Adaptor DAP12 Research Articles

Abstract 1319: In vivo programming of natural killer cells and T cells using mRNA delivered cytotoxic chimeric antigen receptors

Abstract Introduction: Current generation of adoptive cell therapy relies on ex vivo engineering of immune cells with tumor-targeting chimeric antigen receptors. In addition to manufacturing challenges, ex vivo engineered cells such as CAR-NK and CAR-T show limited tumor infiltration and persistency within tumor microenvironment. A method leveraging mRNA-based delivery for in-vivo engineering of these cells could address these issues but has not been established. Conventional mRNA/LNP when infused systemically result in significant accumulation in liver tissue and the CAR has no immune cell specificity in their expression or function. Method: Many immune receptors are heterodimeric complexes composed of a ligand binding subunit (e.g. CD16 and TCR) and a signaling adaptor containing Immunoreceptor Tyrosine-based Activation Motif (ITAM) (e.g. FcRγ and CD3ζ). Notably, the cell surface expression of the ligand binding subunit often depends on the expression of the ITAM adaptor. Based on this we fused HER2-targeting scFv to multiple activating receptors whose expression is restricted to NK cells and T cells, and evaluated their expression and anti-tumor function. Results: We developed a series of Natural Cytotoxic Receptor (NCR)-based in vivo CAR constructs by fusing HER2-targeting scFv to the native extracellular and transmembrane sequences of the receptor NKp30, NKp44 and NKp46, These CARs engage signaling adaptor FcRγ or DAP12 expressed in NK cells, and exhibit potent tumoricidal activity against HER2+ ovarian cancer cells as well as mediate tumor-induced cytokine and chemokine response. Using Huh7 liver cell as a model for off-target expression in hepatocytes, we found that NKp44 and NKp46-based CAR requires their cognate signaling adaptors for stable cell surface expression. We also showed that similar strategy can be used to design receptors for selective expression on the surface of T cells utilizing CD3ε chain fused with tumor targeting scFv. Conclusion: These results demonstrated the feasibility of engineering primary NK cells with CARs based on the NCR family of receptors NKp44 and NKp46, whose expression requires the immune-restricted signaling adaptors. These CAR-NK receptors do not contain intracellular signaling motifs but can engage and activate their signaling natural adaptors to initiate tumor killing and cytokine response. An mRNA/LNP based delivery of NKp44-based CAR for cancer treatment is currently under clinical development. Citation Format: Neha Diwanji, Daniel Getts, Yuxiao Wang. In vivo programming of natural killer cells and T cells using mRNA delivered cytotoxic chimeric antigen receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1319.

Chimeric Antigen Cytotoxic Receptors for In Vivo Engineering of Tumor-Targeting NK Cells.

Chimeric Ag receptor (CAR) NK cells are challenging to manufacture and fail to achieve consistent tumor infiltration and sustained cytolytic function in the tumor microenvironment. In vivo engineering of NK cells using mRNA-based CAR delivery may overcome these issues. In this study, we developed an invivo programming method by designing CARs that leverage the biology of NK cell receptors for cell type-specific expression and function. These CARs were engineered by fusion of a tumor recognition domain with the natural cytotoxic receptor family including NKp30, NKp44, and NKp46. Our results demonstrated that these natural cytotoxic receptor-based CARs can engage endogenous signaling adaptors to effectively activate human NK cells for tumor lysis and cytokine production. Specifically, we discovered that stable expression of an NKp44-based CAR was contingent on the presence of the immune cell-specific signaling adaptor DAP12. This innovative strategy facilitates direct in situ programming of NK cells, enhancing safety and minimizing off-target effects in nontargeted, healthy tissues.

Interleukin-3-dependent potentiation of IgE responsiveness in mouse basophils.

