (Biffo et al., 2018).

Stimulation of protein synthesis is one of the hallmarks of T cell exit from quiescence, beginning of growth and proliferation and is associated with both quantitative (Garcia-Sanz et al., 1998) and qualitative changes (Mikulits et al., 2000) in translation. Stimulation of protein synthesis occurs through the nutrient growth factor receptor cascades that converge on initiation factors eIF4E, through PI3K-mTORC1, ERK-MNK, and eIF6 (Loreni et al., 2014; Bhat et al., 2015). Activation of the T cell receptor leads to a robust activation of the growth factor cascade. In particular, stimulation of the mTORC1-S6K branch is of outmost importance for T cell function. mTORC1 is a major controller of both translation and T cell biology (Bjur et al., 2013; Piccirillo et al., 2014). Inhibition of mTORC1 by rapamycin has profound effects on T cell response and polarization (Powell and Delgoffe, 2010). The relevance of the translational branch in driving mTORC1 responses is demonstrated by the fact that ablation of 4EBP1 and 4EBP2, which are translational modulators phosphorylated by mTORC1, rescues the inhibitory action on proliferation of raptor depletion (Dowling et al., 2010). Several mRNAs are rapidly engaged following T cell stimulation, promoting an immediate translational and glycolytic switch to ramp up the T cell activation program (Wolf et al., 2020). Recently, we found that stimulation of fatty acid synthesis in T cells requires the translational activation of the rate-limiting enzyme ACC1. Intriguingly, ACC1 mRNA was expressed but not translated also in quiescent T cells (Ricciardi et al., 2018). Translational regulation of glycolytic enzymes was seen also in activated CD4⁺ cells (Manfrini et al., 2020b), in line with other experimental models. Another translation factor, eIF5A, promotes the expression of a subset of mitochondrial proteins involved in the TCA cycle and oxidative phosphorylation (Puleston et al., 2019). In short, in vitro, T cells rapidly respond to T cell receptor stimulation by increasing protein synthesis.

The presence of a large layer of translational control in T cells raises the question whether TILs are efficiently translating in vivo, which environmental stimuli regulate their translation, and whether active translation affects their developmental pathway. In this study we demonstrate that, in vivo, suppressive CD4⁺ Tregs and cytotoxic CD8⁺ T cells are the major populations of translating lymphocytes. Hypoxia and mTOR signaling are major modulators of lymphocyte translation, and translating lymphocytes are phenotypically different from translationally inactive lymphocytes. We hypothesize that the predictive value on survival of suppressive CD4⁺ Tregs and cytotoxic CD8⁺ T cells is due to their capability to be translationally active amid a large percentage of not translating lymphocytes.

TILs have been characterized by several transcriptomic studies leading to their extensive multi-lineage phenotypic classification (Paijens et al., 2021). In this study, we found that the translational activity of TILs is not uniformly distributed, being more intense in CD8⁺ T cells and CD4⁺ Tregs. A stratification of the translational capability of T cells associated with phenotypic markers shows that highly translating T cells are characterized by the presence of cytotoxic markers for CD8⁺ and suppressive markers for CD4⁺. Finally, highly translating T cells are characterized by markers for active cycling. The relevance of our observations is consistent with clinical evidence indicating that CD8⁺/Treg ratio is, in general, a strong predictive parameter for survival in cancer (Gooden et al., 2011). We will discuss some issues derived from our data.

Translation is strongly regulated by microenvironmental cues and its impairment acts as a limiting factor in tumorigenesis and tumor growth (Barna et al., 2008; Miluzio et al., 2011). Consequently, tumor genetic lesions converge on the translational machinery, leading to its constitutive activation, and rendering translation relatively independent of the signals posed by the microenvironment (Loreni et al., 2014; Robichaud et al., 2019). The relevance of the translational machinery in the cell autonomous growth of tumors is demonstrated by evidence indicating that genetic depletion of translation factors greatly reduces tumorigenesis and tumor growth (Barna et al., 2008; Miluzio et al., 2011; Truitt et al., 2015). In the tumor microenvironment, tumor cells are therefore taking full advantage of the restrictive nutrient conditions, thanks to the activation of oncogenic mutations. In sharp contrast, TILs lack mutations that alter their capability to synthesize proteins in conditions of high stress, and as such, are bound to permissive conditions in order to translate their transcriptional repertoire. In this context, one important question is whether the unfavorable conditions of the tumor microenvironment are affecting the action of some TILs classes, and whether they lead to specific developmental trajectories.

Hypoxia, far from being surprising, seems to act as a general inhibitor of translation of TILs. It is known since long that hypoxia inhibits translation acting on AMPK (Horman et al., 2002) and eIF2α(Koumenis et al., 2002). Phosphorylation of eIF2α occurs through four distinct kinases, PERK, GCN2, PKR, and HRI, that, together, are part of the integrated stress response, a complex set of events that drives, among others, the shut-off of general translation and the activation of specific mRNA translation (Rios-Fuller et al., 2020). Importantly, all four kinases are expressed in T cells at the mRNA level (Critchley-Thorne et al., 2007). PERK is considered a major regulator of translational control of hypoxia (van den Beucken et al., 2006). Unfortunately, we were not able to efficiently detect the specific activity of single eIF2α kinases in TILs, therefore it remains unresolved whether also in our conditions, PERK is the major regulator of translational shut-off and of eIF2α phosphorylation. It is possible that other pathways can contribute to the translational shut-off, given the sequel of adapting responses, especially in chronic conditions (Schito and Semenza, 2016), and considering that amino acid limitations affect TCR signaling (Ron-Harel et al., 2019). Oxygen sensing in the immune microenvironment shapes immunological responses, very often with confounding reports. In the past, it has been proposed that hypoxia leads to the preferential differentiation of peripheral, intratumor Tregs (Dang et al., 2011). In light of what we observe, it is unlikely that Treg in hypoxic environment can contribute to immune suppression, unless a sequel of phenomena occurs: first, the hypoxic environment causes conversion of conventional T cells in Tregs, which then persist for some time; second, the hypoxic environment induces angiogenesis, thus reviving the suppression and translation capability of converted Tregs. It is obviously possible that a slow adaptation and phenotypic change can occur in human tumors, where the equilibrium between the immune system and tumor growth may last years. Summarizing, we think that our data are more consistent with a model in which the expansion of suppressive, intratumoral Tregs requires active translation and simultaneous increased rate of fatty acid synthesis (Pacella et al., 2018) and, in general, is associated with high glycolytic capability (Procaccini et al., 2016). In contrast, hypoxia may acutely impair the suppressive capability of Tregs.

