Hetero-oligomerization between the TNF receptor superfamily members CD40, Fas and TRAILR2 modulate CD40 signalling

Abstract

TNF receptor superfamily members (TNFRSF) such as CD40, Fas and TRAIL receptor 2 (TRAILR2) participate to the adaptive immune response by eliciting survival, proliferation, differentiation and/or cell death signals. The balance between these signals determines the fate of the immune response. It was previously reported that these receptors are able to self-assemble in the absence of ligand through their extracellular regions. However, the role of this oligomerization is not well understood, and none of the proposed hypotheses take into account potential hetero-association of receptors. Using CD40 as bait in a flow cytometry Förster resonance energy transfer assay, TNFRSF members with known functions in B cells were probed for interactions. Both Fas and TRAILR2 associated with CD40. Immunoprecipitation experiments confirmed the interaction of CD40 with Fas at the endogenous levels in a BJAB B-cell lymphoma cell line deficient for TRAILR2. TRAILR2-expressing BJAB cells displayed a robust CD40-TRAILR2 interaction at the expense of the CD40-Fas interaction. The same results were obtained by proximity ligation assay, using TRAILR2-positive and -negative BJAB cells and primary human B cells. Expression of the extracellular domains of Fas or TRAILR2 with a glycolipid membrane anchor specifically reduced the intrinsic signalling pathway of CD40 in 293T cells. Conversely, BJAB cells lacking endogenous Fas or TRAILR2 showed an increased NF-κB response to CD40L. Finally, upregulation of TRAILR2 in primary human B cells correlated with reduced NF-κB activation and reduced proliferation in response to CD40L. Altogether, these data reveal that selective interactions between different TNFRSF members may modulate ligand-induced responses upstream signalling events.

Figure 1

CD40 interacts with Fas and TRAILR2. (a) Flow cytometry FRET assay between CD40, Fas and TACI. The image shows the combination of the three receptors fused to ECFP and EYFP. The gate used to calculate the percentage of FRET-positive cells was established by using a ECFP-EYFP fusion protein (100% FRET blue dots) together with a ECFP/EYFP co-transfection (0% FRET red dots), bottom left panel. Bottom right panel shows the mean value of FRET-positive cells and SEM of five independent experiments. (b) Flow cytometry FRET screening for different TNFRSF members expressed in B cells. (c) Flow cytometry FRET assay between CD40, Fas, TRAILR2 and TACI lacking the intracellular domain (ΔICD). In all cases, FRET+ corresponds to the positive FRET reporter (ECFP–EYFP fusion protein) and FRET- corresponds to the negative control (ECFP/EYFP co-transfection). The dotted line represents background FRET levels

Figure 2

CD40, Fas and TRAILR2 colocalize at the surface of human B cells. Immunostainings of CD40-FAS and CD40-TRAILR2 in BJAB TRAILR2-positive (pos) and BJAB TRAILR2-negative (neg) cell lines. Scale bars correspond to 20 and 5 μm in the main image and the inset, respectively. Fluorescence intensity plots across sections are shown in Supplementary Figures 1 and 2

Figure 3

CD40 selectively interacts with TRAILR2 over Fas. (a) Co-immunoprecipitation of CD40, Fas and TRAILR2 in BJAB TRAILR2-negative (neg) and TRAILR2-positive (pos) cells followed by WB detection of CD40, Fas and TRAILR2. (WMF: input of whole membrane fraction). (b) FACS expression profile of CD40 and Fas or CD40 and TRAILR2 on BJAB cell line TRAILR2-negative (left panel) together with a confocal image of the same cell type analyzed by PLA (right panel) for both CD40-Fas or CD40-TRAILR2 interactions. (c) Same as panel B, for the TRAILR2-positive BJAB cell line. (d) Mean and S.E.M. of the PLA assay. Spot numbers per cell were counted in the focal plane. The number of cells analyzed by PLA is indicated on each image in panels b and c. Scale bars in panels c and d correspond to 10 μm

