Rapid and high throughput assessment of cellular immunity against SARS-CoV-2 based on the ex vivo activation of genes in leukocyte assay with whole blood.

During the novel coronavirus outbreak and vaccine development, antibody production garnered major focus as the primary immunogenic response. However, cellular immunity's recent demonstration of comparable or greater significance in controlling infection demands the re-evaluation of the importance of T-cell immunity in SARS-CoV-2 infection. Here, we developed a novel assay, the ex vivo activation of genes in leukocytes (EAGL), which employs short-term whole blood stimulation with the LeukoComplete™ system, to measure ex vivo SARS-CoV-2-specific T cell responses (cellular immunity). This assay measures upregulated mRNA expression related to leukocyte activation 4 h after antigen stimulation. LeukoComplete™ system uses whole blood samples, eliminating the need for pretreatment before analysis. Furthermore, this system's high reproducibility is ensured through a series of operations from mRNA extraction to cDNA synthesis on a 96-well plate. In the performance evaluation using fresh blood from previously SARS-CoV-2-infected and COVID-19-vaccinated individuals, the EAGL assay had a comparable sensitivity and specificity to the ELISpot assay (EAGL: 1.000/1.000; ELISpot: 0.900/0.973). As a simple, high-throughput assay, the EAGL assay is also a quantitative test that is useful in studies with large sample numbers, such as monitoring new vaccine efficacies against novel coronaviruses or epidemiologic studies that require cellular immune testing during viral infection.

MEIS1-mediated transactivation of synaptotagmin-like 1 promotes CXCL12/CXCR4 signaling and leukemogenesis.

The TALE-class homeoprotein MEIS1 specifically collaborates with HOXA9 to drive myeloid leukemogenesis. Although MEIS1 alone has only a moderate effect on cell proliferation in vitro, it is essential for the development of HOXA9-induced leukemia in vivo. Here, using murine models of leukemogenesis, we have shown that MEIS1 promotes leukemic cell homing and engraftment in bone marrow and enhances cell-cell interactions and cytokine-mediated cell migration. We analyzed global DNA binding of MEIS1 in leukemic cells as well as gene expression alterations in MEIS1-deficent cells and identified synaptotagmin-like 1 (Sytl1, also known as Slp1) as the MEIS1 target gene that cooperates with Hoxa9 in leukemogenesis. Replacement of SYTL1 in MEIS1-deficent cells restored both cell migration and engraftment. Further analysis revealed that SYTL1 promotes cell migration via activation of the CXCL12/CXCR4 axis, as SYTL1 determines intracellular trafficking of CXCR4. Together, our results reveal that MEIS1, through induction of SYTL1, promotes leukemogenesis and supports leukemic cell homing and engraftment, facilitating interactions between leukemic cells and bone marrow stroma.

A cell-type-specific requirement for IFN regulatory factor 5 (IRF5) in Fas-induced apoptosis.

Apoptosis is a highly regulated process of cell suicide that occurs during development, host defense, and pathophysiology. The transcription factor IFN regulatory factor 5 (IRF5), known to be involved in the activation of innate immune responses, recently has been shown to be critical for DNA damage-induced apoptosis and tumor suppression. Here, we report on a cell-type-specific role of IRF5 in promoting apoptosis upon signaling through the death receptor Fas (CD95/APO-1/TNFRSF6). In particular, we show that mice deficient in the Irf5 gene are resistant to hepatic apoptosis and lethality in response to the in vivo administration of a Fas-activating monoclonal antibody, and that IRF5 is involved in a stage of Fas signaling that precedes the activation of caspase 8 and c-Jun N-terminal kinase (JNK). In addition to hepatocytes, IRF5 is also required for apoptosis in dendritic cells activated by hypomethylated CpG but not in thymocytes and embryonic fibroblasts in vitro. Thus, these findings reveal a cell-type-specific function for IRF5 in the complex regulatory mechanism of death-receptor-induced apoptosis.

Expression of a dominant negative form of Daxx in vivo rescues motoneurons from Fas (CD95)-induced cell death.

Fas-induced death of motoneurons in vitro has been shown to involve two signaling cascades that act together to execute the death program: a Fas-Daxx-ASK-1-p38 kinase-nNOS branch, which controls transcriptional and post-translational events, and the second classical Fas-FADD-caspase-8 branch. To analyze the role of Daxx in the developmental motoneuron cell death, we studied Fas-dependent cell death in motoneurons from transgenic mice that overexpress a dominant-negative form of Daxx. Motoneurons purified from these transgenic mice are resistant to Fas-induced death. This protective effect is specific to Fas because ultraviolet irradiation-triggered death is not affected by the transgene. The Daxx and the FADD pathways work in parallel because only Daxx, but not FADD, is involved in the transcriptional control of neuronal nitric oxide synthase and nitric oxide production. Nevertheless, we do not observe involvement of Daxx in developmental motoneuronal cell death, as the pattern of naturally occurring programmed cell death in vivo is normal in transgenic mice overexpressing the dominant negative form of Daxx, suggesting that Daxx-independent pathways are used during development.

An essential role for membrane rafts in the initiation of Fas/CD95-triggered cell death in mouse thymocytes.

Fas, a member of the tumor necrosis factor receptor family, can upon ligation by its ligand or agonistic antibodies trigger signaling cascades leading to cell death in lymphocytes and other cell types. Such signaling cascades are initiated through the formation of a membrane death-inducing signaling complex (DISC) that includes Fas, the Fas-associated death domain protein (FADD) and caspase-8. We report here that a considerable fraction of Fas is constitutively partitioned into sphingolipid- and cholesterol-rich membrane rafts in mouse thymocytes as well as the L12.10-Fas T cells, and Fas ligation promotes a rapid and specific recruitment of FADD and caspase-8 to the rafts. Raft disruption by cholesterol depletion abolishes Fas-triggered recruitment of FADD and caspase-8 to the membrane, DISC formation and cell death. Taken together, our results provide the first demonstration for an essential role of membrane rafts in the initiation of Fas-mediated cell death signaling.