Pathways of antigen processing.

T cell recognition of antigen-presenting cells depends on their expression of a spectrum of peptides bound to major histocompatibility complex class I (MHC-I) and class II (MHC-II) molecules. Conversion of antigens from pathogens or transformed cells into MHC-I- and MHC-II-bound peptides is critical for mounting protective T cell responses, and similar processing of self proteins is necessary to establish and maintain tolerance. Cells use a variety of mechanisms to acquire protein antigens, from translation in the cytosol to variations on the theme of endocytosis, and to degrade them once acquired. In this review, we highlight the aspects of MHC-I and MHC-II biosynthesis and assembly that have evolved to intersect these pathways and sample the peptides that are produced.

Convergent Structures Illuminate Features for Germline Antibody Binding and Pan-Lassa Virus Neutralization.

Lassa virus (LASV) causes hemorrhagic fever and is endemic in West Africa. Protective antibody responses primarily target the LASV surface glycoprotein (GPC), and GPC-B competition group antibodies often show potent neutralizing activity in humans. However, which features confer potent and broadly neutralizing antibody responses is unclear. Here, we compared three crystal structures of LASV GPC complexed with GPC-B antibodies of varying neutralization potency. Each GPC-B antibody recognized an overlapping epitope involved in binding of two adjacent GPC monomers and preserved the prefusion trimeric conformation. Differences among GPC-antibody interactions highlighted specific residues that enhance neutralization. Using structure-guided amino acid substitutions, we increased the neutralization potency and breadth of these antibodies to include all major LASV lineages. The ability to define antibody residues that allow potent and broad neutralizing activity, together with findings from analyses of inferred germline precursors, is critical to develop potent therapeutics and for vaccine design and assessment.

Systematic Analysis of Monoclonal Antibodies against Ebola Virus GP Defines Features that Contribute to Protection.

Antibodies are promising post-exposure therapies against emerging viruses, but which antibody features and in vitro assays best forecast protection are unclear. Our international consortium systematically evaluated antibodies against Ebola virus (EBOV) using multidisciplinary assays. For each antibody, we evaluated epitopes recognized on the viral surface glycoprotein (GP) and secreted glycoprotein (sGP), readouts of multiple neutralization assays, fraction of virions left un-neutralized, glycan structures, phagocytic and natural killer cell functions elicited, and in vivo protection in a mouse challenge model. Neutralization and induction of multiple immune effector functions (IEFs) correlated most strongly with protection. Neutralization predominantly occurred via epitopes maintained on endosomally cleaved GP, whereas maximal IEF mapped to epitopes farthest from the viral membrane. Unexpectedly, sGP cross-reactivity did not significantly influence in vivo protection. This comprehensive dataset provides a rubric to evaluate novel antibodies and vaccine responses and a roadmap for therapeutic development for EBOV and related viruses.

Reactive Oxygen Species and Neutrophil Function.

Neutrophils are essential for killing bacteria and other microorganisms, and they also have a significant role in regulating the inflammatory response. Stimulated neutrophils activate their NADPH oxidase (NOX2) to generate large amounts of superoxide, which acts as a precursor of hydrogen peroxide and other reactive oxygen species that are generated by their heme enzyme myeloperoxidase. When neutrophils engulf bacteria they enclose them in small vesicles (phagosomes) into which superoxide is released by activated NOX2 on the internalized neutrophil membrane. The superoxide dismutates to hydrogen peroxide, which is used by myeloperoxidase to generate other oxidants, including the highly microbicidal species hypochlorous acid. NOX activation occurs at other sites in the cell, where it is considered to have a regulatory function. Neutrophils also release oxidants, which can modify extracellular targets and affect the function of neighboring cells. We discuss the identity and chemical properties of the specific oxidants produced by neutrophils in different situations, and what is known about oxidative mechanisms of microbial killing, inflammatory tissue damage, and signaling.

Mouse cytomegalovirus-experienced ILC1s acquire a memory response dependent on the viral glycoprotein m12.

Innate lymphoid cells (ILCs) are tissue-resident sentinels that are essential for early host protection from pathogens at initial sites of infection. However, whether pathogen-derived antigens directly modulate the responses of tissue-resident ILCs has remained unclear. In the present study, it was found that liver-resident type 1 ILCs (ILC1s) expanded locally and persisted after the resolution of infection with mouse cytomegalovirus (MCMV). ILC1s acquired stable transcriptional, epigenetic and phenotypic changes a month after the resolution of MCMV infection, and showed an enhanced protective effector response to secondary challenge with MCMV consistent with a memory lymphocyte response. Memory ILC1 responses were dependent on the MCMV-encoded glycoprotein m12, and were independent of bystander activation by proinflammatory cytokines after heterologous infection. Thus, liver ILC1s acquire adaptive features in an MCMV-specific manner.

