Tryptophan potentiates CD8+ T cells against cancer cells by TRIP12 tryptophanylation and surface PD-1 downregulation

Abstract

Background: Tryptophan catabolites suppress immunity. Therefore, blocking tryptophan catabolism with indoleamine 2,3-dioxygenase (IDO) inhibitors is pursued as an anticancer strategy.

Methods: The intracellular level of tryptophan and kynurenine was detected by mass spectrum analysis. The effect of tryptophan and IDO inhibitors on cell surface programmed cell death protein 1 (PD-1) level were measured by flow cytometry. A set of biochemical analyses were used to figure out the underlying mechanism. In vitro co-culture system, syngeneic mouse models, immunofluorescent staining, and flow cytometry analysis were employed to investigate the role of tryptophan and IDO inhibitor in regulating the cytotoxicity of CD8⁺ T cells.

Results: Here, we reported that IDO inhibitors activated CD8⁺ T cells also by accumulating tryptophan that downregulated PD-1. Tryptophan and IDO inhibitors administration, both increased intracellular tryptophan, and tryptophanyl-tRNA synthetase (WARS) overexpression decreased Jurkat and mice CD8⁺ T cell surface PD-1. Mechanistically, WARS tryptophanylated lysine 1136 of and activated E3 ligase TRIP12 to degrade NFATc1, a PD-1 transcription activator. SIRT1 de-tryptophanylated TRIP12 and reversed the effects of tryptophan and WARS on PD-1. Tryptophan or IDO inhibitors potentiated CD8⁺ T cells to induce apoptosis of co-cultured cancer cells, increased cancer-infiltrating CD8⁺ T cells and slowed down tumor growth of lung cancer in mice.

Conclusions: Our results revealed the immune-activating efficacy of tryptophan, and suggested tryptophan supplemental may benefit IDO inhibitors and PD-1 blockade during anticancer treatments.

Keywords: CD8-positive T-lymphocytes; immunotherapy; indoleamine 2,3-dioxygenase.

Figure 1

Tryptophan tryptophanyl-tRNA synthetase (WARS)-dependently downregulated programmed cell death protein 1 (PD-1) expression. (A) Me-Trp administration increases intracellular tryptophan. Anti-CD3 and anti-CD28 antibodies co-stimulated Jurkat T cells were supplemented with 73.5 μM L-methyl-Trp in the culture media. The levels of intracellular tryptophan were detected by liquid chromatography-mass spectrometry. n=3, mean±SD. Knockdown WARS abrogated cell surface PD-1 regulation by Me-Trp. The cell surface PD-1 levels were detected by mean fluorescence intensity (MFI, herein after) and percentage of PD-1⁺ cells in Jurkat cells and Jurkat cells with WARS knocked down by small interfering RNAs (siRNAs) when Me-Trp supplemental (B) or tryptophan-deprivation (C) were applied to cells. All levels were compared with that of Jurkat cells without treatment. n=3, mean±SD. Silencing NFATc1 diminished WARS ability to alter cell surface PD-1. The PD-1 expression levels were detected in Jurkat T cells and NFATc1 knockdown Jurkat T cells under either WARS overexpression (D) or WARS knockdown (E). MFI levels and PD-1-positive cell levels were calculated and compared with that of the controls. n=3, mean±SD. Pyrophosphate (ppi) inhibited Me-Trp supplemental and tryptophan-starvation to alter PD-1. The PD-1 expression levels were detected in stimulated Jurkat T cells that were cultured in media supplemented with Me-Trp (F) or with tryptophan deprived (G) under presence or absence of ppi (10 mM final concentration) in the culture media. n=3, mean±SD. (H) Me-Trp administration decreased mouse CD8⁺ T cells surface PD-1. Mouse-naïve CD8⁺ T cells were isolated and activated by anti-CD3/CD28 antibodies (5 µg/mL). The CD8⁺ T cells surface PD-1 expression levels were detected when they were cultured in media with indicated Me-Trp levels. n=3, mean±SD. *P<0.05, **p<0.01, ***p<0.001, two-tailed Student’s t-test.

