Defective endomembrane dynamics in Rab27a deficiency impairs nucleic acid sensing and cytokine secretion in immune cells.

Endosomal Toll-like receptors (eTLRs) are essential for the sensing of non-self through RNA and DNA detection. Here, using spatiotemporal analysis of vesicular dynamics, super-resolution microscopy studies, and functional assays, we show that endomembrane defects associated with the deficiency of the small GTPase Rab27a cause delayed eTLR ligand recognition, defective early signaling, and impaired cytokine secretion. Rab27a-deficient neutrophils show retention of eTLRs in amphisomes and impaired ligand internalization.

The solute carrier SLC15A4 is required for optimal trafficking of nucleic acid-sensing TLRs and ligands to endolysosomes.

A function-impairing mutation (feeble) or genomic deletion of SLC15A4 abolishes responses of nucleic acid–sensing endosomal toll-like receptors (TLRs) and significantly reduces disease in mouse models of lupus. Here, we demonstrate disease reduction in homozygous and even heterozygous Slc15a4 feeble mutant BXSB male mice with a Tlr7 gene duplication. In contrast to SLC15A4, a function-impairing mutation of SLC15A3 did not diminish type I interferon (IFN-I) production by TLR-activated plasmacytoid dendritic cells (pDCs), indicating divergence of function between these homologous SLC15 family members.

Silica exposure and chronic virus infection synergistically promote lupus-like systemic autoimmunity in mice with low genetic predisposition.

Considerable evidence indicates that autoimmune disease expression depends on both genetic and environmental factors. Among potential environmental triggers, occupational airway exposure to crystalline silica and virus infections have been linked to lupus and other autoimmune diseases in both humans and mouse models. Here, we hypothesized that combined silica and virus exposures synergize and induce autoimmune manifestations more effectively than single exposure to either of these factors, particularly in individuals with low genetic predisposition.

Lupus acceleration by a MAVS-activating RNA virus requires endosomal TLR signaling and host genetic predisposition.

Viruses have long been implicated in the pathogenesis of autoimmunity, yet their contribution remains circumstantial partly due to the lack of well-documented information on infections prior to autoimmune disease onset. Here, we used the lymphocytic choriomeningitis virus (LCMV) as a model to mechanistically dissect the impact of viral infection on lupus-like autoimmunity. Virus persistence strongly enhanced disease in mice with otherwise weak genetic predisposition but not in highly predisposed or non-autoimmune mice, indicating a synergistic interplay between genetic susceptibility and virus infection.

Induction of Systemic Autoimmunity by a Xenobiotic Requires Endosomal TLR Trafficking and Signaling from the Late Endosome and Endolysosome but Not Type I IFN.

Type I IFN and nucleic acid-sensing TLRs are both strongly implicated in the pathogenesis of lupus, with most patients expressing IFN-induced genes in peripheral blood cells and with TLRs promoting type I IFNs and autoreactive B cells. About a third of systemic lupus erythematosus patients, however, lack the IFN signature, suggesting the possibility of type I IFN-independent mechanisms. In this study, we examined the role of type I IFN and TLR trafficking and signaling in xenobiotic systemic mercury-induced autoimmunity (HgIA).

Murine Models for Viral Hemorrhagic Fever.

Hemorrhagic fever (HF) viruses, such as Lassa, Ebola, and dengue viruses, represent major human health risks due to their highly contagious nature, the severity of the clinical manifestations induced, the lack of vaccines, and the very limited therapeutic options currently available. Appropriate animal models are obviously critical to study disease pathogenesis and develop efficient therapies. We recently reported that the clone 13 (Cl13) variant of the lymphocytic choriomeningitis virus (LCMV-Cl13), a prototype arenavirus closely related to Lassa virus, causes in some mouse strains endothelial damage, vascular leakage, platelet loss, and death, mimicking pathological aspects typically observed in Lassa and other HF syndromes.

An agonist antibody that blocks autoimmunity by inducing anti-inflammatory macrophages.

We have devised a method of using intracellular combinatorial libraries to select antibodies that control cell fates. Many agonist antibodies have been selected with this method, and the process appears to be limited only by the availability of a phenotypic selection system. We demonstrate the utility of this approach to discover agonist antibodies that engage an unanticipated target and regulate macrophage polarization by selective induction of anti-inflammatory M2 macrophages.

Interleukin-7 is required for CD4(+) T cell activation and autoimmune neuroinflammation.

IL-7 is known to be vital for T cell homeostasis but has previously been presumed to be dispensable for TCR-induced activation. Here, we show that IL-7 is critical for the initial activation of CD4(+) T cells in that it provides some of the necessary early signaling components, such as activated STAT5 and Akt. Accordingly, short-term in vivo IL-7Rα blockade inhibited the activation and expansion of autoantigen-specific CD4(+) T cells and, when used to treat experimental autoimmune encephalomyelitis (EAE), prevented and ameliorated disease.

