Publications by authors named "Walter Reith"

92 Publications

Tumor-specific cytolytic CD4 T cells mediate immunity against human cancer.

Sci Adv 2021 Feb 26;7(9). Epub 2021 Feb 26.

Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, CH-1211, Switzerland.

CD4 T cells have been implicated in cancer immunity for their helper functions. Moreover, their direct cytotoxic potential has been shown in some patients with cancer. Here, by mining single-cell RNA-seq datasets, we identified CD4 T cell clusters displaying cytotoxic phenotypes in different human cancers, resembling CD8 T cell profiles. Using the peptide-MHCII-multimer technology, we confirmed ex vivo the presence of cytolytic tumor-specific CD4 T cells. We performed an integrated phenotypic and functional characterization of these cells, down to the single-cell level, through a high-throughput nanobiochip consisting of massive arrays of picowells and machine learning. We demonstrated a direct, contact-, and granzyme-dependent cytotoxic activity against tumors, with delayed kinetics compared to classical cytotoxic lymphocytes. Last, we found that this cytotoxic activity was in part dependent on SLAMF7. Agonistic engagement of SLAMF7 enhanced cytotoxicity of tumor-specific CD4 T cells, suggesting that targeting these cells might prove synergistic with other cancer immunotherapies.
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http://dx.doi.org/10.1126/sciadv.abe3348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7909889PMC
February 2021

NLRC5 promotes transcription of genes and Vγ9Vδ2 T cell-mediated killing.

iScience 2021 Jan 7;24(1):101900. Epub 2020 Dec 7.

Università della Svizzera italiana (USI), Faculty of Biomedical Sciences, Institute for Research in Biomedicine, 6500 Bellinzona, Switzerland.

BTN3A molecules-BTN3A1 in particular-emerged as important mediators of Vγ9Vδ2 T cell activation by phosphoantigens. These metabolites can originate from infections, e.g. with or by alterations in cellular metabolism. Despite the growing interest in the genes and their high expression in immune cells and various cancers, little is known about their transcriptional regulation. Here we show that these genes are induced by NLRC5, a regulator of MHC class I gene transcription, through an atypical regulatory motif found in their promoters. Accordingly, a robust correlation between and gene expression was found in healthy, in -infected donors' blood cells, and in primary tumors. Moreover, forcing NLRC5 expression promoted Vγ9Vδ2 T-cell-mediated killing of tumor cells in a BTN3A-dependent manner. Altogether, these findings indicate that NLRC5 regulates the expression of genes and hence open opportunities to modulate antimicrobial and anticancer immunity.
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http://dx.doi.org/10.1016/j.isci.2020.101900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7753138PMC
January 2021

Interplay of RFX transcription factors 1, 2 and 3 in motile ciliogenesis.

Nucleic Acids Res 2020 09;48(16):9019-9036

Univ Lyon, Université Claude Bernard Lyon-1, CNRS UMR-5310, INSERM U-1217, Institut NeuroMyoGène, F-69008, Lyon, France.

Cilia assembly is under strict transcriptional control during animal development. In vertebrates, a hierarchy of transcription factors (TFs) are involved in controlling the specification, differentiation and function of multiciliated epithelia. RFX TFs play key functions in the control of ciliogenesis in animals. Whereas only one RFX factor regulates ciliogenesis in C. elegans, several distinct RFX factors have been implicated in this process in vertebrates. However, a clear understanding of the specific and redundant functions of different RFX factors in ciliated cells remains lacking. Using RNA-seq and ChIP-seq approaches we identified genes regulated directly and indirectly by RFX1, RFX2 and RFX3 in mouse ependymal cells. We show that these three TFs have both redundant and specific functions in ependymal cells. Whereas RFX1, RFX2 and RFX3 occupy many shared genomic loci, only RFX2 and RFX3 play a prominent and redundant function in the control of motile ciliogenesis in mice. Our results provide a valuable list of candidate ciliary genes. They also reveal stunning differences between compensatory processes operating in vivo and ex vivo.
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http://dx.doi.org/10.1093/nar/gkaa625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7498320PMC
September 2020

Toll-like receptor 4 inhibition prevents autoimmune diabetes in NOD mice.

