The impact of heteromer formation on CD40 signalling was studied in cell lines and in primary human B cells, showing that they can negatively regulate CD40L-induced responses. intrinsic signalling pathway of CD40 in 293T cells. Conversely, BJAB I2906 cells lacking endogenous Fas or TRAILR2 showed an increased NF-protein synthesis but was dependent upon active PI3K. Both previously described rescue mechanisms are ligand dependent, raising the question whether the relative amount of receptors may impact directly on CD40 signalling in a ligand-independent way. Several TNFRSF members are able to self-associate, in particular Fas, TRAILRs and CD40.18, 19, 20, 21, 22, 23 We previously reported that CD40 can form non-covalent dimers Cd8a in the absence of ligand, with an important contribution of the extracellular region to establish contacts.23 However, the potential for different TNFR family members to heteromerize has not been investigated. In this study, we identified selective interactions between CD40 and Fas, and between CD40 and TRAILR2. These heteromers also form at the endogenous level, and appear to be dynamic, driven by the preferential association of CD40 with TRAILR2 over Fas. The impact of heteromer formation on CD40 signalling was studied in cell lines and in primary human B cells, showing that they can negatively regulate CD40L-induced responses. Thus, heteromer formation between receptors with opposite functions could represent the most apical regulation of TNFRSF signalling. Results CD40 interacts with Fas and TRAILR2 The first evidence of CD40CFas conversation was obtained by F?rster resonance energy transfer (FRET) by flow cytometry. Fas was initially predicted to serve as a negative control for CD40CCD40 conversation, but yielded high FRET rates when it was co-transfected with CD40 (Physique 1a). Then, we tested the ability of CD40 to interact with other TNFRSF members important for B-cell function such as Fas, TRAILR1, TRAILR2, BCMA (TNFRSF17), BAFFR (BR3 or TNFRSF13C), TACI (TNFRSF13B) and the two unrelated receptors ErbB1 and ErbB2. No conversation could be detected with ErbB1, ErbB2, TRAILR1, TACI, BAFFR or BCMA. However, positive FRET responses were observed between CD40 and Fas and, to a lesser extent, between CD40 and TRAILR2 (Physique 1b). Ligand-independent associations of CD40 with itself, with Fas and with TRAILR2 were readily observed with constructs lacking the intracellular domain name (ICD), indicating that the latter is not required for the observed homo- and hetero-oligomerizations (Physique 1c). No conversation was detected between Fas and TRAILR2. In summary, in transiently transfected 293T cells, CD40 interacts with Fas and TRAILR2 as detected by FRET and these interactions do not require the intracellular domains. Open in a separate windows Physique 1 CD40 interacts with Fas and TRAILR2. (a) Flow cytometry I2906 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 impartial 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 name (ICD). In all cases, FRET+ corresponds to the positive FRET reporter (ECFPCEYFP fusion protein) and FRET- corresponds to the unfavorable control (ECFP/EYFP co-transfection). The dotted line represents background FRET levels CD40 selectively interacts with TRAILR2 over Fas In order to visualize these interactions in cells with endogenous expression levels, the CD40-positive and Fas-positive B-cell lymphoma cell line BJAB, with or without expression of TRAILR2, was used.24 Immunocytochemistry using antibodies against the ectodomains of CD40, Fas and TRAILR2 showed colocalization at the cell surface between CD40 and Fas in TRAILR2-negative BJAB cells. However, colocalization of CD40 and Fas was I2906 reduced in TRAILR2-positive BJAB when compared with TRAILR2-unfavorable BJAB cells, despite comparable Fas expression levels. Under the same conditions, colocalization of CD40 and TRAILR2 was detected in TRAILR2-positive BJAB cells (Physique 2, Supplementary Figures 1 and 2). Open in a separate window Physique 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-unfavorable (neg) cell lines. Scale bars correspond to 20 and.