Interleukin-17D regulates group 3 innate lymphoid cell function through its receptor CD93.

The interleukin (IL)-17 family, consisting of six members, promotes host defense but can in some context promote the development of autoimmune disease. Here, we examined the role of IL-17D, a poorly understood member in the IL-17 family. IL-17D was expressed primarily by colonic epithelial cells. Il17d-/- mice were more susceptible to acute colitis, bacterial infection and experimentally induced colon cancer than their wildtype counterparts. Il17d deficiency impaired IL-22 production by group 3 innate lymphoid cells (ILC3s) and reduced expression of IL-22-dependent antimicrobial peptides, RegIIIß and RegIIIγ, in colon tissue at steady state and in colitis; this was associated with changes in microbial composition and dysbiosis. Protein purification studies revealed that IL-17D bound not canonical IL-17 receptors, but rather CD93, a glycoprotein expressed on mature ILC3s. Mice lacking Cd93 in ILC3s exhibited impaired IL-22 production and aggravated colonic inflammation in experimental colitis. Thus, an IL-17D-CD93 axis regulates ILC3 function to preserve intestinal homeostasis.

The persistence of interleukin-6 is regulated by a blood buffer system derived from dendritic cells.

The interleukin-6 (IL-6) membrane receptor and its circulating soluble form, sIL-6R, can be targeted by antibody therapy to reduce deleterious immune signaling caused by chronic overexpression of the pro-inflammatory cytokine IL-6. This strategy may also hold promise for treating acute hyperinflammation, such as observed in coronavirus disease 2019 (COVID-19), highlighting a need to define regulators of IL-6 homeostasis. We found that conventional dendritic cells (cDCs), defined in mice via expression of the transcription factor Zbtb46, were a major source of circulating sIL-6R and, thus, systemically regulated IL-6 signaling. This was uncovered through identification of a cDC-dependent but T cell-independent modality that naturally adjuvants plasma cell differentiation and antibody responses to protein antigens. This pathway was then revealed as part of a broader biological buffer system in which cDC-derived sIL-6R set the in-solution persistence of IL-6. This control axis may further inform the development of therapeutic agents to modulate pro-inflammatory immune reactions.

Excessive Polyamine Generation in Keratinocytes Promotes Self-RNA Sensing by Dendritic Cells in Psoriasis.

Psoriasis is a chronic inflammatory disease whose etiology is multifactorial. The contributions of cellular metabolism to psoriasis are unclear. Here, we report that interleukin-17 (IL-17) downregulated Protein Phosphatase 6 (PP6) in psoriatic keratinocytes, causing phosphorylation and activation of the transcription factor C/EBP-ß and subsequent generation of arginase-1. Mice lacking Pp6 in keratinocytes were predisposed to psoriasis-like skin inflammation. Accumulation of arginase-1 in Pp6-deficient keratinocytes drove polyamine production from the urea cycle. Polyamines protected self-RNA released by psoriatic keratinocytes from degradation and facilitated the endocytosis of self-RNA by myeloid dendritic cells to promote toll-like receptor-7 (TLR7)-dependent RNA sensing and IL-6 production. An arginase inhibitor improved skin inflammation in murine and non-human primate models of psoriasis. Our findings suggest that urea cycle hyperreactivity and excessive polyamine generation in psoriatic keratinocytes promote self-RNA sensation and PP6 deregulation in keratinocytes is a pivotal event that amplifies the inflammatory circuits in psoriasis.

Immune Signaling in Neurodegeneration.

Neurodegenerative diseases of the central nervous system progressively rob patients of their memory, motor function, and ability to perform daily tasks. Advances in genetics and animal models are beginning to unearth an unexpected role of the immune system in disease onset and pathogenesis; however, the role of cytokines, growth factors, and other immune signaling pathways in disease pathogenesis is still being examined. Here we review recent genetic risk and genome-wide association studies and emerging mechanisms for three key immune pathways implicated in disease, the growth factor TGF-ß, the complement cascade, and the extracellular receptor TREM2. These immune signaling pathways are important under both healthy and neurodegenerative conditions, and recent work has highlighted new functional aspects of their signaling. Finally, we assess future directions for immune-related research in neurodegeneration and potential avenues for immune-related therapies.

