Remodeling Translation Primes CD8⁺ T Cell Antitumor Immunity

Abstract

The requisites for protein translation in T cells are poorly understood and how translation shapes the antitumor efficacy of T cells is unknown. Here we demonstrated that IL15-conditioned T cells were primed by the metabolic energy sensor AMPK to undergo diminished translation relative to effector T cells. However, we showed that IL15-conditioned T cells exhibited a remarkable capacity to enhance their protein translation in tumors, that which effector T cells were unable to duplicate. Studying the modulation of translation for applications in cancer immunotherapy revealed that direct ex vivo pharmacological inhibition of translation elongation primed robust T cell antitumor immunity. Our work elucidates that altering protein translation in CD8⁺ T cells can shape their antitumor capability.

Introduction

The efficacy of T cells to combat tumor growth is intimately tied to bioenergetics (1–3). Glycolytic T effectors are rapidly disarmed in the tumor microenvironment by nutrient deprivation (4) and inhibition of mitochondrial biogenesis (5). In contrast, T cells with memory properties induce tumor control highlighted by an exquisite ability to persist long-term in vivo (6) and the antitumor immunity conferred by memory T cells is attributed in part to their intrinsic metabolic path (7). mRNA translation is an essential feature of cell bioenergetics (8), but has not been applied to T cell biology for applications in tumor immunotherapy.

In response to nutrient and amino acid availability, mammalian target of rapamycin (mTOR) catalyzes mRNA translation through the activation of ribosomal protein S6 kinase beta-1 (p70S6K)), stimulating a component of the 40S ribosomal subunit, ribosomal protein S6 (S6) (9). In response to metabolic stress, AMP-activated protein kinase (AMPK) limits protein synthesis to conserve metabolic energy through two mechanisms. AMPK inhibits mTOR-directed p70S6K protein translation (10), limiting activation of S6. AMPK sequesters the elongation step of protein translation through phosphorylation of the kinase eukaryotic elongation factor 2 (eEF2K), which in turn phosphorylates eEF2 (p-eEF2) (11,12). p-eEF2 restricts protein translation through the inhibition of GTP-dependent translocation of the A-site-bound peptidyl-tRNA to the P-site during the elongation phase (13).

Here, we demonstrated that modulation of translation was a requisite for T cell tumor control. Using IL2 and IL15 cytokine priming to study translation in effector and memory-like T cells, respectively, we demonstrated that AMPK restricted protein translation in IL15-primed T cells through canonical mechanisms. Studying the paths of protein translation in T cells revealed that modulation of protein translation elongation improved T cell antitumor immunity, providing a unique treatment strategy.

Materials and Methods

Mice

Pmel (B6.CgThy1^a/CyTg(TcraTcrb)8Rest/J) and C57BL/6J mice were obtained from the Jackson Laboratory. 6–8 week old mice were used in the study. All animal experiments were approved by the Medical University of South Carolina (MUSC) Institutional Animal Care and Use Committee and the Division of Laboratory Animal Resources at MUSC maintained all mice.

Tumor & T Cell Cultures

For T cell cultures, splenocytes were manually dissociated to single cell suspension, red blood cells were lysed, and whole splenocytes from Pmel mice were activated with hgp100 peptide (1μg/mL, GenScript) and expanded with 200U rhIL2 (NCI) in complete T cell media (RPMI 1640, 10% FBS, 300mg/L L-glutamine, 100U/mL Penicillin, 100μg/mL Streptomycin, 1mM Sodium Pyruvate, 100μM NEAA, 1mM HEPES, 55μM β-mercaptoethanol, 0.2% Plasmocin Mycoplasma Prophylactic). For cytokine differentiation, after 3 days of expansion, T cells were split into complete T cell media containing 200U rhIL2 or 50ng/mL rhIL15 (Shenandoah) and harvested for analysis at time points indicated throughout the text and figure legends. Vehicle (DMSO), Compound C (Sigma), or Rapamycin (Sigma) were added at 1μM and 10nM, respectively. For drug treatments, whole splenocytes from Pmel mice were activated (as described above) in the presence of vehicle, 10μM AICAR (dissolved in H₂O, Sigma), or 20nM Homoharringtonine (dissolved in DMSO, Sigma).

