Cystathionine-gamma-lyase overexpression in T cells enhances antitumor effect independently of cysteine autonomy

Abstract

T cells could be engineered to overcome the aberrant metabolic milieu of solid tumors and tip the balance in favor of a long-lasting clinical response. Here, we explored the therapeutic potential of stably overexpressing cystathionine-gamma-lyase (CTH, CSE, or cystathionase), a pivotal enzyme of the transsulfuration pathway, in antitumor CD8⁺ T cells with the initial aim to boost intrinsic cysteine metabolism. Using a mouse model of adoptive cell transfer (ACT), we found that CTH-expressing T cells showed a superior control of tumor growth compared to control T cells. However, contrary to our hypothesis, this effect was not associated with increased T cell expansion in vivo or proliferation rescue in the absence of cysteine/cystine in vitro. Rather than impacting methionine or cysteine, ACT with CTH overexpression unexpectedly reduced glycine, serine, and proline concentration within the tumor interstitial fluid. Interestingly, in vitro tumor cell growth was mostly impacted by the combination of serine/proline or serine/glycine deprivation. These results suggest that metabolic gene engineering of T cells could be further investigated to locally modulate amino acid availability within the tumor environment while avoiding systemic toxicity.

Keywords: T cell; adoptive cell transfer; amino acid; cysteine; metabolism.

FIGURE 1

Cystathionine gamma‐lyase (CTH) overexpression in T cells. A, Model positing the alteration of cysteine (Cys) metabolism and deprivation in the tumor environment as a major mechanism of T cell dysfunction. Dendritic cells (DC) generate Cys by importing and reducing cystine (Cys₂) or by converting methionine (Met) to Cys through the transsulfuration pathway. Regulatory T cells (Treg) suppress Cys accumulation in the extracellular compartment and interfere with glutathione (GSH)‐mediated redox remodeling by DCs. Myeloid‐derived suppressor cells (MDSC) sequester Cys₂ without returning Cys in the extracellular compartment. Tumor cells consume Cys for their metabolic needs. B, Comparison of amino acid concentrations measured by liquid chromatography‐tandem mass spectrometry between the plasma and the tumor interstitial fluid (TIF) of B16‐OVA melanoma‐bearing mice. *P < .05. C, Simplified view of the transsulfuration pathway. Dotted arrow represents multistep conversions. D, Retroviral contructs for the expression of the surface reporter protein Thy1.1 alone (control) or along with CTH under the PGK promoter. E, Representative flow cytometry analysis of Thy1.1 expression showing the efficacy of mouse CD8⁺ T cell transduction 2 days after infection (left panel) and quantification of seven independent experiments (right panel). F, RT‐qPCR analysis of Cth mRNA expression in mouse CD8⁺ T cells following Cth‐expressing retroviral infection as compared to endogenous expression in control T cells, spleen, and liver of naïve mice. G, Western blot analysis showing overexpression of CTH protein in Cth‐transduced T cells as compared to control T cells overexpressing CD2 or Thy1.1 alone. H, Flow cytometry analysis of the indicated markers. Ki‐67 and TOX expression was assessed following cell permeabilization. I, Flow cytometry analysis of γ‐interferon (IFNγ)⁺ and tumor necrosis factor‐α (TNFα)⁺ cells following restimulation and cell permeabilization. Data (means ± SD) are representative of two independent experiments

FIGURE 2

In vivo antitumor effect of infused cystathionine gamma‐lyase (CTH)‐expressing CD8⁺ T cells. A, Schematic representation and timeline of the preclinical model of antitumor T cell therapy. B, Conditioning regimen requirement for revealing a transient adoptive cell transfer (ACT) effect on tumor growth (mean area ± SEM). C, Rates of tumor growth represented over time in untreated mice (No ACT: total body irradiation [TBI] + interleukin‐2 [IL‐2] alone) or following ACT of OT‐I CD8⁺ T cells transduced with Thy1.1 alone (control) or along with CTH (n = 6‐8 mice per group). Data show one experiment representative of three independent experiments. Statistical difference between control and CTH ACT is shown using area under curve (AUC) comparison D, Relative tumor growth data normalized from three independent experiments (No ACT, n = 19; ACT control, n = 24; ACT CTH, n = 23) where the mean of tumor size of the No ACT group at day 9 was set at 100. E, Mouse survival in the indicated groups and pooled from three independent experiments

