Plasmacytoid dendritic cells (pDC) are rare cells found in peripheral blood and lymphoid tissues. pDC are considered to be “professional” type I interferon (IFN) producing cells and produce 10–100-fold more IFN-α than other cell types in response to enveloped viruses or synthetic TLR-7 and -9 agonists. In this study, purified pDC were found to express high levels of IFN-λ receptor mRNA as well as cell-surface IFN-λ receptor. We have developed intracellular flow cytometry assays using antibodies to IFN-λ1/3 or -λ2 to assess the expression of IFN-λ proteins by pDC. We observed that a subset of human pDC expresses only intracellular IFN-α while another subset produces both IFN-α and IFN-λ after stimulation with virus or the TLR9 agonist, CpGA; the cells that co-expressed IFN-α and IFN-λ were the cells with the highest levels of IFN-α expression. Antibody cross-linking of CD4 or BDCA-2 molecules on pDC inhibited both HSV-induced IFN-λ and IFN-α production. Like the production of IFN-α, the HSV-induced IFN-λ production in pDC was mediated through TLR9 and independent of virus replication. Exogenous IFN-λ treatment of pDC resulted in increased virus-induced expression of both IFN-α and IFN-λ. In addition, both exogenous IFN-λ and –α inhibited dexamethasone-induced apoptosis of pDC. We conclude that pDC are major producers of IFN-λ1 and –λ2 in response to viral stimulation and also express functional receptors for this cytokine. Thus, IFN-λ can serve as an autocrine signal to strengthen the antiviral response of pDC by increasing IFN-α and IFN-λ production, resulting in prolonged pDC survival.

Chimeric Antigen Receptor (CAR) T cells are recognized as efficacious therapies with demonstrated ability to produce durable responses in blood cancer patients. Regulatory approvals and acceptance of these unique therapies by patients and reimbursement agencies have led to a significant increase in the number of next generation CAR T clinical trials. Flow cytometry is a powerful tool for comprehensive profiling of individual CAR T cells at multiple stages of clinical development, from product characterization during manufacturing to longitudinal evaluation of the infused product in patients. There are unique challenges with regard to the development and validation of flow cytometric methods for CAR T cells; moreover, the assay requirements for manufacturing and clinical monitoring differ. Based on the collective experience of the authors, this recommendation paper aims to review these challenges and present approaches to address them. The discussion focuses on describing key considerations for the design, optimization, validation and implementation of flow cytometric methods during the clinical development of CAR T cell therapies.

Human pDC recognize Influenza/Flu and HSV through TLR7 and -9, respectively, resulting in production of type I and III IFN and pro-inflammatory cytokines. We observed that TLR7 is constitutively present in the ER and LAMP1-positive late endosomes in pDC, and upon stimulation with Flu, TLR7 co-localization with late endosomes increased. Synthetic small molecule TLR7 ligands from the imidazoquinoline family are being used for anti-viral therapy and as immune adjuvants. We investigated the mechanisms by which 3M003 induces type I and III IFN production in pDCs. We found that 3M003 induced IFNs much more rapidly than Flu or HSV, but at much lower levels. Surprisingly, neither the TLR7 inhibitor IRS661 nor cysteine endolysosomal protease inhibitor Z-FM-FMK affected 3M003 induced IFN production although both effectively inhibited Flu-induced IFN production. In addition, 3M003 inhibited Flu, HSV, HIV and CpGA induced IFN production in pDC when used together with these TLR7/9 agonists. However, 3M003 did not affect uptake by pDC. Interestingly, 3M003 inhibited virus-induced IRF7 nuclear translocation while enhancing its phosphorylation. Taken together, these results suggest that in pDCs, 3M003 utilizes signaling pathways distinct from HSV or Flu resulting in the different levels of IFN production.

Human plasmacytoid dendritic cells (pDC) recognize single stranded RNA and double stranded DNA viruses through endosomal TLR7 and -9, respectively, resulting in production of type I and III interferons (IFN). pDC produce both IFN-α and IFN-λ upon Influenza (Flu) virus stimulation; this induction of IFN is dependent on signaling through TLR7 since the TLR7 inhibitor, IRS661, inhibited Flu-induced IFN production in pDC, but not HSV-induced IFN, which signals through TLR9. Cathepsins (Cat), cysteine endolysosomal proteases, are essential for antigen processing in flu-stimulated murine DCs. We found that the broad Cat inhibitor, Z-FM-FMK, inhibited flu and HIV-induced IFN-α production by pDC, both which require processing to make their RNA accessible to TLR7, but had no effect on the small molecule TLR7 ligand Imiquimod-induced IFN production. Flu is known to enter cells via the endocytic pathway, with viral membrane fusion with the endolysosomal membrane required for the release of its viral RNA into the cytoplasm and its subsequent replication. Using ImageStream imaging flow cytometry, we observed that TLR7 is constitutively present in the EEA1 (early)- and LAMP1 (late)-positive endosomes in pDC, and, upon stimulation with flu, TLR7 co-localization with LAMP1-positive endosomes increases. Taken together, these results indicate that recognition of Flu in pDC requires virus endocytosis, Cat processing, and results in TLR7 movement from early to late endosomes and IFN production.