B16F1 tumor cells obtained from ATCC were maintained in RMPI complete T cell media and passaged three times prior to in vivo inoculations. Tumor cells were determined to be mycoplasma free in November 2019. The tumor cell line has not been authenticated in the past year.

Protein Synthesis and Flow Cytometry

Fluorochrome-conjugated monoclonal antibodies CD8-APC (53–6.7), CD62L-FITC (MEL-14), CD44-Pe-Cy7 (IM7), and Thy1.1-Percp-Cy5.5 (HIS51) and respective isotype controls were purchased from Thermo Fisher. Live-or-Dye-PE Fixable Viability Stain was purchased from Biotium. Extracellular stains were performed in FACS buffer (PBS, 10% FBS). p-S6 staining was performed using Intracellular Fixation & Permeabilization Buffer Set (eBioscience, 88–8824-00). Cells were fixed then stained with p-S6-APC (cupk43k, Thermo Fisher) or isotype control in permeabilization buffer. Protein Synthesis was measured using the Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit according to the manufacturers’ protocol (Thermo Fisher, C10428). Briefly, cells were incubated in methionine-free media and control cells were treated with 100μg/ 5×10⁵ cells cycloheximide (Cayman Chemical, 601105). Cells were washed with PBS and stained extracellularly for 15 minutes and subsequently incubated in 50μM L-homopropargylglycine for 1 hour. Live-or-Dye Fixable Viability Stain (Biotium, 32005-T) was added for 10 minutes at the end of this incubation. Cells were fixed using 4% paraformaldehyde, washed with 3% BSA, and permeabilized using 0.5% Triton X-100 solution. Cells were washed in 3% BSA, followed by staining with Alexa Fluor 488 azide to label active protein synthesis. Isotype or CHX controls were used in all experiments. Samples were run directly on a BD Accuri C6 flow cytometer and analysis was performed with FlowJo software (TreeStar).

Immunoblotting

T cells were lysed in RIPA buffer (Sigma) supplemented with protease inhibitor cocktail (Cell Signaling) and phosphatase inhibitors I and II (Sigma). Protein concentrations were normalized using Pierce BCA Kit (Thermo Fisher, 23227) and loaded to 4–10% agarose gels (BioRad, 4568084). p-eEF2 (Thr56), eEF2, p-p70S6K (Thr389), p70S6K, p-AMPK (Thr172), AMPK, p-mTOR (S2448), p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), Ubiquitin (P4D1), β-actin, and HRP-linked anti-rabbit and mouse secondaries were obtained from Cell Signaling Technologies. p-p90RSK was obtained from Biolegend. Phospho proteins were developed with Pierce ECL Plus Western Blotting Substrate (Thermo Fisher, 32134). Total proteins and p-p44/42 MAPK (Erk1/2) were probed with Pierce ECL Western Blotting Substrate (Thermo Fisher, 32109).

RNA Analysis

RNA was isolated with RNeasy Mini Kit (QIAGEN, 74104) and concentration was measured using the SpectraDrop Micro-volume Microplate (Molecular Devices). Single-strand cDNA was made with 500ng RNA using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, 4387406, Thermo Fisher Scientific).Mouse Taqman gene probes (Applied Biosystems, Thermo Fisher) were used to perform real-time PCR in triplicate using the StepOnePlus Real-Time PCR system (Applied Biosystems, Thermo Fisher). Gene expression for Tcf7 was normalized to Gapdh and calculated using ΔΔCT method.

T Cell-Tumor Cell Co-culture

For transwell co-culture assays (Corning), B16F1 tumor cells were seeded for 24 hours, then T cells cultured in IL2 or IL15 were added to transwells in complete T cell media supplemented with respective cytokine, and harvested 36 hours later. Vehicle or p-p44/42 MAPK (Erk1/2) inhibitor U0126 (10μM, Cell Signaling Technology) was added to transwell co-cultures 4 hours prior to T cell harvest.

