L-Arginine Modulates T Cell Metabolism and Enhances Survival and Anti-tumor Activity

Abstract

Metabolic activity is intimately linked to T cell fate and function. Using high-resolution mass spectrometry, we generated dynamic metabolome and proteome profiles of human primary naive T cells following activation. We discovered critical changes in the arginine metabolism that led to a drop in intracellular L-arginine concentration. Elevating L-arginine levels induced global metabolic changes including a shift from glycolysis to oxidative phosphorylation in activated T cells and promoted the generation of central memory-like cells endowed with higher survival capacity and, in a mouse model, anti-tumor activity. Proteome-wide probing of structural alterations, validated by the analysis of knockout T cell clones, identified three transcriptional regulators (BAZ1B, PSIP1, and TSN) that sensed L-arginine levels and promoted T cell survival. Thus, intracellular L-arginine concentrations directly impact the metabolic fitness and survival capacity of T cells that are crucial for anti-tumor responses.

Keywords: L-arginine; LiP-MS; T cell; T cell survival; cancer immunotherapy; metabolism; metabolite sensing; metabolome; proteome.

Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract

Figure 1

Metabolic and Proteomic Profiling Reveals Distinct Changes in L-Arginine Metabolism in Activated Human T Cells (A) Schematic view of the experimental approach. (B) Comparison of protein abundances between 72-hr-activated (CD3 + CD28 antibodies) and freshly isolated non-activated human naive CD4+ T cells. Closed circles indicate proteins that changed significantly (FDR = 0.05, S0 = 1). Colored dots are enzymes of the arginine and proline metabolism that changed significantly. (C) Comparison of metabolite abundances in 72 hr-activated and freshly isolated non-activated human naive CD4+ T cells. Closed circles indicate metabolites that changed significantly (|Log2 fc| > 1, p < 0.01). Colored dots are metabolites of the arginine and proline metabolism that changed significantly. Similar changes were observed when 72 hr-activated CD4+ T cells were compared with naive CD4+ T cells cultured overnight in the absence of TCR stimulation. See also Figure S1 and Tables S1, S2, and S3.

Figure 2

L-Arginine Is Rapidly Metabolized upon Activation (A) Intracellular abundance of L-arginine in non-activated (non-act) and activated naive CD4+ T cells (CD3 + CD28 antibodies). Boxplot, n = 30 from three donors, each in a different color. (B) Kinetics of 3H-L-arginine uptake during a 15-min pulse. Box plot, n = 5 from three donors. (C) Uptake, proteome incorporation and intracellular abundance of the indicated amino acids. Barplot (left): 5 × 104 cells were activated for 4 days and consumption of amino acids from medium was analyzed. Essential amino acids are in gray; n = 4 from four donors, error bars represent SEM. Barplot (center): proteome incorporation of amino acids estimated from the copy numbers of each protein. Heat map (right): intracellular amino acid abundance relative to naive T cells over time as determined by mass spectrometry (MS) n = 30 from three donors. Leucine and isoleucine could not be distinguished as they have the same mass. (D) Changes in the abundance of metabolites and proteins of the arginine and proline metabolism between non-activated and 72 hr-activated CD4+ T cells. Log2 fold changes of proteins and metabolites are color-coded. Significant changes are in bold (FDR = 0.05, S0 = 1 for proteins; and p < 0.05 [two-tailed unpaired Student’s t test], |Log2 fc| > 1 for metabolites). Black dots are metabolites that were not detected by MS. Only enzymes that were detected by MS are shown. (E) Metabolic tracing of L-arginine. Ninety-six hour-activated T cells were pulsed with 13C6-L-arginine and the metabolic fate was analyzed by LC-MS/MS at different time points. AFL, apparent fractional labeling; n = 4 from two donors. 13C Citrulline was not detected. Error bars represent SEM. For (A) and (B), upper whisker = min(max(x), Q_3 + 1.5 ∗ IQR) and lower whisker = max(min(x), Q_1 – 1.5 ∗ IQR).