The pathways that connect to translation are several, and their description is beyond the limits of this paper (Roux and Topisirovic, 2018). Our data indicate that the mTORC1 pathway is a major controller of TIL translation. In addition, we found that the Mnk/eIF4E pathway even if it was not massively detectable in most T cells, led to a subset of p-eIF4E positive cells correlating with high puromycin incorporation. From this perspective, it will be interesting to characterize subset of highly translating cells, in relationship to the activated pathways. This effort may require the establishment of new technologies to combine the detection of p-eIF4E with sequencing strategies, but it will be important in order to understand the impact of Mnk inhibition on cancer growth (Bramham et al., 2016). We were unable to effectively characterize the eIF6 pathway in TILs, due to the absence of antibodies detecting its phosphorylation (Ceci et al., 2003). Overall, each lymphocyte can differentially regulate its transcriptional repertoire depending on the specific pathways that are activated.

The observation of a different phenotype between T cells incorporating high levels of puromycin and T cells incorporating low levels has far-reaching implications. The correlation between translation and Ki-67 expression is logically linked to the fact that cell cycle progression requires de novo protein synthesis to sustain cell growth. Several studies at the single-cell level, combining mRNAseq and TCR sequencing, have unveiled the clonal expansion of subset of T cells. Interestingly, one major expanding subset is the CD4⁺ Tregs. TCR sequencing of breast cancer-associated Tregs revealed that Tregs have little TCR sharing with conventional T cells, confirming that Tregs in tumors are mainly generated through local expansion (Plitas et al., 2016). Local expansion of Tregs is supported by our data according to which translating T cells are Ki-67 positive. In general, we did not find differences between the analysis of the B16 mouse model and the MC38 one. We speculate that the local expansion of a translating phenotype is a general phenomenon and it occurs in areas that are not hypoxic.

The phenotypic identity of translating cells extends beyond the co-expression of Ki-67 and puromycin incorporation. CD8⁺ cells with prolonged exposure to antigens enter a state of exhaustion characterized by the elevated expression of inhibitory receptors (e.g., PD-1, CTLA-4, TIM-3, TIGIT, and LAG3). TILs expressing markers of exhaustion (PD-1, LAG3, and TIM3) are more likely to express IFN-γ (Gros et al., 2014). While exhausted CD8⁺ T cells have reduced function as compared to those elicited by an acute infection, several data suggest that are the exhausted CD8⁺ T cells that are exerting residual control over tumor growth (Li et al., 2019). Exhausted CD8⁺ T cells still play a critical role in cancer. Indeed, it is still confused how exhaustion is related to various properties, such as cell proliferation and effector functions. According to the operational definition given above, exhaustion is clearly not associated with the loss of translational activity, which results, for instance, in the enrichment of Ki-67 and TIM3 expression in Puro⁺ cells. The higher expression of CTLA-4 and TGF-β (Sakaguchi et al., 2020) in CD4⁺ translating cells fully correlates with suppressive properties.

In conclusion, the tumor microenvironment has been shown, in decades of studies, to provide a full range of stimuli (Maman and Witz, 2018) that may regulate translation, like hypoxia or nutrients. We suggest that two T cells that have an identical ‘transcriptome’ and protein repertoire infiltrate a tissue in two different microenvironment niches and are subject to rapid and differential translational regulation. If translational regulation persists, these two cells will have different developmental trajectories, as clearly shown by our study in which high puromycin incorporation goes hand in hand with the expression of specific markers. Last, our data suggest that T cell receptor stimulation and hypoxia are two main microenvironment clues that regulate the T cell trajectory. This further layer of complexity, however, rather than increasing the number of players in the process of tumor immunoediting enlightens the opposing role of CD8⁺ cytotoxic lymphocytes and CD4⁺ Tregs. Further characterization of translational control in these cells is required.

Limitations of our study: Quantitative puromycin incorporation was used to arbitrarily discriminate two T cell populations, highly translating and lowly translating. It is evident that this rough subdivision may not completely describe the tumor microenvironment. It is also evident that time may change the translational profile of a T cell, and a highly translating cell may shift to a lowly translating one if the environmental conditions change: accordingly, we have currently no clues on whether the highly translating phenotype is stable.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Decision letter after peer review:

Thank you for submitting your article "mTOR-dependent translation drives tumor infiltrating CD8⁺ Effector and CD4⁺ Treg cells expansion" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Ivan Topisirovic as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Carla Rothlin as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Ola Larsson (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) It was thought that the focus on mTOR signaling is too narrow and that other relevant pathways impinging on translation apparatus should be considered. These include integrated stress response and MNK/eIF4E axis, as these pathways have been implicated in adaptation to hypoxia and/or immune responses in neoplasia.

2) In some parts of the manuscript methodology/analysis should be improved. Specifically, anti-phospho-S240/244 ribosomal protein S6 antibody should be used instead of the antibody recognizing phosphorylated S235/236, as the latter phosphoacceptor sites are not exclusively phosphorylated in a mTOR-dependent manner. In addition, it was found that the statistical tests used were in some cases inappropriate, as illustrated in reviews below.

3) Assessing co-expression of the markers presented in figures 4D and 5D and relating their levels to protein synthesis activity was thought to be warranted in order to grasp full heterogeneity of tumor-infiltrating T cells.

4) Finally, it was thought that the manuscript may benefit from additional editing.

