CD301b + migratory cDC2 cells temporarily retain polyclonal CD4 T cells during priming to scan their antigen specificity.

Plasmacytoid dendritic cells (pDC) are potent producers of IFN-α in response to DNA and RNA viruses and subsequently mature into antigen presenting cells. We investigated the role of DNA methyltransferase 1 (DNMT1) and Ten-eleven translocation methyl-cytosine dioxygenase 2 (TET2), which are generally known as gene silencers and activators, respectively, in pDC activation. PBMC from healthy donors were stimulated with Herpes Simplex Virus (HSV) or Influenza-A virus (IAV) for 6- and 24h. PBMC were surface-stained for pDC markers CD123, HLA-DR and CD11C and co-stimulatory markers CD80 and CD86, then intracellularly for DNMT1, TET2 and IFN-α, and acquired by flow cytometry. mRNA expression of DNMT1 in purified pDC was determined by qRT-PCR. At 6h, DNMT1 protein was expressed at equivalent levels in pDC with and without stimulation and in IFN-α+ and IFN-α-pDC, and in unstimulated controls. In contrast, TET2 proteins were elevated in IFN-α+ but not IFN-α-pDC and controls at 6h. At 24h, DNMT1 increased while TET2 was reduced in stimulated pDC, correlating with high IFN-α levels at 6h and low levels at 24h. There was no change in DNMT1 gene expression in virus-stimulated vs. unstimulated controls. CD80 and CD86 were elevated at 24h compared to 6h in stimulated pDC, confirming pDC differentiation from IFN-α producing to antigen presenting cells. At the point of pDC differentiation, TET2 levels were decreased and DNMT1 increased. These results suggest that TET2 is demethylating and causing activation of genes associated with pDC IFN-α production, while DNMT1 methylates and silences genes in this pathway. This results in an initial induction of TET2 and IFN-α production followed by upregulation of co-stimulatory markers, DNMT1 and loss of IFN-α. Supported by 1. AAI trainee fellowship Careers in Immunology Fellowship Program AAI EIN #: 52-2317193 2. R01Al106125

Plasmacytoid dendritic cells (pDC) are innate immune cells and potent producers of interferon alpha (IFN-α). Sars-CoV-2, an RNA virus that causes Coronavirus Disease 2019 (COVID-19), has taken the lives of more than 400,000 people in the United States. Reports indicate that COVID-19 patients have reduced plasma IFN-α, suggesting a potential use of IFN-α as a disease therapeutic. However, investigations on pDC function and phenotype in COVID-19 patients are needed. We isolated peripheral blood mononuclear cells from fifty hospitalized COVID-19 patients. PBMC were stimulated with HSV-1 or Influenza A virus and IFN-α production and phenotype were assessed by flow cytometry. After stimulation, there were fewer IFN-α+ pDC from COVID-19 patients compared to controls. To reduce inflammation, COVID-19 patients may be treated with dexamethasone, a corticosteroid that has negative effects on pDC function. Although there was more impairment of pDC numbers and function in dexamethasone-treated subjects, the pDC dysregulation was also seen prior to dexamethasone treatment. Phenotypically, we identified reduced expression of pDC markers BDCA2 and CD123, and an upregulation of co-stimulatory markers on pDC from COVID-19 patients. We also observed an increased proportion of Ki67+ pDCs, which indicates increased turnover of pDCs during moderate to severe COVID-19 disease. In summary, pDC from COVID-19 patients produce significantly less IFN-α, express costimulatory ligands used to stimulate adaptive immunity, and may be undergoing rapid turnover. Overall, these changes may compromise the antiviral and adaptive immune response or represent pDC exhaustion during SARS-CoV-2 infection.

