Extracellular redox modulation by regulatory T cells

Abstract

We demonstrate that the mechanism of redox remodeling during mouse T-cell activation involves secretion of glutathione by dendritic cells and its subsequent cleavage to cysteine. Extracellular cysteine accumulation results in a lower redox potential, which is conducive to proliferation, and changes the net redox status of exofacial protein domains. Regulatory T cells inhibit this redox metabolite signaling pathway, which represents a previously unrecognized mechanism for immunosuppression of effector T cells.

Figure 1. Mechanism of Cys_ex accumulation

(a) DCs were co-cultured with Tn cells at a 1:1, 1:2 or 1:4 ratio for different durations, and the [Cys]_ex was measured. (b) Changes in the extracellular Cys/cystine redox potential at 36 h. The extracellular Cys/cystine redox potential was calculated according to the Nernst equation: E_h = E_o + RT/2ℱ ln ([cystine]/[Cys]2), using E_o=−250 mV (pH=7.4). (c) Effect of NF-κB pathway inhibitors on [Cys]_ex. (d) Cys metabolism during DC-T cell interaction and the effects of inhibitors. Propargylglycine (PPG), butathionine sulfoximine (BSO), sulfasalzine (SAS), L-aspartic acid β-hydroxamate (AβH), acivicin (ACV) and MK-571 inhibit γ-cystathionase, γ-glutamylcysteinyl synthetase, the x_C⁻ and the X_AG transporter, γ-glutamyltranspeptidase, and the multidrug resistance protein 1 (MRP1) respectively. (e) Inhibition of [Cys]_ex by various inhibitors. DCs were co-cultured for 36 h with Tn cells (1:4) ± 400 μM AβH, or ± 500 μM SAS, or ± 250 μM ACV or ± SAS+ACV. [Cys]_ex is expressed as a percent of the concentration in untreated DC-T cell co-culture medium. The data represent the mean ± SD of at least 4 experiments with different batches of cells. (f) xCT expression in DCs cultured ± Tn cells for 36 h. xCT migrates as 35 and 55 kDa bands. (g) [GSH]_in in Teffs cells co-cultured with DCs (1:4) for 16 h ± SAS or ACV. Representative data from one of three independent experiments are shown for a, b, c, f, g. (*, p<0.05; **, p<0.005; ***, p<0.0005; two tailed Student’s t-test).

Figure 2. Treg-mediated extracellular redox remodeling

(a) [Cys]_ex during co-culture of DCs with Tn cells (1:4) or with Tn+Treg cells (1:4:1 or 1:4:2). (b) [Cys]_ex in DC+LPS±Treg (1:2). (c) Treg-mediated suppression of [GSH]_in in Teffs measured by labeling with chloromethylfluorescein diacetate (CMFDA) and (d) quantitative analysis. Student’s t-test revealed a significant reduction in DC+T cell- and DC+LPS-induced [Cys]_ex (panels a, b, n=4) and GSH_in labeling in T cells (panel d, n=2) in the presence of Treg cells. (e) Teff proliferation ± Tregs and with the addition of Cys to the medium as measured by the [³H]-thymidine incorporation assay. Student’s t-test revealed significant inhibition of proliferation of Teffs by Tregs, which was abrogated by addition of exogenous Cys (n=3). (f) Cell surface thiol levels on DCs and T cells as a function of co-culture using Alexa-maleimide 488 (ALM-488) staining followed by FACS analysis. (g) Quantification of the mean fluorescence intensity (MFI) data shown in panel f (n=4). (h) Confocal microscopy using Alexa-maleimide 594 staining shows an increase in cell surface thiol levels on DCs and T cells as a function of co-culture as compared to single culture. (i) Tregs suppress surface thiol levels on DCs, T cells and LPS-activated DCs. (j) Quantitative analysis of FACS data shown in panel i (n =3). Data represent the mean ± SD of independent experiments (n as indicated in each section). (*, p<0.05; **, p<0.005; two tailed Student’s t-test).