Basophils produce interleukins (IL)-4 in response to various stimuli and may contribute to type 2 immune responses to various infections and allergens. We found that resting basophils freshly isolated from mice produce IL-4 in response to IL-3 but not to high-affinity Fc receptor (FcεRI) cross-linking (CL), yet both required the immunoreceptor tyrosine-based activation motif (ITAM) containing adaptor Fc receptor γ-chain (FcRγ), while basophils activated in vitro by IL-3 become responsive to FcεRI CL. Acquisition of responsiveness to FcεRI CL occurred upon infection with Trichinella spiralis or administration of superantigen. Because cultured basophils return to a quiescent state upon starvation with IL-3 with surface FcεRI levels unchanged, this acquisition is reversible and probably reflects intracellular events requiring protein synthesis. Interestingly, similar activation-associated acquisition was observed for responsiveness to other stimuli, including CD200R3 CL, which is known to signal via DAP-12, and the allergen protease papain. This acquisition of responsiveness to FcεRI CL was inhibited by Jak inhibitor. Thus, the IL-3 signal bifurcates downstream of Jak, into two distinct pathway, one leading to IL-4 production and the other to render basophils competent to respond to stimuli dependent on ITAM-containing adaptors DAP12 and FcRγ for IL-4 production.

TREM2 drives microglia response to amyloid-β via SYK-dependent and -independent pathways

Genetic studies have highlighted microglia as pivotal in orchestrating Alzheimer's disease (AD). Microglia that adhere to Aβ plaques acquire a transcriptional signature, "disease-associated microglia" (DAM), which largely emanates from the TREM2-DAP12 receptor complex that transmits intracellular signals through the protein tyrosine kinase SYK. The human TREM2R47H variant associated with high AD risk fails to activate microglia via SYK. We found that SYK-deficient microglia cannot encase Aβ plaques, accelerating brain pathology and behavioral deficits. SYK deficiency impaired the PI3K-AKT-GSK-3β-mTOR pathway, incapacitating anabolic support required for attaining the DAM profile. However, SYK-deficient microglia proliferated and advanced to an Apoe-expressing prodromal stage of DAM; this pathway relied on the adapter DAP10, which also binds TREM2. Thus, microglial responses to Aβ involve non-redundant SYK- and DAP10-pathways. Systemic administration of an antibody against CLEC7A, a receptor that directly activates SYK, rescued microglia activation in mice expressing the TREM2R47H allele, unveiling new options for AD immunotherapy.

Abstract P104: Therapeutic targeting of TREM1 with PY159 promotes myeloid cell reprogramming and unleashes anti-tumor immunity

Abstract Myeloid cells present in the tumor microenvironment can exist in immunosuppressive states that impede productive anti-tumor immunity. One strategy for targeting these immunosuppressive mechanisms is reprogramming of myeloid cells from immunosuppressive to immunostimulatory, resulting in the removal of the immune inhibition and unleashing of anti-tumor immunity. Triggering receptor expressed on myeloid cells-1 (TREM1) is an immunoglobulin superfamily cell surface receptor expressed primarily on neutrophils and subsets of monocytes and tissue macrophages. TREM1 signals through the association with DAP12 adaptor protein and mediates proinflammatory signaling, amplifies the host immune response to microbial pathogens, and has been implicated in the development of acute and chronic inflammatory diseases. TREM1 is also enriched in tumors, specifically on tumor-associated myeloid cells. To investigate the potential of TREM1 modulation as an anti-cancer therapeutic strategy, we developed PY159, an afucosylated humanized anti-TREM1 monoclonal antibody. We found that PY159 does not deplete TREM1-expressing cells, but rather acts as a TREM1 agonist. In vitro human blood assays showed that PY159 treatment upregulated activation markers on monocytes and stimulated neutrophil chemotaxis, as assayed by flow cytometry, transcriptional analysis, and in vitro migration assays. Furthermore, PY159 induced a selective set of proinflammatory cytokines and chemokines, which was dependent on PY159 afucosylation. We validated TREM1 expression in human tumors by single-cell RNAseq, immunohistochemistry, and flow cytometry, and found that it is expressed on myeloid populations, including tumor-associated neutrophils (TAN), tumor-associated macrophages (TAM), and monocytic myeloid-derived suppressive cells (mMDSC). We showed that PY159 can also induce proinflammatory cytokines and chemokines in dissociated human tumors in vitro, demonstrating that PY159 can reprogram tumor-associated myeloid cells. Finally, in vivo treatment of mice with a surrogate anti-mouse TREM1 antibody, PY159m, promoted anti-tumor efficacy in several syngeneic mouse tumor models, both as single-agent and in combination with checkpoint inhibitors, such as anti-PD-1 antibody. Together, these results demonstrate that therapeutic targeting of TREM1 with a TREM1 agonist antibody, PY159, promotes myeloid cell reprogramming and anti-tumor immunity. PY159 safety and tolerability have been demonstrated in non-human primates, and safety and efficacy of PY159 are currently being evaluated in first-in-human clinical trial (NCT04682431) including solid tumors that are resistant and refractory to standard of care therapies. Citation Format: Erin Mayes, Vladi Juric, Mikhail Binnewies, Pamela Canaday, Tian Lee, Subhadra Dash, Joshua L. Pollack, Joshua Rudolph, Vicky Huang, Xiaoyan Du, Nadine Jahchan, Asa J. Ramoth, Shilpa Mankikar, Manith Norng, Carlos Santamaria, Kevin P. Baker, Linda Liang. Therapeutic targeting of TREM1 with PY159 promotes myeloid cell reprogramming and unleashes anti-tumor immunity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P104.

