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mTOR-dependent translation drives tumor infiltrating CD8+ effector and CD4+ Treg cells expansion

  1. Benedetta De Ponte Conti
  2. Annarita Miluzio
  3. Fabio Grassi
  4. Sergio Abrignani
  5. Stefano Biffo  Is a corresponding author
  6. Sara Ricciardi  Is a corresponding author
  1. Institute for Research in Biomedicine, Università della Svizzera Italiana (USI), Switzerland
  2. Istituto Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi", Italy
  3. Department of Medical Biotechnology and Translational Medicine, Universita` degli Studi di Milano, Italy
  4. Department of Clinical Sciences and Community Health, Università degli Studi di Milano, Italy
  5. Bioscience Department, Università degli Studi di Milano, Italy
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Cite this article as: eLife 2021;10:e69015 doi: 10.7554/eLife.69015

Abstract

We performed a systematic analysis of the translation rate of tumor-infiltrating lymphocytes (TILs) and the microenvironment inputs affecting it, both in humans and in mice. Measurement of puromycin incorporation, a proxy of protein synthesis, revealed an increase of translating CD4+ and CD8+ cells in tumors, compared to normal tissues. High translation levels are associated with phospho-S6 labeling downstream of mTORC1 activation, whereas low levels correlate with hypoxic areas, in agreement with data showing that T cell receptor stimulation and hypoxia act as translation stimulators and inhibitors, respectively. Additional analyses revealed the specific phenotype of translating TILs. CD8+ translating cells have enriched expression of IFN-γ and CD-39, and reduced SLAMF6, pointing to a cytotoxic phenotype. CD4+ translating cells are mostly regulatory T cells (Tregs) with enriched levels of CTLA-4 and Ki67, suggesting an expanding immunosuppressive phenotype. In conclusion, the majority of translationally active TILs is represented by cytotoxic CD8+ and suppressive CD4+ Tregs, implying that other subsets may be largely composed by inactive bystanders.

Editor's evaluation

By employing melanoma and colon cancer allograft mouse models, the authors show that tumor-infiltrating T-cells exhibit heterogeneity in levels of protein synthesis that correlates with their immunophenotypes. Moreover, some evidence is provided that the observed heterogeneity in protein synthesis rates in tumor-infiltrating T-cells levels is driven by distinct conditions in different parts of tumor microenvironment. Overall, these findings further corroborate the importance of mRNA translation in immune cell plasticity and suggest that relying on monitoring steady-state mRNA levels may not provide the full picture of immunophenotypes in tumor niche.

https://doi.org/10.7554/eLife.69015.sa0

Introduction

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.

Results

Translational efficiency is increased by TCR signaling and inhibited by hypoxia

Puromycin, by entering the acceptor site of ribosomes and incorporating into nascent polypeptide chains, represents a valid tool to quantify protein synthesis within cells in vivo. We used puromycin measurement in combination with quantitative immunoblotting to capture global protein synthesis rate in human primary lymphocytes (Figure 1A). We have previously shown that naïve T cells have a poised translational machinery, with pre-accumulated mRNAs that are efficiently translated only after T cell receptor stimulation (Ricciardi et al., 2018). This prompted us to further evaluate the relationship between T cell receptor stimulation and translation. T cell receptor stimulation leads to a progressive, temporal, steady increase of protein synthesis (Figure 1B). Next, we checked whether the strength of T cell receptor stimulation affected the global output of proteins. To this end, primary lymphocytes were briefly cultivated in the presence of two different amounts of anti-CD3/CD28, and protein synthesis was measured by puromycin incorporation (Figure 1C). We saw higher puromycin incorporation in cells stimulated with the higher concentration of anti-CD3/CD28 (Figure 1C). Phosphorylation of ribosomal protein rpS6 occurs in serine 235/236 by S6K1-2 downstream of mTORC1 and by RSKs kinases downstream of ERK, as well as in serine 240/244 by S6K1-2 kinases (Meyuhas, 2015). All these pathways are activated by TCR stimulation (Piccirillo et al., 2014). Consistently, rpS6 phosphorylation increased from undetectable to strong levels after anti-CD3/CD28 stimulation (Figure 1C). rpS6 itself was detectable before TCR stimulation and increased upon stimulation, in line with previous data (Wolf et al., 2020; Araki et al., 2017).

T cell receptor stimulation through mTORC1 is a strong stimulator of translation counterbalanced by hypoxia in primary lymphocytes.

(A) Schematic diagram for the experiment. (B) Immunoblot of puromycin incorporation in CD4+ T cells following stimulation by anti-CD3/CD28 at the indicated time points shows that T cell receptor stimulation leads to a progressive increase of puromycin in cultured lymphocytes. The immunoblot shows two replicates with cells isolated from one healthy donor per experiment. Densitometry normalized to vinculin. (C) The strength of T cell receptor stimulation correlates with puromycin incorporation. The immunoblot shows two replicates with cells isolated from one healthy donor per experiment. (D) mTOR inhibition reduces rpS6 phosphorylation and translation. Stimulated CD4+ T cells were treated for 30 min with either 2 μM PP242 or 3 μM MNK inhibitor before collecting extracts. Puromycin incorporation and phosphorylation of rpS6 were measured by Western blotting. The immunoblot is representative of the pool of two replicates with cells isolated from one healthy donor per experiment. (E and F) Hypoxic environment sharply reduces translation. Stimulated CD4+ T cells were transferred from 20% O2 to 1% O2 for the indicated times (E), and translation was measured by flow cytometry (F). Data are mean ± s.d. p Values are determined by ANOVA with Dunnett’s post hoc test. ****p < 0.0001. (G) Hypoxia induces phosphorylation of eIF2α. Stimulated CD4+ T cells were incubated for 4 hr under normoxia or hypoxia and the phosphorylation of eIF2α was determined by ELISA assay as described in Materials and methods using an anti-phospho-eIF2α-specific antibody. The data represents the pool of three independent experiments. Data are mean ± s.d. p Values are determined by two-tailed Student’s t-tests. ****p < 0.001.

