ATP promotes immunosuppressive capacities of mesenchymal stromal cells by enhancing the expression of indoleamine dioxygenase

Abstract

Introduction: MSCs are often found within tumors, promote cancer progression and enhance metastasis. MSCs can act as immuosuppressive cells, partially due to the expression of the enzyme indoleamine dioxygenase (IDO) which converts tryptophan to kynurenine. Decreased concentration of tryptophan and increased kynurenine, both interfere with effective immune response. Damage associated molecular patterns (DAMPs) including ATP are found within the tumor microenvironment, attract MSCs, and influence their biology.

Methods: Bone marrow derived MSCs were exposed to ATP for 4 days, in the presence of 100 ng IFNγ/mL. Intracellular expression of IDO in MSCs was assessed by FACS. Conditioned media from thus stimulated MSCs was analyzed for kynurenine content and its suppressive effect on lymphocyte proliferation. Apyrase or P2 × 7-receptor antagonist (AZ 11645373) were applied in order to inhibit ATP induced effect on MSCs.

Results: We demonstrate, that ATP at concentrations between 0.062 and 0.5 mM increases dose dependently the expression of IDO in MSCs with subsequent increased kynurenine concentrations within the supernatant at about 60%. This effect could be abolished completely in the presence of ATP degrading enzyme (apyrase) or when MSCs were pretreated with a P2 × 7-receptor antagonist (AZ 11645373). Consistently, supernatants from MSCs stimulated with ATP, inhibited lymphocyte proliferation from 65% to 16%.

Conclusions: We characterized ATP as a DAMP family member responsible for necrosis-induced immunomodulation. Given the increased concentration of DAMPs within tumor tissue and the fact that DAMPs can act as chemotattractants to MSCs, our results have implications for therapeutic strategies targeting the tumor microenvironment.

Keywords: DAMPs; cancer; inflammation; mesenchymal stromal cells; wound healing.

Figure 1

ATP dose‐dependently enhances kynurenine production by MSCs. (A) In the presence of 100 ng IFNγ/mL culturing media containing also 100 μg tryptophan/mL, bone marrow derived MSCs (15 × 10³ cells/cm²) were stimulated with indicated concentrations of ATP for 4 days and kynurenine concentration was assessed within the supernatant by a colorimetric assay. Shown is one out of eight independent experiments with mean values and error bars indicating standard deviation. (B) In the presence of indicated concentration of ATP‐degrading enzyme apyrase in culturing media supplemented with 100 μg tryptophan/mL, bone marrow derived MSCs (15 × 10³ cells/cm²) were stimulated for 4 days with IFNγ (100 ng/mL) and ATP (500 μM). Apyrase dose‐dependently inhibits ATP‐enhanced kynurenine production in MSCs. Shown are representative results (mean ± standard deviation) from one out of four individual experiments

Figure 2

ATP promotes immunosuppressive MSCs by acting through P2 × 7‐Receptor. Bone marrow derived MSCs were cultured for four days in tryptophan saturated media in the presence of IFNγ alone or in combination with ATP ± P2 × 7 receptor antagonist (AZ11645373). MSCs were fixed, permeabilized and assessed for intracellular IDO expression by FACS (A), while culturing supernatant was analyzed for kynurenine (B), or used as conditioned media for culturing fluorenscent‐labelled PBL stimulated with anti‐CD3/anti‐CD28‐beads to induce their proliferation within 4 days (C). Shown are results from one representative out of four independent experiments

Figure 3

ATP enhances IFNγ‐induced inhibition of lymphocyte proliferation by MSCs. Bone marrow derived MSCs were cultured for four days in tryptophan saturated media in the presence of IFNγ (100 ng/mL) alone or in combination with ATP (250 μM). Thus obtained conditioned media was used to culture PBL in the presence of anti‐CD3/anti‐CD28‐beads (1 bead per 12.5 PBLs), Lymphocyte proliferation after 4 days was assessed by performing flow cytometry. Shown is the ratio of proliferating lymphocytes from one representative out of at least three independent experiments

Figure 4

Kynurenine plays an important role in MSC‐induced inhibition of lymphocyte proliferation. (A) In the presence of anti‐CD3/anti‐CD28‐beads, CFSE‐labelled lymphocytes were incubated for 4 days in fresh media or conditioned media obtained from non‐stimulated MSCs cultured in media containing 100 μg/mL tryptophan. Lymphocyte proliferation was assessed by performing flow cytometry (FACS). Shown is the ratio of proliferated lymphocytes. Conditioned media from non‐stimulated MSCs does not inhibit lymphocyte proliferation. Shown are results from one representative out of at least three independent experiments. (B) Culturing media was supplemented with 200 μM kynurenine and added at indicated ratios to conditioned media from non‐stimulated MSCs, in order to mimic kynurenine production by stimulated MSCs. PBL were resuspended in above described kynurenine‐supplemented conditioned media and stimulated by anti‐CD3/anti‐CD28‐beads to induce proliferation. Kynurenine dose‐dependently inhibits PBL proliferation within 4 days. Shown is one representative experiment out of two independent experiments with asterisks indicating P values (*P ≤ 0.05 and **P ≤ 0.01)

Figure 5

ATP does not influence MSC Proliferation. Bone marrow derived MSCs were cultured in the presence of indicated concentration of ATP for 4 days. Shown are results (mean ± standard deviation) form one representative out of seven independent experiments