Sci Rep 2019 12 18;9(1):19350. Epub 2019 Dec 18.

Cell Isolation and Transplantation Center, Diabetes Center, University of Geneva Hospitals, Geneva, Switzerland.

TLR4 is a transmembrane receptor of the innate immune system that recognizes LPS from gram-negative bacteria. Its stimulation induces pro-inflammatory responses and modulates adaptive immunity. Our aim is to determine the role of TLR4 in the activation and proliferation of T lymphocytes in the onset of autoimmune diabetes, using the non-obese diabetic (NOD) mouse model. Antigen-specific activation and proliferation of diabetogenic T cells were assessed in vitro by Carboxyfluorescein succinimidyl ester (CFSE) dilution, in presence of vehicle or CLI-095, a cyclohexene derivative that inhibits TLR4 signaling. NOD mice were treated with vehicle or CLI-095 and sacrificed either before or after the onset of autoimmune diabetes. T lymphocyte activation and proliferation were evaluated in treated and control mice. Insulitis was analyzed by histology and diabetes incidence was determined in treated and control mice. Our results demonstrate that TLR4 blockade decreases CD4+ T lymphocyte activation and auto-antigen-specific proliferation both in vitro and in vivo, decreases the infiltrative insulitis and finally prevents the onset of spontaneous diabetes. Taken together, our data demonstrate that TLR4 signaling contributes to the development and maintenance of autoimmune diabetes. The immunomodulatory effect of CLI-095 could be part of a preventive strategy targeting patients at risk for type 1 diabetes.
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http://dx.doi.org/10.1038/s41598-019-55521-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920362PMC
December 2019

Specific enhancer selection by IRF3, IRF5 and IRF9 is determined by ISRE half-sites, 5' and 3' flanking bases, collaborating transcription factors and the chromatin environment in a combinatorial fashion.

Nucleic Acids Res 2020 01;48(2):589-604

Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen H-4032, Hungary.

IRF3, IRF5 and IRF9 are transcription factors, which play distinct roles in the regulation of antiviral and inflammatory responses. The determinants that mediate IRF-specific enhancer selection are not fully understood. To uncover regions occupied predominantly by IRF3, IRF5 or IRF9, we performed ChIP-seq experiments in activated murine dendritic cells. The identified regions were analysed with respect to the enrichment of DNA motifs, the interferon-stimulated response element (ISRE) and ISRE half-site variants, and chromatin accessibility. Using a machine learning method, we investigated the predictability of IRF-dominance. We found that IRF5-dominant regions differed fundamentally from the IRF3- and IRF9-dominant regions: ISREs were rare, while the NFKB motif and special ISRE half-sites, such as 5'-GAGA-3' and 5'-GACA-3', were enriched. IRF3- and IRF9-dominant regions were characterized by the enriched ISRE motif and lower frequency of accessible chromatin. Enrichment analysis and the machine learning method uncovered the features that favour IRF3 or IRF9 dominancy (e.g. a tripartite form of ISRE and motifs for NF-κB for IRF3, and the GAS motif and certain ISRE variants for IRF9). This study contributes to our understanding of how IRF members, which bind overlapping sets of DNA sequences, can initiate signal-dependent responses without activating superfluous or harmful programmes.
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http://dx.doi.org/10.1093/nar/gkz1112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954429PMC
January 2020

Mitochondrial arginase-2 is a cell‑autonomous regulator of CD8+ T cell function and antitumor efficacy.

JCI Insight 2019 11 21;4(24). Epub 2019 Nov 21.

Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland.

