signature=60eebcc1ed34c5404f4e111ad8dc7291,Talk: Identification of a Unique Subset DC-STAMP+ (Dendri...

Background/Purpose: The contribution of Th17 cells to disease pathogenesis in psoriatic arthritis (PsA) is not well understood. We previously demonstrated that the expression of DC-STAMP (Dendritic Cell-Specific Transmembrane Protein), a 7-pass transmembrane protein required for monocyte fusion during osteoclast (OC) development, is increased in freshly isolated monocytes from PsA patients and may be a marker of osteoclast precursors. We also noted that a subset of CD3+/CD4+ T cells expressed DC-STAMP. Herein, we examined if DC-STAMP+/CD3+/CD4+ cells have a Th17 cell signature.

Method: Peripheral blood mononuclear cells (PBMC) were isolated from PsA patients and healthy controls (HC), stained with an antibody cocktail composed of Th17-specific antibodies including IL-23R, CD161, CCR4, CCR6, and 1A2, a monoclonal antibody to DC-STAMP. The intracellular expression of IL-17, IFN-γ and IL-4 was also examined. A portion of PBMC was cultured in the presence of Th17-promoting cytokines and examined by the same antibody cocktail after 8 days in culture. IL-17 concentration in culture supernatants was evaluated by ELISA and the expression of Th17-specific genes was analyzed by real-time PCR (RT-PCR). Cellular localization of DC-STAMP on DC-STAMP+/CD3+/CD4+ T cells and DC-STAMP+/CD14+ monocytes was visualized and compared by Amnis ImageStream.

Result:Analysis of flow data from PsA patients (n=32) revealed a positive correlation between the percentage of DC-STAMP+ and DC-STAMP+/CD3+ cells (Pearson correlation=0.506, p=0.003). We identified a unique subset of CD3+/CD4+/DC-STAMP+ cells with a Th17 cell signature (CCR4+/CCR6+/IL23R+/IL17A+/CD161+). These cells expressed low levels of intracellular IL-4 and IFN-γ. Importantly, this subset was detected in PsA patients but not in HC. After 8-day culture in Th17-promoting conditions, the percentage of DC-STAMP+ cells was increased 2-fold and the Th17 phenotype was greatly enhanced. These cells secreted increased levels of IL-17 compared to unstimulated cells. RT-PCR analysis revealed an increase in CCR4, CCR6, IL23R and RORgammaT gene expression, which was associated with an elevated DC-STAMP signal after culture. DC-STAMP+/CD3+ cells from tonsil were distinct from those in PBMC and did not have Th17 signature. DC-STAMP was mainly expressed on the cell surface of DC-STAMP+/CD14+ monocytes but was found evenly distributed in the cytoplasm of DC-STAMP+/CD3+/CD4+ T cells.

Conclusion:A novel CD3+/CD4+/DC-STAMP+ cell population with a distinct Th17 cell signature was identified in a subset of PsA patients. This T cell phenotype was enhanced in the presence of Th17-promoting cytokines in vitro and was associated with increased secretion of IL-17. The intracellular localization of DC-STAMP on CD3+/CD4+ T cells suggests internalization of receptor/antibody complexes, a phenomenon commonly observed in chemokine receptors. Distinct cellular localization of DC-STAMP in CD14+ monocytes and CD3+/CD4+ T cells suggests divergent biological functions of DC-STAMP in these 2 cell lineages. Future studies on this unique DC-STAMP+ T cell subset may provide new insights into the mechanisms of inflammation and bone destruction regulated by T cells in PsA.