Figure 3

L-Arginine Globally Influences Metabolism of Activated Human T Cells (A) Human naive CD4+ T cells were activated in control medium (Ctrl) or in medium supplemented with 3 mM L-arginine (L-Arg) or 3 mM L-ornithine (L-Orn) and harvested at different time points. The heat map shows the difference between the abundance of metabolites in T cells cultured in L-Arg or L-Orn-medium and controls. Shown are only metabolites with a Log2 fc > 1 and an adjusted p value of < 0.05; n = 12 from two donors. (B) Differential analysis of the glycolytic pathway between naive CD4+ T cells cultured in L-Arg medium or Ctrl medium, 96 hr after activation. Log2 fold changes of proteins and metabolites are color-coded. Proteins or metabolites whose abundance changed significantly are in bold (for proteins FDR = 0.005, S0 = 5, |Log2 fc| > 1 and for metabolites p < 0.05 (Student’s t test), |Log2 fc| > 1). 3-P-glycerate and 2-P-glycerate could not be distinguished as they have the same mass. (C) Seventy-two hour-activated T cells were plated in fresh medium and glucose consumption was determined enzymatically after 24 hr; n = 9 from three donors. Error bars represent SEM. (D) Seahorse experiment performed with activated (96 hr) T cells from one donor. Oligomycin was injected after 56 min, FCCP after 96 min, and antimycin (to inhibit the respiratory chain) after 136 min. Data are representative of five independent experiments with different donors; n = 4. Error bars represent SEM. (E and F) Relative oxygen consumption rate (OCR) (E) and relative spare respiratory capacity (SRC) (F) of activated (96 hr) T cells; n = 12 from three donors. ∗∗∗∗p < 0.0001 (Student’s t test). Error bars represent SEM. See also Figure S2 and Table S4.

Figure 4

L-Arginine Limits Human T Cell Differentiation and Endows Cells with a High Survival Capacity In Vitro (A and B) Human naive CD4+ T cells were activated in L-Arg medium or Ctrl medium in the presence of 10 ng/mL IL-12. IFN-γ was quantified in culture supernatants after 5 days (A) or after re-activation for 5 hr with PMA/ionomycin (B); n = 9 from three donors. (C) Naive CD4+ T cells were labeled with CellTrace Violet (CTV) and activated in L-Arg medium or Ctrl medium. On day 10, proliferating CTVlo T cells were stained with an antibody to CCR7 and analyzed by flow cytometry; n = 15 from three donors. (D) Naive CD4+ T cells were activated for 5 days in L-Arg or Ctrl medium in the presence of exogenous IL-2, washed extensively, and cultured in Ctrl medium in the absence of IL-2. Shown is the percentage of living T cells as determined by Annexin V staining at different time points after IL-2 withdrawal. One representative experiment out of three performed. (E) Same experiment as in (D). Shown is the difference of living activated CD4+ and CD8+ T cells 5 days after withdrawal of IL-2; n = 46, from 16 donors (CD4+ T cells); n = 13, from four donors (CD8+ T cells). (F) Difference of living activated CD4+ T cells 5 days after IL-2 withdrawal. Naive CD4+ T cells were activated and L-Arg (3 mM) was added to the culture medium at the indicated time points; n = 12 from four donors. (G) Difference of living activated CD4+ T cells 5 days after IL-2 withdrawal. Naive CD4+ T cells were activated in Ctrl medium or medium supplemented with the indicated metabolites (3 mM, except for spermidine 0.1 mM). Ctrl, n = 21; D-Arg, n = 9; L-lysine, n = 18; L-Arg-HCl, n = 10; L-Arg + L-Lys, n = 12; L-Orn, n = 20; L-Cit, L-Pro, n = 12; urea, creatine, agmatine, n = 6; putrescine, n = 18; spermidine, n = 8, from at least three donors. (H) Difference of living activated CD4+ T cells 5 days after IL-2 withdrawal. Naive CD4+ T cells were activated in the presence or absence of nitric oxide synthase inhibitors dimethylarginine (DiMeArg) or L-NG-nitroarginine methyl ester (L-NAME), both used at 1 mM. Ctrl and L-Arg, n = 26; DiMeArg and L-NAME, n = 16; DiMeArg + L-Arg and L-NAME + L-Arg, n = 12, from at least three donors. (I) Difference of living activated CD4+ T cells 5 days after IL-2 withdrawal. Naive CD4+ T cells were activated in absence (Ctrl) or presence of the arginase inhibitors Nω-Hydroxy-nor-L-arginine (norNOHA, 300 μM) or S-(2-boronoethyl)-L-cysteine (BEC, 300 μM); n = 21, from seven donors. (J) Same as in (I) but cultures were performed in medium containing 150 μM L-arginine. (K) Effect of norNOHA and BEC on proliferation of CTV-labeled naive T cells measured 72 hr after activation. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (Student’s t test). (A–J) Error bars represent SEM throughout. See also Figures S3 and S4.

