Myeloid-derived suppressor cells inhibit T-cell activation by depleting cystine and cysteine

Abstract

Myeloid-derived suppressor cells (MDSC) are present in most cancer patients and are potent inhibitors of T-cell-mediated antitumor immunity. Their inhibitory activity is attributed to production of arginase, reactive oxygen species, inducible nitric oxide synthase, and interleukin-10. Here we show that MDSCs also block T-cell activation by sequestering cystine and limiting the availability of cysteine. Cysteine is an essential amino acid for T-cell activation because T cells lack cystathionase, which converts methionine to cysteine, and because they do not have an intact xc- transporter and therefore cannot import cystine and reduce it intracellularly to cysteine. T cells depend on antigen-presenting cells (APC), such as macrophages and dendritic cells, to export cysteine, which is imported by T cells via their ASC neutral amino acid transporter. MDSCs express the xc- transporter and import cystine; however, they do not express the ASC transporter and do not export cysteine. MDSCs compete with APC for extracellular cystine, and in the presence of MDSCs, APC release of cysteine is reduced, thereby limiting the extracellular pool of cysteine. In summary, MDSCs consume cystine and do not return cysteine to their microenvironment, thereby depriving T cells of the cysteine they require for activation and function.

Figure 1. β-mercaptoethanol facilitates antigen-driven T cell activation

A, Transgenic CD4⁺ DO11.10 and TS1, and CD8⁺ Clone 4 and OT-I splenocytes were co-cultured with their respective peptides in the presence or absence of 5×10⁻⁵ M β-ME. Data are from one of four (TS1 and Clone 4) and one of five (DO11.10 and OT-I) independent experiments. Results of the pooled experiments for each transgenic population are significant at p <0.01 (Wilcoxon paired-sample test). B, Average percent increase ± SD in T cell proliferation for DO11.10, TS1, Clone 4, and OT-I splenocytes activated in the presence of β-ME shown in panel A.

Figure 2. MDSC express the heterodimeric x_c⁻ transporter (xCT + 4F2), and do not express cystathionase or the ASC neutral amino acid transporter

A, Peritoneal macrophages, purified CD4⁺ splenic T cells, and DC (all from naive BALB/c mice), and 4T1-induced blood MDSC were stained with mAbs to Gr1, CD11b, F4/80, CD4, and/or CD11c and analyzed by flow cytometry. Purity of each population is shown. B, Total RNA was isolated from the MDSC, macrophages, and CD4⁺ T cells shown in panel A, reverse transcribed, and PCR amplified for the x_c⁻ (xCT and 4F2) and ASC transporters, cystathionase, and GAPDH. PCR for cystathionase, 4F2, xCT, and ASC was performed on 2, 3, 4, and 3 independent cell preparations, respectively, for each cell type, C, Peritoneal macropahges, bone marrow DC, purified splenic CD4⁺ T cells, and blood Gr1⁺CD11b⁺ cells (all from naive mice), and 4T1-induced blood MDSC were stained with antibodies to xCT, 4F2, Gr1, CD11b, F4/80, CD11c, and CD4. Gated populations (MDSC: Gr1⁺CD11b⁺; macrophages: F4/80⁺; DC: CD11c⁺; and T cells: CD4⁺) were analyzed for expression of xCT or 4F2. Data are from one of three independent cell preparations. D, Splenic macrophages, DC, purified T cells, and blood MDSC from tumor-free (naive) or 4T1-tumor-bearing mice were stained with antibodies to Gr1, CD11b, F4/80, CD11c, CD4, and ASC, and the gated Gr1⁺CD11b⁺, F4/80⁺, CD4⁺, or CD11c⁺CD11b⁺ populations analyzed by flow cytometry.

