Toxoplasma gondii Inactivates Human Plasmacytoid Dendritic Cells by Functional Mimicry of IL-10.

Plasmacytoid dendritic cells (pDCs) are the major producers of IFN-α, an antiviral cytokine involved in immunomodulation and control of HIV type 1 replication, whereas Toxoplasma gondii is a life-threatening opportunistic infection in AIDS patients. During infection with HIV type 1, human pDCs decrease in circulation and remaining pDC produce lower amounts of IFN-α in response to viral stimulation. In this study, we investigated the impact of coinfection with T. gondii on the innate virus-directed responses of human pDCs. Using intracellular flow cytometry and fluorescence microscopy, we determined that T. gondii invaded but did not induce IFN-α or TNF-α in human pDC. However, T. gondii inhibited IFN-α and TNF-α produced in response to HSV and HIV, thus functionally inactivating pDC. IFN-α production was inhibited only in cells infected by T. gondii, which inhibited neither uptake of GFP-HSV nor localization of TLR9 in CD71+ endosomes, directing us to investigate downstream events. Using imaging flow cytometry, we found that both T. gondii and IL-10 inhibited virus-induced nuclear translocation, but not phosphorylation, of IFN response factor 7. Blockade of IFN response factor 7 nuclear translocation and inhibition of the IFN-α response was partially reversed by a deficiency in the T. gondii-derived ROP16 kinase, known to directly phosphorylate STAT3, a critical mediator of IL-10's anti-inflammatory effects. Taken together, our results indicate that T. gondii suppresses pDC activation by mimicking IL-10's regulatory effects through an ROP16 kinase-dependent mechanism. Our findings further imply a convergent mechanism of inhibition of TLR signaling by T. gondii and IL-10 and suggest potential negative consequences of HIV/T. gondii coinfection.

Best practices for the development, analytical validation and clinical implementation of flow cytometric methods for chimeric antigen receptor T cell analyses.

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.