Immunization with Components of the Viral Fusion Apparatus Elicits Antibodies That Neutralize Epstein-Barr Virus in B Cells and Epithelial Cells.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with epithelial-cell cancers and B cell lymphomas. An effective EBV vaccine is not available. We found that antibodies to the EBV glycoprotein gH/gL complex were the principal components in human plasma that neutralized infection of epithelial cells and that antibodies to gH/gL and gp42 contributed to B cell neutralization. Immunization of mice and nonhuman primates with nanoparticle vaccines that displayed components of the viral-fusion machinery EBV gH/gL or gH/gL/gp42 elicited antibodies that potently neutralized both epithelial-cell and B cell infection. Immune serum from nonhuman primates inhibited EBV-glycoprotein-mediated fusion of epithelial cells and B cells and targeted an epitope critical for virus-cell fusion. Therefore, unlike the leading EBV gp350 vaccine candidate, which only protects B cells from infection, these EBV nanoparticle vaccines elicit antibodies that inhibit the virus-fusion apparatus and provide cell-type-independent protection from virus infection.

Class-switched memory B cells remodel BCRs within secondary germinal centers.

Effective vaccines induce high-affinity memory B cells and durable antibody responses through accelerated mechanisms of natural selection. Secondary changes in antibody repertoires after vaccine boosts suggest progressive rediversification of B cell receptors (BCRs), but the underlying mechanisms remain unresolved. Here, the integrated specificity and function of individual memory B cell progeny revealed ongoing evolution of polyclonal antibody specificities through germinal center (GC)-specific transcriptional activity. At the clonal and subclonal levels, single-cell expression of the genes encoding the costimulatory molecule CD83 and the DNA polymerase Polη segregated the secondary GC transcriptional program into four stages that regulated divergent mechanisms of memory BCR evolution. Our studies demonstrate that vaccine boosts reactivate a cyclic program of GC function in class-switched memory B cells to remodel existing antibody specificities and enhance durable immunological protection.

The CLEC-2-podoplanin axis controls the contractility of fibroblastic reticular cells and lymph node microarchitecture.

In lymph nodes, fibroblastic reticular cells (FRCs) form a collagen-based reticular network that supports migratory dendritic cells (DCs) and T cells and transports lymph. A hallmark of FRCs is their propensity to contract collagen, yet this function is poorly understood. Here we demonstrate that podoplanin (PDPN) regulates actomyosin contractility in FRCs. Under resting conditions, when FRCs are unlikely to encounter mature DCs expressing the PDPN receptor CLEC-2, PDPN endowed FRCs with contractile function and exerted tension within the reticulum. Upon inflammation, CLEC-2 on mature DCs potently attenuated PDPN-mediated contractility, which resulted in FRC relaxation and reduced tissue stiffness. Disrupting PDPN function altered the homeostasis and spacing of FRCs and T cells, which resulted in an expanded reticular network and enhanced immunity.

Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells.

The local regulation of type 2 immunity relies on dialog between the epithelium and the innate and adaptive immune cells. Here we found that alarmin-induced expression of the co-stimulatory molecule OX40L on group 2 innate lymphoid cells (ILC2s) provided tissue-restricted T cell co-stimulation that was indispensable for Th2 and regulatory T (Treg) cell responses in the lung and adipose tissue. Interleukin (IL)-33 administration resulted in organ-specific surface expression of OX40L on ILC2s and the concomitant expansion of Th2 and Treg cells, which was abolished upon deletion of OX40L on ILC2s (Il7raCre/+Tnfsf4fl/fl mice). Moreover, Il7raCre/+Tnfsf4fl/fl mice failed to mount effective Th2 and Treg cell responses and corresponding adaptive type 2 pulmonary inflammation arising from Nippostrongylus brasiliensis infection or allergen exposure. Thus, the increased expression of OX40L in response to IL-33 acts as a licensing signal in the orchestration of tissue-specific adaptive type 2 immunity, without which this response fails to establish.

An Antibody Targeting the Fusion Machinery Neutralizes Dual-Tropic Infection and Defines a Site of Vulnerability on Epstein-Barr Virus.