Figure 2

Indoleamine 2,3-dioxygenase (IDO) inhibitors decreases programmed cell death protein 1 (PD-1) through accumulating intracellular tryptophan. (A) IDO inhibitors altered intracellular levels of both tryptophan and kynurenine. Intracellular levels of tryptophan and kynurenine of Jurkat T cells and IDO inhibitors-treated Jurkat T cells were detected by liquid chromatography-mass spectrometry. The final concentrations of IDO inhibitors for indoximod (Ind) were: 0, 10, 50 µM; for epacadostat (Epa) were: 0, 1, 3 µM; and for NLG919 analog (NLG) were: 0, 0.1, 1 µM, respectively. (B) NFATc1 knockdown prevented IDO inhibitors to decrease PD-1. Cell surface PD-1 mean fluorescence intensity (MFI) and PD-1-positive levels were measured for Jurkat T cells and NFATc1 knockdown Jurkat T cells that were cultured with presence of indicated levels IDO inhibitors in the culture media. The final concentrations of IDO inhibitors for indoximod (Ind) were: 0, 1, 10, 50 µM; for epacadostat (Epa) were: 0, 0.3, 1, 3 µM; and for NLG919 analog (NLG) were: 0, 0.025, 0.1, 1 µM, respectively (herein after for all dose-dependent experiments of IDO inhibitors in Jurkat T cells). (C) Kynurenine did not affect surface PD-1 expression. Relative cell surface PD-1 MFI levels were measured for Jurkat T cells that were cultured in media supplemented with indicated levels of kynurenine. (D) IDO inhibitors decreased PD-1 in mouse CD8⁺ T cells. Relative cell surface PD-1 MFI levels and PD-1-positive levels were measured for mouse CD8⁺ T cells that were cultured with presence of different levels IDO inhibitors in the culture media. The final concentrations of IDO inhibitors for indoximod (Ind) were: 0, 1, 10, 50 µM; for epacadostat (Epa) were: 0, 0.3, 1, 3 µM; and for NLG919 analog (NLG) were: 0, 0.025, 0.1, 0.2 µM, respectively (herein after for all dose-dependent experiments of IDO inhibitors in mouse CD8⁺ T cells). IDO inhibitors increased interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α level in T cells. The IL-2, IFN-γ, and TNF-α levels were detected in stimulated Jurkat T cells, IDO inhibitors-treated Jurkat T cells by flow cytometry (E), in mice CD8⁺ T cells, and IDO inhibitors-treated mice CD8⁺ T cells by ELISA (F). *P<0.05, **p<0.01, ***p<0.001; ns, not significant.

Figure 3

Tryptophan and tryptophanyl-tRNA synthetase (WARS) downregulates programmed cell death protein 1 (PD-1) transcription factor NFATc1. (A) Me-Trp and indoleamine 2,3-dioxygenase (IDO) inhibitors decreased the messenger RNA (mRNA) level of PD-1 in Jurkat T cells. The PD-1 mRNA levels of anti-CD3 and anti-CD28 antibodies co-stimulated Jurkat T cells were detected with reverse transcription quantitative PCR (RT-qPCR) when the cells were treated with different levels of indoximod, epacadostat, NLG919 analog, or Me-Trp. Me-Trp supplemental and WARS overexpression decreased the protein level of NFATc1. The protein (B) and mRNA (C) levels of NFATc1 were detected for activated Jurkat T cells that were cultured in media supplemented with indicated levels of Me-Trp. n = 3, mean±SD. Tryptophan starvation increased protein levels of NFATc1. The protein (D) and mRNA (E) levels of NFATc1 were detected at indicated time for activated Jurkat T cells that were cultured in tryptophan-deprived media. n=3, mean±SD. WARS overexpression decreased protein levels of NFATc1. The protein (F) and mRNA (G) levels of NFATc1 were detected in both activated Jurkat T cells and WARS-overexpression activated Jurkat T cells. WARS knockdown increased protein levels of NFATc1. The protein (H) and mRNA (I) levels of NFATc1 were detected in both activated Jurkat T cells and WARS-knockdown activated Jurkat T cells. (J) NFATc1 knockdown abrogated Me-Trp to decrease the cell surface PD-1. The MFI and PD-1-positive Jurkat T cells were detected in Jurkat and NFATc1 knockdown Jurkat cells that were cultured under different levels of Me-Trp. *P<0.05, **p<0.01, ***p<0.001; ns, not significant.