Reviewer #1:

In this study, mouse models are employed to systematically establish protein synthesis activity of tumor-infiltrating CD4⁺ and CD8⁺ T lymphocytes in vivo. The effects of stimuli emanating from tumor niche on translational activity of CD4⁺ and CD8⁺ T lymphocytes were also studied. Using ex vivo experiments, the evidence is provided that T cell receptor stimulation induces protein synthesis and mTOR signaling in tumor-infiltrating T cells, whereas hypoxia exerts the opposite effect. Next, the authors used mouse melanoma and colorectal cancer models to confirm these findings. Using the same models, the authors show that tumor-infiltrating CD4⁺ and CD8⁺ T lymphocytes translate more than their counterparts in the spleen. Importantly, it was also observed that tumor-infiltrating lymphocytes also exhibit significant intercellular heterogeneity in translational activity. This was followed by establishing the correlations between levels of protein synthesis and T cell phenotypes using flow cytometry, which revealed strong correlation between levels of protein synthesis and immunophenotypes. Collectively, these findings support the tenet that translation plays a major role in establishing tumor-infiltrating T cell phenotypes, and demonstrate that there is pronounced intracellular heterogeneity in their protein synthesis activity. This heterogeneity in the levels of mRNA translation between tumor-infiltrating T cells may at least in part be explained by the difference in stimuli that these cells are exposed in distinct parts of tumor microenvironment. In long-term, this study may inspire single-cell proteomics and/or similar approaches that do not rely on steady-state mRNA levels to monitor the plasticity of tumor-infiltrating lymphocytes and thus provide molecular bases for more efficient immunotherapies.

Strengths: Presented findings further support the critical role of the protein synthesis apparatus in establishing lymphocyte phenotypes, and emphasize the importance of considering alterations that occur downstream of changes in mRNA levels when determining the lymphocyte subtypes in the tumors. This is of particular interest considering recent increase in the number of single cell sequencing studies aiming to address composition of tumor-infiltrating lymphocytes, wherein correlations with phenotypes are driven based on steady-state mRNA composition of the cell. Methodologically, the clear advantage of this study, is that the most of the work was done in vivo, and is therefore of high physiological relevance. In addition, both melanoma and colon cancer models were employed, thereby suggesting that reported observations are likely to be generally applicable, and not cancer type-specific. In addition, the authors monitored the effects of cancer-relevant perturbations on protein synthesis, including T cell receptor stimulation and hypoxia. Overall, this study provides solid evidence that translation plays a major role in establishment of immunophenotypes of the tumor-infiltrating lymphocytes, and that interaction between tumor microenvironment and translational machinery may be responsible for intercellular heterogeneity of T cells in tumor niche.

Weaknesses: The major drawback of this study is that it relies on correlation and lacks experiments aiming to decipher mechanisms underlying the interactions between tumor niche and translational machinery of tumor infiltrating lymphocytes. Moreover, it was thought that the focus on mTOR may be too narrow and that other signaling mechanisms known to modulate translation rates in response to environmental cues should also be investigated. Finally, it remains unclear whether the changes in protein synthesis rates affect all the mRNAs uniformly or induce selective perturbations in translational activity of specific subsets of mRNAs.

Notwithstanding these weaknesses, it was thought that with appropriate revision, provided evidence is sufficient to support the major conclusion of the manuscript which is that the interactions between tumor microenvironment and translational machinery play a major role in determining immunophenotypes of tumor-infiltrating lymphocytes and that relying on steady-state mRNA levels may not be sufficient to grasp full plasticity of tumor-infiltrating T cells.

– Sole focus of the study is on mTOR signaling. This is somewhat surprising as other mechanisms have been also shown to play a central role in translational reprogramming under hypoxia and T cell receptor stimulation (e.g. ISR, MNK-dependent phosphorylation of eIF4E, etc). Based on this, it was thought that more comprehensive assessment of alterations in signaling pathways that impinge on translational machinery is warranted to pinpoint signaling pathways that mediate the observed effects of tumor microenvironment on T lymphocytes.

– Conclusions on alterations in mTOR signaling are based solely on monitoring phospho-rpS6 levels. Importantly, the antibody that the authors used recognizes S235/236 on rpS6 which are phosphorylated in cells lacking S6K1 and 2 (PMID: 15060135) and are phosphorylated by other AGC kinases (e.g. RSKs). The phoposho-acceptor site on rpS6 that is exclusively sensitive to alterations in the mTORC1/S6K signaling axis is S240/244 (PMID: 15060135) and thus, antibody recognizing S240/244 should be employed. To strengthen their conclusions, the authors should also add appropriate total antibody controls and monitor other mTORC1 targets (e.g. 4E-BPs, eEF2K).

– Considering the general lack of mechanistic data to explain heterogeneity in translational activity of tumor-infiltrating T cells, it was thought that monitoring the impact of mTOR inhibitors as well as compounds targeting other signaling mechanisms that adjust protein synthesis in accordance to changes in cellular environment (e.g. ISRIB, MNK inhibitors) on T-cell immunophenotypes and heterogeneity in vivo would be informative.

– It is well-established that in addition to changes in global protein synthesis levels, stressors such as hypoxia selectively affect translation of different mRNA subsets, whereby the latter changes are likely to significantly contribute to observed immunophenotypes. I understand that translatome-wide translational profiling is out of the scope of this short report and also technically very challenging, but perhaps authors should monitor mRNA and protein levels of selected number of factors that are known to be translationally regulated under these conditions and/or implicated in T cell biology in their system.

– The article may benefit from some careful editing.

Reviewer #2:

The tumor microenvironment includes the extracellular matrix (ECM) and a multitude of cells including immune cells. During cancer progression, the tumor associated ECM and cell composition/activity is altered. For example, the activity of immune cells towards tumor cells is commonly reduced which facilitates e.g. tumor growth and metastasis. It is well established that, in vitro, activation of T lymphocytes leads to a dramatic upregulation of protein synthesis and increased proliferation. Yet, to what extent this is true in vivo is not yet characterized. In this manuscript, Benedetta De Ponte Conti et. al address this using a FACS-based approach whereby global protein synthesis (quantified by puromycin incorporation) is related to expression of established activation markers for CD4⁺ and CD8⁺ T cells in tumors from the B16/F10 melanoma model. The conclusions are largely in agreement with studies of in vitro models as activation of CD4 or CD8 cells induces proliferate and bolsters protein synthesis. This study thereby pinpoints the close relationship between protein synthesis and immune cell activity also in vivo. One particularly interesting finding is the links between protein synthesis and Treg phenotype. In aggregate, the claims are well supported and important. This study will likely fuel many additional studies trying to in greater detail understand how protein synthesis in immune cells relate to their function within the tumor microenvironment.