Figure 3. MDSC suppression is reversed by β-ME or NAC)

A, Transgenic splenocytes and their respective peptides were co-cultured at varying ratios with 4T1-induced blood MDSC (90% Gr1⁺CD11b⁺ cells) with or without β-ME, and T cell activation measured by ³H-thymidine uptake. Data are plotted as percent suppression relative to T cells plus peptide without MDSC, and are from one of six independent experiments. Values for percent suppression in the presence of β-ME vs. no β-ME for the pooled experiments are significant at p <0.0005 (Wilcoxon paired-sample test). B, Transgenic splenocytes were co-cultured as in A in the presence of 0.5mM NAC and without β-ME. Ratio of splenocytes to MDSC for panel B was 1:0.5. Data are from one of three independent experiments. Pooled experiments are significant at p <0.005 (Wilcoxon paired-sample test).

Figure 4. MDSC import cystine and do not export cysteine

A and B, Peritoneal macrophages (92% F4/80⁺), T cells (85% CD4⁺) and bone marrow DC (85% CD11c⁺) (all from tumor-free BALB/c mice), and 4T1-induced blood MDSC (92% Gr1⁺CD11b⁺) were cultured in sodium-free buffer for 20 min, followed by incubation in buffer with ³H-glutamate in the presence or absence of cold competitors L-cystine or L-leucine. Data for panels A and B are from one of five and two independent experiments, respectively. Results of the pooled experiments are significant at p <0.0005 and p<0.025, respectively. C. Higher concentrations of cold competitor L-cystine are required to inhibit glutamate uptake by MDSC relative to peritoneal macrophages. Peritoneal macropahges from tumor-free mice and 4T1-induced MDSC were cultured with ³H-glutamate as in panels A and B in the presence of cold L-cystine. Data are from one of four independent experiments. D, Macrophages and DC, but not MDSC, export cysteine. Peritoneal macrophages (85% F4/80⁺), DC (82% CD11c⁺), and purified CD4⁺ T cells (95% CD4⁺) (all from tumor-free mice), and 4T1-induced blood MDSC (97% Gr1⁺CD11b⁺) were incubated in serum-free HL-1 medium and cysteine content of the culture supernatants measured by the DTNB colorimetric assay. Data are from one of five independent experiments. Results of the pooled experiments are significant at p<0.0005. E, MDSC prevent macrophages from releasing cysteine. Macrophages (86.7% F4/80⁺) and titered numbers of 4T1-induced MDSC (90.6% Gr1⁺CD11b⁺) were co-cultured in serum-free HL-1 medium, and cysteine release measured by the DTNB colorimetric assay. Data are from one of two independent experiments and are significant at p<0.005 for the pooled. F, Serum was obtained from tumor-free BALB/c mice or from BALB/c mice with 4T1 tumors (primary mammary tumors of >5mm in diameter and >60% Gr1⁺CD11b⁺ leukocytes). Serum levels of cystine were determined by HPLC. Each symbol represents an individual mouse; horizontal bars indicate the mean. The two groups are significantly different at p<0. Statistics for all pooled results used the Wilcoxon paired-sample test.

Figure 5

MDSC suppress T cell activation by sequestering cystine and cysteine. A, DC and macrophages generate cysteine by reducing cystine imported through their x_c⁻ transporter, or by the conversion of methionine (Met) to cysteine through the action of cystathionase. Excess intracellular cysteine is exported by DC and macrophages through the ASC neutral amino acid transporter. Extracellular cysteine is also generated by the thioredoxin-mediated reduction of cystine to cysteine. During antigen presentation and T cell activation, T cells, which are unable to produce their own cysteine because they lack cystathionase and the x_c⁻ transporter, import cysteine through their ASC neutral amino acid transporter. B, MDSC must obtain all of their cysteine by importing cystine and reducing it to cysteine since they do not synthesize cystathionase and do not express the ASC neutral amino acid transporter. Therefore, as tumor burden and MDSC levels increase, MDSC consume increasing quantities of cystine. Since MDSC do not contain the ASC neutral amino acid transporter, they do not return cysteine to their surroundings. The competition between macrophages, DC, and MDSC for cystine leads to reduced uptake of cystine by macrophages and DC, and the concomitant decrease in cysteine released by these cells. Because of the lower levels of extracellular cystine, thioredoxin-mediated generation of extracellular cysteine is also reduced. Collectively, these effects result in the local depletion of cysteine and the inhibition of T cell activation.