Figure 5

Increased Intracellular L-Arginine Levels Endow Mouse T Cells with a High Survival Capacity In Vitro and In Vivo (A) BALB/c CD90.1+ CD4+ TCR transgenic T cells specific for the influenza HA110–119 peptide were adoptively transferred into CD90.2+ host mice that were then immunized subcutaneously (s.c.) with HA110–119 in complete Freund’s adjuvant (CFA). Mice were fed with L-arginine-HCl (1.5 mg/g body weight) or PBS, administrated daily starting 1 day before immunization. Fifteen days later, the amount of CD44hi CD90.1+ CD4+ TCR transgenic T cells in draining lymph nodes was measured by fluorescence-activated cell sorting (FACS) analysis; n = 9 from two independent experiments. (B and C) In vitro T cell survival experiment with C57BL/6 wild-type (WT) or Arg2–/– T cells. Naive CD62Lhi CD44lo CD4+ T cells and CD8+ T cells were activated for 4 days in L-Arg or Ctrl medium in the absence or presence of the arginase inhibitor norNOHA (500 μM). On day 2 exogenous IL-2 was added to the cultures, on day 4 cells were washed extensively and cultured in medium without IL-2. Shown is the difference in the percentage of living CD4+ (B) and CD8+ (C) T cells relative to WT T cells as determined by Annexin V staining 2 days after IL-2 withdrawal. WT, n = 6-19; WT norNOHA, n = 6–8; Arg2–/–, n = 4–6; Arg2–/– norNOHA, n = 4. (D) Equal numbers of CD45.1+ WT and CD45.2+ CD90.2+Arg2–/– naive CD8+ T cells were transferred into CD45.2+ CD90.1+ host mice. Mice were immunized with the OVA257–264 peptide in CFA. Fifteen days after immunization, the amount of OVA257–264-specific CD44hi CD8+ T cells was measured in draining lymph nodes by flow cytometry using OVA257–264/H-2Kb multimers; n = 4. One representative experiment out of two performed. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (Student’s t test). Error bars represent SEM throughout. See also Figure S5.

Figure 6

BAZ1B, PSIP1, and TSN Mediate the L-Arginine-Dependent Reprogramming of T Cells toward Increased Survival Capacity (A) Scheme of the limited proteolysis workflow. (B) Proteins that experience a structural change in response to 1 mM L-arginine but not to 1 mM D-arginine or L-ornithine. Transcriptional regulators are in orange, proteins are grouped according to their functions. Known interactions are indicated based on http://string-db.org/ and http://www.genemania.org/. (C) Survival experiment with human CD4+ T cell clones devoid of the indicated proteins. Control (Ctrl), n = 39; Cas9-transduced control (Cas9 Ctrl), n = 45; BAZ1B-KO, PSIP1-KO, and PTPN6-KO, n = 46, n = 9, and n = 29, respectively. Each T cell clone was analyzed in triplicate. Bars represent the mean ± SEM. (D) Same as in (C). Cas9 Ctrl, n = 20; TSN-KO and B2M-KO, n = 23 and n = 3, respectively. (E) Percentage of living cells after IL-2 withdrawal of T cells cultured in Ctrl medium. Ctrl, n = 39; Cas9 Ctrl, n = 45; BAZ1B-KO, PSIP1-KO, and TSN-KO, n = 46, n = 9, and n = 29, respectively. (F–I) Western blots or FACS analysis of T cell clones showing deletion of target proteins. C refers to Cas9 Ctrl clones. Unspecific bands are marked with asterisk. An antibody to tubulin (Tub) was used as a loading control. B2M-KO was verified by staining cells with an antibody against MHC-I. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (Student’s t test). (C–E) Error bars represent SEM throughout. See also Figure S6 and Table S5.