As sufficient extracellular arginine is crucial for T cell function, depletion of extracellular arginine by elevated arginase 1 (Arg1) activity has emerged as a hallmark immunosuppressive mechanism. However, the potential cell-autonomous roles of arginases in T cells have remained unexplored. Here, we show that the arginase isoform expressed by T cells, the mitochondrial Arg2, is a cell-intrinsic regulator of CD8+ T cell activity. Both germline Arg2 deletion and adoptive transfer of Arg2-/- CD8+ T cells significantly reduced tumor growth in preclinical cancer models by enhancing CD8+ T cell activation, effector function, and persistence. Transcriptomic, proteomic, and high-dimensional flow cytometry characterization revealed a CD8+ T cell-intrinsic role of Arg2 in modulating T cell activation, antitumor cytoxicity, and memory formation, independently of extracellular arginine availability. Furthermore, specific deletion of Arg2 in CD8+ T cells strongly synergized with PD-1 blockade for the control of tumor growth and animal survival. These observations, coupled with the finding that pharmacologic arginase inhibition accelerates activation of ex vivo human T cells, unveil Arg2 as a potentially new therapeutic target for T cell-based cancer immunotherapies.
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http://dx.doi.org/10.1172/jci.insight.132975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6975264PMC
November 2019

Macrophage migration inhibitory factor regulates TLR4 expression and modulates TCR/CD3-mediated activation in CD4+ T lymphocytes.

Sci Rep 2019 06 28;9(1):9380. Epub 2019 Jun 28.

Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals and University of Geneva, Geneva, Switzerland.

Toll-like receptor 4 (TLR4) is involved in CD4+ T lymphocyte-mediated pathologies. Here, we demonstrate that CD4+ T lymphocytes express functional TLR4 that contributes to their activation, proliferation and cytokine secretion. In addition, we demonstrate that TLR4-induced responses are mediated by macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine. We also demonstrate that MIF regulates suboptimal TCR/CD3-mediated activation of T lymphocytes. On one hand, MIF prevents excessive TCR/CD3-mediated activation of CD4+ T lymphocytes under suboptimal stimulation conditions and, on the other hand, MIF enables activated CD4+ T lymphocytes to sense their microenvironment and adapt their effector response through TLR4. Therefore, MIF appears to be a major regulator of the activation of CD4+ T lymphocytes and the intensity of their effector response. TLR4-mediated activation is thus an important process for T cell-mediated immunity.
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http://dx.doi.org/10.1038/s41598-019-45260-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599059PMC
June 2019

High-throughput Screening of Human Tumor Antigen-specific CD4 T Cells, Including Neoantigen-reactive T Cells.

Clin Cancer Res 2019 07 23;25(14):4320-4331. Epub 2019 Apr 23.

Ludwig Institute for Cancer Research and Department of Oncology, University of Lausanne, Lausanne, Switzerland.

Purpose: Characterization of tumor antigen-specific CD4 T-cell responses in healthy donors and malignant melanoma patients using an amplified T-cell library screening procedure.

Patients And Methods: A high-throughput, human leukocyte antigen (HLA)-independent approach was used to estimate at unprecedented high sensitivity level precursor frequencies of tumor antigen- and neoantigen-specific CD4 T cells in healthy donors and patients with cancer. Frequency estimation was combined with isolation and functional characterization of identified tumor-reactive CD4 T-cell clones.

Results: In healthy donors, we report frequencies of naïve tumor-associated antigen (TAA)-specific CD4 T cells comparable with those of CD4 T cells specific for infectious agents (). Interestingly, we also identified low, but consistent numbers of memory CD4 T cells specific for several TAAs. In patients with melanoma, low frequencies of circulating TAA-specific CD4 T cells were detected that increased after peptide-based immunotherapy. Such antitumor TAA-specific CD4 T-cell responses were also detectable within the tumor-infiltrated tissues. TAA-specific CD4 T cells in patients displayed a highly polyfunctional state, with partial skewing to Type-2 polarization. Finally, we report the applicability of this approach to the detection and amplification of neoantigen-specific CD4 T cells.

Conclusions: This simple, noninvasive, high-throughput screening of tumor- and neoantigen-specific CD4 T cells requires little biologic material, is HLA class II independent and allows the concomitant screening for a large number of tumor antigens of interest, including neoantigens. This approach will facilitate the immunomonitoring of preexisting and therapy-induced CD4 T-cell responses, and accelerate the development of CD4 T-cell-based therapies.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-1356DOI Listing
July 2019

Absence of MHC-II expression by lymph node stromal cells results in autoimmunity.

