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Experimental autoimmune encephalomyelitis (EAE), a Th1-mediated inflammatory disease of the central nervous system (CNS), is a model of human multiple sclerosis. Cytosolic phospholipase A2α (cPLA2α), which initiates production of prostaglandins, leukotrienes, and platelet-activating factor, is present in EAE lesions. Using myelin oligodendrocyte glycoprotein (MOG) immunization, as well as an adoptive transfer model, we showed that cPLA2α-/- mice are resistant to EAE. Histologic examination of the CNS from MOG-immunized mice revealed extensive inflammatory lesions in the cPLA2α-/- mice, whereas the lesions in cPLA2α-/- mice were reduced greatly or completely absent. MOG-specific T cells generated from WT mice induced less severe EAE in cPLA2α-/- mice compared with cPLA2α-/- mice, which indicates that cPLA2α-/- plays a role in the effector phase of EAE. Additionally, MOG-specific T cells from cPLA2α-/- mice, transferred into WT mice, induced EAE with delayed onset and lower severity compared with EAE that was induced by control cells; this indicates that cPLA2α-/- so plays a role in the induction phase of EAE. MOG-specific T cells from cPLA2α-/- mice were deficient in production of Th1-type cytokines. Consistent with this deficiency, in vivo administration of IL-12 rendered cPLA2α-/- mice susceptible to EAE. Our data indicate that cPLA2α plays an important role in EAE development and facilitates differentiation of T cells toward the Th1 phenotype. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells kill target cells following triggering via germline-encoded receptors interacting with target cell-expressed ligands (direct killing), or via antibody-dependent cellular cytotoxicity (ADCC) mediated by FcγRIIIa. NK cytotoxicity is modulated by signaling through activating or inhibitory receptors. A major checkpoint is mediated by the NK inhibitory receptor NKG2A/CD94 and its target cell ligand, HLA-E, which is complexed with HLA signal sequence-derived peptides termed VL9 (HLA-E-VL9). We have previously reported the isolation of a murine HLA-E-VL9-specific IgM antibody 3H4 and the generation of a higher affinity IgG version (3H4v3). Here we have used phage display library selection to generate a high affinity version of 3H4v3, called 3H4v31, with an ∼700 fold increase in binding affinity. We show using an HLA-E-VL9+ K562 tumor model that, in vitro, the addition of 3H4v31 to target cells increased direct killing of targets by CD16-negative NK cell line NK-92 and also mediated ADCC by NK-92 cells transfected with CD16. Moreover, ADCC by primary NK cells was also enhanced in vitro by 3H4v31. 3H4v31 was also able to bind and enhance target cell lysis of endogenously expressed HLA-E-VL9 on human cervical cancer and human pancreatic cancer cell lines. In vivo, 3H4v31 slowed the growth rate of HLA-E-VL9+ K562 tumors implanted into NOD/SCID/IL2rγ null mice compared to isotype control when injected with NK-92 cells intratumorally. Together, these data demonstrate that mAb 3H4v31 can enhance NK cell killing of HLA-E-VL9-expressing tumor cells in vitro by both direct killing activity and by ADCC. Moreover, mAb 3H4v31 can enhance NK cell control of tumor growth in vivo. We thus identify HLA-E-VL9 monoclonal antibodies as a promising novel anti-tumor immunotherapy., One Sentence Summary: A high affinity monoclonal antibody against HLA-E-VL9 enhances natural killer cell anti-tumor killing by checkpoint inhibition and antibody dependent cellular cytotoxicity.

Lattice light-sheet microscopy (LLSM) is valuable for its combination of reduced photobleaching and outstanding spatiotemporal resolution in 3D. Using LLSM to image biosensors in living cells could provide unprecedented visualization of rapid, localized changes in protein conformation or posttranslational modification. However, computational manipulations required for biosensor imaging with LLSM are challenging for many software packages. The calculations require processing large amounts of data even for simple changes such as reorientation of cell renderings or testing the effects of user-selectable settings, and lattice imaging poses unique challenges in thresholding and ratio imaging. We describe here a new software package, named ImageTank, that is specifically designed for practical imaging of biosensors using LLSM. To demonstrate its capabilities, we use a new biosensor to study the rapid 3D dynamics of the small GTPase Rap1 in vesicles and cell protrusions., (© 2019 O'Shaughnessy et al.)

Experimental autoimmune encephalomyelitis (EAE), a Th1-mediated inflammatory disease of the central nervous system (CNS), is a model of human multiple sclerosis. Cytosolic phospholipase A2alpha (cPLA2alpha), which initiates production of prostaglandins, leukotrienes, and platelet-activating factor, is present in EAE lesions. Using myelin oligodendrocyte glycoprotein (MOG) immunization, as well as an adoptive transfer model, we showed that cPLA2alpha-/- mice are resistant to EAE. Histologic examination of the CNS from MOG-immunized mice revealed extensive inflammatory lesions in the cPLA2alpha+/- mice, whereas the lesions in cPLA2alpha-/- mice were reduced greatly or completely absent. MOG-specific T cells generated from WT mice induced less severe EAE in cPLA2alpha-/- mice compared with cPLA2alpha+/- mice, which indicates that cPLA2alpha plays a role in the effector phase of EAE. Additionally, MOG-specific T cells from cPLA2alpha-/- mice, transferred into WT mice, induced EAE with delayed onset and lower severity compared with EAE that was induced by control cells; this indicates that cPLA2alpha also plays a role in the induction phase of EAE. MOG-specific T cells from cPLA2alpha-/- mice were deficient in production of Th1-type cytokines. Consistent with this deficiency, in vivo administration of IL-12 rendered cPLA2alpha-/- mice susceptible to EAE. Our data indicate that cPLA2alpha plays an important role in EAE development and facilitates differentiation of T cells toward the Th1 phenotype.