T Cell Transfers and Tumor Model

2.5×10⁵ B16F1 melanomas injected 200μl sterile PBS were established subcutaneously on the right flank of female C57BL/6 mice. Tumor-bearing mice were 5Gy irradiated 24 hours prior to T cell transfer. After 7 days of tumor growth 2×10⁶ Pmel T cells were infused via tail vein in 100μl of sterile PBS to melanoma-bearing mice. Tumor growth was measured every other day with calipers, area was calculated by tumor length x tumor width and survival was monitored with an experimental endpoint of tumor size ≥ 400mm². Vehicle, AICAR, Lys6K2 S6 Kinase Inhibitor (DMSO, 10μM) (Focus Biomolecules), or HHT-treated T cells were transferred after 4 days of ex vivo expansion following the aforementioned protocol. For ex vivo analysis of transferred T cells, mice were scarified 5 days after transfer and tumors and spleens were processed to single cell suspensions. Tumors were digested using the Mouse Tumor Dissociation Kit (Miltenyi, 130–096-730) according to manufacturer’s protocol.

Seahorse Bioanalysis

Oxygen consumption rate (OCR) was measured in Seahorse XF medium (Agilent) supplemented with 100nM insulin, 1mM sodium pyruvate, 5.6mM glucose, 4mM glutamine, 1% FCS under basal conditions and in response to 1μM oligomycin, 1.5 μM FCCP, and 2μM rotenone + 1μM Antimycin. A using the XFe96 Extracellular Flux Analyzer (Seahorse Bioscience). Cell-Tak (Corning) was used for adherence of 4×10⁵ T cells per well.

Proteomics

Sample preparation

T cells were lysed in 9M urea, 50 mM Tris pH 8, and 100 units/mL Pierce Universal Nuclease (ThermoFisher) and the concentration of protein was measured using a BCA assay (ThermoFisher). Protein was trypsin (Sigma) digested at 37°C for 18 hours, and the resulting peptides were desalted using C18 ZipTips (Millipore).

Liquid Chromatography and Mass Spectrometry Data Acquisition Parameters

Peptides were separated and analyzed on an EASY nLC 1200 System (ThermoScientific) in-line with the Orbitrap Fusion Lumos Tribrid mass spectrometer (ThermoScientific) with instrument control software v. 4.2.28.14. Peptides were pressure loaded at 1,180 bar, and separated on a C18 reversed phase column (Acclaim PepMap RSLC, 75 μm x 50 cm (C18, 2 μm, 100 Å)) (ThermoFisher) using a gradient of 2% to 35% B in 120 min (Solvent A: 0.1% FA; Solvent B: 80% ACN/ 0.1% FA) at a flow rate of 300 nL/min at 45 °C.

Mass spectra were acquired in data-dependent mode with a high resolution (60,000) FTMS survey scan, mass range of m/z 375–1575, followed by tandem mass spectra (MS/MS) of the most intense precursors with a cycle time of 3 s. The automatic gain control target value was 4.0e5 for the survey MS scan. Fragmentation was performed with a precursor isolation window of 1.6 m/z, a maximum injection time of 50 ms, and HCD collision energy of 35%; the fragments were detected in the Orbitrap at a 15,000 resolution. Monoisotopic-precursor selection was set to “peptide”. Apex detection was not enabled. Precursors were dynamically excluded from resequencing for 20 sec and a mass tolerance of 10 ppm. Advanced peak determination was not enabled. Precursor ions with charge states that were undetermined, 1, or > 7 were excluded.