Rhesus Macaque Activating Killer Immunoglobulin-Like Receptors Associate With Fc Receptor Gamma (FCER1G) and Not With DAP12 Adaptor Proteins Resulting in Stabilized Expression and Enabling Signal Transduction.

Activating killer cell immunoglobulin-like receptors (KIR) in macaques are thought to be derived by genetic recombination of the region encoding the transmembrane and intracellular part of KIR2DL4 and a KIR3D gene. As a result, all macaque activating KIR possess a positively charged arginine residue in the transmembrane region. As human KIR2DL4 associates with the FCER1G (also called Fc receptor-gamma, FcRγ) adaptor, we hypothesized that in contrast to human and great ape the activating KIRs of macaques associate with FcRγ instead of DAP12. By applying co-immunoprecipitation of transfected as well as primary cells, we demonstrate that rhesus macaque KIR3DS05 indeed associates with FcRγ and not with DAP12. This association with FcRγ results in increased and substantially stabilized surface expression of KIR3DS05. In addition, we demonstrate that binding of specific ligands of KIR3DS05, Mamu-A1*001 and A1*011, resulted in signal transduction in the presence of FcRγ in contrast to DAP12.

Complement receptor 3 mediates both sinking phagocytosis and phagocytic cup formation via distinct mechanisms.

A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. Alternatively, CRs have been reported to induce membrane ruffles or phagocytic cups, akin to those induced by Fcγ receptors (FcγRs), but the details of these events are unclear. Here we used real-time 3D imaging and knockout mouse models to clarify how particles (human red blood cells) are internalized by resident peritoneal F4/80+ cells (macrophages) via CRs and/or FcγRs. We first show that FcγRs mediate highly efficient, rapid (2-3 min) phagocytic cup formation, which is completely abolished by deletion or mutation of the FcR γ-chain or conditional deletion of the signal transducer Syk. FcγR-mediated phagocytic cups robustly arise from any point of cell-particle contact, including filopodia. In the absence of CR3, FcγR-mediated phagocytic cups exhibit delayed closure and become aberrantly elongated. Independent of FcgRs, CR3 mediates sporadic ingestion of complement-opsonized particles by rapid phagocytic cup-like structures, typically emanating from membrane ruffles and largely prevented by deletion of the immunoreceptor tyrosine-based activation motif (ITAM) adaptors FcR γ-chain and DAP12 or Syk. Deletion of ITAM adaptors or Syk clearly revealed that there is a slow (10-25 min) sinking mode of phagocytosis via a restricted orifice. In summary, we show that (1) CR3 indeed mediates a slow sinking mode of phagocytosis, which is accentuated by deletion of ITAM adaptors or Syk, (2) CR3 induces phagocytic cup-like structures, driven by ITAM adaptors and Syk, and (3) CR3 is involved in forming and closing FcγR-mediated phagocytic cups.

Osteoclast Signal Transduction During Bone Metastasis Formation.