Figure 4

NFATc1 degradation is catalyzed by E3 ligase TRIP12. (A) Proteasome inhibitor MG-132 increased NFATc1 protein levels. The NFATc1 protein levels were detected in Jurkat cells and MG132-tretaed (20 µM) Jurkat cells. (B) Lysine 1136 of TRIP12 was tryptophanylated. A MS/MS spectrum from human liver protein library matched a tryptophanylated TRIP12 peptide (DTISNNREK_TrpIK). (C, D) TRIP12 interacted with NFATc1. TRIP12 was co-overexpressed with NFATc1 in HEK293T cells. Co-immunoprecipitation experiments were carried out when Flag-tagged NFATc1 or TRIP12 was used as bait. (E) Knockdown TRIP12 prolonged the half-life of endogenous NFATc1 protein. The protein levels of NFATc1 were measured for Jurkat T cells and TRIP12 knockdown Jurkat T cells at different time points after cycloheximide (CHX) treatments to block protein synthesis. Cells were transfected with siTRIP12 for 48 hours, followed by 50 µg/mL CHX treatment for 1, 2, and 4 hours. Relative NFATc1 protein levels were quantified against those of the untreated cells. (F) TRIP12 knockout decreased ubiquitination of ectopically expressed NFATc1. The ubiquitination levels of overexpressed NFATc1 were detected in HEK293T cells and TRIP12 knockout HEK293T cells. (G) TRIP12 knockdown increased endogenous NFATc1 protein levels. The protein levels of endogenous NFATc1 were detected in HEK293T cells and TRIP12 knockdown Jurkat T cells. (H) Impairing TRIP12 catalytic activity abrogated its ability to ubiquitinate NFATc1. NFATc1 was overexpressed alone, or co-overexpressed with TRIP12 or TRIP12 lacking HECT domain (TRIP12-ΔHECT) in HEK293T cells, and ubiquitination levels of NFATc1 were detected. (I) TRIP12 knockdown abrogated Me-Trp to decrease the cell surface programmed cell death protein 1 (PD-1). The mean fluorescence intensity (MFI) and PD-1positive cells were detected in Jurkat T cells and TRIP12 knockdown Jurkat T cells when Me-Trp supplemental was applied to cells. n=3, mean±SD. Silencing TRIP12 elevated cell PD-1 messenger RNA (mRNA) and surface PD-1 protein. The PD-1 mRNA levels (J) and the MFI and PD-1-positive cells (K) were detected in Jurkat T cells and TRIP12 knockdown Jurkat T cells. *P<0.05, **p<0.01, ***p<0.001.