Mass Spectrometry Data Processing

Protein identification and quantification were extracted from raw LC-MS/MS data using the MaxQuant platform v.1.6.3.3 with the Andromeda database searching algorithm and label free quantification (LFQ) algorithm (14, 15,16). Data were searched against a mouse Uniprot reference database UP000000589 with 54,425 proteins (March, 2019) and a database of common contaminants. The false discovery rate, determined using a reversed database strategy, was set at <1% at the protein and peptide level. Fully tryptic peptides with a minimum of 7 residues were required including cleavage between lysine and proline. Two missed cleavages were permitted. LC-MS/MS analyses were performed in triplicate (IL2 and IL15) or duplicate (vehicle and HHT-primed T cells) for each biological replicate with match between runs enabled. The “fast LFQ” was disabled and “stabilize large ratios” features were enabled. The first search was performed with a 20 ppm mass tolerance, after recalibration a 4.5 ppm tolerance was used for the main search. A minimum ratio count of 2 was required for protein quantification with at least one unique peptide. Parameters included static modification of cysteine with carbamidomethyl and variable N-terminal acetylation.

The protein groups text file from MaxQuant was processed in Perseus v. 1.6.5.0 (16). Identified proteins were filtered to remove proteins only identified by a modified peptide, matches to the reversed database, and potential contaminants.

Normalized LFQ intensities were log2 transformed. The LFQ intensities of technical replicates were averaged for each biological replicate. Quantitative measurements were required in at least three of five biological replicates in each treatment group.

Statistical Analysis

For all experiments in which protein synthesis was measured, mean fluorescent intensity (MFI) of HPG incorporation was divided by MFI of HPG incorporation in a condition-matched cycloheximide-treated well. Tumor growth was analyzed by linear regression of growth curves of vehicle versus drug-treated T cells. Survival to 40 days or tumor size of 400mm² was the experimental endpoint with Log-rank test for survival proportions of mice treated with vehicle versus drug-treated T cells used for analysis. Statistical analyses were performed with GraphPad Prism v8.3.0. Statistical parameters can be found in the figure legends.

For proteomics, Log2 transformed, protein LFQ intensities, normalized in MaxQuant, exhibited normal distributions. Binary comparisons of each LC-MS/MS analysis yielded Pearson correlation coefficients >0.90 between all technical and biological replicates within each experiment. For comparison of IL2 and IL15-conditioned T cells, quantitative measurements were required in at least three out of five biological replicates in each group yielding 1,521 quantified protein groups (Supplemental Table S1). Using a Student’s t-test 634 protein groups had a pval <0.05, with more stringent criteria of a permutation-based FDR of 5% and S0 of 0.1, 546 proteins were considered significantly different. For comparison of vehicle and HHT-primed T cells, quantitative measurements were required in all four biological replicates in each group yielding 2,217 protein groups (Supplemental Table S2). Using a Student’s t-test, 426 proteins had a pval <0.05. Volcano and enrichment plots were generated using R software. The -log10 pval versus the difference in Log2 protein intensities (IL15 - IL12) or (HHT – Vehicle) are shown. Unless otherwise noted, significance was assessed by 2-tailed student’s t-test.

Results and Discussion

IL15-conditioning restricted protein synthesis in T cells

IL2 and IL15 cytokine-mediated T cell differentiation is a strategy to study the molecular mechanisms that affect effector and memory-associated T cell lineages, respectively (7,17,18). Here we used IL2 and IL15 conditioning to assess the canonical mechanisms that regulate protein translation in effector and memory-like T cells as depicted in Figure 1A. Culture of T cells with IL15 resulted in a pool of T cells with memory traits, specifically CD62L/CD44 co-expression and transcription factor 7 (Tcf7) gene expression (Supplemental Figs. S1A–B) (19). Consistent with reports that the energy sensor AMPK is integral for memory T cell formation (20), AMPK activation was observed within 4 hours of IL15 addition to T cells (Fig. 1B). Concordant with activation of p-AMPK, canonical mechanisms of AMPK that modulated protein translation (Fig. 1A) were evident. Specifically, reduced p-p70S6K and enhanced p-eEF2 were observed in IL15-treated T cells relative to IL2 effector cells (Fig. 1B). Since p-p70S6K generates activation of S6 ribosomal subunit (pS6), we measured pS6 using flow cytometry. Within 6 hours of cytokine addition pS6 was diminished in IL15-treated T cells relative to IL2 effectors (Fig. 1C).