Type I interferon is a therapeutic target for virus-induced lethal vascular damage.

The outcome of a viral infection reflects the balance between virus virulence and host susceptibility. The clone 13 (Cl13) variant of lymphocytic choriomeningitis virus--a prototype of Old World arenaviruses closely related to Lassa fever virus--elicits in C57BL/6 and BALB/c mice abundant negative immunoregulatory molecules, associated with T-cell exhaustion, negligible T-cell-mediated injury, and high virus titers that persist. Conversely, here we report that in NZB mice, despite the efficient induction of immunoregulatory molecules and high viremia, Cl13 generated a robust cytotoxic T-cell response, resulting in thrombocytopenia, pulmonary endothelial cell loss, vascular leakage, and death within 6-8 d.

Essential requirement for IRF8 and SLC15A4 implicates plasmacytoid dendritic cells in the pathogenesis of lupus.

In vitro evidence suggests that plasmacytoid dendritic cells (pDCs) are intimately involved in the pathogenesis of lupus. However, it remains to be determined whether these cells are required in vivo for disease development, and whether their contribution is restricted to hyperproduction of type I IFNs. To address these issues, we created lupus-predisposed mice lacking the IFN regulatory factor 8 (IRF8) or carrying a mutation that impairs the peptide/histidine transporter solute carrier family 15, member 4 (SLC15A4).

Anti-IFN-α/β receptor antibody treatment ameliorates disease in lupus-predisposed mice.

The demonstration in humans and mice that nucleic acid-sensing TLRs and type I IFNs are essential disease mediators is a milestone in delineating the mechanisms of lupus pathogenesis. In this study, we show that Ifnb gene deletion does not modify disease progression in NZB mice, thereby strongly implicating IFN-α subtypes as the principal pathogenic effectors. We further document that long-term treatment of male BXSB mice with an anti-IFN-α/β receptor Ab of mouse origin reduced serologic, cellular, and histologic disease manifestations and extended survival, suggesting that disease acceleration by the Tlr7 gene duplication in this model is mediated by type I IFN signaling.

Transmethylation in immunity and autoimmunity.

The activation of immune cells is mediated by a network of signaling proteins that can undergo post-translational modifications critical for their activity. Methylation of nucleic acids or proteins can have major effects on gene expression as well as protein repertoire diversity and function. Emerging data indicate that indeed many immunologic functions, particularly those of T cells, including thymic education, differentiation and effector function are highly dependent on methylation events.

Systemic autoimmunity and lymphoproliferation are associated with excess IL-7 and inhibited by IL-7Rα blockade.

Lupus is characterized by disturbances in lymphocyte homeostasis, as demonstrated by the marked accumulation of activated/memory T cells. Here, we provide evidence that proliferation of the CD8+ precursors for the accumulating CD4⁻CD8⁻ T cells in MRL-Fas(lpr) lupus-predisposed mice is, in part, driven by commensal antigens. The ensuing lymphadenopathy is associated with increased production of IL-7 due to expansion of fibroblastic reticular cells, the primary source of this cytokine.

Deletion of IgG-switched autoreactive B cells and defects in Fas(lpr) lupus mice.

During a T cell-dependent Ab response, B cells undergo Ab class switching and V region hypermutation, with the latter process potentially rendering previously innocuous B cells autoreactive. Class switching and hypermutation are temporally and anatomically linked with both processes dependent on the enzyme, activation-induced deaminase, and occurring principally, but not exclusively, in germinal centers. To understand tolerance regulation at this stage, we generated a new transgenic mouse model expressing a membrane-tethered gamma2a-reactive superantigen (gamma2a-macroself Ag) and assessed the fate of emerging IgG2a-expressing B cells that have, following class switch, acquired self-reactivity of the Ag receptor to the macroself-Ag.

Sensors of the innate immune system: their link to rheumatic diseases.

Evidence strongly suggests that excessive or protracted signaling, or both, by cell-surface or intracellular innate immune receptors is central to the pathogenesis of most autoimmune and autoinflammatory rheumatic diseases. The initiation of aberrant innate and adaptive immune responses in autoimmune diseases can be triggered by microbes and, at times, by endogenous molecules--particularly nucleic acids and related immune complexes--under sterile conditions. By contrast, most autoinflammatory syndromes are generally dependent on germline or de novo gene mutations that cause or facilitate inflammasome assembly.

Sensors of the innate immune system: their mode of action.