Osteoclasts are myeloid lineage-derived bone-resorbing cells of hematopoietic origin. They differentiate from myeloid precursors through a complex regulation process where the differentiation of preosteoclasts is followed by intercellular fusion to generate large multinucleated cells. Under physiological conditions, osteoclastogenesis is primarily directed by interactions between CSF-1R and macrophage colony-stimulating factor (M-CSF, CSF-1), receptor activator of nuclear factor NF-κB (RANK) and RANK ligand (RANKL), as well as adhesion receptors (e.g., integrins) and their ligands. Osteoclasts play a central role in physiological and pathological bone resorption and are also required for excessive bone loss during osteoporosis, inflammatory bone and joint diseases (such as rheumatoid arthritis) and cancer cell-induced osteolysis. Due to the major role of osteoclasts in these diseases the better understanding of their intracellular signaling pathways can lead to the identification of potential novel therapeutic targets. Non-receptor tyrosine kinases and lipid kinases play major roles in osteoclasts and small-molecule kinase inhibitors are emerging new therapeutics in diseases with pathological bone loss. During the last few years, we and others have shown that certain lipid (such as phosphoinositide 3-kinases PI3Kβ and PI3Kδ) and tyrosine (Src−family and Syk) kinases play a critical role in osteoclast differentiation and function in humans and mice. Some of these signaling pathways shows similarity to immunoreceptor-like receptor signaling and involves important other enzymes (e.g., PLCγ2) and adapter proteins (such as the ITAM−bearing adapters DAP12 and the Fc-receptor γ-chain). Here, we review recently identified osteoclast signaling pathways and their role in osteoclast differentiation and function as well as pathological bone loss associated with osteolytic tumors of the bone. A better understanding of osteoclast signaling may facilitate the design of novel and more efficient therapies for pathological bone resorption and osteolytic skeletal metastasis formation.

Different Calcium and Src Family Kinase Signaling in Mac-1 Dependent Phagocytosis and Extracellular Vesicle Generation.

Encountering opsonized particles by neutrophils results in phagocytosis of the particle and generation of extracellular vesicles with antibacterial property (aEV). The aim of the present study is to compare the involvement of different receptors and receptor-proximal signaling pathways in these two parallel processes. Investigating human neutrophils from peripheral blood, we show that complement receptors are decisive for both processes whereas immunoglobulin binding Fc receptors (FcR) only participate moderately in phagocytosis and pattern recognition receptors induce mild EV production but only minimal phagocytosis. Studying bone marrow derived neutrophils of genetically modified animals we verify that the involved complement receptor is CR3, also known as the β2 integrin Mac-1. We show that genetic deletion of the adaptor molecules FcRγ chain or DAP12 does not influence either process, suggesting potential redundant function. Combined absence of the Src family kinases Hck, Fgr, and Lyn drastically impairs phagocytosis but does not influence aEV production. In contrast, deletion of PLCγ2 has no influence on phagocytosis, but reduces aEV formation. In accord with the essential role of PLCγ2, aEV biogenesis both from murine and from human neutrophils is dependent on presence of extracellular calcium. Absence of external calcium prevented the generation of antibacterial EVs, whereas the spontaneous EV formation was not influenced. We thus show that phagocytosis and biogenesis of antibacterial EVs are independent processes and proceed on different signaling pathways although the same receptor plays the critical role in both. Our data reveal the possibility in neutrophilic granulocytes to modulate aEV production without disturbing the phagocytic process.

MICA Regulates the Expression of DAP10 and Signals through an Independent PI3K Pathway in NKG2D Positive Cervical Cancer Cells

NKG2D receptor engages ligands such as MICA and MICB, which activates cytotoxicity in NK cells leading to the destruction of tumour cells expressing these ligands. In normal human lymphoid cells the association of DAP10 with NKG2D is essential for signalling and important for its cell surface expression. However, the mechanism of the NKG2D/DAP10 complex upregulation is not completely understood in cancer. Also, the role of DAP10 in the activation of the PI3/AKT signaling pathway in cervical cancer has not been fully elucidated. In the present study, we investigated the role of MICA in the regulation of DAP10 in cervical cancer cells. First, we demonstrate the presence of the NKG2D/DAP10 complex in different tumour cell lines by flow cytometry. Also, we demonstrate that MICA upregulates the expression of DAP10 in cervical cancer cells in a time dependent manner by immunoblotting. We found that the AKT kinase is constitutively phosphorylated and MICA induced an increase in tyrosine phosphorylation. Furthermore, this activation is independent of the PI3K in cervical cancer cell lines as determined by immunoblotting and flow cytometry. Our results provide evidence supporting the notion that MICA functions as a stimulatory molecule to regulate the expression of the receptor adapter DAP10 in cervical cancer cells and thus may contribute to their proliferation and survival. The possibility that the NKG2D-DAP10 complex is widely expressed in different types of cancer may confer an advantage to transformed cells to survive in the tumour microenvironment and escape from the immune surveillance.