Epstein-Barr virus (EBV) is a causative agent of infectious mononucleosis and is associated with 200,000 new cases of cancer and 140,000 deaths annually. Subunit vaccines against this pathogen have focused on the gp350 glycoprotein and remain unsuccessful. We isolated human antibodies recognizing the EBV fusion machinery (gH/gL and gB) from rare memory B cells. One anti-gH/gL antibody, AMMO1, potently neutralized infection of B cells and epithelial cells, the two major cell types targeted by EBV. We determined a cryo-electron microscopy reconstruction of the gH/gL-gp42-AMMO1 complex and demonstrated that AMMO1 bound to a discontinuous epitope formed by both gH and gL at the Domain-I/Domain-II interface. Integrating structural, biochemical, and infectivity data, we propose that AMMO1 inhibits fusion of the viral and cellular membranes. This work identifies a crucial epitope that may aid in the design of next-generation subunit vaccines against this major public health burden.

The Microglial Innate Immune Receptor TREM2 Is Required for Synapse Elimination and Normal Brain Connectivity.

The triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial innate immune receptor associated with a lethal form of early, progressive dementia, Nasu-Hakola disease, and with an increased risk of Alzheimer's disease. Microglial defects in phagocytosis of toxic aggregates or apoptotic membranes were proposed to be at the origin of the pathological processes in the presence of Trem2 inactivating mutations. Here, we show that TREM2 is essential for microglia-mediated synaptic refinement during the early stages of brain development. The absence of Trem2 resulted in impaired synapse elimination, accompanied by enhanced excitatory neurotransmission and reduced long-range functional connectivity. Trem2-/- mice displayed repetitive behavior and altered sociability. TREM2 protein levels were also negatively correlated with the severity of symptoms in humans affected by autism. These data unveil the role of TREM2 in neuronal circuit sculpting and provide the evidence for the receptor's involvement in neurodevelopmental diseases.

Self-Renewal and Toll-like Receptor Signaling Sustain Exhausted Plasmacytoid Dendritic Cells during Chronic Viral Infection.

Although characterization of T cell exhaustion has unlocked powerful immunotherapies, the mechanisms sustaining adaptations of short-lived innate cells to chronic inflammatory settings remain unknown. During murine chronic viral infection, we found that concerted events in bone marrow and spleen mediated by type I interferon (IFN-I) and Toll-like receptor 7 (TLR7) maintained a pool of functionally exhausted plasmacytoid dendritic cells (pDCs). In the bone marrow, IFN-I compromised the number and the developmental capacity of pDC progenitors, which generated dysfunctional pDCs. Concurrently, exhausted pDCs in the periphery were maintained by self-renewal via IFN-I- and TLR7-induced proliferation of CD4- subsets. On the other hand, pDC functional loss was mediated by TLR7, leading to compromised IFN-I production and resistance to secondary infection. These findings unveil the mechanisms sustaining a self-perpetuating pool of functionally exhausted pDCs and provide a framework for deciphering long-term exhaustion of other short-lived innate cells during chronic inflammation.

Loss of TREM2 rescues hyperactivation of microglia, but not lysosomal deficits and neurotoxicity in models of progranulin deficiency.

Haploinsufficiency of the progranulin (PGRN)-encoding gene (GRN) causes frontotemporal lobar degeneration (GRN-FTLD) and results in microglial hyperactivation, TREM2 activation, lysosomal dysfunction, and TDP-43 deposition. To understand the contribution of microglial hyperactivation to pathology, we used genetic and pharmacological approaches to suppress TREM2-dependent transition of microglia from a homeostatic to a disease-associated state. Trem2 deficiency in Grn KO mice reduced microglia hyperactivation. To explore antibody-mediated pharmacological modulation of TREM2-dependent microglial states, we identified antagonistic TREM2 antibodies. Treatment of macrophages from GRN-FTLD patients with these antibodies led to reduced TREM2 signaling due to its enhanced shedding. Furthermore, TREM2 antibody-treated PGRN-deficient microglia derived from human-induced pluripotent stem cells showed reduced microglial hyperactivation, TREM2 signaling, and phagocytic activity, but lysosomal dysfunction was not rescued. Similarly, lysosomal dysfunction, lipid dysregulation, and glucose hypometabolism of Grn KO mice were not rescued by TREM2 ablation. Synaptic loss and neurofilament light-chain (NfL) levels, a biomarker for neurodegeneration, were further elevated in the Grn/Trem2 KO cerebrospinal fluid (CSF). These findings suggest that TREM2-dependent microglia hyperactivation in models of GRN deficiency does not promote neurotoxicity, but rather neuroprotection.