Figure 7

CD8+ T Cells with Increased L-Arginine Levels Display Improved Anti-tumor Activity In Vivo (A) Survival of activated mouse CD8+ OT-I T cells (4 days) after IL-2 withdrawal. Data points represent the difference between the percentage of living T cells from cultures performed in L-Arg medium or Ctrl medium; n = 11. (B) CD90.1+ CD45.1/2+ and CD90.1+ CD45.1+ naive CD8+ OT-I T cells were activated for 4 days in Ctrl medium or L-Arg medium, respectively. Equal numbers of the congenically marked activated OT-I cells were co-transferred into Cd3e–/– mouse and the number of living T cells was measured in pooled spleen and lymph nodes at the indicated time points; n = 3 at each time point. (C) Naive CD8+ OT-I T cells were activated with CD3 + CD28 antibodies in L-Arg medium or Ctrl medium. Five days after activation, the percentage of Tcm-like cells (CD44hi, CD62L+) was measured by flow cytometry; n = 15. (D) Naive OT-I CD8+ T cells were activated in L-Arg medium or Ctrl medium and IFN-γ was quantified in culture supernatants after 5 days; n = 15. (E) Same as in (D) but T cells were re-activated on day 5 day with PMA/Ionomycin; n = 15. (F and G) B16.OVA melanoma cells were injected into C57BL/6 mice and tumors were allowed to grow for 10 days. Naive OT-I CD8+ T cells were activated in vitro in L-Arg medium or Ctrl medium and injected into tumor bearing mice. Tumor burden (F) and survival (G) were assessed over time. Data are representative of three independent experiments, each performed with seven to nine mice per group. (H) B16.OVA melanoma cells were injected into C57BL/6 mice and tumors were allowed to grow for 6 days. At day 6, naive CD8+ OT-I T cells were transferred into tumor bearing mice and at day 7 mice were immunized with OVA peptide. Starting one day before the T cell transfer, PBS or L-arginine (1.5 mg/g body weight) was orally administered daily; n = 19 from three independent experiments. Bars represent the SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (Student’s t test). In (G), ∗p < 0.05 as determined by log-rank test comparison between curves. Error bars represent SEM throughout.

Figure S1

Quality Control of the Proteome Dataset, Related to Figure 1 (A) Sorting of human naive CD4+ T cells. Shown are FACS plots of cells after enrichment with anti-CD4 magnetic beads. Cells were sorted as CD4+ CCR7+ CD45RA+ and CD8–CD25–. (B) Expression kinetics of indicated marker proteins. Bars represent the SEM of data from different donors, n = 7 (for resting cells), n = 3 (for 12h, 72h), n = 2 (for 96h, 48h), n = 1 (for 24h). CD25 and CD8 were not identified in resting cells. After activation, expression of CD25 increased whereas CD8 was never detected. (C) Identified protein groups per condition. Taking all conditions together, a total of 9,718 proteins were identified. Per condition two numbers are indicated; the higher number indicates the total identifications and the lower number the mean of the single shots. Samples in blue were measured on a different instrument than samples in black. L-arg refers to 3 mM L-arginine. (D) Relative protein abundance over time shown as a heat map. Log2 fold changes (FC) are relative to naive resting T cells. The marker for proliferating cells Ki-67 increased abruptly after 48h, when cells started to proliferate. CD40L expression increased immediately after activation and then decreased to initial levels. A similar expression pattern was observed for CD69, which inhibits egress from lymph nodes (Shiow et al., 2006). The expression of integrins α4 and β7 increased at later time points. (E) Copy numbers of individual subunits of well-characterized protein complexes were plotted against each other. As the Sec23 subfamily includes Sec23A and Sec23B, their copy numbers were added up. The same was done for the subfamily members of Sec24 (A-D). (F) Copy numbers of components of the nuclear pore complex (NPC). The stoichiometry of subunits measured using targeted quantitative proteomics (Ori et al., 2013) is indicated on the graph in red. Shown are copy numbers measured in naive resting T cells from seven donors. (G) Same as in (F) but shown are copy numbers measured from activated cells (72h). n = 3 from three donors. Note that the numbers of Nup107 increased from 11,464 ± 1620 to 53,091 ± 1471. (A and E–G) Error bars represent SEM throughout.