NK cell receptor NKG2D sets activation threshold for the NCR1 receptor early in NK cell development.

The activation of natural killer (NK) cells depends on a change in the balance of signals from inhibitory and activating receptors. The activation threshold values of NK cells are thought to be set by engagement of inhibitory receptors during development. Here, we found that the activating receptor NKG2D specifically set the activation threshold for the activating receptor NCR1 through a process that required the adaptor DAP12. As a result, NKGD2-deficient (Klrk1-/-) mice controlled tumors and cytomegalovirus infection better than wild-type controls through the NCR1-induced production of the cytokine IFN-γ. Expression of NKG2D before the immature NK cell stage increased expression of the adaptor CD3ζ. Reduced expression of CD3ζ in Klrk1-/- mice was associated with enhanced signal transduction through NCR1 and CD3ζ-deficiency resulted in hyper-responsiveness to stimulation via NCR1. Thus, an activating receptor developmentally set the activity of another activating receptor on NK cells and determined NK cell-reactivity to cellular threats.

TREM2 Is a Receptor for β-Amyloid that Mediates Microglial Function

Mutations in triggering receptor expressed on myeloid cells 2 (TREM2) have been linked to increased Alzheimer's disease (AD) risk. Neurobiological functions of TREM2 and its pathophysiological ligands remain elusive. Here we found that TREM2 directly binds to β-amyloid (Aβ) oligomers with nanomolar affinity, whereas AD-associated TREM2 mutations reduce Aβ binding. TREM2 deficiency impairs Aβ degradation in primary microglial culture and mouse brain. Aβ-induced microglial depolarization, K+ inward current induction, cytokine expression and secretion, migration, proliferation, apoptosis, and morphological changes are dependent on TREM2. In addition, TREM2 interaction with its signaling adaptor DAP12 is enhanced by Aβ, regulating downstream phosphorylation of SYK and GSK3β. Our data demonstrate TREM2 as a microglial Aβ receptor transducing physiological and AD-related pathological effects associated with Aβ.

FcεRI γ-Chain Negatively Modulates Dectin-1 Responses in Dendritic Cells.

The inhibitory effect of immunoreceptor tyrosine-based activation motif (ITAM)-containing adapters DAP12 and FcεRI γ-chain (FcRγ) has been found in many immune functions. Herein, we have further explored the role of these adapters in C-type lectin receptors response. We identified that FcRγ, but not DAP12, could negatively regulate the Dectin-1 responses in dendritic cells (DCs). Loss of FcRγ or both DAP12 and FcRγ enhanced the maturation and cytokine production in DCs upon Dectin-1 activation compared to normal cells, whereas DCs lacking only DAP12 showed little changes. In addition, increments of T cell activation and T helper 17 polarization induced by FcRγ-deficient DCs were observed both in vitro and in vivo. Examining the Dectin-1 signaling, we revealed that the activations of several signaling molecules were augmented in FcRγ-deficient DCs stimulated with Dectin-1 ligands. Furthermore, we demonstrated that the association of phosphatases SHP-1 and PTEN with FcRγ may contribute to the negative regulation of FcRγ in Dectin-1 activation in DCs. These results extend the inhibitory effect of ITAM-containing adapters to Dectin-1 response in immune functions, even though Dectin-1 contains an ITAM-like intracellular domain. According to the role of Dectin-1 in responding to microbes and tumor cells, our finding may have applications in the development of vaccine and cancer therapy.

Phagocytic Receptors Activate Syk and Src Signaling during Borrelia burgdorferi Phagocytosis.

Phagocytosis of the Lyme disease-causing pathogen Borrelia burgdorferi has been shown to be important for generating an inflammatory response to the pathogen. As a result, understanding the mechanisms of phagocytosis has been an area of great interest in the field of Lyme disease. Several cell surface receptors that participate in B. burgdorferi phagocytosis have been reported, including the scavenger receptor MARCO and integrin α3β1. We sought to define the mechanisms by which these receptors mediate phagocytosis and to identify signaling pathways activated downstream of these receptors upon contact with B. burgdorferi We identified both Syk and Src signaling pathways as ones that participate in B. burgdorferi phagocytosis and the resulting cytokine activation. In our studies, we found that both MARCO and integrin β1 play a role in the activation of the Src kinase pathway. However, only integrin β1 participates in the activation of Syk. Interestingly, the integrin activates Syk without the help of the signaling adaptor Dap12 or FcRγ. Thus, we report that multiple pathways participate in B. burgdorferi internalization and that different cell surface receptors act simultaneously in cooperation and independently to mediate phagocytosis.