Activation of protein synthesis correlates with rpS6 phosphorylation (Figure 1C). Inhibition of mTOR pathway by the mTOR inhibitor PP242 leads to both the reduction of rpS6 phosphorylation and a conspicuous inhibition of puromycin incorporation, that is, protein synthesis (Figure 1D). The ERK pathway converges on Mnk1/2 that phosphorylate Ser209 of eIF4E (Roux and Topisirovic, 2018). In vitro, Mnk inhibition did not consistently reduce puromycin incorporation, indicating that this pathway is not massively involved in global translation of T cells (Figure 1D).

Since infiltrating lymphocytes may encounter an environment rich in hypoxic areas, we wondered if hypoxia was able to rapidly modify the capability of lymphocytes to translate in response to T cell receptor stimulation. We therefore quantitated the rate of puromycin incorporation in the presence of hypoxic environment (Figure 1E). Hypoxia resulted in a rapid reduction of the translation rate of primary lymphocytes (Figure 1F, Figure 1—source data 3). Several translation pathways are modulated by hypoxia, including the one driven by PERK. PERK phosphorylates Ser51 of eIF2α (Koumenis et al., 2002). We did not detect eIF2α phosphorylation by Western blotting, likely due to the limited number of human cells. We used a modified ELISA procedure and found that hypoxia induced eIF2α phosphorylation (Figure 1G, Figure 1—source data 4). On the basis of these data, we hypothesize that, in vivo, the combination of T cell receptor stimulation and hypoxic conditions is a major determinant of the translational capability of lymphocytes.

Only a minority of TILs actively translates and is characterized by an mTORC1 activated phenotype

Next, we addressed the question of whether, in vivo, we could observe heterogeneity in the translational efficiency of TILs. To answer this question, we engrafted subcutaneously C57BL/6 mice with B16F10 melanoma cells (Figure 2A). We administered a single injection of puromycin (50 mg/kg body mass) and after 1 hr, mice were sacrificed and T cells isolated for analysis in flow cytometry (Figure 2A). Then, the amount of protein synthesized was quantified on the basis of puromycin incorporation by FACS. The same gating strategy was applied for spleen lymphocytes and TILs (Figure 2—figure supplement 1). Intriguingly, we found that at the single-cell level, in vivo, the translational efficiency of both CD4+ and CD8+ TILs was highly variable (Figure 2B and C, Figure 2—source data 1), namely some TILs had no puromycin incorporation, others were highly positive for puromycin. On the basis of the gating shown in Figure 2B and C, we defined two categories of infiltrating T cells, high puromycin (Puro+) and low puromycin (Puro-) T cells, reflecting translating T cells and translationally silent T cells, respectively. This simple analysis unveiled that CD4+ TILs translated more than spleen-resident CD4+ T cells (Figure 2B). The number of highly translating CD4+ TILs in different animals ranged between 20% and 60% (Figure 2B). Similar to CD4+ TILs, the number of highly translating CD8+ TILs was more than twofold higher than in the spleen (Figure 2C). Within individual tumors, Puro+ CD8+ TILs ranged between 20% and 90% (Figure 2C). Taken together these data demonstrate the presence of a strong layer of translational control, driven by the microenvironment, that affects the biological activity of individual lymphocytes.

Figure 2 with 2 supplements see all
The tumor microenvironment stimulates translation in a limited number of tumor-infiltrating lymphocytes (TILs) with activated mTORC1 pathway.

(A) Experimental outline. Tumor cells were injected in recipient mice. Puromycin was injected intraperitoneally, and T cells were collected 1 hr later for the analysis. (B and C) The amount of incorporated puromycin was determined by FACS analysis in CD4+ (B) or CD8+ lymphocytes (C). Representative plots and statistical analysis (mean ± SEM) show that the number of Puro+ TILs is always higher in tumors versus spleen and that only parts of TILs are translationally active. Percentages of positive cells in each gate are shown. Data from three experiments pooled together (n = 5–8 mice per experiment) *p < 0.05; ***p < 0.001; ****p < 0.0001. (D) Immunohistochemical analysis of puromycin in CD4+ TILs. Puro+ cells (pink) are concentrated in some tumor areas, where clusters of highly translating CD4+ cells (brown) are found. Scale bars, 10 μm. (E) Representative plots (left) and statistical analysis (mean ± SEM) for pS6(S235/236) within Puro+ and Puro- CD4+ and CD8+ TILs shows that, in vivo, most translating TILs have the mTORC1-S6K pathway active. Percentages of positive cells in each gate are shown. Data from two experiments pooled together (n = 5 mice per experiment) ****p < 0.0001. (G) Experimental outline. Tumor cells were injected in recipient mice that were given intraperitoneal injections of 5 mg/kg everolimus for 2 consecutive days. Puromycin was injected 1 hr before sacrificing mice and collecting TILs cells for subsequent flow cytometry analysis. (H) Representative flow cytometry plots and statistical analysis (mean ± SEM) for puromycin within CD4+ and CD8+ TILs showing that the mTOR inhibitor everolimus reduces translation. Percentages of positive cells in each gate are shown. Data from two experiments pooled together (n = 10 mice per experiment) **p < 0.01; ***p < 0.001.

IHC on paraffin-embedded B16 tumor sections also confirmed that (i) the levels of incorporated puromycin were heterogenous inside the tumor, with areas having high expression mixed with areas devoid of expression (Figure 2D), and that (ii) scattered lymphocytes show a robust puromycin immunoreactivity (Figure 2D). To substantiate these results within a different tumor microenvironment, we engrafted C57BL/6 mice with MC38 colon adenocarcinoma cells. Similar to what was obtained for B16 TILs, we found that TILs have significant higher levels of puromycin signal over spleen-resident T cells, and that, in vivo, within the tumor, only a fraction of lymphocytes is translationally active (Figure 2—figure supplement 2A).