The discovery of molecular sensors that enable eukaryotes to recognize microbial pathogens and their products has been a key advance in our understanding of innate immunity. A tripartite sensing apparatus has developed to detect danger signals from infectious agents and damaged tissues, resulting in an immediate but short-lived defense response. This apparatus includes Toll-like receptors, retinoid acid-inducible gene-I-like receptors and other cytosolic nucleic acid sensors, and nucleotide-binding and oligomerization domain-like receptors; adaptors, kinases and other signaling molecules are required to elicit effective responses.

Commitment to the regulatory T cell lineage requires CARMA1 in the thymus but not in the periphery.

Regulatory T (T(reg)) cells expressing forkhead box P3 (Foxp3) arise during thymic selection among thymocytes with modestly self-reactive T cell receptors. In vitro studies suggest Foxp3 can also be induced among peripheral CD4(+) T cells in a cytokine dependent manner. T(reg) cells of thymic or peripheral origin may serve different functions in vivo, but both populations are phenotypically indistinguishable in wild-type mice.

Impaired negative regulation of homeostatically proliferating T cells.

Acute lymphopenia-induced homeostatic proliferation (HP) of T cells promotes antitumor immunity, but the mechanism is unclear. We hypothesized that this is due to a lack of inhibitory signals that allows activation of T cells with low affinity for self-antigens. Tumors resist immunity in part by expressing inhibitory molecules such as PD-1 ligand 1 (PD-L1), B7-H4, and TGF-beta.

Gamma delta T cell homeostasis is controlled by IL-7 and IL-15 together with subset-specific factors.

Among T cell subsets, gamma delta T cells uniquely display an Ag receptor-based tissue distribution, but what defines their preferential homing and homeostasis is unknown. To address this question, we studied the resources that control gamma delta T cell homeostasis in secondary lymphoid organs. We found that gamma delta and alpha beta T cells are controlled by partially overlapping resources, because acute homeostatic proliferation of gamma delta T cells was inhibited by an intact alpha beta T cell compartment, and both populations were dependent on IL-7 and IL-15.

Tumor immunity via homeostatic T cell proliferation: mechanistic aspects and clinical perspectives.

Efforts to develop effective anti-tumor immunotherapies are hampered by the difficulty of overcoming tolerance against tumor antigens, which in most instances are normal gene products that are over-expressed, preferentially expressed or re-expressed in cancer cells. Considering that lymphopenia-induced homeostatic T cell proliferation is mediated by self-peptide/MHC recognition and that the expanded cells acquire some effector functions, we hypothesized that this process could be used to break tolerance against tumor antigens. Studies by us and others in several mouse models demonstrated that availability of tumor antigens during homeostatic T cell proliferation indeed leads to effective anti-tumor autoimmunity with specificity and memory.

Human adult tonsil xenotransplantation into SCID mice for studying human immune responses and B cell lymphomagenesis.

Objective: To generate a human-mouse xenochimeric model where human cells remain clustered in the animal to optimize their interactions and recovery.

Materials And Methods: Severe combined immune deficient mice (SCID) were xenografted subcutaneously with human adult tonsil pieces (hu-ton-SCID mice). Such animals were: (a) compared with those receiving tonsil cells in suspension, and (b) immunized with de novo and recall antigens.

Identification of clonally expanded T cells in rheumatoid arthritis using a sequence enrichment nuclease assay.

Identification of expanded clones engaged in immune and autoimmune responses is still imperfect, since they are often diluted by irrelevant cells expressing diverse specificities. To efficiently characterize T cell receptors expressed by clonally expanded lymphocytes in rheumatoid arthritis (RA) and other inflammatory conditions, we developed an assay system, termed sequence enrichment nuclease assay (SENA). Key elements of SENA are the efficiency of heat-denatured DNA strand reassociation, which increases exponentially with concentration, and the elimination of unhybridized sequences by single-strand-specific DNase.

Fas (CD95) participates in peripheral T cell deletion and associated apoptosis in vivo.

Following exposure to some types of antigen (superantigens), responsive T cells expand and then decline in numbers, a phenomenon that has been called 'peripheral deletion'. This process may play a role in limiting autoimmune reactions and in the maintenance of immune homeostasis. Here we describe experiments on peripheral deletion in mice carrying the lpr/lpr defect, which has been shown to be due to defective production of the CD95/Fas molecule.

V beta gene repertoire in the aging mouse: a developmental perspective.

To define age-associated alterations in the immune system at the molecular level, we have analyzed TCR V beta gene expression patterns at the fetal, neonatal, adult, and advanced ages of mice. In contrast to V gamma and VH genes, V beta genes rearranged without any preference related to their chromosomal organization. Endogenous superantigen-mediated clonal deletions were registered for the first time at the neonatal stage, presumably reflecting the late developmental appearance of these molecules.