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Inhibitory effect of tumor cell–derived lactic acid on human T cells

Karin Fischer, Petra Hoffmann, Simon Voelkl, Norbert Meidenbauer, Julia Ammer, Matthias Edinger, Eva Gottfried, Sabine Schwarz, Gregor Rothe, Sabine Hoves, Kathrin Renner, Birgit Timischl, Andreas Mackensen, Leoni Kunz-Schughart, Reinhard Andreesen, Stefan W. Krause and Marina Kreutz

Data supplements

Article Figures & Data

Figures

  • Figure 1

    Lactate levels measured in the serum of patients with different malignant diseases. Lactate serum concentrations were determined in 140 patients with different malignancies. Patients were grouped for disease burden into 3 groups describing the stage of disease (“Material and methods”). The normal range of lactate serum levels determined in healthy individuals (0-2.2 mM) is indicated (gray background).

  • Figure 2

    Lactic acid suppresses proliferation and partially induces cell death in CTLs. (A) 3H-thymidine uptake in CTLs during short-term (6.5 hours) incubation in 5 to 20 mM lactic acid or sodium lactate. Data are means ± SEM (n = 3). (B) CFSE-labeled CTLs were stimulated for 24 hours with PHA/ionomycin, and cell division was determined by FACS on day 6 in the absence or presence of 5, 10, and 20 mM lactic acid. Gated viable cells from a representative experiment are shown (n = 3). (C) CTLs were treated with 10 or 20 mM lactic acid or HCl (pH 6.5) for 6.5 hours or 24 hours, and viable cells were determined by flow cytometry as annexinV-FITC/PI-negative cells. Data are means ± SEM; n = 8; *P < .02; **P < .003.

  • Figure 3

    Lactic acid does not alter surface antigen profile in CTLs. CTLs were treated for 24 hours with 20 mM lactic acid or were left untreated. After staining with the indicated mAbs, the phenotype of CTLs was determined by FACS. Grey indicates isotype control of untreated cells; dotted line, control; and solid line, with lactic acid. Results are representative of 3 independent experiments.

  • Figure 4

    Lactic acid strongly diminishes cytokine production in CTLs. (A) CTLs were incubated for 16 hours with or without lactic acid, lactic acid/buffered with NaOH, or sodium lactate and then stimulated for another 5 hours with PMA/ionomycin in the presence of the substances. Cells were stained intracellularly with mAbs for IL-2 and IFN-γ and analyzed by flow cytometry. Results are representative of 3 independent experiments. (B) CTLs were cultured in the absence or presence of 5 to 20 mM lactic acid, sodium lactate, or HCl (∼ pH 6.8). Data represent means ± SEM; n = 4. (C) IL-2, IFN-γ, and CD8 mRNA were determined by quantitative RT-PCR after stimulation for 3 hours with PMA/ionomycin in the presence of 20 mM lactic acid, 20 mM sodium lactate, or HCl (∼ pH 6.8). Data are means ± SEM; n = 4. (D) CTLs were treated for 24 hours with 20 mM lactic acid (LA), washed, and recovered for 24 hours in normal medium (med). Intracellular cytokines were determined by flow cytometry. Results are representative of 3 independent experiments. **P < .0016; ***P < .0001.

  • Figure 5

    Lactic acid affects the cytotoxic activity and the availability of intracellular perforin and granzyme-B. (A) The cytotoxic activity of Melan-A–specific CTLs was determined by incubating CTLs with PKH26+ Melan-A peptide–loaded T2 target cells at indicated effector-target ratios. The cytotoxicity assay was performed for 3 hours in the presence of 15 mM lactic acid, 15 mM sodium lactate, HCL, or in normal medium. (B) Untreated CTLs (gray) or CTLs that were treated for 24 hours with 20 mM lactic acid (solid line) were analyzed for their intracellular content of human perforin and granzyme-B. Appropriate isotype control of untreated CTLs is shown (dashed line). Results are representative of 3 independent experiments. Mean of triplicate ± SEM. *P < .05.

  • Figure 6

    Tumor spheroid–infiltrating CTLs exhibit a diminished ability for cytokine production, and oxamic acid is able to prevent this suppression. (A) MCTSs were generated in the presence or absence of oxamic acid from exponentially growing MelIm tumor cells. After 4 to 5 days, medium was replaced by a suspension containing 0.4 × 106/mL CTLs. Infiltrating CTLs were visualized in 5-μm paraffin sections by staining for the T-cell marker CD3 (bar = 100 μm). (B) Day-4 to day-11 MCTSs were generated in the presence or absence of 60 mM oxamic acid (OA) and supernatants analyzed for lactate content. Horizontal bars represent the mean values. (C) Reduction of IFN-γ–positive CTLs relative to control cells without MCTSs against the lactate content of the MCTS coculture. (D) After 24 hours of coculture, day-5 MCTSs were disintegrated, and cells were stained intracellularly for IL-2 and IFN-γ. CTLs cultured in medium without MCTSs were used as control. Shown are representative data of 3 independent experiments.

  • Figure 7

    MCT-1 transport activity and lactic acid influx in CTLs. (A) Expression of MCT-1 and MCT-2 in CTL lines and unstimulated human CD8+ T cells (from the same donor, respectively) analyzed by Western blot. Hela cells were used as a positive control. (B) Blocking of MCT transport activity by treating CTLs for 5 hours with 3 or 9 mM CINN or 20 mM lactic acid. Intracellular cytokines IL-2 and IFN-γ were determined by FACS. Results are normalized to untreated CTLs. (C) Uptake of 13C-labeled lactate by CTLs. CTLs were incubated for 30 minutes with 20 mM external 13C-lactate in the presence or absence of HCl (*P < .05; n = 4). Endogenous lactate and the uptake of exogenous lactate and was determined in the cell lysates by mass spectrometry. (D) Decrease of the intracellular pH in CTLs 30 minutes after addition of lactic acid was determined flow cytometrically with SNARF-1. Shown are means ± SEM from 3 independent experiments with different donors.