We then addressed whether puromycin incorporation, in vivo, was associated with mTORC1 signaling. TCR stimulation causes an increase of mTORC1 activity that can be detected by the analysis of rpS6 phosphorylation (Ricciardi et al., 2018). Notably, both CD4+ (Figure 2E, Figure 2—source data 1) and CD8+ (Figure 2F, Figure 2—source data 1) cells containing high levels of puromycin were consistently positive for rpS6 phosphorylation. We found that phosphorylation of rpS6 in the puromycin-positive cells was significantly higher both at Ser235/236 (Figure 2E–F) and at Ser240/244 (Figure 2—figure supplement 2B-C, Figure 2—figure supplement 2—source data 1). In order to verify that also in vivo, as in vitro (Figure 1), inhibition of the mTORC1 pathway affected the translation of TILs, we treated mice with mTOR inhibitor everolimus (Figure 2G). In short, treatment of mice with everolimus caused a decrease in the percentage of puromycin-positive TILs (Figure 2H, Figure 2—source data 1), and a slight but significant decrease in rpS6 phosphorylation (Figure 2—figure supplement 2D, Figure 2—figure supplement 2—source data 1).

Mnk inhibition in vitro did not result in significant changes in puromycin incorporation (Figure 1). However, in vivo, a very small pool of TILs was positive for p-eIF4E, and clearly segregated with high puromycin incorporation (Figure 2—figure supplement 2E, (Figure 2—figure supplement 2—source data 1)). In conclusion, the activation of specific signaling pathways, at the single-cell level, explains high puromycin incorporation. Among them, the mTORC1 pathway affects the global translation rate of a substantial percentage of TILs.

Hypoxic niches reduce puromycin incorporation

Next, the relationship between puromycin incorporation and hypoxia, in vivo, was analyzed. We injected mice with pimonidazole (PMO), a chemical probe that forms protein adducts in viable hypoxic cells (Rademakers et al., 2011) and puromycin (Figure 3A) and performed stainings on tissue sections for the endothelial marker CD31. It was found that the amount of tumor areas that were not vascularized was limited, the maximal distances between vessels being in the range of 150–200 µm (Figure 3B). Subsequently, we purified both CD4+ (Figure 3C) and CD8+ (Figure 3D) TILs and analyzed by FACS analysis the relationship between PMO and puromycin incorporation. In spite of the relative absence of highly hypoxic tumor areas, the data show that, in vivo, an inverse relationship between PMO staining and puromycin exists (Figure 3C and D, Figure 3—source data 1). Next, we performed immunofluorescence analysis. We clearly detected cells triple-stained for PMO, CD4+ and p-eIF2α−like immunoreactivity in hypoxic areas (Figure 3E), but not in non-hypoxic areas (Figure 3—figure supplement 1). We suggest that hypoxia acts as a repressor of translation also in vivo, in TILs. These data raise the question whether the phenotype of TIL is specific.

Figure 3 with 1 supplement see all
Hypoxia limits translation of tumor-infiltrating lymphocytes (TILs) in vivo.

(A) Experimental outline. Tumor cells were injected in recipient mice. Puromycin was injected intraperitoneally together with the hypoxia marker pimonidazole (PMO). T cells were collected 1 hr later for the analysis. (B) CD31 staining in tumor specimens shows ample vascularization with limited areas far from blood vessels suggesting the absence of truly hypoxic areas and limited hypoxic gradients. Scale bars, 15 μm. (C and D) Representative plots (left) and statistical analysis (mean ± SEM) for PMO+ within Puro+ and Puro- CD4+ and CD8+ TILs show, in vivo, the inverse correlation between hypoxia and translational efficiency. Percentages of positive cells in each gate are shown. Data from two experiments pooled together (n = 7–8 mice per experiment) **p < 0.01. (E) Representative immunofluorescence images showing that in tumor specimens the majority of PMO+ CD4+ TILs are p-eIF2α(S51)+. Scale bars, 10 μm.

High translation rate in CD8+ cells correlates with IFN-γ production and CD-44 expression

We verified the existence of a relationship between high translation and the CD8+ phenotype by measuring through flow cytometry the expression of markers of memory and effector T cell differentiation, activation, as well as of relevant costimulatory and coinhibitory molecules. The experimental design is shown in Figure 4A. First of all, we observed a maximum of puromycin incorporation in CD44+ CD8+ T cells, indicating that Puro+ CD8+ TILs largely retain an activated-like phenotype (Figure 4B, Figure 4—source data 1). We co-stained freshly isolated CD8+ TILs for puromycin and the following sets of markers: (i) PD-1, TIM3, CTLA-4, and TIGIT that characterize CD8 T cell exhaustion within the tumor microenvironment (Anderson et al., 2016); (ii) the T cell receptor costimulatory proteins CD28, ICOS, SLAMF6, and CD27, pro-inflammatory IFN-γ and TNF-α cytokines, Granzyme B; (iii) tissue-resident T cells markers CD103, CD69, and CCR5 (Golubovskaya and Wu, 2016); (iv) ectonucleoside triphosphate diphosphohydrolase-1 CD39 (Takenaka et al., 2019). We decided to apply a statistical design in which we pooled the number of events observed in all animals, rather than a direct comparison of Puro+ versus Puro-, in a single animal. By this approach, we may underestimate statistical significance, but we obtain relationships that may have a general significance. First, we found a strong positive relationship between puromycin incorporation and Ki67 staining, suggesting clonal amplification (proliferation) of actively translating cells (Figure 4C, Figure 4—source data 1). Notably, we did not detect statistical differences in the expression of PD-1, TIGIT, CTLA-4, CD28, CD69, CCR5, CD103, and TNF-α between high and low puromycin incorporating cells (Figure 4D, Figure 4—source data 1). Conversely, we found a positive relationship between puromycin incorporation and the expression of TIM3, SLAMF6, CD39, Granzyme B, CD27, and IFN-γ. In turn, ICOS expression was selectively decreased in highly translating CD8+ cells (Figure 4D). Next, we analyzed co-marker expression. We found that highly translating cells were characterized by having high TIM3-low SLAMF6, high TIM3-high PD-1 (Figure 4E–F, Figure 4—source data 1). Consistently, high PD1 and high TIM3 expression associated with Ki67+ cells (Figure 4—figure supplement 1A-B, Figure 4—figure supplement 1—source data 1). Altogether, these data show that translationally active CD8+ T cells are confined in non-hypoxic areas, characterized by the activation of the mTORC1 pathway and can be defined by a specific immunophenotype. Taken together the data suggest the existence of clonally expanding CD8+ cells, highly translating, and moving versus the exhaustion status.