Figure 1. IL15-conditioning restricted protein synthesis in T cells.

A, Schematic of AMPK-directed inhibition of translation B, Immunoblotting and C, Flow cytometric plots and quantification of pS6 and D, protein synthesis in Pmel T cells activated with peptide and expanded for 3 days then differentiated with IL2 or IL15 for 4–6 hours. T cells described in C-D were treated with E, vehicle or Compound C and F, vehicle or Rapamycin for 6 hours and assessed for protein synthesis. G, Flow cytometric plot and quantification of protein synthesis in Pmel T cells described in C-D then differentiated with IL2 or IL15 for 3 days. Data are presented as ±SEM, **p<0.01, ***p<0.001, ****p<0.0001 (two-tailed t test). Data points represent biological replicates. Individual experiments were performed three times. Immunoblots were repeated twice. H, Volcano plot, where differentially expressed (DE) proteins (FDR < 0.05%) are marked as red. Canonical genes associated with translation, cell stress response, and unfolded protein response are annotated I, Enrichment plot of DE proteins for Gene Ontology (GO) biological processes in IL2 (blue) or IL15 (red) T cells, x axis indicates –1*log10 transformed p-values of enrichment. All of the reported p-values were statistically significant under the Bonferroni correction. Proteomics performed on T cell preparations were from five individual mice.

To measure how protein translation was altered in T cell subsets, we employed a rigorously validated FACS-based protein synthesis assay in which a fluorescent amino acid analogue of methionine (homopropargylglycine, HPG) is incorporated into actively translating cells (21–23). IL15 T cells exhibited reduced protein synthesis relative to IL2-treated effectors within 6 hours of cytokine addition (Fig. 1D). To test if AMPK directly inhibits translation in IL15-primed T cells, we added the AMPK inhibitor Compound C to T cell cultures at the time of cytokine addition. Translation was restored in IL15 primed T cells treated with Compound C (Fig. 1E). In a similar manner, treatment with mTOR inhibitor Rapamycin illustrated that translation in T cells at the time of cytokine addition was dependent on mTOR (Fig. 1F).

We confirmed that IL2 and IL15 T cells sustained increased and reduced translation, respectively, after 3 days of cytokine differentiation (Fig. 1G). Protein degradation assessed by ubiquitination was modestly reduced in IL15-treated T cells relative to IL2 (Supplemental Fig. S1C). These data prompted us to use LC-MS/MS analysis of IL2 and IL15-differentiated T cells to assess global protein differences among T cell groups (Supplemental Table S1). Proteins necessary for translation initiation (eukaryotic initiation factor 4a, eIF4a) and elongation (eEF2) were increased in IL2-derived T cells relative to IL15 as were proteins associated with an increased unfolded/misfolded protein burden (endoplasmic reticulum oxidoreductase, ERO1α and Bax) (24,25) (Fig. 1H). Gene ontology analysis of biological functions demonstrated that IL2 effector T cells harbored a proteome committed to sustaining translation (Fig. 1I). Consistent with memory T cell metabolism, IL15-treated T cells were enriched for β-oxidation of fatty acids (20). Metabolic processes of generation of precursor metabolites, cellular respiration, and ATP synthesis coupled proton transport were biological processes enriched in IL15-treated T cells relative to IL2 (Fig. 1I).

Stimulation of AMPK Improved T Cell Tumor Control

To test if stimulation of AMPK in T cells modulated protein translation, we treated T cells with AMPK stimulant 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). This treatment increased p-AMPK and enhanced and reduced the AMPK targets p-eEF2 and p-p70S6K, respectively (Fig. 2A). Protein synthesis and pS6 were diminished in AICAR-treated T cells (Figs. 2B–C). To test the effect of AMPK stimulation on T cell-mediated tumor control we activated and expanded Pmel T cells in the presence of AICAR. Protein synthesis and pS6 were diminished relative to vehicle controls (Figs. 2D–E). We infused B16 melanoma-bearing mice with vehicle or AICAR-treated T cells. Infusion of AMPK-primed T cells improved tumor control and extended animal survival relative to vehicle controls (Figs. 2F–G).