There are some parts I think could be more developed:

1. There is a close link between protein synthesis and proliferation following activation of CD4 and CD8 T cells. This raises the question of how close puromycin incorporation associates with proliferation. In figure 5 S1, Ki67 vs protein synthesis is assessed in CD4⁺ T cells. This suggests a very close relationship. IS there a corresponding relationship for CD8⁺ T cells? Are there any aspects of the protein synthesis profile which do not associate with proliferation? What would the phenotype of such cells be?

2. All analyses are performed on populations of cells but there are no cross-comparisons between marker expression. I.e. is there one subset of T cells with high protein synthesis which express most of these markers (Figure 4D) or are there subsets of high protein synthesis cells which express subsets of markers? I am not sure if it is technical possible, but it would be interesting to assess co-expression of the identified markers in figure 4D and 5D and how their expression relates to protein synthesis.

3. Analysis is largely qualitative when assessing expression of makers and protein synthesis. Perhaps it would be informative to also perform a quantitative analysis within single tumors?

4. It is unclear to me what the relationship between T cell exhaustion and protein synthesis (and also proliferation) is based on the presented data. It seems logical to expect reduced protein synthesis in exhausted T cells, but this may not be the case.

Reviewer #3:

Overall the figures and the text were clear and concise and the data well described. The data shown support the claims made, but the authors have not examined the other major pathway for regulation of translation. My main issues relate to the first three figures.

1. The authors focus on the regulation of protein synthesis through mTOR, however translation initiation is also regulated by eIF2. Moreover, the PERK-eIF2-ATF4 axis has been shown to be important for tumour cell adaptation to stress – therefore, as all four eIF2 kinases are expressed in these models, it is important to determine any effect from eIF2 on translation activation/inhibition:

– What are the levels of eIF2α phosphorylation in the -/+puromycin cell populations (Figure 1 and 2)?

– As hypoxia is known to regulate eIF2 kinases what is the activation status of the four kinases in these model systems during hypoxia (Figure 1 – 3)?

– It is known that mTOR signalling can crosstalk to eIF2 signalling, and vice-versa, so how do the levels of eIF2α phosphorylation (and activity of eIF2 kinases) correlate with mTOR activity in these models during hypoxia (Figure 1 – 3)?

– To support the author's conclusions it would be necessary to show that the regulation of translation in this model is independent of eIF2 signalling.

2. The author's state at various points that the data shows mTOR activity is increased in the puromycin positive proportion of cells, however they do not directly show this:

– The antibody used for phospho-RPS6 recognises residues (Ser235/236), which can be phosphorylated in the absence of S6K1 and S6K2, therefore, could this regulation be independent of mTOR? Although RPS6 is downstream of mTOR signalling it is not a direct substrate of mTOR, therefore, to fully support these conclusions, blots for direct substrates of mTOR (p-S6K1, p-4EBP1), with corresponding blot for all total proteins, should be shown.

3. Figure 1C – Puromycin incorporation demonstrates that protein synthesis is increased yet only the phospho-RPS6 blot is included. A total RPS6 blot must be included to demonstrate that the increase in phospho-RPS6 signal is not simply increased synthesis of total RPS6. This is especially important as the abundance of vinculin (used as the loading control) varies across samples in Figure 1B and 1C.

4. Figure 1F – statistical analysis – multiple comparisons have been made to the 0 hr time point. Running multiple t-tests in this manner increases the chance of error, therefore, statistical analysis should take these multiple comparisons into account by using ANOVA with Dunnett's post hoc test.

Essential revisions:

1) It was thought that the focus on mTOR signaling is too narrow and that other relevant pathways impinging on translation apparatus should be considered. These include integrated stress response and MNK/eIF4E axis, as these pathways have been implicated in adaptation to hypoxia and/or immune responses in neoplasia.

We understand. The focus on the mTORC1 pathway was due to the overwhelming role that it has within both immune system biology and translation, perhaps also for the existence of excellent reagents and a good comprehension of its basic function. We included statements on translation pathways in the Introduction and in the Discussion, and added several experiments.

To comply (experimentally) with this criticism, we have started to analyse in vitro in primary T cells the effects of: 1. hypoxia on Ser51 eIF2α phosphorylation, 2. the effects of Mnk inhibition on puromycin incorporation and on Ser209 eIF4E phosphorylation.

1. eIF2a phosphorylation was below reliable detection limit by Western Blotting in primary human T cells. Next, we attempted to set up an ELISA assay: the assay was successful for phospho-eIF2α and demonstrates an increase of eIF2α phosphorylation in hypoxia conditions (Figure 1G). Next, we attempted FACS analysis of eIF2α phosphorylation in mouse TILs. In our hands, we cannot detect in lymphocytes reliable, specific 2α phosphorylation by FACS analysis (Author response image 1). We then turned to immunohistochemistry: starting with the observation that PMO hypoxic labelling correlates with low puromycin incorporation (Figure 3D), we were able to set up triple staining methods for CD4⁺, PMO, P-eIF2α. We were able to demonstrate that PMO staining clusters with eIF2α staining in CD4⁺ cells (Figure 3 and Figure 3 —figure supplement 1). Overall, a negative correlation between puromycin and PMO staining/eIF2α/phosphorylation seems evident.

Author response image 1

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2. eIF4E phosphorylation was below reliable detection limit by Western Blotting in primary human T cells. We also performed ELISA assay for phosho-eIF4E, but we did not see changes in eIF4E phosphorylation that we could confirm as specific. There are two explanations, (a) eIF4E is not phosphorylated in activated T cells, (b) the extent of phospo/dephospho eIF4E is below reliable detection, either because a minority of cells is affected or due to low levels. We think that (a) is unlikely, given that TCR activation stimulates Mnk and, in mouse spleen lymphocytes, after CD3 crosslinking, p-eIF4E can be detected (Gorentla et al., 2013). We think that (b) is likely as the primary cells we are dealing with, are from a circulating subset, and consistently other MS studies do not report p-eIF4E in human T cells, example (Howden et al., 2019) (and related dataset). In addition, in classic reports (Kleijn and Proud, 2002) changes of p-eIF4e were not evident upon stimulation of lymphocytes similar to what we used. In the same context, we found that acute Mnk inhibition did not reduce puromycin incorporation in cultured primary human lymphocytes stimulated with anti-CD3/CD28 (Figure 1D). Concerning infiltrating CD3+ cells, the labelling of p-eIF4E by FACS analysis can be gated to a small proportion of cells, 1-2% (Figure 2 —figure supplement 2E). These p-eIF4E positive cells were, interestingly, puromycin positive (same as above) (2). Taken together the data support a model in which eIF4E phosphorylation is important in a specific cellular context.