Life Sci Alliance 2018 Dec 17;1(6):e201800164. Epub 2018 Dec 17.

Department of Pathology and Immunology, School of Medicine, University of Geneva, Geneva, Switzerland.

How lymph node stromal cells (LNSCs) shape peripheral T-cell responses remains unclear. We have previously demonstrated that murine LNSCs, lymphatic endothelial cells (LECs), blood endothelial cells (BECs), and fibroblastic reticular cells (FRCs) use the IFN-γ-inducible promoter IV (pIV) of the MHC class II (MHCII) transactivator CIITA to express MHCII. Here, we show that aging mice (>1 yr old) in which MHCII is abrogated in LNSCs by the selective deletion of pIV exhibit a significant T-cell dysregulation in LNs, including defective Treg and increased effector CD4 and CD8 T-cell frequencies, resulting in enhanced peripheral organ T-cell infiltration and autoantibody production. The proliferation of LN-Tregs interacting with LECs increases following MHCII up-regulation by LECs upon aging or after exposure to IFN-γ, this effect being abolished in mice in which LECs lack MHCII. Overall, our work underpins the importance of LNSCs, particularly LECs, in supporting Tregs and T-cell tolerance.
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http://dx.doi.org/10.26508/lsa.201800164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297861PMC
December 2018

Spatiotemporal expression of endogenous TLR4 ligands leads to inflammation and bone erosion in mouse collagen-induced arthritis.

Eur J Immunol 2016 11 6;46(11):2629-2638. Epub 2016 Sep 6.

NovImmune SA, Plan-Les-Ouates, Geneva, Switzerland.

Increased expression of endogenous Toll-like receptor 4 (TLR4) ligands (e.g., Tenascin-C, S100A8/A9, citrullinated fibrinogen (cFb) immune complexes) has been observed in patients with rheumatoid arthritis (RA). However, their roles in RA pathogenesis are not well understood. Here, we investigated the expression kinetics and role of endogenous TLR4 ligands in the murine model of collagen-induced arthritis (CIA). Tenascin-C was upregulated in blood early in CIA, and correlated positively with the clinical score at day 56. Levels of S100A8/A9 increased starting from day 28, peaking at day 42, and correlated positively with joint inflammation. Levels of anti-cFb antibodies increased during the late phase of CIA and correlated positively with both joint inflammation and cartilage damage. Blockade of TLR4 activation at the time of the first TLR4 ligand upregulation prevented clinical and histological signs of arthritis. A TLR4-dependent role was also observed for Tenascin-C and cFb immune complexes in osteoclast differentiation in vitro. Taken together, our data suggests that the pathogenic contribution of TLR4 in promoting joint inflammation and bone erosion during CIA occurs via various TLR4 ligands arising at different stages of disease. The data also suggests that Blockade of TLR4 with monoclonal antibodies is a promising strategy in RA treatment.
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http://dx.doi.org/10.1002/eji.201646453DOI Listing
November 2016

Mammalian Innate Immune Response to a Leishmania-Resident RNA Virus Increases Macrophage Survival to Promote Parasite Persistence.

Cell Host Microbe 2016 Sep 1;20(3):318-328. Epub 2016 Sep 1.

Department of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland. Electronic address:

Some strains of the protozoan parasite Leishmania guyanensis (L.g) harbor a viral endosymbiont called Leishmania RNA virus 1 (LRV1). LRV1 recognition by TLR-3 increases parasite burden and lesion swelling in vivo. However, the mechanisms by which anti-viral innate immune responses affect parasitic infection are largely unknown. Upon investigating the mammalian host's response to LRV1, we found that miR-155 was singularly and strongly upregulated in macrophages infected with LRV1+ L.g when compared to LRV1- L.g. LRV1-driven miR-155 expression was dependent on TLR-3/TRIF signaling. Furthermore, LRV1-induced TLR-3 activation promoted parasite persistence by enhancing macrophage survival through Akt activation in a manner partially dependent on miR-155. Pharmacological inhibition of Akt resulted in a decrease in LRV1-mediated macrophage survival and consequently decreased parasite persistence. Consistent with these data, miR-155-deficient mice showed a drastic decrease in LRV1-induced disease severity, and lesional macrophages from these mice displayed reduced levels of Akt phosphorylation.
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http://dx.doi.org/10.1016/j.chom.2016.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5493041PMC
September 2016

IDO-orchestrated crosstalk between pDCs and Tregs inhibits autoimmunity.