Figure 2. Stimulation of AMPK Improved T Cell Tumor Control.

A, Immunoblotting and B, flow cytometric plots and quantification of protein synthesis and C, pS6 in Pmel T cells activated with peptide and expanded for 3 days then differentiated in the presence of vehicle or AICAR for 4–6 hours. D, Flow cytometric plots and quantification protein of synthesis and E, pS6 in Pmel T cells activated and expanded as in B-C in the presence of vehicle or AICAR. Data are presented as ±SEM, *p<0.05, ***p<0.001, ****p<0.0001 (two-tailed t test). Data points represent biological replicates. Individual experiments performed three times. Immunoblot repeated twice. F, Pmel T cells activated and expanded as in D-E and infused to sublethally irradiated mice bearing 7 day established subcutaneous B16 melanomas, G, Survival of mice treated as in F (n=3 mice treated with no T cells and n=7 mice per group infused with vehicle or AICAR-treated T cells). Data are presented as ±SEM. F, Linear regression of tumor growth, ****p<0.0001. G, Comparison of survival curves between mice infused with vehicle and AICAR T cells, Log-rank (Mantel-Cox) test, **p=0.0027. Adoptive transfer experiments were performed twice.

IL15-primed T Cells Increase Translation in Tumors

In tumors, memory T cells can acquire effector cell functions (26), but how translation is regulated in this process is unknown. We transferred IL2 or IL15-differentiated T cells into tumor-bearing mice and measured translation in transferred T cells. In tumors IL15-primed T cells exhibited enhanced translation relative to IL2-derived tumor infiltrating lymphocytes (TILs) (Fig. 3A).

Figure 3. IL15-primed T Cells Increased Translation in Tumors.

Flow cytometric analysis with quantification of protein synthesis or pS6 in IL2 or IL15-derived Pmel T cells (Thy1.1+) A, transferred to tumor-bearing mice as described in Fig. 1D. Indicated organs were harvested 5 days later or B–C, harvested from transwell co-culture with B16 tumor cells. D, Immunoblotting of T cells harvested as in B-C. E, Immunoblotting of IL2 or IL15-derived T cells treated with vehicle or decreasing doses of U0126 p-p44/42 MAPK (Erk1/2) inhibitor. F, Flow cytometric analysis with quantification of protein synthesis and G, pS6 in IL2 or IL15-derived T cells from transwell co-culture with B16 tumors cells treated with vehicle or 10μM U0126. Data are presented as ±SEM, *p<0.05, **p<0.01, ***p<0.001(two-tailed t test). A, Data points represent individual mice. B-G, Data points represent biological replicates. Experiments A-D performed three times, E-G performed twice.

To assess molecular changes associated with translation in IL2 and IL15-derived T cells in tumors, we created a transwell co-culture tumor-T cell assay in which T cells and tumors cells shared media without contact. This assay assessed the effect of nutrient competition or secreted factors on T cell translation, but does not measure the effect of antigen encounter or contact-dependent tumor inhibition on T cell translation. Within 36 hours of co-culture we found that translation in IL2 T cells was impaired. Similar to IL15-primed T cell patterns in vivo (Fig. 3A), IL15-treated T cells exhibited a remarkable capacity to increase translation in co-culture with tumor cells (Fig. 3B). In accordance with enhanced translation, IL2 T cells co-cultured with tumor cells diminished pS6; whereas, IL15-conditioned T cells from tumor co-culture increased pS6 relative to tumor-free controls (Fig. 3C).