Figure S2

Impact of L-citrulline on Metabolism, Related to Figure 3 (A) Human naive CD4+ T cells were activated in normal medium or in L-Arg medium. Nitric oxide formation was measured using DAF-FM diacetate at different time points. (B) T cells were activated in control medium (Ctrl, containing 1mM L-arginine), or in medium supplemented with 3mM L-arginine (L-Arg) or 3mM L-citrulline (L-Cit) and harvested at different time points. The heat map shows the difference in the abundance of metabolites in T cells cultured in L-Arg- or L-Cit-medium compared to controls. Shown are only metabolites with a log2 fold change > 1 and an adjusted p value of 

Figure S3

L-Arginine Delays the Onset of Proliferation, Related to Figure 4 (A) Kinetics of T cell proliferation. Human naive CD4+ T cells were labeled with CellTraceViolet (CTV) and activated in Ctrl medium or in L-Arg medium or in medium supplemented with 3 mM D-arginine or 3 mM L-arginine together with 3 mM L-lysine. Cell divisions were monitored at 48h and 72h by flow cytometry. (B) CTV-labeled CD4+ T cells were activated in normal medium or L-Arg medium and the dilution of CTV was measured over time by flow cytometry. n = 5 from two donors. (C) 3H-L-arginine uptake by 3 day-activated CD4+ T cells during a 15 min pulse. Where indicated, 3 mM L-arginine, D-arginine or L-lysine was added to the culture medium as a competitive uptake inhibitor. n = 7 for control, n = 9 for L-Arg, n = 5 for D-Arg, and n = 9 for L-Lys. Error bars represent SEM throughout.

Figure S4

L-Arginine Increases the Survival of Activated T Cells Independent of mTOR Signaling, Related to Figure 4 (A) Human naive CD4+ T cells were activated for 4 days, lysed and the phosphorylation levels of S6K1 (pThr389) and 4E-BP (pThr37/46) were analyzed by western blot. Rapamycin inhibited the phosphorylation of the mTOR targets, while DMSO or supplementation of the culture medium with 3 mM L-arginine had no effect. T cells hardly proliferated upon activation in culture medium containing no or 20 μM L-lysine and therefore phosphorylation of the target proteins could not be assessed. (B) T cell survival experiment. Human naive CD4+ T cells were activated in Ctrl medium or in medium containing 100 nM rapamycin. On day 5, cells were washed to withdraw IL-2 and cell survival was measured at different time points. (C) Same as in (B) but cell survival was only measured 5 days after IL-2 withdrawal. n = 7 from seven donors. Boxplot. Same as in Figures 2A and 2B. (D) Metabolic profiling of CD4+ T cells activated in medium containing 100 nM rapamycin. The heat map shows the difference of metabolite abundances between rapamycin-treated cells and controls. n = 10 from two donors.

Figure S5

Oral Administration of L-Arginine Increases L-Arginine Levels in Mouse Sera and T Cells, Related to Figure 5 (A) BALB/c mice were administered L-arginine (1.5 mg/g body weight) and sera were collected after 30 min. L-arginine and, as a control, L-threonine concentrations were analyzed on a MassTrak amino acid analyzer. n = 4. (B) BALB/c mice were immunized with ovalbumin in CFA. Sixty hours later, activated T cells from draining lymph nodes were enriched using magnetic beads coated with antibodies to CD44. Metabolites were extracted using hot 70% ethanol and L-arginine and L-glutamine levels (as an internal standard) were measured using LC-MS/MS. Shown is the ratio between L-arginine and L-glutamine intensities. n = 14. (C) Intracellular L-arginine levels of wild-type and Arg2–/– CD4+ and CD8+ T cells 4 days after activation. n = 3. For statistical tests, a two-tailed unpaired Student’s t test was used throughout, n.s. non significant; ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005; ∗∗∗∗p < 0.0001. Error bars represent SEM throughout.

Figure S6

L-arginine Upregulates Sirtuin-1, Related to Figure 6 (A) Copy numbers of Sirtuin-1 (SIRT1) as determined by quantitative MS in human naive CD4+ T cells activated in normal medium or L-Arg-medium. n = 3 from three donors. (B) T cell survival experiment. The Sirtuin-1 inhibitor Ex-527 was added at the time point of activation at a concentration of 5 μM. n = 16 from four donors. (C) T cell survival experiments with clones expressing Cas9 only, or clones devoid of Sirtuin-1. n = 16 from 6 clones. Right panel: western blot of two different Sirtuin-1 knockout clones generated with different sgRNAs. ∗ unspecific band. For statistical tests, a two-tailed unpaired Student’s t test was used throughout, n.s. non significant; ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005; ∗∗∗∗p < 0.0001. (B and C) Error bars represent SEM throughout.