Abstract
Type I interferons (IFN-I) are key innate immune effectors predominantly produced by activated plasmacytoid dendritic cells (pDCs). By modulating immune responses at their foundation, IFNs can widely reshape immunity to control infectious diseases and malignancies. Nevertheless, their biological activities can also be detrimental to surrounding healthy cells, as prolonged IFN-I signaling is associated with excessive inflammation and immune dysfunction. The interaction of the human pDC receptor immunoglobulin-like transcript 7 (ILT7) with its IFN-I-regulated ligand, bone marrow stromal cell antigen 2 (BST2) plays a key role in controlling the IFN-I amounts produced by pDCs in response to Toll-like receptor (TLR) activation. However, the structural determinants and molecular features of BST2 that govern ILT7 engagement and activation are largely undefined. Using two functional assays to measure BST2-stimulated ILT7 activation as well as biophysical studies, here we identified two structurally-distinct regions of the BST2 ectodomain that play divergent roles during ILT7 activation. We found that although the coiled-coil region contains a newly defined ILT7-binding surface, the N-terminal region appears to suppress ILT7 activation. We further show that a stable BST2 homodimer binds to ILT7, but post-binding events associated with the unique BST2 coiled-coil plasticity are required to trigger receptor signaling. Hence, BST2 with an unstable or a rigid coiled-coil fails to activate ILT7, whereas substitutions in its N-terminal region enhance activation. Importantly, the biological relevance of these newly defined domains of BST2 is underscored by the identification of substitutions having opposing potentials to activate ILT7 in pathological malignant conditions.
Highlights
Type I interferons (IFN-I) are key innate immune effectors predominantly produced by activated plasmacytoid dendritic cells
To identify the determinants in bone marrow stromal cell antigen 2 (BST2) ectodomain required for immunoglobulin-like transcript 7 (ILT7) activation we used two previously described co-culture assays, namely a reconstituted ILT7 reporter assay and a PBMCs at a ratio of 3:1 (PBMC)-based assay (Fig. 1B)
Selected BST2 mutants were tested in a PBMC-based coculture assay, which directly measures the quantity of IFN-I produced by PBMCs after TLR7 agonist stimulation following engagement and activation of the ILT7 pathway in plasmacytoid dendritic cells (pDCs) by BST2 [30, 34]
Summary
We previously demonstrated that the BST2 ectodomain is sufficient to interact with ILT7 and activate its inhibitory signaling cascade [30]. When tested in the ILT7 reporter assay, substitution of leucine at position 70 for aspartic acid significantly enhanced activation (Fig. 4, A and B) This mutant behaved just as BST2 WT in the PBMC-based assay (Fig. 4C). In agreement with the ILT7 reporter cell assay, the BST2 C91A mutant was unable to significantly trigger the ILT7-dependent IFN-I repression pathway in PBMCs, whereas no significant differences were observed between BST2 WT and the C91A/N92Q mutant (Fig. 5C) These results indicate that the structural stability of the BST2 dimer conferred by the disulfide bonds is critical for BST2-mediated ILT7 activation, the disulfide bond formed through Cys-91 might contribute to the proper orientation of a neighboring highly glycosylated site that otherwise could impair BST2–ILT7 engagement. These results validate that post-binding events are governing the extent of ILT7 activation by BST2
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