FOXP3+ Tregs play key roles in maintenance of immune tolerance and autoimmune reactions. TNFRSF25 (also known as DR3) is one of the more recently discovered TNFRSF member, which is expressed by mouse Tregs, naïve CD4, CD8, NKT cells and TLR4-induced DC and macrophages. A significant Treg in vivo expansion by TNFRSF25 stimulation has been reported. Here, we show that by single dose i.p. injection of an agonistic mAb, 4C12, Treg expansion starts to be observed on day 3, reached a peak (up to 30-40% of total CD4+ T-cells) on day 5-6, and then returned to baseline (5-7%) by day 10. Upon repeated 3-day interval injections, high level (about 20%) of expanded Tregs can be sustained more than 10 days. As reported by other authors, followed by one dose 4C12 injection, the absolute number of CD8+T cells, B cells, dendritic cells and macrophages was not affected. In the in vitro study, 4C12 antibody did not significantly induce Treg expansion in CD3/CD28/IL-2 culture system. Moreover, we characterized the TNFRSF25 expanded Tregs in vivo, we found that the expression level of CD304, CD152, Helio, GARP, LAP (TGF-b1), FR4, GITR was significantly increased in comparison to the control. These data suggest that multiple injections of 4C12 can long term sustain expanded Tregs at certain level, the biological significance of Treg need to be further studied.

The brain and dendritic cell (BAD)-associated lysosome-associated membrane protein (LAMP)-like molecule, also known as BAD-LAMP, c20orf103, UNC-46, is a newly identified LAMP family member, which has been recently officially designated as LAMP5. It is a transmembrane glycosylated protein localized in the ER-Golgi intermediate compartment (ERGIC). LAMP5 shares sequence and structural homology with other LAMP family members. In mouse, LAMP5 is expressed in postnatal cortical neurons of particular layers, where it is enriched in defined zones along neuronal projections. In human, like its murine homolog, LAMP5 is principally expressed in brain, and it was recently found in primary plasmacytoid dendritic cells (pDCs). However, LAMP5 expression profile and function have not been extensively studied in pDCs. By using anti-human LAMP5 monoclonal antibody (clone 124-40B), we report that LAMP5 is specifically expressed in peripheral blood pDCs which are characterized by surface expression of CD303+/CD304+/CD123+/HLA-DR+/CXCR3+/CD11c-/CD14-/CD209-, but is not expressed by GM-CSF plus FL3 or IL-4 induced monocyte-derived CD303+/CD123+/HLA-DR+/CD209+/CD14+/CXCR3dim pDCs or CD209+/CD123dim/CXCR3dim/HLA-DR+/CD11c+/CD14+ mDCs. Like other LAMP members, LAMP5 is rapidly relocated on the cell surface of peripheral blood pDCs after 24 hours stimulation by IL-3. These data suggest that LAMP5 may be a useful marker for studying pDC development and activation.

Monitoring populations of immune cell subsets, such as B lymphocytes, T lymphocytes, NK cells, NKT cells, Treg cells, monocytes and dendritic cells, is important in many different infectious diseases, including HIV/AIDS, to assess response and recovery to treatment and therapy. In the last decade, multicolor flow cytometry has greatly increased the capabilities and consistency of immunophenotyping and immunomonitoring. However, limitations still preclude a wider adaption of >10 color flow cytometry, largely due to the limited availability of fluorophores with flexible utility in a complex panel. Although instruments with 18-20 parameters are now increasingly common, until recently, the available reagents had significant practical limitations, such as limited brightness and permeability, and undesirable cross-beam excitation, resulting in reduced sensitivity when detecting lowly abundant antigens in these assaulted channels. The Brilliant Violet™ family of fluorescent probes, released in 2011, overcomes these limitations, with significantly increased overall brightness and signal-to-noise. Here, we present a 15 color/ 17 parameter flow cytometry assay, introducing Brilliant Violet™ 605 and Brilliant Violet 650™ in our expanding family of fluorescent polymers. This family of fluorescent probes will be instrumental in making a wide array of commercially available conjugates with a broad range of brightness and no limitations for intracellular detection.

Abstract Podoplanin is a mucin-type transmembrane glycoprotein containing extensive Oglycosylation in extracellular domain and two phosphorylation sites in a short cytoplasmic tail. It is expressed in kidney glomerular epithelial cells, lymphatic endothelium and subcapsular epithelial cells in murine thymus. Recently, podoplanin was found on murine F4/80 positive peritoneal macrophages and human follicular dendritic cells (FDCs) indicating that podoplanin is involved in immune response. We have reported that podoplanin is induced during Th9 and Th17 polarization. Here, we studied podoplanin expression on different types of murine myeloid cells. We found that podoplanin was not detectable on circulating and bone marrow derived neutrophils, monocytes, basophils and esinophils, its expression level on neutrophil and monocytes was not inducible followed by fMLP or TNF-a stimulation. Podoplanin was also not detectable on spleen and lymph node resided CD11b+macrophages, CD11c+ dendritic cells (cDCs) and CD8+ plasmacytoid dendritic cells (pDCs). However, podoplanin expression level has been dramatically increased on GM-CSF driven bone marrow-derived dendritic cells. These data suggest that podoplanin can be induced during GM-CSF driven dendritic cell differentiation, indicating that podoplanin may be a transient marker during dendritic cells differentiation and maturation. The mechanism of GM-CSF induced podoplanin and it significance need to be further investigated.

Interleukin-27 (IL-27) is a novel IL-6/IL-12 cytokine family member that consists of EBV-induced gene-3 (EBI3) and p28 subunits. IL-27 triggers expansion of antigen specific naïve CD4+ T cells, promotes polarization of Th1 cells, and inhibits the development of Th2 and Th17 cells. It is known that IL-27 is an early product of activated antigen-presenting cells. But the regulation of IL-27 production has not been well documented. Here, we studied the effects of IFN-γ and LPS on IL-27 production by mouse macrophages. Thioglycollate-elicited peritoneal cells from Balb/c mice were stimulated with IFN-γ and LPS with various combinations and different time periods. IL-27 expression was examined by flow cytometric analyses of intracellular staining with fluorochrome-conjugated anti-mouse IL-27 mAb (MM27.7B1) and bead-based analyses of the cultured supernatants. The data showed that LPS alone-stimulated macrophages yielded less than 10% of IL-27 positive cells on 24 hrs after stimulation. However, if the cells were primed with IFN-γ for 2 hours and then stimulated with LPS, higher frequency of IL-27 producing cells was observed (25% to 45%). Kinetic studies demonstrated that a significant level of IL-27 secretion was detected as early as 2 hours and the peak production was observed around 22 hours post stimulation.