Given the clear effects of the mTORC1 pathway in vitro, we tested in vivo whether acute Everolimus (mTORC1 blocker) administration affected phosphorylation of rpS6 and puromycin incorporation in infiltrating TILs. Figures 2G-H and Figure 2 —figure supplement 2D show a reduction in both. The results confirm the importance of the mTORC1 pathway in sustaining, in vivo, TILs translation rates.

2) In some parts of the manuscript methodology/analysis should be improved. Specifically, anti-phospho-S240/244 ribosomal protein S6 antibody should be used instead of the antibody recognizing phosphorylated S235/236, as the latter phosphoacceptor sites are not exclusively phosphorylated in a mTOR-dependent manner. In addition, it was found that the statistical tests used were in some cases inappropriate, as illustrated in reviews below.

Several experiments have been repeated with anti-phospho-S240-244. The results are almost overlapping with the ones obtained with anti-phospho-S235-236 (Figures 1C-D; Figure 2—figure supplement 2B-C; Figure 5 —figure supplement 1B). Statistic tests have been checked and amended where necessary (methods).

(3) Assessing co-expression of the markers presented in figures 4D and 5D and relating their levels to protein synthesis activity was thought to be warranted in order to grasp full heterogeneity of tumor-infiltrating T cells.

This point is intriguing. The analysis has been re-done through co-stainings. The results were revealing for CD8⁺ cells, whose relevance in tumor and phenotypic stratification are more clear than for CD4⁺. We found: SLAM6-/TIM3+ cells co-segregating with high puromycin; TIM3+/PD1+ co-segregating with high puromycin (Figures 4E-F); Ki67 and PD-1/TIM3 coexpression (Figure 4 —figure supplement 1). The interpretation is straightforward. Dysfunctional CD8 cells show the highest degree of clonal expansion and proliferation among tumor infiltrating cells (Li, van der Leun, Yofe et al., Cell 2019). Expression of Slamf6 in Tim3+ cells characterizes progenitor exhausted cells that retain polyfunctionality and are responsive to immune checkpoint blockade (ICB) during cancer immunotherapy. Conversely, loss of Slamf6 is associated with terminal exhaustion and loss of responsiveness to ICB, albeit Slamf6-Tim3+ cells maintain high secretion of IFN-γ and proliferative activity (Miller et al., 2019). Our results suggest that these cell subsets are still actively translating and therefore exhaustion is not associated with the loss of translational activity. Accordingly, TIM3+PD-1+ cells exhibited high puromycin incorporation and are an emerging target (Daver et al., 2021). In spite of the semantic confusion these data imply that exhaustion is a continuum and “exhausted” cells are exerting antitumor function, in line with recent works showing that it is the exhausted CD8⁺ T cells that are exerting residual control over tumor growth (Li et al., 2019). See also from line 333, discussion.

Concerning the CD4⁺ cells, the analysis was less meaningful, given their heterogeneity. However, we confirm the contribution of TREGs/CTLA4/TGFα (Shevyrev and Tereshchenko, 2019) to the puromycin pool (Figure 6), which would suggest that immunosuppression is an active cellular activity requiring translation.

4) Finally, it was thought that the manuscript may benefit from additional editing.

Done.

Reviewer #1:

In this study, mouse models are employed to systematically establish protein synthesis activity of tumor-infiltrating CD4⁺ and CD8⁺ T lymphocytes in vivo. The effects of stimuli emanating from tumor niche on translational activity of CD4⁺ and CD8⁺ T lymphocytes were also studied. Using ex vivo experiments, the evidence is provided that T cell receptor stimulation induces protein synthesis and mTOR signaling in tumor-infiltrating T cells, whereas hypoxia exerts the opposite effect. Next, the authors used mouse melanoma and colorectal cancer models to confirm these findings. Using the same models, the authors show that tumor-infiltrating CD4⁺ and CD8⁺ T lymphocytes translate more than their counterparts in the spleen. Importantly, it was also observed that tumor-infiltrating lymphocytes also exhibit significant intercellular heterogeneity in translational activity. This was followed by establishing the correlations between levels of protein synthesis and T cell phenotypes using flow cytometry, which revealed strong correlation between levels of protein synthesis and immunophenotypes. Collectively, these findings support the tenet that translation plays a major role in establishing tumor-infiltrating T cell phenotypes, and demonstrate that there is pronounced intracellular heterogeneity in their protein synthesis activity. This heterogeneity in the levels of mRNA translation between tumor-infiltrating T cells may at least in part be explained by the difference in stimuli that these cells are exposed in distinct parts of tumor microenvironment. In long-term, this study may inspire single-cell proteomics and/or similar approaches that do not rely on steady-state mRNA levels to monitor the plasticity of tumor-infiltrating lymphocytes and thus provide molecular bases for more efficient immunotherapies.

Strengths: Presented findings further support the critical role of the protein synthesis apparatus in establishing lymphocyte phenotypes, and emphasize the importance of considering alterations that occur downstream of changes in mRNA levels when determining the lymphocyte subtypes in the tumors. This is of particular interest considering recent increase in the number of single cell sequencing studies aiming to address composition of tumor-infiltrating lymphocytes, wherein correlations with phenotypes are driven based on steady-state mRNA composition of the cell. Methodologically, the clear advantage of this study, is that the most of the work was done in vivo, and is therefore of high physiological relevance. In addition, both melanoma and colon cancer models were employed, thereby suggesting that reported observations are likely to be generally applicable, and not cancer type-specific. In addition, the authors monitored the effects of cancer-relevant perturbations on protein synthesis, including T cell receptor stimulation and hypoxia. Overall, this study provides solid evidence that translation plays a major role in establishment of immunophenotypes of the tumor-infiltrating lymphocytes, and that interaction between tumor microenvironment and translational machinery may be responsible for intercellular heterogeneity of T cells in tumor niche.