These data prompted us to assess molecular changes that affect translation in tumor co-cultures within T cell groups. In IL15-derived T cells, p-AMPK was modestly reduced in tumor co-culture and p-eEF2 was released (Fig. 3D). P-p70S6K was further reduced in IL15-primed T cells upon co-culture with tumor cells (Fig. 3D). Given that pS6 was increased in IL15 T cells co-cultured with tumor cells, we assessed activation of other molecular factors known to stimulate pS6 at S235/S236. A non-canonical signaling pathway that activates pS6 occurs through p90-ribosomal S6 kinase (p-p90RSK) that lies downstream of extracellular signal-related kinase (ERK) (27). Memory T cells are poised to signal Erk1/2 and IL15 conditioning enhances Erk1/2 activation (28,29). We observed that IL15 T cells express increased p-p90RSK relative to IL2 effectors. We were unable to detect differences in p-p90RSK in IL15 T cells responding to tumor cell co-culture (Supplemental Fig. S2). To establish that re-invigoration of translation of IL15-primed T cells in the tumor microenvironment was dependent on ERK1/2, we treated T cells in transwell co-cultures with the p-p44/42 MAPK (Erk1/2) inhibitor U0126. IL15-derived T cells expressed more p-p44/42 MAPK (Erk1/2) relative to IL2-treated cells (Fig. 3E) and 10μM U0126 abrogated p-p44/42 MAPK (Erk1/2) completely in IL2 and IL15 primed T cells. Inhibition of p-p44/42 MAPK (Erk1/2) reduced translation and p-S6 in IL15-derived T cells co-cultured with tumor cells relative to controls (Fig. 3F–G). Our data indicate that the tumor microenvironment dysregulates protein synthesis in effector T cells, suggesting that IL15-primed T cells harbor alternate mechanisms to support translation in tumors.

Inhibition of Translation Elongation Primed T Cell Antitumor Immunity

p-p70S6K appeared dispensable for enhanced translation in IL15-primed T cells in co-culture (Fig. 3). These data propelled us to measure how p-p70S6K-mediated activation of S6 affects T cell tumor control. We activated T cells in the presence of p70S6K inhibitor Lys6K2 (30) and confirmed that pS6 was extinguished (Supplemental Fig. S3A). Unexpectedly, protein synthesis was not impaired in p70S6K-treated T cells relative to vehicle (Supplemental Fig. S3B). Transfer of vehicle or Lys6K2-treated T cells to melanoma-bearing mice revealed that inhibition of p70S6K activation of S6 did not improve tumor control (Supplemental Fig. S3C).

In IL15 and AICAR-treated T cells, we observed a robust increase in p-eEF2 (Figs. 1–2). p-eEF2 inhibits the elongation step of protein translation through blockade of GTP-dependent translocation of the A-site-bound peptidyl-tRNA to the P-site (13). We reasoned that protein translation elongation may be a critical metabolic process that inhibits T cell function in tumors. We targeted protein translation elongation using the protein synthesis inhibitor Homoharringtonine (HHT) which interacts with the A-site of the ribosome to prohibit A-site binding of peptidyl-tRNA (31). T cells treated with HHT underwent reduced translation relative to controls (Fig. 4A). Live cell numbers of T cells were reduced in the HHT-treated condition 24 and 48 hours after activation (Fig. 4B). We found that T cells treated with HHT at the time of activation continued to undergo limited translation relative to vehicle controls after HHT was removed from cultures and T cells were re-expanded in vitro (Fig. 4C).

Figure 4. Inhibition of Translation Elongation Primed T Cell Antitumor Immunity.

