DYSREGULATION OF THE ILT7/BST2 PDC NEGATIVE FEEDBACK BY HIV-1:IMPLICATIONS FOR HIV-1 TRANSMISSION AND IMMUNOPATHOGENESIS

Introduction: The immunoglobulin like transcript 7 (ILT7) is a surface molecule selectively expressed by human plasmacytoid dendritic cell (pDC). ILT7 cross-linking inhibits Toll like receptor (TLR) 7/9-mediated pDC activation and type I interferon (IFNI) production. The bone marrow stromal cell antigen 2 (BST2) is a natural ligand for ILT7, is expressed on several cell types and encoded by an IFN I-stimulated gene. BST2/ ILT7 interaction may provide a negative feedback for pDC activation. Alterations of the BST2/ILT7 negative feedback may contribute to HIV1-induced pDC over-activation and pathogenesis. We tested: 1) if BST2/ILT7 expression inperipheral blood mononuclear cell (PBMC) correlates with TLR-mediated pDC activation; 2) which stimuli can influence BST2 expression and IFN-I production by PBMC; 3) if TLR-induced pDC activation is directly modulated by BST2-expressing cells in vitro. Methods: PBMC from healthy donors were cultured overnight with or without imiquimod (TLR7L), CpG ODN (TLR9L), AT2-HIV1, TNFα, IFNγ, IL4 IL10, Anti-Human Interferon Alpha/Beta Receptor Chain 2 (IFNAR2), BST2-GST fusion protein. T cells were stimulated using CD3 antibody. The effect of BST2 blockade and ILT7 cross-linking were tested using an anti-BST2 and cross linking-ILT7 monoclonal antibodies (mAbs), respectively. ILT7, BST2, CD83 and CCR7 expression was analyzed by flow cytometry. 293T cell lines transfected with BST2WT, or BST2 mutants, were used to test anti-BST2 mAb efficiency of binding or co-cultured with purified pDC to test the biologic effect of BST2. IFNα production was quantified by ELISA. Statistical analyses were performed using SPSS 19.0. Results:pDC exclusively expressed ILT7, which was rapidly downregulated in vitro as part of a first step of pDC differentiation, characterized by an increase of the pDC morphological complexity and CCR7 expression. CD83 expression, indicative of full pDC activation and maturation, occurred only following TLR stimulation. Conversely, BST2 expression was not affected by in vitro culture; it was highest in monocytes, mDC and B cells compared to pDC and T cells and it was modulated by TLR7/9L-induced IFNα production. BST2 expression on pDC was highest at intermediate stimuli concentrations but modestly increased at maximum concentrations; a profile which correlated with CD83 expression and IFNα production but not with indoleamine 2,3dioxygenase (IDO) activity after HIV stimulation. PBMC pre-treatment with ILT7 cross-linking mAbs reduced both TLR9L/HIV-induced IFNα production and HIV-induced IDO activity. In contrast, pre-treatment with blocking BST2 Abs did not increase IFNα production or IDO activity. The lack of biological effect of BST2 was not due to inefficient αBST2 Ab binding, as PE-labelled αBST2 Ab efficiently stained BST2-transfected 293T cell lines. No change in IFNα production were observed using either a soluble BST2 protein or a co-culture system based on purified pDC and BST2WT transfected 293T cells. T cell receptor engagement resulted in maximum BST2 expression on T cells, but BST2-blocking mAbs did not affect IFNα release even when BST2 expression on T cell was enhanced. IL10 and TNFα inhibited TLR9L-induced IFNα production but BST2 blockade did not restored IFNα responses. Conclusions: Our data suggest that ILT7 cross-linking may acts as homeostatic mechanism on circulating pDC rather than a negative feedback for activated mature pDC, and argue against the role of BST2 as a biologically active ILT7 ligand.