TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor

Abstract

TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine) is a new member of the TNF family that is induced upon T cell receptor engagement and activates c-Jun N-terminal kinase (JNK) after interaction with its putative receptor (TRANCE-R). In addition, TRANCE expression is restricted to lymphoid organs and T cells. Here, we show that high levels of TRANCE-R are detected on mature dendritic cells (DCs) but not on freshly isolated B cells, T cells, or macrophages. Signaling by TRANCE-R appears to be dependent on TNF receptor-associated factor 2 (TRAF2), since JNK induction is impaired in cells from transgenic mice overexpressing a dominant negative TRAF2 protein. TRANCE inhibits apoptosis of mouse bone marrow-derived DCs and human monocyte-derived DCs in vitro. The resulting increase in DC survival is accompanied by a proportional increase in DC-mediated T cell proliferation in a mixed leukocyte reaction. TRANCE upregulates Bcl-xL expression, suggesting a potential mechanism for enhanced DC survival. TRANCE does not induce the proliferation of or increase the survival of T or B cells. Therefore, TRANCE is a new DC-restricted survival factor that mediates T cell-DC communication and may provide a tool to selectively enhance DC activity.

Figure 1

TRANCE-R expression in various cell types. Cells were prepared as described in the Materials and Methods section and stained with 10 μg/ml of the hCD8-TRANCE recombinant protein (solid lines) or with secondary reagents alone (dotted line). Only viable cells as determined by propidium iodide (PI) exclusion were gated and analyzed for TRANCE-R expression. Fresh DCs were analyzed by two-color staining after gating on CD11c^high cells. Each staining was reproduced at least twice.

Figure 2

TRANCE is a DC survival factor that upregulates Bcl-x_L. (A) BMDCs were cultured in complete medium in the presence or absence of recombinant TRANCE (1 μg/ml) for 48 h and were then visualized under an inverted light microscope. (B) Duplicate wells containing 3 × 10⁴ BMDCs were cultured with increasing doses of recombinant TRANCE in complete medium in flat-bottomed 96-well plates. The percentage of cell survival was assessed 48 h later by trypan blue exclusion. The average of three experiments, and the SEMs, are shown. (C) 3 × 10⁴ BMDCs were cultured in complete medium in the presence or absence of recombinant TRANCE (1 μg/ml) or mCD8-CD40L (1/1,000 of the culture supernatants). Cell viablity was assessed daily by trypan blue exclusion. Representative data of three independent experiments are shown. (D) 3 × 10⁴ GM-CSFs and IL-4 stimulated human monocyte-derived DCs were cultured for 2 d in monocyte conditioned medium to generate mature DCs (26). Thereafter, DCs were cultured in the presence or absence of recombinant TRANCE (1 μg/ml) and cell viability was assessed each day by trypan blue exclusion. (E) 50 μg of protein extracted from BMDCs that had been cultured for 24 h as described in Fig. 2 C were analyzed for Bcl-2 and Bcl-x_L protein expression by Western blot analysis. Basal levels of Bcl-2 and Bcl-x_L were determined in day 8 BMDCs (0 h).

Figure 3

Cell surface marker expression and T cell stimulatory function of TRANCE-treated BMDCs. (A) 2.5 × 10³ BMDCs were cultured with increasing doses of TRANCE in a final volume of 100 μl in triplicate in flat-bottomed 96-well plates. After 48 h, 10⁵ purified allogeneic T cells in 100 μl were added in each well and [³H]thymidine incorporation was assessed after 3 d of culture. One experiment out of three is shown. (B) 2.5 × 10⁴ BMDCs were cultured in the presence or absence of TRANCE or CD40L for 48 h. After washing and counting the cells, dilutions of live cells were cultured with 10⁵ purified allogeneic T cells and [³H]thymidine incorporation was assessed after 3 d of culture. (C) BMDCs were cultured in complete medium for 24 h in the presence (solid lines) or absence (dotted lines) of soluble FLAG-TRANCE (1 μg/ml) and analyzed for the indicated surface markers expression by FACS^® after gating the live cells. Similar results were obtained after 48 h of culture.

Figure 4

TRANCE does not induce the proliferation of B or T cells. Triplicate wells of 2 × 10⁴ purified B cells were cultured in complete medium in the presence of increasing doses of soluble TRANCE or CD40L in flat-bottomed 96-well plates. 10⁵ purified T cells were cultured in complete medium containing Con A (2.5 μg/ml) in the presence of increasing doses of soluble TRANCE. [³H]thymidine incorporation was assessed after 2 d of culture.

Figure 5

TRANCE-R signaling is dependent on TRAF2. Thymocytes from transgenic mice expressing TRAF2.DN or control littermates were stimulated with hCD8-TRANCE (1 μg/ml) on OKT8-coated (10 μg/ml) plates for the indicated amount of time then assayed for JNK activity. The degree of JNK activation was analyzed on a phosphorimager (Molecular Imager System; Bio-Rad Laboratories, Hercules, CA) and plotted as fold induction over time 0. Representative results of three independent experiments are shown.