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Plasmacytoid dendritic cells [pDCs] represent a rare innate immune subset uniquely endowed with the capacity to produce substantial amounts of type-I interferons. This function of pDCs is critical for effective antiviral defenses and has been implicated in autoimmunity. While IFN-I and select cytokines have been recognized as pDC secreted products, a comprehensive agnostic profiling of the pDC secretome in response to a physiologic stimulus has not been reported. We applied LC-MS/MS to catalogue the repertoire of proteins secreted by pDCs in the unperturbed condition and in response to challenge with influenza H1N1. We report the identification of a baseline pDC secretome, and the repertoire of virus-induced proteins including most type-I interferons, various cytokines, chemokines and granzyme B. Additionally, using single-cell RNA-seq [scRNA-seq], we perform multidimensional analyses of pDC transcriptional diversity immediately ex vivo and following stimulation. Our data evidence preexisting pDC heterogeneity, with subsequent highly specialized roles within the pDC population upon stimulation ranging from dedicated cytokine super-producers to cells with APC-like traits. Dynamic expression of transcription factors and surface markers characterize subclusters within activated pDCs. Integrating the proteomic and transcriptomic datasets confirms the pDC-subcluster origin of the proteins identified in the secretome. Our findings represent the most comprehensive molecular characterization of primary human pDCs at baseline, and in response to influenza virus, reported to date. [ABSTRACT FROM AUTHOR]

Plasmacytoid dendritic cells [pDCs] represent a rare innate immune subset uniquely endowed with the capacity to produce substantial amounts of type-I interferons. This function of pDCs is critical for effective antiviral defenses and has been implicated in autoimmunity. While IFN-I and select cytokines have been recognized as pDC secreted products, a comprehensive agnostic profiling of the pDC secretome in response to a physiologic stimulus has not been reported. We applied LC-MS/MS to catalogue the repertoire of proteins secreted by pDCs in the unperturbed condition and in response to challenge with influenza H1N1. We report the identification of a baseline pDC secretome, and the repertoire of virus-induced proteins including most type-I interferons, various cytokines, chemokines and granzyme B. Additionally, using single-cell RNA-seq [scRNA-seq], we perform multidimensional analyses of pDC transcriptional diversity immediately ex vivo and following stimulation. Our data evidence preexisting pDC heterogeneity, with subsequent highly specialized roles within the pDC population upon stimulation ranging from dedicated cytokine super-producers to cells with APC-like traits. Dynamic expression of transcription factors and surface markers characterize subclusters within activated pDCs. Integrating the proteomic and transcriptomic datasets confirms the pDC-subcluster origin of the proteins identified in the secretome. Our findings represent the most comprehensive molecular characterization of primary human pDCs at baseline, and in response to influenza virus, reported to date.

Kayoko Kibata,1 Tomoki Ito,1 Muneo Inaba,1 Akihiro Tanaka,1 Ryoichi Iwata,2 Noriko Inagaki-Katashiba,1 Vien Phan,1 Atsushi Satake,1 Shosaku Nomura11Kansai Medical University, First Department of Internal Medicine, Osaka, Japan; 2Kansai Medical University, Department of Neurosurgery, Osaka, JapanBackground: Lenalidomide (LEN), an immunomodulatory drug (IMiD), is currently used for treatment of multiple myeloma (MM). LEN potentiates T cell and natural killer cell functions. However, the cellular and molecular mechanisms underlying the immunomodulatory effects of LEN remain unclear. We focused on the effects of LEN on human plasmacytoid dendritic cells (pDCs), which are the major source of interferon (IFN)-α in the blood and play a central role in innate immune responses.Results: We found that bortezomib, a proteasome inhibitor used to treat MM, killed pDCs but that 0.1–3 μM LEN (covering clinical plasma concentration range) did not affect pDC survival or CD86 expression. Bortezomib inhibited pDC-derived IFN-α production in a dose-dependent fashion, but 0.1–3 μM LEN sustained pDC-derived IFN-α production when stimulated with an optimal concentration of CpG-ODN 2216 (3 μM). In pDCs stimulated with a low concentration of CpG-ODN (0.1 μM), LEN enhanced IFN-α production. These results indicated that LEN, when used at a clinically relevant concentration, can potentially enhance IFN-α production by pDCs.Conclusion: Collectively, our findings unveiled a novel target of LEN and extend the repertoire of the drug’s known immunomodulatory effects. These effects may explain the low incidence of herpes zoster viral infection observed during LEN treatment compared with bortezomib treatment. LEN may function as an IMiD affecting a wide array of immune cells, including pDCs, leading to amplification of a positive immune axis able to eliminate MM cells.Keywords: lenalidomide, IMiDs, plasmacyotid DCs, type I IFNs, multiple myeloma