Figure 4 with 1 supplement see all
Puro+ translating CD8+ tumor-infiltrating lymphocytes (TILs) retain an activated-like phenotype.

(A) Schematic diagram for the experiment. (B) Representative plots (left) and statistical analysis (mean ± SEM) of gated CD44+ CD8+ TILs analyzed for puromycin incorporation. Quantitation shows an enrichment for the expression of CD44. Percentages of positive cells in each gate are shown. Data from three experiments pooled together (n = 6 mice per experiment) **p < 0.01. (C) Representative plots (left) and statistical analysis (mean ± SEM) of Ki67+ within Puro+ and Puro- CD8+ TILs. Quantitation shows a positive correlation between translation rate and proliferation. Percentages of positive cells in each gate are shown. Data from two experiments pooled together (n = 5 mice per experiment) **p < 0.01. (D) Statistical analysis (mean ± SEM) for the indicated markers within Puro+ and Puro- CD8+ TILs shows that Puro+ translating CD8+ TILs are enriched for TIM3, SLAMF6, CD39, CD27, ICOS, and IFN-γ expression. Percentages of positive cells in each gate are shown. Data from two experiments pooled together (n = 5 mice per experiment) **p < 0.01; ****p < 0.0001. (E) Representative plots and statistical analysis (mean ± SEM) for SLAMF6 and TIM3 within Puro+ and Puro- CD8+ TILs shows that Puro+ translating CD8+ TILs are enriched for TIM3 expression. Percentages of positive cells in each gate are shown. Data from two experiments pooled together (n = 5 mice per experiment) *p < 0.05. (F) Representative plots and statistical analysis (mean ± SEM) for PD1 and TIM3 within Puro+ and Puro- CD8+ TILs show that Puro+ translating CD8+ TILs are enriched for TIM3 and PD1 expression. Percentages of positive cells in each gate are shown. Data from two experiments pooled together (n = 5 mice per experiment) *p < 0.05.

Efficiently translating CD4+ TILs are enriched for CTLA-4 expression and suppressor Tregs

We asked whether translating Puro+ CD4+ T cells had specific phenotypes. Next, we measured by flow cytometry the expression of markers of memory and effector T cell differentiation, activation, as well as of relevant costimulatory and coinhibitory molecules (Figure 5A). Herein, we summarize the main findings. We observed an increase in puromycin incorporation in CD44+ cells (Figure 5B, Figure 5—source data 1). Next, we analyzed the expression of costimulatory and coinhibitory proteins, that is, PD-1, TIGIT, TIM3, CTLA-4, SLAMF6, CD39, CD27, ICOS, CD28, CD69, CCR5, and CD103 (Figure 5C, Figure 5—source data 1). Some of these markers clustered with puromycin incorporation as shown in Figure 5C. Ki-67 labeling was robustly enriched in cells incorporating higher levels of puromycin (Figure 5—figure supplement 1A).

Figure 5 with 1 supplement see all
Regulatory T cells (Tregs) represents the majority of CD4+ tumor infiltrating Puro+ cells.

(A) Schematic diagram for the experiment. (B) Representative plots (left) and statistical analysis (mean ± SEM) of gated CD44+ CD4+ tumor-infiltrating lymphocytes (TILs) analyzed for puromycin incorporation. Quantitation shows an enrichment for the expression of CD44. Percentages of positive cells in each gate are shown. Data from three experiments pooled together (n = 6 mice per experiment) **p < 0.01. (C) Statistical analysis (mean ± SEM) for the indicated markers within Puro+ and Puro- CD4+ TILs shows that Puro+ translating CD4+ TILs are enriched for CTLA4, CD39, CD27, ICOS, CCR5, and CD103 expression. Data from two experiments pooled together (n = 5 mice per experiment) *p < 0.05; **p < 0.01; ****p < 0.0001. (D) Representative plots (left) and statistical analysis (mean ± SEM) of gated CD25+ Foxp3+ CD4+ lymphocytes show that the number of Puro+ Tregs is higher in tumors versus spleen. Percentages of positive cells in each gate are shown. Data from two experiments pooled together (n = 4–5 mice per experiment) ***p < 0.001. (E) Immunohistochemical analysis of puromycin in Foxp3+ TILs shows that clusters of highly translating Foxp3+ cells are found. Scale bar, 20 μm. (F) Representative plots (left) and statistical analysis (mean ± SEM) for pS6(S235/236) within Puro+ and Puro- Tregs showing that, in vivo, most of translating Tregs have the mTORC1-S6K pathway active. Percentages of positive cells in each gate are shown. Data from one experiment representative of two (n = 7) ***p < 0.001.

Tregs can be identified by CD25-FoxP3. We analyzed the difference in translation between tumor infiltrating Tregs and spleen-resident Tregs. Notably, only 4–6% Tregs of the spleen incorporated puromycin (Figure 5D, Figure 5—source data 1), whereas approximately 40% of tumor-infiltrating Tregs incorporated high levels of puromycin (Figure 5D, Figure 5—source data 1). In keeping with these findings, IHC on paraffin-embedded tumor sections showed that puromycin signals overlap with FoxP3 staining (Figure 5E). Measurement of intracellular levels of phosphorylated S6 ribosomal protein (pS6) in gated Puro+ and Puro- Tregs showed, in line with previous observations, that pS6 labeling partitioned with high levels of puromycin, both for rpS6 S235/S236 (Figure 5F, Figure 5—source data 1) and for rpS6 Ser240/244 (Figure 5—figure supplement 1, Figure 5—figure supplement 1—source data 1).