OH MYeloid! Immune cells wreaking havoc on brain homeostasis

Genetic mutations responsible for neurodegenerative Nasu‐Hakola disease have been localized to the gene TREM2 and its adaptor DAP12, but it remained unclear what causes the brain to deteriorate. In this issue of The EMBO Journal, Kleinberger et al (2017) provide intriguing evidence suggesting a TREM2 mutation alone can lead to striking microglial dysfunction and precipitate changes in cerebral blood flow and metabolism in mice.

Modulation of Human Leukocyte Antigen-C by Human Cytomegalovirus Stimulates KIR2DS1 Recognition by Natural Killer Cells.

The interaction of inhibitory killer cell Ig-like receptors (KIRs) with human leukocyte antigen (HLA) class I molecules has been characterized in detail. By contrast, activating members of the KIR family, although closely related to inhibitory KIRs, appear to interact weakly, if at all, with HLA class I. KIR2DS1 is the best studied activating KIR and it interacts with C2 group HLA-C (C2-HLA-C) in some assays, but not as strongly as KIR2DL1. We used a mouse 2B4 cell reporter system, which carries NFAT-green fluorescent protein with KIR2DS1 and a modified DAP12 adaptor protein. KIR2DS1 reporter cells were not activated upon coculture with 721.221 cells transfected with different HLA-C molecules, or with interferon-γ stimulated primary dermal fibroblasts. However, KIR2DS1 reporter cells and KIR2DS1+ primary natural killer (NK) cells were activated by C2-HLA-C homozygous human fetal foreskin fibroblasts (HFFFs) but only after infection with specific clones of a clinical strain of human cytomegalovirus (HCMV). Active viral gene expression was required for activation of both cell types. Primary NKG2A−KIR2DS1+ NK cell subsets degranulated after coculture with HCMV-infected HFFFs. The W6/32 antibody to HLA class I blocked the KIR2DS1 reporter cell interaction with its ligand on HCMV-infected HFFFs but did not block interaction with KIR2DL1. This implies a differential recognition of HLA-C by KIR2DL1 and KIR2DS1. The data suggest that modulation of HLA-C by HCMV is required for a potent KIR2DS1-mediated NK cell activation.

Avoidance of On-Target Off-Tumor Activation Using a Co-stimulation-Only Chimeric Antigen Receptor

Chimeric antigen receptors (CARs) combine Tcell activation with antibody-mediated tumor antigen specificity, bypassing the need for Tcell receptor (TCR) ligation. A limitation of CAR technology is on-target off-tumor toxicity caused by target antigen expression on normal cells. Using GD2 as a model cancer antigen, we hypothesized that this could be minimized by using Tcells expressing Vγ9Vδ2 TCR, which recognizes transformed cells in a major histocompatibility complex (MHC)-unrestricted manner, in combination with a co-stimulatory CAR that would function independently of the TCR. An anti-GD2 CAR containing a solitary endodomain derived from the NKG2D adaptor DAP10 was expressed in Vγ9Vδ2+ Tcells. Differential ligation of the CAR and/or TCR using antibody-coated beads showed that pro-inflammatory cytokine response depended on activation of both receptors. Moreover, in killing assays, GD2-expressing neuroblastoma cells that engaged the Vγ9Vδ2 TCR were efficiently lysed, whereas cells that expressed GD2 equivalently but did not engage the Vγ9Vδ2 TCR were untouched. Differentiation between X-on tumor and X-off tumor offers potential for safer immunotherapy and broader target selection.

