Antigen-presenting dendritic cells provide the reducing extracellular microenvironment required for T lymphocyte activation

Abstract

T lymphocytes are defective in cystine uptake and thus require exogenous thiols for activation and function. Here we show that monocyte-derived human dendritic cells (DCs) release cysteine in the extracellular space. Cysteine generation is increased by lipopolysaccharide and tumor necrosis factor alpha, and by contact with T cells specifically recognizing soluble or alloantigens. These stimuli also induce thioredoxin (TRX) accumulation in DCs. However, only the contact with antigen-specific T cells triggers TRX secretion by the antigen-presenting cells. Fewer extracellular thiols are recovered after DC-T cell interactions when cystine uptake or TRX activity are inhibited. In addition, glutamate (Glu) and anti-TRX-inactivating antibodies inhibit antigen-dependent T lymphocyte proliferation. These findings indicate that, during antigen presentation, DCs uptake cystine and release cysteine and TRX, thus providing a reducing microenvironment that facilitates immune response.

Figure 1

Thiol release by DCs is induced by T cells. Eight-day DCs (7 × 10⁴), untreated or treated for the last 24 h with LPS, were incubated 6 h alone (DCs; DCs-LPS) or with alloreactive T cells from a parallel primary 7-day MLR (DCs + alloT; DCs-LPS + alloT) or with T cells from a 7-day MLR between two uTs (DCs + uT) at a DC/T cell ratio of 1:10. As a control, DCs were fixed for 10 min with 1% glutaraldehyde before coculture with alloreactive T cells (GA-DCs + alloT). In a separate experiment (filled columns), DCs were cultured alone (DCs) or loaded overnight with tetox (DCs + tetox) and then incubated for 6 h with autologous tetox-specific T cells at the same DC/T cell ratio (DCs + tetox + tetoxT). At the end of the incubation, thiols present in the supernatants were evaluated by DTNB colorimetric assay. As a control, thiol release by the same number of alloreactive (alloT) or tetox-specific T cells (tetoxT) is shown. One representative experiment of six is shown ± SD.

Figure 2

Cys is the major small thiol released by DCs. The supernatants from 5 × 10⁵ nonstimulated (DCs), LPS-stimulated DCs (DCs + LPS), or DCs incubated with alloreactive T cells at a DC/T cell ratio of 1:10 (DCs + alloT), were analyzed for the presence of Cys, Cys₂, or GSH by HPLC. Open columns show the amounts present in fresh medium.

Figure 3

Regulation of TRX synthesis and secretion in human DCs by LPS, TNF-α, or antigen-specific T cells. (A) Aliquots of the lysates (cyt, Upper) and supernatants (sec, Lower) from untreated DCs (lane 1), or DCs treated for the last 24 h with LPS (lane 2) or TNF-α (lane 3), cultured for 6 h alone (lanes 1–3) or with alloreactive (lane 4, alloT), or nonspecific T cells (lane 5, uT) or CD40L-transfected cells (lane 6, CD40L), were resolved under reducing conditions and transferred to nitrocellulose filters. Filters were decorated with polyclonal anti-TRX antibodies. Cell lysates and supernatants from the same number of alloreactive T cells (lane 7) and CD40L-transfected cells (lane 8) cultured alone are included as controls. (B) Lysates (cyt, Upper) and supernatants (sec, Lower) of 8-day DCs cultured alone for 6 h (lane 1), or loaded with tetox and incubated for 6 h alone (lane 2, tetox), with autologous anti-tetox T cells (lane 3, tetox + tetoxT) or with nonspecific activated T cells (lane 4, tetox + uT) were analyzed by Western blotting with anti-TRX as in A.

Figure 4

Glutamate and antibodies inactivating TRX (9G9) inhibit the accumulation of extracellular thiols. LPS-stimulated DCs (7 × 10⁴; DCs + LPS) were incubated alone, and the same number of immature DCs were incubated with alloreactive T cells (DCs + alloT). Incubations were performed 6 h in the presence of 2 mM Glu, 50 μM BCNU, 100 μM DCNB, 5 μg/ml of 2B1 or 9G9 mAb, or 2 mM Glu plus 5 μg/ml of 9G9 mAb. Thiols present in supernatants at the end of the culture period were determined by DTNB assay. Results are expressed as the percent of thiol release relative to untreated 6-h cultures of LPS-DCs (open columns) or DCs-alloreactive T cells (gray columns). One representative experiment of three is shown ± SD.

Figure 5

Glutamate and 9G9 inhibit T cell proliferation induced by antigen-presenting DCs. Eight-day DCs (5 × 10⁴) were incubated with alloreactive T cells from a parallel primary 7-day MLR (DCs + alloT) or loaded with tetox and incubated with autologous tetox-specific T cells (DCs + tetoxT), at a DC/T cell ratio of 1:4, in the presence or absence of 2 mM Glu or 5 μg/ml of 9G9 or 2B1 mAb, or 5 μg/ml of 9G9 and 50 μM Cys (this condition was tested on DC + alloT cocultures only), as indicated. [³H]Thymidine incorporation was evaluated at day 3. Results are expressed as the percent of [³H]thymidine incorporation relative to control cocultures of DC-alloreactive T cells (filled columns, 55,826 ± 6,895 cpm) or of DCs-tetox-specific T cells (open columns, 66,318 ± 3,153 cpm). No significant differences in [³H]thymidine incorporation were observed when LPS-treated DCs were used (not shown). Mean of three different experiments ± SD.