CTLA-4 and TGF-β are Treg markers that correlate with the suppression activity of Tregs (Sakaguchi et al., 2020). We therefore gated Tregs and analyzed the correlation between puromycin incorporation, CTLA-4 and TGF-β. Puro+ Tregs showed the selective upregulation of CTLA-4 with respect to the Puro- counterpart (Figure 6A, Figure 6—source data 1). Intracellular staining for TGF-β revealed enhanced secretion by Puro+ Tregs (Figure 6—source data 1, ). Puro+ Tregs had also higher levels of Ki67 staining (Figure 6C, Figure 6—source data 1). Overall, our data confirm the hypothesis that, within tumors, the main translating and expanding CD4+ subset is represented by suppressive Tregs with mTORC1 activation. If the hypothesis is true, then the expression of Ki67 should be higher in Puro+ Tregs, compared to Puro+ Tconv. Indeed, Puro+ Tregs expressed significantly more Ki67 than Puro+ Tconv cells (Figure 6D, Figure 6—source data 1), confirming that the tumor microenvironment preferentially fosters cell cycling of immunosuppressive cells.

Highly translating regulatory T cells (Tregs) are functionally active and are more proliferative than highly translating Tconv.

(A) Representative plots (left) and statistical analysis (mean ± SEM) for CTLA-4+ (B), TGF-β+ (C), Ki67+ (C), within Puro+ and Puro- Tregs shows that, in vivo, Puro+ translating Tregs are more proliferative but also exhibit a more activated phenotype than Puro- Tregs. Percentages of positive cells in each gate are shown. Data from one experiment representative of two (n = 6–9) *p < 0.05; **p < 0.01. (D) Representative plots (left) and statistical analysis (mean ± SEM) for Ki67+ within Puro+ Tregs and Puro+ Tconv shows that, in vivo, Puro+ Tregs expressed significantly more Ki67 than Puro+ Tconv (C), indicating that the former are more proliferative than the latter. Percentages of positive cells in each gate are shown. Data from one experiment representative of two (n = 6) **p < 0.01.

Discussion

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.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Mus musculus)B16F10PMID:32699136Cells were obtained from Dr Matteo Bellone, HSR Scientific Institute, Milan, Italy
Cell line (Mus musculus)MC38PMID:32699136Cells were obtained from Dr Maria Rescigno, Humanitas University, Rozzano (MI), Italy
AntibodyMouse monoclonal anti-Puromycin, clone 12D10MilliporeCat#: MABE343, RRID:AB_2566826WB (1:10000), IHC (1:500)
AntibodyMouse monoclonal anti-Vinculin, clone V284MilliporeCat#: 05–386, RRID:AB_11212640WB (1:1000)
AntibodyMouse monoclonal anti-ActinSigma-AldrichCat#: A4700, RRID:AB_476730WB (1:1000)
AntibodyRabbit polyclonal anti phospho-rpS6 Ser235/236Cell SignalingCat#: 2211, RRID:AB_331679WB (1:1000)
AntibodyRabbit polyclonal anti phospho-rpS6 Ser240/244Cell SignalingCell Signaling Technology Cat#: 2215, RRID:AB_331682WB (1:1000)
AntibodyRabbit polyclonal anti-phospho-eIF2α Ser51Cell SignalingCat#: 3597, RRID:AB_390740IF (1:400), ELISA (1:500)
AntibodyRabbit monoclonal anti-FoxP3Cell SignalingCat#: 98377, RRID:AB_2747370IHC (1:100)
AntibodyRabbit monoclonal anti-CD4Cell SignalingCat#: 25229, RRID:AB_2798898IHC (1:100)
AntibodyRat monoclonal anti-CD4BioLegendBioLegend Cat#: 100401, RRID:AB_312686IF (1:400)
AntibodyRabbit monoclonal anti-CD31Cell SignalingCat#: 77699, RRID:AB_2722705IHC (1:100)
AntibodyMouse monoclonal APC conjugated anti-CD62LeBioscienceClone: MEL-14, Cat#: 17-0621-83FC (1:200)
AntibodyMouse monoclonal PE conjugated anti-CCR5eBioscienceClone: 7A4, Cat#: 12-1951-82FC (1:200)
AntibodyMouse monoclonal FITC conjugated anti-ICOSeBioscienceClone: C398.4A, Cat#: 11-9949-82FC (1:200)
AntibodyMouse monoclonal PE/Cy7 conjugated anti-CD39eBioscienceClone: 24DMS1, Cat#: 25-0391-30FC (1:200)
AntibodyMouse monoclonal PerCP/EF710 conjugated anti-CD3eBioscienceClone: 17A2, Cat#: 46-0032-80FC (1:200)
AntibodyMouse monoclonal PE conjugated anti-phospho-S6eBioscienceClone: cupk43k, Cat#: 12-9007-41FC (1:100)
AntibodyMouse monoclonal FITC conjugated anti-CD44BioLegendClone: IM7, Cat#: 103008FC (1:200)
AntibodyMouse monoclonal PE/Cy7 conjugated anti-CD25BioLegendClone: PC61, Cat# 102016 FC (1:200)
AntibodyMouse monoclonal APC/Cy7 conjugated anti-CD4BioLegendClone: RM4-5, Cat#: 100526FC (1:200)
AntibodyMouse monoclonal PE/Cy7 conjugated anti-CD4BioLegendClone GK1.5, Cat#: 100422FC (1:200)
AntibodyMouse monoclonal PE/Cy7 conjugated anti-CD3BioLegendClone: 145–2C11, Cat#: 100320FC (1:200)
AntibodyMouse monoclonal Pacific Blue conjugated anti-CD8αBioLegendClone: 53–6.7, Cat#: 100725FC (1:200)
AntibodyMouse monoclonal PE conjugated anti-CTLA-4BioLegendClone: UC10-4B9, Cat#: 106305FC (1:100)
AntibodyMouse monoclonal APC conjugated anti-Tim3BioLegendClone: B8.2C12, Cat#: 134007FC (1:200)
AntibodyMouse monoclonal PerCP/Cy5.5 conjugated anti-PD1BioLegendClone: RMPI-30, Cat#: 109120FC (1:200)
AntibodyMouse monoclonal PerCP/Cy5.5 anti-CD27BioLegendClone: LG.3A10, Cat#: 124214FC (1:200)
AntibodyMouse monoclonal FITC conjugated anti-CD28BioLegendClone: E18, Cat#: 122007FC (1:200)
AntibodyMouse monoclonal PE conjugated anti-CD103BioLegendClone: 2E7, Cat#: 121406FC (1:200)
AntibodyMouse monoclonal PE/Cy7 conjugated anti-CD69BioLegendClone: H1.2F3, Cat#:104526FC (1:200)
AntibodyMouse monoclonal PE/Cy7 conjugated anti-TIGITBioLegendClone: 1G9, Cat#:142107FC (1:200)
AntibodyMouse monoclonal PE conjugated anti-SLAMF6BioLegendClone: 330-AJ, Cat#:134605FC (1:200)
AntibodyMouse monoclonal APC conjugated anti- FoxP3eBioscienceClone: FJK-16s, Cat#: 17-5773-82FC (1:100)
AntibodyMouse monoclonal PE conjugated anti-Ki67eBioscienceClone: SolA15, Cat#: 12-5698-80FC (1:100)
AntibodyMouse monoclonal RedMab 549 conjugated anti-PMOHypoxiprobeCat#: 5914FC (1:100)
AntibodyMouse monoclonal APC of FITC conjugated anti-PuromycinMilliporeClone: 12D10FC (1:100)
AntibodyMouse monoclonal PE conjugated anti-IFN-γBioLegendClone: XMG1.2 Cat#: 505808FC (1:100)
AntibodyMouse monoclonal APC conjugated anti- TNF-αeBioscienceClone: MP6-XT22, Cat#: 506308FC (1:100)
AntibodyMouse monoclonal APC conjugated anti-Granzyme BBioLegendClone: GB11, Cat#: 515303FC (1:100)
AntibodyMouse monoclonal BV421 conjugated anti-TGF-βBiolegendClone: TW7-16B4, Cat#: 141407FC (1:100)
Commercial assay or kitBD Fixation Permeabilization KiteBioscienceCat#: 554714
Commercial assay or kiteBioscience FoxP3 staining buffer seteBioscienceCat#:: 00-5523-00
Commercial assay or kitCD4+ T Cell Isolation KitMiltenyi Biotec130-091-155
Chemical compound, drugHuman T-Activator CD3/CD28Thermo Fisher Scientific11131D
Chemical compound, drugPuromycinSigma-AldrichP8833
Chemical compound, drugPMOHydroxyprobeCat#: HP1-XXX
Chemical compound, drugEverolimusSigma-AldrichSML2282
Chemical compound, drugPPP242Sigma-AldrichP0037
Chemical compound, drugMNK inhibitorSigma-Aldrich454861