IL-15 stimulates NKG2D while promoting IgM expression of B-1a cells

Interleukin (IL)-15, a key manipulator of T-cell function also modulates B-1a cell activity by augmenting activation markers, turning them towards type 1 polarization and immunoglobulin (Ig) expression which is significant in the context of gut immunity. Here we show, for the first time, IL-15 mediated up-regulation of the activation receptor NKG2D and its adaptor DAP10 in B-1a cells indicating their essential coupling with IL-15 receptor signaling pathway. Our results demonstrate IL-15 treatment increases phosphorylation of STAT5 and p38 leading to translocation of NF-κB onto the nucleus, an attribute that delineates activation of B-1a cells and its role in inflammation. In parallel, increase of anti-apoptotic Bcl-xL suggests its role in long term survival of B-1a cells in culture by IL-15. The cytokine induced overexpression of the plasma cell differentiation transcription factor BLIMP-1 while reducing PAX-5a that could be responsible for the spontaneous Ig secretion by B-1a cells. Up-regulation of IgM transcripts in presence of IL-15 validates mucosal response of the cells through natural Abs to counter pathogens.

Pertussis toxin targets the innate immunity through DAP12, FcRγ, and MyD88 adaptor proteins

Activation of the innate immunity by adjuvants, such as pertussis toxin (PTX), in the presence of autoreactive lymphocytes and antigen mimicry is sufficient to trigger autoimmunity. Toll-like, C-type lectin, and immunglobulin-like receptors play an important role in the innate immunity by sensing a variety of microbial products through several adaptor proteins, including MyD88, DAP12, and FcRγ. This study investigated the interaction between PTX and innate immune components. The direct interactions between coated PTX and receptor-fusion proteins were examined using ELISA-based binding assays. Functionally, PTX-binding receptors could be classified into two groups: inhibition (DAP12-coupled TREM2, ITIM-bearing SIRPα, SIGNR1/SIGNR3/DCSIGN) and activation (MyD88-associated TLR4, DAP12-coupled LMIR5/CD300b, FcRγ-coupled LMIR8/CD300c, CLEC9A, MGL-1). DAP12, MyD88, and FcRγ were selected for further investigation. A comprehensive analysis of gene transcription showed that PTX up-regulated the expression of various inflammatory mediators. DAP12 deficiency resulted in reduction or enhancement of inflammatory responses in a cytokine-specific manner. PTX was able to activate the TREM2-DAP12 signalling pathway. PTX induced lower expression of inflammatory mediators in the absence of FcRγ alone and substantially lost its inflammatory capacity in the absence of both FcRγ and MyD88. PTX was able to activate the MyD88-NF-κB signalling pathway in the presence of TLR2 or TLR4. The inflammatory activity of PTX was completely lost by heating. These results demonstrate that PTX targets the innate immunity through DAP12, FcRγ, and MyD88 providing new insights into the immunobiology of PTX.

Measles virus nucleocapsid protein modulates the Signal Regulatory Protein-β1 (SIRPβ1) to enhance osteoclast differentiation in Paget's disease of bone

Paget's disease of bone (PDB) is a chronic localized bone disorder in an elderly population. Environmental factors such as paramyxovirus are implicated in PDB and measles virus nucleocapsid protein (MVNP) has been shown to induce pagetic osteoclasts (OCLs). However, the molecular mechanisms underlying MVNP stimulation of OCL differentiation in the PDB are unclear. We therefore determined the MVNP regulated gene expression profiling during OCL differentiation. Agilent microarray analysis of gene expression identified high levels of SIRPβ1 (353-fold) expression in MVNP transduced human bone marrow mononuclear cells stimulated with RANKL. Real-time PCR analysis further confirmed that MVNP alone upregulates SIRPβ1 mRNA expression in these cells. Also, bone marrow mononuclear cells derived from patients with PDB showed high levels of SIRPβ1 mRNA expression compared to normal subjects. We further show that MVNP increases SIRPβ1 interaction with DAP12 adaptor protein in the presence and absence of RANKL stimulation. shRNA knockdown of SIRPβ1 expression in normal human bone marrow monocytes decreased the levels of MVNP enhanced p-Syk and c-Fos expression. In addition, SIRPβ1 knockdown significantly decreased MVNP stimulated dendritic cell–specific transmembrane protein (DC-STAMP) and connective tissue growth factor (CTGF) mRNA expression during OCL differentiation. Furthermore, we demonstrated the contribution of SIRPβ1 in MVNP induced OCL formation and bone resorption. Thus, our results suggest that MVNP modulation of SIRPβ1 provides new insights into the molecular mechanisms which control high bone turnover in PDB.