Weaknesses: The major drawback of this study is that it relies on correlation and lacks experiments aiming to decipher mechanisms underlying the interactions between tumor niche and translational machinery of tumor infiltrating lymphocytes. Moreover, it was thought that the focus on mTOR may be too narrow and that other signaling mechanisms known to modulate translation rates in response to environmental cues should also be investigated. Finally, it remains unclear whether the changes in protein synthesis rates affect all the mRNAs uniformly or induce selective perturbations in translational activity of specific subsets of mRNAs.

We acknowledge the mechanistic weakness of this manuscript, given the complexity of dealing with the small numbers and heterogeneity of tumor infiltrating lymphocytes. We analysed eIF2α pathways (Figure 1G; Figure 3E and its supplemental), Mnk-eIF4E (Figure 1D; Figure 2 —figure supplement 2) and manipulated the mTOR pathway with Everolimus (Figure 2H). In this manuscript we address whether (a) high translation in TILs is ubiquitous or selective for some cells, (b) whether it correlates with gene expression changes and (c) is regulated by signalling pathways/environmental clues. It is likely that protein synthesis changes (also) specific mRNA translation given the current body of evidence of how translation works (Hershey et al., 2019). Formal proof, in our work, is lacking. The fact that translating cells have a specific phenotype (Figures 5-6), however, supports the idea of selective mRNA translation.

Notwithstanding these weaknesses, it was thought that with appropriate revision, provided evidence is sufficient to support the major conclusion of the manuscript which is that the interactions between tumor microenvironment and translational machinery play a major role in determining immunophenotypes of tumor-infiltrating lymphocytes and that relying on steady-state mRNA levels may not be sufficient to grasp full plasticity of tumor-infiltrating T cells.

– Sole focus of the study is on mTOR signaling. This is somewhat surprising as other mechanisms have been also shown to play a central role in translational reprogramming under hypoxia and T cell receptor stimulation (e.g. ISR, MNK-dependent phosphorylation of eIF4E, etc). Based on this, it was thought that more comprehensive assessment of alterations in signaling pathways that impinge on translational machinery is warranted to pinpoint signaling pathways that mediate the observed effects of tumor microenvironment on T lymphocytes.

The point has been addressed in the general reply (1). We performed an analysis of other pathways, hypoxia-eIF2 and Mnk-eIF4E, and new data have been inserted as detailed in (1) and in the Public Review.

– Conclusions on alterations in mTOR signaling are based solely on monitoring phospho-rpS6 levels. Importantly, the antibody that the authors used recognizes S235/236 on rpS6 which are phosphorylated in cells lacking S6K1 and 2 (PMID: 15060135) and are phosphorylated by other AGC kinases (e.g. RSKs). The phoposho-acceptor site on rpS6 that is exclusively sensitive to alterations in the mTORC1/S6K signaling axis is S240/244 (PMID: 15060135) and thus, antibody recognizing S240/244 should be employed. To strengthen their conclusions, the authors should also add appropriate total antibody controls and monitor other mTORC1 targets (e.g. 4E-BPs, eEF2K).

The point on S240/244 has been addressed in the general reply (2). New data have been inserted in new Figures 1C-D; Figure 2 —figure supplement 2B-C; Figure 5 —figure supplement 1B.

4EBPs: Human and mouse lymphocytes do not express 4E-BP1, but 4E-BP2 (our data; FPKM 4E-BP1: 100-300; FPKM 4E-BP2 3000-5000; (So et al., 2016)). There is an antibody described in the literature that functions in the detection of p-4E-BP (Yi et al., 2017) by FACS. In our hands, it does not give reliable staining in tumor infiltrating lymphocytes (Cell Signalling). Note: the manufacturer shows use of this antibody for the tumor Jurkat T cell line +/- PI3K inhibition, whereas Yi et al., (2017) used it for splenocytes after in vitro stimulation. Both experimental approaches have better cell homogeneity/size/number etc… compared to TILs. In short, it is not that surprising that we fail to see specific staining in infiltrating lymphocytes because they are heterogenous, small, and not stimulated with aCD3/CD28. Similarly, we have not found antibodies for p-eEFK that work effectively by FACS.

– Considering the general lack of mechanistic data to explain heterogeneity in translational activity of tumor-infiltrating T cells, it was thought that monitoring the impact of mTOR inhibitors as well as compounds targeting other signaling mechanisms that adjust protein synthesis in accordance to changes in cellular environment (e.g. ISRIB, MNK inhibitors) on T-cell immunophenotypes and heterogeneity in vivo would be informative.

We tested first in vitro the effects of Mnk inhibitors on global translation, and we did not see differences (Figure 1D). Given the data with mTORC inhibitor Everolimus, we tested its effect in vivo. Since mTORC1 inhibition might have an effect both on tumor cells and T cells, chronic mTORC1 treatment would be confounding. Therefore, we briefly pulsed with Everolimus and assessed the effect on T cells. We found that Everolimus reduces the number of TILs incorporating puromycin, and rpS6 phosphorylation (Figure 2H and its supplemental). ISRIB study will be part of a new project in which we will study eIF2B/eIF2α. We expect that eIF2α phosphorylation is essential for T cell viability and adaptation in vivo, but we have no solid data yet (Rashidi et al., 2020).

– It is well-established that in addition to changes in global protein synthesis levels, stressors such as hypoxia selectively affect translation of different mRNA subsets, whereby the latter changes are likely to significantly contribute to observed immunophenotypes. I understand that translatome-wide translational profiling is out of the scope of this short report and also technically very challenging, but perhaps authors should monitor mRNA and protein levels of selected number of factors that are known to be translationally regulated under these conditions and/or implicated in T cell biology in their system.

We are working on this issue since a long time and it is more complex than what it seems, i.e. since we must score for divergent mRNA/protein expression, and take in account many aspects, mRNA/protein stability, detection threshold, double staining ISH-IHC etc. We are still, unfortunately, (very) far from a decent solution that can be applied to TILs. As this reviewer probably knows, we know some “metabolic” targets regulated at the translational level in cultured CD4⁺ cells, such as ACC1 and PKM (Manfrini et al., 2020; Ricciardi et al., 2018). However, we need to define secreted cytokines, a physiologically relevant endpoint in the tumor microenvironment. IL-2 is one of such (Garcia-Sanz and Lenig, 1996) that may be regulated but we have no formal proof in vivo.