A, Flow cytometric analysis with quantification of protein synthesis in Pmel T cells activated with peptide in the presence of vehicle or Homoharringtonine (HHT) for 48 hours or B (Data are combined results from 5 separate experiments), Live cell numbers of T cells described in A harvested at indicated timepoints. C, Flow cytometric analysis with quantification of protein synthesis in Pmel T cells activated and expanded as in A then expanded for 3 more days in the absence of drug. A, C, Data points represent biological replicates, experiments performed at least 3 times. B, D, Adoptive transfer of Pmel T cells activated as in A and infused to tumor-bearing mice as described in Fig. 1D, (n=3 mice treated with no T cells and n=8 mice per group infused with vehicle or HHT-treated T cells). Linear regression of tumor growth between mice infused with vehicle and HHT T cells, ****p<0.0001. E, Survival of mice treated as in D. D, Survival analysis between mice infused with vehicle and HHT T cells, Log-rank (Mantel-Cox) test, ***p=0.0002. Adoptive transfer experiments were performed twice. F, Flow cytometric analysis with quantification of protein synthesis in vehicle or HHT-conditioned Thy1.1+ Pmel T cells in indicated organs 5 days after transfer as described in D. Data points represent individual mice and experiments were performed twice. G, Oxygen consumption rate (OCR) trace in T cells activated, as in A, in response to the indicated injections (gray box indicates measure of ATP-linked respiration, blue box indicates measure of proton leak). H, Quantification of indicated bioenergetics parameters from G. Data points represent biological replicates, experiment performed three times. Data are presented as ±SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (two-tailed t test), ns=not significant. I, Enrichment plot of DE proteins for GO biological processes in vehicle (blue) or HHT (red) T cells, where x axis indicates –1*log10 transformed p-values of enrichment. J, Volcano plot, where DE proteins (FDR < 0.05%) are marked as red. Canonical genes associated with mitochondrial ATP synthesis, cell survival, and deubiquitylation are annotated. All of the reported p-values were statistically significant under the Bonferroni correction. Proteomic analysis performed on four separate T cell preparations from four individual mice. K, Immunoblot of Pmel T cells treated as in C. Immunoblots were performed 3 times.

We confirmed by immunoblot that HHT-mediated translation inhibition was not due to stimulation of AMPK, as HHT -primed T cells expressed reduced p-AMPK. increased p-p70S6K, and reduced p-eEF2. In line with these data, the metabolic energy sensor p-mTOR and pS6 were enhanced in HHT-treated T cells (Supplemental Fig. S4A–B). These data suggested that inhibition of the elongation step of translation overrided p70S6K-mediatead activation of S6 to control polypeptide chain formation in T cells. To measure the capacity to control tumors we transferred vehicle or HHT-treated Pmel T cells to mice bearing 7-day established B16F1 melanomas. We found that HHT T cells exhibited profound tumor control in contrast to vehicle-matched controls (Fig. 4D) and extended animal survival (Fig. 4E). In vivo HHT-primed TILs exhibited more translation than vehicle-matched TILs (Fig. 4F).

We next measured the bioenergetic traits that may account for the ability of HHT T cells to produce more protein in response to tumor antigen. HHT T cells did not harbor a greater spare respiratory capacity. However, compared to vehicle controls, HHT-treated T cells exhibited increased mitochondrial coupling efficiency and reduced proton leak synonymous with a heighted efficiency to generate ATP from the available metabolic pool (Fig. 4G–H). We used LC-MS/MS proteomics to profile HHT-primed T cells (Supplemental Table 2). In line with the bioenergetics data, generation of precursor metabolites, cellular respiration, and ATP synthesis coupled proton transport were enriched biological processes in HHT-primed T cells relative to vehicle controls (Fig. 4I). These data bear a striking resemblance to the enriched processes found in IL15-primed T cells (Fig. 1); however, a memory phenotype was not evident after expansion of HHT-treated T cells relative to vehicle (Supplemental Fig. 4C). The proteome showed an abundance of deubiquitylation enzymes increased in HHT-treated T cells relative to vehicle cells and increased anti-apoptotic mitochondrial hallmark Bcl-2 (Fig. 4J) (7). Synonymous with reduced protein degradation, western blotting confirmed that HHT-primed T cells harbored fewer ubiquitinated proteins (Fig. 4K). The data show that translation in T cells is tied to bioenergetics that shape tumor control. Targeting translation is thus a potential strategy to augment antitumor immunity.