Human samples and mice

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Human blood samples from healthy male or female donors were collected with written informed consent, and collection was performed according to protocols approved by Ethics Committee of Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Ca’Granda Ospedale Maggiore Policlinico. All animal experiments were performed in accordance with the Swiss Federal Veterinary Office guidelines and approved by the Ethical Committee of the Cantonal Veterinary with authorization number TI 37/2016. C57BL/6J mice were bred in specific pathogen-free facility at the Institute for Research in Biomedicine (Bellinzona, Switzerland). Mice were housed, five per cage, in ventilated cages under standardized conditions (20°C ± 2° C, 55% ± 8% relative humidity, and 12 hr light/dark cycle). Food and water were available ad libitum, and mice were examined daily.

Cell isolation and culture from human blood

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PBMCs were isolated from blood samples by Ficoll-Hypaque density-gradient centrifugation. CD4+ T cells were enriched from PBMCs by magnetic separation (AutoMACS, Miltenyi Biotec) using human CD4+ T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions before flow sorting on a FACSAria II (BD Biosciences).

CD4+ T cells were activated with anti-CD3/CD28-coated beads (Life Technologies Dynabeads T-Activator) and IL-2 (20 IU/mL) and cultured in RPMI 1640 medium (Life Technologies) supplemented with 1% penicillin-streptomycin (Life Technologies), 2 mM GlutaMAX (Life Technologies), 1 mM sodium pyruvate (STEMCELL Technologies) for the indicated time intervals.

Typical cell culture conditions (37°C, 5% CO2, and environmental [21%] oxygen) were used and the conditions referred to as normoxia. Hypoxia was generated with an oxygen/CO2 controller incubator (Galaxy 48R; Eppendorf). It was set at 1% oxygen and 5% CO2.

Cell isolation from mice specimens

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T cells were obtained from cell suspensions of spleen. For TILs isolation, tumors were cut in small pieces and resuspended in RPMI1640 with 1.5 mg/mL Type I Collagenase (Sigma), 100 mg/mL DNase I (Roche), and 5% FBS, digested for 45 min at 37°C under gentle agitation. The digestion product was then passed through a 70 μm cell strainer to obtain a single-cell suspension. Lymphocytes were then enriched by Percoll density gradient following the manufacturer’s protocol.

Murine tumor cell lines

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B16F10 and MC38 cells were cultured in RPMI1640 supplemented with 10% heat-inactivate FBS, 100 U/mL penicillin/streptomycin, and 100 U/mL kanamycin. Cells were tested for the absence of Mycoplasma and maintained in 5% CO2 at 37°C. Frozen aliquots were thawed for each in vivo experiment and passaged in vitro for the minimum time required. Tumor cells at 70–80% confluency were harvested by diluting them 1:5 in 0.25% trypsin.