Figure 4

CD40 interacts with Fas and TRAILR2 in primary human B cells. (a) FACS expression profile of CD40 and Fas on primary human B cells non stimulated (NS), activated with PMA-ionomycin (PI) or activated with anti-BCR plus CD40L (BC) (left panels) together with a confocal image of the same cell type analyzed by PLA for CD40-Fas interaction (right panels) (one representative image out of three independent donors is shown). (b) Percentage of CD40-Fas double-positive cells in five different donors treated as described in panel a. (c) Mean and S.E.M. of the PLA assay for the three donors analyzed by PLA. Spot numbers were counted in positive cells in the focal plane. (d, e and f) Same as a–c, but for CD40–TRAILR2 interactions. The number of cells analyzed in the example is indicated on each image. Scale bars correspond to 5 μm. (g) HLA-DR and isotype control mean fluorescence intensity (MFI) and S.E.M. of five different human primary B cells samples that were either NS, activated with PI or activated with anti-BCR plus CD40L (BC). (h) Same as g, but for CD86 detection

Figure 5

Heteromer formation with signalling-incompetent TRAILR2 or Fas has a negative impact on CD40 signalling. (a) NF-κB luciferase assay in 293T cells transiently transfected with 0.5 ng of CD40, 1 ng of CD40L (left panel) or with 0.5 ng of TACI, 1 ng of BAFF (right panel) and increasing amounts of GPI-anchored CD40, TRAILR2, Fas or TACI. One out of three independent assays with similar results is shown. (b) Flow cytometry analysis of TRAILR2-GPI, CD40 full-length and TACI full-length surface expression. (c) Left: surface expression levels of CD40 full-length, CD40-GPI and CD40 full-length in the presence of TRAILR2, detected by staining with Flag-CD40L. Right, surface expression levels of CD40-GPI and TRAILR2-GPI, detected by staining with an anti-TRAILR3 mAb recognizing the GPI-proximal region of the fusion receptors. MFI, mean of fluorescence intensity. (d) NF-κB luciferase assay in 293T cells transiently transfected with 0.5 ng of CD40, 1 ng of CD40L and increasing amounts of GPI-anchored Fas, Fas-ΔCRD1, CD40 and TACI. One out of two independent assays with similar results is shown. (e) Flow cytometry analysis of Fas and Fas-ΔCRD1 surface expression

Figure 6

Endogenous TRAILR2 or Fas modulate CD40 signalling. (a) NF-κB response of wild-type, Fas KO, TRAILR2 KO and CD40 KO BJAB cell lines induced by the indicated concentrations of Flag-ACRP-CD40L (mega-CD40L). One out of two independent experiments is shown (b). Average of the different clones and replicates of each KO cell line stimulated with 1 μg/ml of mega-CD40L. (c) Ex vivo expression profile of CD40 in marginal zone (MZ) and switched memory (SM) primary human B cells. (d) Expression profile of CD40 in MZ and SM along 6 days of CD40L+IL-21 stimulation. (e and f) Same as c and d, but for Fas expression. (g and h) Same as c and d, but for TRAILR2 expression. MZ B cells are gated as CD27+, IgD+ and SM B cells are gated as CD27+ IgD-. The mean and S.E.M. of six independent experiments is shown. (i) TRAILR2 expression in CD27+ B cells of one representative donor out of four tested after six days of stimulation with CD40L+IL-21. The same gating was used in panel j. (j) CFSE proliferation profile of TRAILR2 low CD27+ (grey histogram) and TRAILR2 high CD27+ (black line) primary human B cells of one representative donor out of four tested after six days of stimulation with CD40L+IL-21. (k) Average proliferative response for the four independent donors analyzed. (l) Flow cytometry analysis of phospho-p65 (RelA) after 5-min stimulation with CD40L. The figure shows five independent donors analyzed in duplicate