A CD38-directed, single-chain T-cell engager targets leukemia stem cells through IFN-γ-induced CD38 expression.

ABSTRACT: Treatment resistance of leukemia stem cells (LSCs) and suppression of the autologous immune system represent major challenges to achieve a cure in acute myeloid leukemia (AML). Although AML blasts generally retain high levels of surface CD38 (CD38pos), LSCs are frequently enriched in the CD34posCD38neg blast fraction. Here, we report that interferon gamma (IFN-γ) reduces LSCs clonogenic activity and induces CD38 upregulation in both CD38pos and CD38neg LSC-enriched blasts. IFN-γ-induced CD38 upregulation depends on interferon regulatory factor 1 transcriptional activation of the CD38 promoter. To leverage this observation, we created a novel compact, single-chain CD38-CD3 T-cell engager (BN-CD38) designed to promote an effective immunological synapse between CD38pos AML cells and both CD8pos and CD4pos T cells. We demonstrate that BN-CD38 engages autologous CD4pos and CD8pos T cells and CD38pos AML blasts, leading to T-cell activation and expansion and to the elimination of leukemia cells in an autologous setting. Importantly, BN-CD38 engagement induces the release of high levels of IFN-γ, driving the expression of CD38 on CD34posCD38neg LSC-enriched blasts and their subsequent elimination. Critically, although BN-CD38 showed significant in vivo efficacy across multiple disseminated AML cell lines and patient-derived xenograft models, it did not affect normal hematopoietic stem cell clonogenicity and the development of multilineage human immune cells in CD34pos humanized mice. Taken together, this study provides important insights to target and eliminate AML LSCs.

Reconstruction of LPS Transfer Cascade Reveals Structural Determinants within LBP, CD14, and TLR4-MD2 for Efficient LPS Recognition and Transfer.

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, binds Toll-like receptor 4 (TLR4)-MD2 complex and activates innate immune responses. LPS transfer to TLR4-MD2 is catalyzed by both LPS binding protein (LBP) and CD14. To define the sequential molecular interactions underlying this transfer, we reconstituted in vitro the entire LPS transfer process from LPS micelles to TLR4-MD2. Using electron microscopy and single-molecule approaches, we characterized the dynamic intermediate complexes for LPS transfer: LBP-LPS micelles, CD14-LBP-LPS micelle, and CD14-LPS-TLR4-MD2 complex. A single LBP molecule bound longitudinally to LPS micelles catalyzed multi-rounds of LPS transfer to CD14s that rapidly dissociated from LPB-LPS complex upon LPS transfer via electrostatic interactions. Subsequently, the single LPS molecule bound to CD14 was transferred to TLR4-MD2 in a TLR4-dependent manner. The definition of the structural determinants of the LPS transfer cascade to TLR4 may enable the development of targeted therapeutics for intervention in LPS-induced sepsis.

Integrin αvß3 Limits Cytokine Production by Plasmacytoid Dendritic Cells and Restricts TLR-Driven Autoimmunity.

Plasmacytoid dendritic cells (pDCs) are strongly implicated as a major source of IFN-I in systemic lupus erythematosus (SLE), triggered through TLR-mediated recognition of nucleic acids released from dying cells. However, relatively little is known about how TLR signaling and IFN-I production are regulated in pDCs. In this article, we describe a role for integrin αvß3 in regulating TLR responses and IFN-I production by pDCs in mouse models. We show that αv and ß3-knockout pDCs produce more IFN-I and inflammatory cytokines than controls when stimulated through TLR7 and TLR9 in vitro and in vivo. Increased cytokine production was associated with delayed acidification of endosomes containing TLR ligands, reduced LC3 conjugation, and increased TLR signaling. This dysregulated TLR signaling results in activation of B cells and promotes germinal center (GC) B cell and plasma cell expansion. Furthermore, in a mouse model of TLR7-driven lupus-like disease, deletion of αvß3 from pDCs causes accelerated autoantibody production and pathology. We therefore identify a pDC-intrinsic role for αvß3 in regulating TLR signaling and preventing activation of autoreactive B cells. Because αvß3 serves as a receptor for apoptotic cells and cell debris, we hypothesize that this regulatory mechanism provides important contextual cues to pDCs and functions to limit responses to self-derived nucleic acids.