In vitro measurement of protein synthesis

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For in vitro measurements of protein synthesis, CD4+ T cells stimulated in vitro for the indicated time intervals were treated with 5 μg/mL puromycin for 10 min. Puromycin incorporation was then determined by flow cytometry or Western blotting as described later.

In vivo measurement of protein synthesis

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To measure protein synthesis in vivo, mice were intraperitoneal injected with puromycin at a concentration of 50 mg/kg dissolved in PBS at pH 6.4–6.6; 1 hr later, mice were sacrificed, and mice specimens rapidly collected. Spleen or cutaneous tumors were further processed for flow cytometry or histology analysis as described later. The sample size was estimated according to the following parameters: power 80%, alpha 0.05, average means of Puro+ cells 60% ± 20% for the tumor group and 30% for the non-tumor sample. Consequently, the minimal number was fixed to N = 5/group.

For the analysis of the effect of everolimus on translation rate and mTORC1 signaling, B16 mice were given intraperitoneal injections of 5  mg/kg everolimus (Sigma-Aldrich) dissolved in PBS for 2 consecutive days. Puromycin was injected 1 hr before sacrificing mice and collecting TILs for subsequent flow cytometry analysis.

In vitro stimulation of lymphocytes and hypoxia

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CD4+ T cells stimulated in vitro for 36 hr were transferred from 20% O2 to 1% O2 for the indicated times. Translation and eIF2α phosphorylation were measured by flow cytometry and ELISA, respectively, as described later.

In vivo detection of tissue hypoxia

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PMO (Hydroxyprobe) was injected intraperitoneally at a concentration of 60 mg/kg dissolved in PBS at pH 6.4–6.6. After 1 hr, mice were killed and processed further for flow cytometry or fluorescence microscopy analysis, as described later.

In vitro eIF2α phosphorylation detection with ELISA assay

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Phosphorylation of eIF2α was measured by ELISA assay. For the assay, each well of a 96-well microtiter plate was coated overnight at 4°C in a humidified chamber with 20 μg of protein extracts of stimulated CD4+ T cellsì either incubated for 4 hr under normoxia or hypoxia. Plate was then probed with either rabbit anti-total-eIF2α antibody (1:500, Cell Signaling) or anti-phospho-specific eIF2α(S51) antibody (1:500, Cell Signaling), for 1 hr at room temperature, followed by incubation with HRP-conjugate secondary antibody for 30 min at room temperature and addition of ELISA colorimetric TMB reagent as a soluble substrate for the detection of peroxidase activity. The absorption intensity was obtained using the Model 680 Microplate Reader (Bio-Rad).

Histology, tissue stainings, antibodies, and imaging

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Spleen or tumor samples were either embedded in OCT and frozen or fixed overnight with 4% paraformaldehyde, transferred to 70% EtOH and embedded in paraffin as previously described (Rosso et al., 2004). Samples were then cut at 4 μm (paraffin) or 10 μm (frozen) and stained with hematoxylin and eosin (Sigma-Aldrich) for morphological analysis. Immunohistochemical and immunofluorescence analyses were performed as described previously (Manfrini et al., 2020a).

Primary antibodies were used at the following dilutions: mouse monoclonal to Puromycin 12D10 (1:500, Millipore), rabbit polyclonal to phospho-eIF2α(S51) (1:400, Cell Signaling), rabbit monoclonal to FoxP3 D2W8ETM (1:100, Cell Signaling), rabbit monoclonal to CD31 D8V9E (1:100, Cell Signaling), rabbit monoclonal to CD4 D7D2Z (1:100, Cell Signaling), rat monoclonal to CD4 GK1.5 (1:400, BioLegend). Secondary Alexa Fluor conjugated goat anti-mouse, donkey anti-rat, and donkey anti-rabbit antibodies were used at the 1:500 dilution (Thermo Fisher Scientific).

Slides were mounted in glycerol supplemented with Mowiol 4–88 mounting medium (Sigma-Aldrich). White field images were acquired using a Leica DM1600 microscope. Fluorescence images were acquired using a confocal microscope (Leica TCS SP5) at 1024 Å, ~1,024 dpi resolution. All the images were further processed with Photoshop CS6 (Adobe, Berkeley, CA) software.

Western blotting

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SDS-PAGE was performed on protein extracts obtained from human CD4+ T cell activated in vitro as previously described. Western blotting was performed as previously described (Ricciardi et al., 2018). The following antibodies were used for Western blotting: rabbit polyclonal to phospho-rpS6 Ser235/236 (1:1000, Cell Signaling), rabbit polyclonal to phospho-rpS6 Ser240/244 (1:1000, Cell Signaling), mouse monoclonal anti-Vinculin (1:1000, Millipore), mouse monoclonal anti-Actin (1:1000, Sigma), mouse monoclonal anti-Puromycin (1:10000, Millipore). Chemiluminescent signals were detected using Amersham ECL Prime (GE Healthcare Life Sciences) and images were acquired using the iBright CL750 Imaging System (Thermo Fisher Scientific).

Where indicated, cells were treated with either 2 μM PP242 (Sigma-Aldrich) or 3 μM MNK inhibitor (Sigma-Aldrich) for 30  min after 48 hr of Dynabeads stimulation.

Flow cytometry

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For assessment of puromycin incorporation on human blood-derived samples, CD4+ T cells stimulated in vitro for the indicated times were fixed and permeabilized with Cytofix and Perm Buffer III buffers (both BD Biosciences) according to the manufacturer’s protocol, and stained with FITC conjugated anti-Puromycin antibody (Millipore). Finally, cells were analyzed by flow cytometry, and the relative amount of protein synthesized quantified with FlowJo software.

The following anti-mouse mAbs were purchased from eBioscience: APC conjugated anti-CD62L (clone: MEL-14, Cat#: 17-0621-83), PE conjugated anti-CCR5 (clone: 7A4, Cat#: 12-1951-82), FITC conjugated anti-ICOS (clone: C398.4A, Cat#: 11-9949-82), PE/Cy7 conjugated anti-CD39 (clone: 24DMS1, Cat#: 25-0391-30) PerCP/EF710 conjugated anti-CD3 (clone: 17A2, Cat#: 46-0032-80), PE conjugated anti-phospho-S6 (clone: cupk43k, Cat#: 12-9007-41).