FIGURE 3

In vivo analysis of infused T cells and endogenous immune cells. Adoptive cell transfer (ACT) was carried out 14 (instead of 10) days after tumor cell inoculation to allow recovery and analysis by flow cytometry of significant numbers of cells in each tumor. Tumor and draining lymph nodes (dLN) were harvested 1 week later for analysis. A‐D, In vivo tracking and analysis of adoptively transferred CD45.1⁺ (OT‐1) T cells by flow cytometry. A, Thy1.1 expression shows control and cystathionine gamma‐lyase (CTH)‐expressing cells within the infused CD45.1⁺ (OT‐1) T cells. B, Quantification of total CD45.1⁺ or Thy1.1⁺ cells in tumors (cell numbers per milligram of tumor) and in the dLNs. Dots show values from individual mice (n = 5‐6 mice per group) and bars show means. C, Expression of the indicated markers among CD45.1⁺ Thy1.1⁺ cells. D, Expression of γ‐interferon (IFNγ), tumor necrosis factor‐α (TNFα), and granzyme B among CD45.1⁺ Thy1.1⁺ cells following in vitro restimulation with PMA‐ionomycin. Data are representative of two independent experiments. E‐H, In vivo analysis of endogenous T and myeloid cells. E, Representative flow cytometry analysis of endogenous hematopoietic CD45.1^– cells. F, Quantification of endogenous CD45⁺CD45.1^– cells in tumors (cell numbers per milligram of tumor) and in the dLNs. G, H, Percentages of endogenous T and myeloid cells as indicated by their respective markers. Data are representative of two independent experiments

FIGURE 4

Cystathionine gamma‐lyase (CTH) overexpression does not affect in vitro T‐cell cytotoxicity. A, Schematic representation of the cytotoxicity assay using transduced CD8⁺ OT‐1 T cells and B16‐OVA tumor cells as target cells. B, After 6 h of incubation of T cells with or without B16‐OVA cells (1:2.5 ratio, B16‐OVA:T), T cell phenotype was analyzed by flow cytometry using the indicated markers. TOX and granzyme B expression was assessed following cell permeabilization. Data (means ± SD) are representative of two independent experiments. C, D, After 18 h of incubation of T cells with B16‐OVA cells at the indicated ratios, live CD8^–DAPI^–annexin V^– cells were quantified by flow cytometry and the percentage of cytotoxicity was deduced. IL‐2, interleukin‐2; MFI, mean fluorescence intensity

FIGURE 5

Cystathionine gamma‐lyase (CTH) overexpression in T cells does not rescue proliferation inhibition induced by cystine (Cys₂) deprivation in vitro. A, Schematic representation of T cell infection (day 2 after stimulation) by control or CTH‐encoding retrovirus followed by Cys₂ removal from the medium (day 4). Cell viability and proliferation were assessed after 2 days (day 6). B, Flow cytometry analysis showing identification of DAPI^– live cells. C, D, Absolute live cell quantification (mean ± SD) (C) and cell proliferation dye (CPD) fluorescence (D), inversely reflecting cell proliferation, in the presence of the indicated concentrations of Cys₂ in the medium. E, Quantification by liquid chromatography‐tandem mass spectrometry of methionine and cysteine (Cys)/Cys₂ at the end of the culture (day 6) in the supernatants of wells supplemented or not with Cys₂ at day 4. Data are representative of at least two independent experiments. FSC‐A, forward scatter area

FIGURE 6

Analysis of amino acid changes induced by cystathionine gamma‐lyase (CTH) expression in vitro and in vivo. A, Schematic representation of T cell stimulation and infection by control (Ctrl) or CTH‐encoding retrovirus followed by in vitro expansion in complete medium supplemented with 500 IU/mL interleukin‐2 (IL‐2). Cells and supernatants were collected on day 7. B, Volcano plot depicting the log₂ fold change (FC) in proteogenic amino acid concentration in cell lysates (left panel) or supernatants (right panel) between Ctrl and CTH‐expressing T cell in vitro cultures. C, Schematic representation of B16‐OVA melanoma cell inoculation followed by indicated adoptive cell transfer (ACT; along with total body irradiation + IL‐2 injections). Six days after ACT, circulating plasma and tumor were harvested (n = 4 mice per group). D, Volcano plot depicting the log₂ FC in proteogenic amino acid concentration in the plasma (left panel) or tumor interstitial fluid (TIF; right panel) between Ctrl and CTH ACT‐treated mice. E, Concentration (mean ± SD, four mice in each group) of indicated amino acids in TIF of control and CTH ACT‐treated mice. *P < .05

FIGURE 7

In vitro effect of serine (Ser), proline (Pro), and glycine (Gly) deprivation on tumor cell viability. A, B, B16‐OVA cells were cultured for 4 days using the indicated concentrations for each of the three amino acids Ser, Pro, and Gly. Live DAPI^–annexin V^– cells were quantified by flow cytometry. Percentages show increased cell death induced by the concomitant deprivation of Ser, Pro, and Gly. C, D, B16‐OVA cells were cultured for 4 days with different deprivation combinations: absence of only one (–Pro, –Gly, or –Ser) or two amino acids at the time. Live cells were quantified as described above. Data (means ± SD, four replicates per condition) are representative of three independent experiments. *P < .05; **P < .01