Plasmacytoid dendritic cells (pDC) are innate immune cells that represent 0.2–0.5% of human PBMC and produce IFN-α upon TLR7 or -9 activation by virus to stimulate innate and adaptive responses. Because of its potency and propensity to induce autoimmunity, IFN-α production must be tightly controlled. We investigated the role of histone 3 lysine 9 di-methylation (H3K9me2) and DNA Methyltransferase 1 (DNMT1) in IFN-α gene silencing in virus-activated pDC. DNMT1 recruits histone deacetylases, which are known to regulate cytokines. H3K9me2 is methylated by G9a methyltransferase, which suppresses IFN-α in fibroblasts. However, in virus-activated pDC, H3K9me2 may induce IFN-α. PBMCs were isolated from healthy donors and stimulated with IAV, HIV, or HSV for 24-hrs. We surface stained for pDC markers CD123 and HLA-DR, and CD11c, then intracellular stained for DNMT1. We also treated pDC with G9a methyltransferase inhibitor, BIX01294, and measured IFN-α and DNMT1 6-hr post stimulation with HSV and IAV. qRT-PCR was performed to measure DNMT1, demethylases, and G9a mRNA in purified pDC. In IAV and HSV stimulated pDC, DNMT1 was significantly upregulated at 24-hrs compared to 8-hr stimulated and unstimulated controls. IFN-α production and DNMT1 expression in IAV-activated pDC, but not in HSV, was significantly inhibited by BIX01294. Furthermore, G9a methyltransferase mRNA was elevated in IAV and HIV-1 purified pDC, but absent in HSV-stimulated cells. The elevation of DNMT1 may assist with limiting IFN-α production in pDC at 24-hrs to prevent excessive immune activation. Further, the reduction of DNMT1 in IAV, but not HSV-activated pDC upon treatment with BIX01294 implies a virus or TLR pathway (TLR7 vs -9) specific role of DNMT1.

Background/ Objective Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver, with over half a million new cases diagnosed annually worldwide. The fatality of HCC is high despite administration of traditional chemotherapies. In recent years, immunotherapy has become a promising strategy to treat cancer, such as PD‐1/PDL‐1 immune checkpoint blockade. However, the interactions between immune cells and HCC and how immunotherapies work, in vivo, in HCC are still unclear. Non‐alcoholic steatohepatitis (NASH) is an extremely severe form of fatty liver disease and often associated with heavy infiltration of immune cells. Natural Killer (NK) cells and plasmacytoid dendritic cells (pDC) are critical in the innate immune response, and are known for their cytotoxic and interferon producing abilities in response to cancer cells and viral pathogens, respectively. To learn more about the role of innate immunity in liver diseases, we evaluated and characterized pDC and NK cells in HCC and NASH compared to their background hepatic tissue. Methods NK cells and pDC were isolated from HCC, NASH and hepatic tissue, stained with CD3, CD56, and CXCR6 or BDCA2 and CD123, respectively. The flow cytometry data were acquired and analyzed using FlowJo. E2‐2 immunostain for pDC was performed on FFPE tissue of HCC and NASH. Results The presence of pDC was identified in the lymphoid aggregates of HCC and background cirrhotic areas (6/9 cases). In contrast, in 3 HCC cases, no pDCs were appreciated either in tumors or their backgrounds, including two patients who were smokers and with a history of coronary artery disease. In addition, using flow cytometry we demonstrated that there were more pDC in tumor infiltrating lymphocytes (TILs) of HCC compared to adjacent and distant hepatic tissues (Figure 1). We also found that NK cells isolated from NASH livers do not express CXCR6, a marker for liver‐resident NK cells, in contrast to NK cells obtained from HCC that express high levels of CXCR6 in both tumor and non‐tumor areas. The NK cells from NASH appear similar to those isolated and expanded from peripheral blood as both show low levels of CXCR6 (Figure 2). This suggests that NK cells in NASH originated from the blood and completely outnumber those in the liver, while the NK cells in HCC are residents of the liver. Conclusions We have shown that both pDC and NK cells are recruited to the liver in patients with NASH or HCC. More pDC were found in HCC tumors compared to surrounding hepatic tissue. NK cells isolated from NASH do not express CXCR6, a marker for liver‐resident NK cells. This study will further uncover the roles of innate immunity in liver disease and assist in the establishment of novel immunotherapies. A. pDC were present in the lymphoid aggregates of HCC and infiltrating tumor tissue. B. The numbers of pDC in TILs of HCC, adjacent and distant hepatic tissue. Reduced expression of CXCR6 in NASH liver