The following mAbs were purchased from BioLegend: FITC conjugated anti-CD44 (clone: IM7, Cat#: 103008), PE/Cy7 conjugated anti-CD25 (clone: PC61, Cat# 102016), APC/Cy7 conjugated anti-CD4 (clone: RM4-5, Cat#: 100526), PE/Cy7 conjugated anti-CD4 (clone GK1.5, Cat#: 100422) PE/Cy7 conjugated anti-CD3 (clone: 145–2 C11, Cat#: 100320), Pacific Blue conjugated anti-CD8α (clone: 53–6.7, Cat#: 100725) PE conjugated anti-CTLA-4 (clone: UC10-4B9, Cat#: 106305), APC conjugated anti-Tim3 (clone: B8.2C12, Cat#: 134007), PerCP/Cy5.5 conjugated anti-PD1 (clone: RMPI-30, Cat#: 109120), PerCP/Cy5.5 anti-CD27 (clone: LG.3A10, Cat#: 124214), FITC conjugated anti-CD28 (clone: E18, Cat#: 122007), PE conjugated anti-CD103 (clone: 2E7, Cat#: 121406), PE/Cy7 conjugated anti-CD69 (clone:H1.2F3, Cat#: 104526), PE/Cy7 conjugated anti-TIGIT (clone: 1G9, Cat#:142107), PE conjugated anti-SLAMF (clone: 330-AJ, Cat#: 134605).

Intracellular staining was performed using the BD Cytofix/Cytoperm and Perm/Wash buffers or, for intracellular FoxP3 (APC, clone: FJK-16s, eBioscience, Cat#: 17-5773-82), Ki-67 (PE, clone: SolA15, eBioscience, Cat#: 12-5698-80), PMO (Hypoxiprobe Red Mab 549, Cat#5914), and Puromycin (APC or FITC, clone#12D10, Millipore) staining, the eBioscience FoxP3 staining buffer set. For intracellular staining of IFN-γ (PE, clone: XMG1.2, Biolegend, Cat#: 505808), TNF-α (APC, clone: MP6-XT22, eBioscience, Cat#: 506308), Granzyme B (APC, clone: GB11, Biolegend, Cat#: 515303), and TGF-β (BV421-labeled, clone:TW7-16B4, Biolegend, Cat#:141407) T cells were incubated for 4 hr at 37°C in ionomycin (Sigma-Aldrich,750 ng/mL) and PMA (Sigma-Aldrich, 20 ng/mL). For the last 3 hr, Brefeldin (eBioscience, 1000× Solution) was added to the cultures.

Statistical analysis

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The data are expressed as mean ± SEM. Student’t t test was used to compare pairs of data. In mice experiments, the statistical analysis was performed with the Prism Software (GraphPad). Comparisons of two groups were calculated using nonparametric Mann-Whitney test or ANOVA with Dunnett’s post hoc test. Error bars, p values, and statistical tests are reported in the corresponding figure legends. All experiments were based on biological replicates, that is, different groups of mice or T cells from different donors. No samples were excluded from the analysis.

Source data

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Excel files are provided for FACS and ELISA analysis. Raw gels as Figure 1—source data 1. Gels with cropped images as Figure 1—source data 2.

Data availability

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

References

Decision letter

  1. Ivan Topisirovic
    Reviewing Editor; Jewish General Hospital, Canada
  2. Carla V Rothlin
    Senior Editor; Yale School of Medicine, United States
  3. Ivan Topisirovic
    Reviewer; Jewish General Hospital, Canada
  4. Ola Larsson
    Reviewer; Karolinska Institutet, Sweden

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

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.

https://doi.org/10.7554/eLife.69015.sa1

Author response

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
Representative flow cytometry data of unstained CD4+ TILs used as a negative control and labelled CD4+ TILs with Alexa Fluor 405 anti-human phospho-eIF2α proving the almost totally absence of a specific 2α phosphorylation signal.

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.

References

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https://doi.org/10.7554/eLife.69015.sa2

Article and author information

Author details

  1. Benedetta De Ponte Conti

    Institute for Research in Biomedicine, Università della Svizzera Italiana (USI), Bellinzona, 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
    ORCID icon "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
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8124-432X

Funding

Associazione Italiana per la Ricerca sul Cancro (IG 19973)

  • Stefano Biffo

Fondazione Romeo ed Enrica Invernizzi (001)

  • Stefano Biffo

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The generous contribution of Fondazione Invernizzi for the Centro Organoidi and of AIRC IG 19973 to SB is acknowledged. We thank for initial help with the immunohistochemistry Giulia Mensa. This manuscript is dedicated to the memory of Prof. Fabrizio Loreni.

Ethics

Human subjects: Informed consent, and consent to publish, was obtained. "Studio comparativo del sistema immunitario tissutale in patologietumorali e autoimmuni con tecnologie multi-omiche (immunom/2020)." September 15, 2020.

This study was performed in strict accordance with the recommendations of the Ethical Commette of the Cantonal Veterinary of Switzerland. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (TI 37/2016) of the Cantonal Veterinary of Switzerland. No surgery was performed. Animals were sacrificed by CO2 euthanasia and every effort was made to minimize suffering.

Senior Editor

  1. Carla V Rothlin, Yale School of Medicine, United States

Reviewing Editor

  1. Ivan Topisirovic, Jewish General Hospital, Canada

Reviewers

  1. Ivan Topisirovic, Jewish General Hospital, Canada
  2. Ola Larsson, Karolinska Institutet, Sweden

Publication history

  1. Received: April 1, 2021
  2. Accepted: November 6, 2021
  3. Version of Record published: November 17, 2021 (version 1)

Copyright

© 2021, De Ponte Conti et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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