Figure 5

Tryptophanylation activates the E3 ligase activity of TRIP12. (A) Tryptophanyl-tRNA synthetase (WARS) increased TRIP12, but not TRIP12^K1136R tryptophanylation. Myc-tagged TRIP12 or TRIP12^K1136R was expressed alone or co-overexpressed with WARS-HA in HEK293T cells. The tryptophanylation levels of anti-myc beads purified TRIP12 and TRIP12^K1136R were examined. (B) WARS knockout decreased NFATc1 ubiquitination levels. NFATc1 was overexpressed alone or co-overexpressed with TRIP12 in WT or WARS-KO HEK293T cells. The ubiquitination levels of purified NFATc1 were detected. (C) WARS knockout increased endogenous NFATc1 levels. The NFATc1 levels in HEK293T cells and WARS-KO HEK293T cells were detected. Ablating TRIP12 tryptophanylation site abrogated Me-Trp supplemental or WARS overexpression to decrease NFATc1. NFATc1 levels were compared between HEK293T cells and TRIP12^K1136R knockin HEK293T cells that were subject to Me-Trp supplemental (D) or WARS overexpression (E). (F) Tryptophanylation mimetic TRIP12^K1136W had higher affinity to NFATc1 than wild-type TRIP12. Myc-tagged TRIP12 or TRIP12^K1136W was each co-overexpressed with NFATc1-flag in HEK293T cells. The amount of TRIP12 or TRIP12^K1136W that was co-immunoprecipitated with NFATc1 were detected. (G) Tryptophan altered the affinity between TRIP12 and WARS. FLAG-tagged TRIP12 was co-overexpressed with WARS-HA in HEK293T cells that were cultured with or without Me-Trp supplementation. The amount of WARS that was co-precipitated with TRIP12 was detected. (H) Tryptophan altered the affinity between TRIP12 and NFATc1. Flag-tagged NFATc1 was co-overexpressed with TRIP12-myc in HEK293T cells that were cultured with or without tryptophan starvation. The amount of TRIP12 that was co-precipitated with NFATc1 was detected. Tryptophan regulated the ubiquitination levels of NFATc1. NFATc1 was co-overexpressed with TRIP12 in HEK293T cells. The ubiquitination levels of NFATc1 from Me-Trp supplemented (I) and tryptophan starved (J) cells were compared with those of untreated cells. (K) WARS knockdown decreased the affinity between TRIP12 and NFATc1. Myc-tagged TRIP12 was co-overexpressed with NFATc1-Flag in HEK293T cells and in HEK293T cells with WARS knockdown. The amount of TRIP12 co-immunoprecipitated with NFATc1 was detected. (L) WARS knockdown decreased ubiquitination of NFATc1. NFATc1 was overexpressed alone or co-overexpressed with TRIP12 in HEK293T cells and in WARS knockdown HEK293T cells. The ubiquitination levels of purified NFATc1 were detected. (M) Tryptophanylation site ablation abrogated WARS to enhance TRIP12-NFATc1 interaction. The amount of Myc-tagged TRIP12 co-precipitated with NFATc1-flag was detected when they were co-overexpressed in WARS-KO HEK293T cells that were with or without WARS overexpression. Quantitation of the western blot analysis was shown. (N) WARS overexpression increased ubiquitination of NFATc1. NFATc1 was overexpressed alone or co-overexpressed with TRIP12 in WARS-KO HEK293T cells in the presence or absence of WARS overexpression. Ubiquitination levels of NFATc1 were compared.

Figure 6

SIRT1 reverses TRIP12 K1136 tryptophanylation. Nicotinamide (NAM) decreased cell surface programmed cell death protein 1 (PD-1) and PD-1 messenger RNA (mRNA) of Jurkat cells. Activated Jurkat T cells were treated with 5 mM NAM for 6 hours. PD-1 mean fluorescence intensity (MFI) and PD-1 positive cells were measured by flow cytometry (A). PD-1 mRNA was examined by reverse transcription quantitative PCR (RT-qPCR) (B). (C) NAM decreased mouse CD8⁺ T cells surface PD-1. Naïve CD8⁺ T cells were isolated from the spleen of BALB/c wild-type (WT) mouse and stimulated with anti-CD3/CD28 antibodies. PD-1 MFI and PD-1 positive cells were compared between NAM-untreated and NAM-treated cells. (D) TRIP12-interacting SIRTs screening. HA-tagged SIRTs was individually co-expressed with TRIP12-flag in HEK293T cells. Interaction between TRIP12 and SIRTs was assayed by co-immunoprecipitation. (E) SIRT1 decreased the tryptophanylation of isolated TRIP12. HA-tagged SIRT1, SIRT2, SITR6, and SIRT7 were individually co-expressed with TRIP12-flag in HEK293T cells and the tryptophanylation levels of TRIP12 purified from each SIRT-expression cell were detected. (F) SIRT1 increased cell surface PD-1 expression. HA-tagged SIRT1, SIRT6, and SIRT7 were individually overexpressed in Jurkat T cells, relative PD-1 MFI and percentage of PD-1⁺ cells were measured. (G) SIRT1 ablation in mice prevented NAM to decrease surface PD-1 expression. Naïve CD8⁺ T cells were isolated from the spleen of WT or SIRT1^−/− C57BL/6 mice and activiated. Relative PD-1 MFI and percentage of PD-1⁺ cells were compared between NAM-treated and NAM-untreated mouse CD8⁺ T cells. *P<0.05, ***p<0.001; ns, not significant.

