Activated human CD4+ T cells express transporters for both cysteine and cystine

Abstract

Because naïve T cells are unable to import cystine due to the absence of cystine transporters, it has been suggested that T cell activation is dependent on cysteine generated by antigen presenting cells. The aim of this study was to determine at which phases during T cell activation exogenous cystine/cysteine is required and how T cells meet this requirement. We found that early activation of T cells is independent of exogenous cystine/cysteine, whereas T cell proliferation is strictly dependent of uptake of exogenous cystine/cysteine. Naïve T cells express no or very low levels of both cystine and cysteine transporters. However, we found that these transporters become strongly up-regulated during T cell activation and provide activated T cells with the required amount of cystine/cysteine needed for T cell proliferation. Thus, T cells are equipped with mechanisms that allow T cell activation and proliferation independently of cysteine generated by antigen presenting cells.

Figure 1. Early T cell activation is independent of Cys and Met whereas T cell proliferation is strictly dependent on Cys.

Purified naïve CD4⁺ T cells were incubated in Met- and Cys-free DMEM medium without stimulation (black columns and lines in A–G) or were stimulated with Dynabeads Human T-Activator CD3/CD28 in Met- and Cys-free DMEM medium (red columns and lines in A–G) or in Met- and Cys-free DMEM medium supplemented with either 33 µM Met (blue columns and lines in A–G), 66 µM Cys₂ (orange columns and lines in A–G), or 33 µM Met plus 66 µM Cys₂ (green columns and lines in A–G). The expression levels of CD25 and CD69 were determined by flow cytometry after 24 hours of cell culture and depicted as mean fluorescence intensity (MFI) (A and C) and as FACS profiles (B and D). The production of IL-2 was measured by ELISA following 24 (E) and 48 (F) hours of cell culture. T cell proliferation was measured asH-thymidine incorporation for the last six hours of a total of 72 hours of cell culture (G and H). The results shown are representative for at least three separate experiments.

Figure 2. Activated T cells strongly up-regulate the x_c⁻ cystine/glutamate antiporter and the neutral amino acid transporters ASCT1 and ASCT2.

Purified naïve CD4⁺ T cells were incubated in X-VIVO 15 medium and stimulated with Dynabeads Human T-Activator CD3/CD28 for the time indicated. The cells were harvested and the expression of xCT (55 and 35 kDa31) and ASCT2 (75 kDa) was determined by Western blot analyses using TCRζ (16 kDa) as loading control (A) or by quantitative RT-PCR (B and C). The gene expression levels of the target genes are given relative to the housekeeping gene GAPDH (B) or as fold increase of the target gene expression relative to GAPDH (C). CD98 expression was measured by FACS analyses following zero (black line), 24 (red line) and 48 (blue line) hours of cell culture (D). The results shown are representative for at least two separate experiments.

Figure 3. Both the x_c⁻ cystine/glutamate antiporter and the neutral amino acid transporters can supply T cells with the required amounts of Cys.

(A and C) Purified naïve CD4⁺ T cells were incubated in X-VIVO 15 medium supplemented with the indicated concentrations of Glu and stimulated with Dynabeads Human T-Activator CD3/CD28 for 72 h (A) or 48 h (C). T cell proliferation was measured by incorporation ofH-thymidine for the last 6 h of cell culture (A). The expression levels of activation markers were measured by FACS analyses (C). (B) Purified naïve CD4⁺ T cells were incubated in X-VIVO 15 medium supplemented with 20 mM of Glu and the indicated concentrations of 2-ME and stimulated with Dynabeads Human T-Activator CD3/CD28 for 72 h. T cell proliferation was measured by incorporation ofH-thymidine for the last 6 h of incubation. (D) Purified naïve CD4⁺ T cells were incubated in X-VIVO 15 medium that was either not supplemented (lane 1) or supplemented with 20 mM of Glu (lane 2) and stimulated with Dynabeads Human T-Activator CD3/CD28 for 72 h. The expression of xCT and ASCT2 was determined by Western blot analyses using TCRζ as loading control. The results shown in A and B represent mean values ± SEM of triplicates determinations and are representative for three separate experiments. The results shown in C and D are representative for two separate experiments.