– The article may benefit from some careful editing.

Done.

Reviewer #2:

The tumor microenvironment includes the extracellular matrix (ECM) and a multitude of cells including immune cells. During cancer progression, the tumor associated ECM and cell composition/activity is altered. For example, the activity of immune cells towards tumor cells is commonly reduced which facilitates e.g. tumor growth and metastasis. It is well established that, in vitro, activation of T lymphocytes leads to a dramatic upregulation of protein synthesis and increased proliferation. Yet, to what extent this is true in vivo is not yet characterized. In this manuscript, Benedetta De Ponte Conti et. al address this using a FACS-based approach whereby global protein synthesis (quantified by puromycin incorporation) is related to expression of established activation markers for CD4⁺ and CD8⁺ T cells in tumors from the B16/F10 melanoma model. The conclusions are largely in agreement with studies of in vitro models as activation of CD4 or CD8 cells induces proliferate and bolsters protein synthesis. This study thereby pinpoints the close relationship between protein synthesis and immune cell activity also in vivo. One particularly interesting finding is the links between protein synthesis and Treg phenotype. In aggregate, the claims are well supported and important. This study will likely fuel many additional studies trying to in greater detail understand how protein synthesis in immune cells relate to their function within the tumor microenvironment.

There are some parts I think could be more developed:

1. There is a close link between protein synthesis and proliferation following activation of CD4 and CD8 T cells. This raises the question of how close puromycin incorporation associates with proliferation. In figure 5 S1, Ki67 vs protein synthesis is assessed in CD4⁺ T cells. This suggests a very close relationship. IS there a corresponding relationship for CD8⁺ T cells? Are there any aspects of the protein synthesis profile which do not associate with proliferation? What would the phenotype of such cells be?

We performed the analysis of Ki67 in CD8⁺ T cells and found a positive correlation with proliferation (new Figure 4C). In addition, we found a positive relationship in CD8⁺ cells between PD-1 expression and Ki-67 labelling, as well as with TIM3 and Ki67 (new Figure 4C and Figure 4 —figure supplement 1). The markers that we identified (Figure 4), in the context of CD8⁺ tumor infiltrating lymphocytes can be compared to the ones found in other studies. It is evident that clonal expansion, as detected, by TCR sequencing (Liu et al., 2020; Zhang et al., 2018) is a major phenomenon that coincides with our observations on protein synthesis (discussion, line 320).

CD4⁺ helper cells are highly heterogenous and a similar conclusion, e.g. whether high translation coincides with clonal expansion, is more difficult. However, Puro+ Tregs far exceed Puro + Tconv (Figure 6D) and, together with the relatively limited EOMES+ Tr1 subset, constituting 1/20 of the FOXP3+ repertoire, they are highly expanded in vivo (Bonnal et al., 2021). In conclusion, highly translating CD4⁺ cells are mostly associated with clonal expansion of Tregs.

Thus, clonal expansion driven by TCR stimulation is a major source of translating TILs.

2. All analyses are performed on populations of cells but there are no cross-comparisons between marker expression. I.e. is there one subset of T cells with high protein synthesis which express most of these markers (Figure 4D) or are there subsets of high protein synthesis cells which express subsets of markers? I am not sure if it is technical possible, but it would be interesting to assess co-expression of the identified markers in figure 4D and 5D and how their expression relates to protein synthesis.

Multiple labelling was performed. We repeated stainings and analysis, maintaining a rigid gating. This allows the definition of CD8⁺ TIM3+PD1+ and CD8⁺, TIM3+, SLAMF6- subsets described in point 3. Data indicate also CD4⁺, FOXP3+, CTLA4+, as highly translating (Figures 5 and 6).

3. Analysis is largely qualitative when assessing expression of makers and protein synthesis. Perhaps it would be informative to also perform a quantitative analysis within single tumors?

Perhaps, we missed the point. The analysis has been quantitative in single tumors. Example: Figure 2, Supp. 2B, right, each dot represents the percentage of cells positive for pS6/Puro+ and pS6/Puro- in a given tumor. The 2D-plot on the left is the representative distribution of density in a single tumor. Data can be alternatively represented as in Figure 3D, where the trajectory of single individuals can be seen, although we thought that this was reasonable only for PMO distribution, since it is a spatial parameter in the tissue (histological). I hope we have clarified. Rephrasing: Figure 2, Figure supplement 2B, right derives from 40 single stainings, same for 2C, etc.

4. It is unclear to me what the relationship between T cell exhaustion and protein synthesis (and also proliferation) is based on the presented data. It seems logical to expect reduced protein synthesis in exhausted T cells, but this may not be the case.

Please see point (3) of mandatory changes. We inserted a discussion statement. Exhaustion is not associated with the loss of translational activity. The topic is in itself confusing because exhaustion may be a continuum or poorly defined. Exhausted CD8⁺ T cells still play a critical role in cancer. TILs expressing markers of exhaustion (PD-1, LAG3 and TIM3) are more likely to express IFN-γ(Gros et al., 2014). While exhausted CD8⁺ T cells have reduced function as compared to those elicited by an acute infection, several data suggest that it is the exhausted CD8⁺ T cells that are exerting residual control over tumor growth (Li et al., 2019). In short, in vivo, exhaustion is a developmental stage that can be reactivated. Unfortunately, the terminology is confusing.

Reviewer #3:

Overall the figures and the text were clear and concise and the data well described. The data shown support the claims made, but the authors have not examined the other major pathway for regulation of translation. My main issues relate to the first three figures.

1. The authors focus on the regulation of protein synthesis through mTOR, however translation initiation is also regulated by eIF2. Moreover, the PERK-eIF2-ATF4 axis has been shown to be important for tumour cell adaptation to stress – therefore, as all four eIF2 kinases are expressed in these models, it is important to determine any effect from eIF2 on translation activation/inhibition:

– What are the levels of eIF2α phosphorylation in the -/+puromycin cell populations (Figure 1 and 2)?