Figure 7

Increased tryptophanylation potentiates T cells to eliminate cancer cells. Tryptamine increased cell surface PD-1 and PD-1 mRNA in Jurkat cells. Relative PD-1 MFI and percentage of PD-1⁺ cells (A) and PD-1 mRNA (B) were measured in Jurkat T cells and tryptamine-treated Jurkat T cells. n=3, mean ± SD. (C) Tryptamine increased cell surface PD-1 in mouse CD8⁺ T cells. Naïve CD8⁺ T cells were isolated from the spleen of BALB/c WT mouse and activated with anti-CD3/CD28 antibodies. Relative PD-1 MFI and percentage of PD-1⁺ cells were measured in cells treated with tryptamine as indicated. n=3, mean±SD. Tryptophan dose-dependently increased the cytotoxicity of CD8⁺ T cells. CD8⁺ T cells were isolated from OVA-immuned BALB/c mice by flow cytometry.The apoptotic rate of LLC cells (D) and CT26 cells (E) that were co-cultured with CD8⁺ T cells (CD8⁺ T cell: LLC/CT26=10:1) in media contained different levels of Me-Trp were measured. n=3, mean±SD. Intraperitoneal dose of Trp and/or indoximod elicited regression of tumor growth. Mice inoculated with the same number of LLC cells were treated by intraperitoneal dose of Me-Trp or indoximod, both Me-Trp and indoximod, or PBS (control), the growth of tumors volume (F) and the tumor sizes and weights of sacrificed mice (G) were shown. n=10, mean±SD. Intraperitoneal dose of Trp and/or indoximod decreased the PD-1 levels. Spleen CD8⁺ T cells (H) and CD8⁺ T cells of tumor-infiltrating lymphocytes (TILs, I) were isolated from PBS, intraperitoneal dose of Me-Trp (Me-Trp), indoximod, and both Me-Trp and indoximod mice groups and analyzed on day 22. Relative MFI of surface PD-1 and percentage of PD-1⁺ cells were measured by flow cytometry. See also online supplemental Figure 7G-H for the sorting of CD8⁺ T cells. n=10, mean±SD. (J) Intraperitoneal dose of Trp or indoximod increased tumor-infiltrating CD8⁺ T cells. Confocal microscopy images of CD4 (red) or CD8 (red) staining on mouse tumor sections from PBS, intraperitoneal dose of Trp (Me-Trp), indoximod, and both Me-Trp and indoximod mice groups. DAPI (blue) showed the staining of nuclear. The density per volume of the CD4 or CD8-positive cells in the tumor sections corresponding to p value was calculated. Scale bar 50 µm, n=30 (three images randomly acquired from each mouse, and ten mice each group were included), mean±SD. (K) Intraperitoneal dose of Trp and/or anti-PD-1 elicited regression of tumor growth. Mice inoculated with B16F10 cells were treated by intraperitoneal dose of Me-Trp or anti-PD-1, both Me-Trp and anti-PD-1, or PBS (control), the tumor sizes and weights of sacrificed mice were shown. n=10, mean±SD. Intraperitoneal dose of Trp or Trp combining anti-PD-1 decreased the PD-1 levels. Spleen CD8⁺ T cells (L) and CD8⁺ T cells of tumor-infiltrating lymphocytes (TILs, M) were isolated from PBS, intraperitoneal dose of Me-Trp (Me-Trp), anti-PD-1, and Me-Trp and anti-PD-1 combined mice groups and analyzed on day 14. Relative MFI of surface PD-1 and percentage of PD-1⁺ cells were measured by flow cytometry. n=10, mean±SD.