– As hypoxia is known to regulate eIF2 kinases what is the activation status of the four kinases in these model systems during hypoxia (Figure 1 – 3)?

Please see also the general response to mandatory corrections. We have now demonstrated by ELISA eIF2α phosphorylation in hypoxic, lowly translating CD4⁺ cells (new Figure 1G). in vivo, we can see that hypoxia-rich regions contain CD4⁺/peIF2a+ cells (new Figure 3 and its supplemental). We call them peIF2a+ immunoreactive-like cells. We cannot perform parallel eIF2 total staining in immunohistochemistry (antibodies of same species). Available Anti-phospho-eIF2α antibodies were not good for FACS analysis in TILs.

The point on the eIF2α kinases is intriguing. Our RNAseq data on human TILs show that all 4 kinases are expressed (Bonnal et al., 2021), with PKR and HRI predominant, and gcn2, PERK 5-fold less. For protein validation, concerning the four kinases, issues of antibody quality and FACS suitability are even worse than for p-eIF2α. Phospho HRK to my knowledge has never been produced. Phospho-PKR by CST has been discontinued. Phospho-GCN2 worked only in large cells. We agree that PERK axis is probably the more relevant. According to the literature, hypoxia leads to phosphorylation of eIF2α by PERK, for instance (Koumenis et al., 2002). In addition, in vivo, hypoxia is accompanied by AMPK activation, which in turn has a feedback on translation (Horman et al., 2002). In short, we think that this issue must be (and will be) treated as a new project on eIF2 kinases (see discussion).

– It is known that mTOR signalling can crosstalk to eIF2 signalling, and vice-versa, so how do the levels of eIF2α phosphorylation (and activity of eIF2 kinases) correlate with mTOR activity in these models during hypoxia (Figure 1 – 3)?

– To support the author's conclusions it would be necessary to show that the regulation of translation in this model is independent of eIF2 signalling.

We agree. It is known that mTOR crosstalks with eIF2 and the extent of potential cross-talk of signalling is huge (Roux and Topisirovic, 2018). However, please note that we never concluded that translation is independent of eIF2 signalling, but we stated that hypoxia in combination with TCR stimulation were the main regulators of the TILs rate of translation, and high translation was associated with specific TIL phenotypes. We hope that this is now clear and that the new data on eIF2a that were added, at least partly, contribute to improving the issue.

2. The author's state at various points that the data shows mTOR activity is increased in the puromycin positive proportion of cells, however they do not directly show this:

– The antibody used for phospho-RPS6 recognises residues (Ser235/236), which can be phosphorylated in the absence of S6K1 and S6K2, therefore, could this regulation be independent of mTOR? Although RPS6 is downstream of mTOR signalling it is not a direct substrate of mTOR, therefore, to fully support these conclusions, blots for direct substrates of mTOR (p-S6K1, p-4EBP1), with corresponding blot for all total proteins, should be shown.

We agree with the comment. We have inserted data on S240/244 which is dependent on S6K1/2 and we found that it behaves similarly to S235/236 (Figures 1C-D; Figure 2 —figure supplement 2B-C; Figure 5 —figure supplement 1B). The advantage of detecting S235/236 rpS6 phosphorylation was its outstanding performance by FACS.

4EBPs: Human and mouse lymphocytes do not express 4E-BP1, but 4E-BP2 (our data; FPKM 4E-BP1: 100-300; FPKM 4E-BP2 3000-5000; (So et al., 2016)). There is an antibody described in the literature that functions in the detection of p-4E-BP (Yi et al., 2017) by FACS. In our hands, it does not give reliable staining in tumor infiltrating lymphocytes.

3. Figure 1C – Puromycin incorporation demonstrates that protein synthesis is increased yet only the phospho-RPS6 blot is included. A total RPS6 blot must be included to demonstrate that the increase in phospho-RPS6 signal is not simply increased synthesis of total RPS6. This is especially important as the abundance of vinculin (used as the loading control) varies across samples in Figure 1B and 1C.

This is completely true and biologically relevant. Total S6 has been added. In line with the literature also rpS6 protein levels increase with stimulation (Howden et al., 2019), which is not surprising since they are TOP mRNAs (Loreni et al., 2014). It is the total absence of phospho-rpS6 in unstimulated cells the real feature of quiescent human T cells. Total S6 does not work in FACS.

4. Figure 1F – statistical analysis – multiple comparisons have been made to the 0 hr time point. Running multiple t-tests in this manner increases the chance of error, therefore, statistical analysis should take these multiple comparisons into account by using ANOVA with Dunnett's post hoc test.

We now performed the suggested tests with similar results.

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Author details

1. Benedetta De Ponte Conti

   1. Institute for Research in Biomedicine, Università della Svizzera Italiana (USI), Bellinzona, Switzerland
   2. Graduate School of Cellular and Molecular Sciences, University of Bern, Bern, Switzerland

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

2. Annarita Miluzio

   Istituto Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi", Milan, Italy

   Contribution

   Data curation, Investigation, Methodology

   Competing interests

   No competing interests declared

3. Fabio Grassi

   1. Institute for Research in Biomedicine, Università della Svizzera Italiana (USI), Bellinzona, Switzerland
   2. Department of Medical Biotechnology and Translational Medicine, Universita` degli Studi di Milano, Milan, Italy

   Contribution

   Investigation, Supervision, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

4. Sergio Abrignani

   1. Istituto Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi", Milan, Italy
   2. Department of Clinical Sciences and Community Health, Università degli Studi di Milano, Milan, Italy

   Contribution

   Conceptualization, Writing - review and editing

   Competing interests

   No competing interests declared

5. Stefano Biffo

   1. Istituto Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi", Milan, Italy
   2. Bioscience Department, Università degli Studi di Milano, Milan, Italy

   Contribution

   Conceptualization, Project administration, Resources, Supervision, Writing - original draft, Writing - review and editing

   For correspondence

   biffo@ingm.org

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3780-1050

6. Sara Ricciardi

   1. Istituto Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi", Milan, Italy
   2. Bioscience Department, Università degli Studi di Milano, Milan, Italy

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Validation, Visualization, Writing - original draft, Writing - review and editing

   For correspondence

   ricciardi@ingm.org

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8124-432X

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