J Autoimmun 2016 Dec 26;75:39-49. Epub 2016 Jul 26.

Department of Pathology and Immunology, University of Geneva, 1211 Geneva 4, Switzerland. Electronic address:

Plasmacytoid dendritic cells (pDCs) have been shown to both mediate and prevent autoimmunity, and the regulation of their immunogenic versus tolerogenic functions remains incompletely understood. Here we demonstrate that, compared to other cells, pDCs are the major expressors of Indoleamine-2,3-dioxygenase (IDO) in steady-state lymph nodes (LNs). IDO expression by LN pDCs was closely dependent on MHCII-mediated, antigen-dependent, interactions with Treg. We further established that IDO production by pDCs was necessary to confer suppressive function to Tregs. During EAE development, IDO expression by pDCs was required for the generation of Tregs capable of dampening the priming of encephalitogenic T cell and disease severity. Thus, we describe a novel crosstalk between pDCs and Tregs: Tregs shape tolerogenic functions of pDCs prior to inflammation, such that pDCs in turn, promote Treg suppressive functions during autoimmunity.
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http://dx.doi.org/10.1016/j.jaut.2016.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127883PMC
December 2016

T Cell Priming by Activated Nlrc5-Deficient Dendritic Cells Is Unaffected despite Partially Reduced MHC Class I Levels.

J Immunol 2016 Apr 4;196(7):2939-46. Epub 2016 Mar 4.

Department of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland;

NLRC5, a member of the NOD-like receptor (NLR) protein family, has recently been characterized as the master transcriptional regulator of MHCI molecules in lymphocytes, in which it is highly expressed. However, its role in activated dendritic cells (DCs), which are instrumental to initiate T cell responses, remained elusive. We show in this study that, following stimulation of DCs with inflammatory stimuli, not only did NLRC5 level increase, but also its importance in directing MHCI transcription. Despite markedly reduced mRNA and intracellular H2-K levels, we unexpectedly observed nearly normal H2-K surface display in Nlrc5(-/-) DCs. Importantly, this discrepancy between a strong intracellular and a mild surface defect in H2-K levels was observed also in DCs with H2-K transcription defects independent of Nlrc5. Hence, alongside with demonstrating the importance of NLRC5 in MHCI transcription in activated DCs, we uncover a general mechanism counteracting low MHCI surface expression. In agreement with the decreased amount of neosynthesized MHCI, Nlrc5(-/-) DCs exhibited a defective capacity to display endogenous Ags. However, neither T cell priming by endogenous Ags nor cross-priming ability was substantially affected in activated Nlrc5(-/-) DCs. Altogether, these data show that Nlrc5 deficiency, despite significantly affecting MHCI transcription and Ag display, is not sufficient to hinder T cell activation, underlining the robustness of the T cell priming process by activated DCs.
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http://dx.doi.org/10.4049/jimmunol.1502084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797726PMC
April 2016

Btn2a2, a T cell immunomodulatory molecule coregulated with MHC class II genes.

J Exp Med 2016 Feb 25;213(2):177-87. Epub 2016 Jan 25.

Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland

Evidence has recently emerged that butyrophilins, which are members of the extended B7 family of co-stimulatory molecules, have diverse functions in the immune system. We found that the human and mouse genes encoding butyrophilin-2A2 (BTN2A2) are regulated by the class II trans-activator and regulatory factor X, two transcription factors dedicated to major histocompatibility complex class II expression, suggesting a role in T cell immunity. To address this, we generated Btn2a2-deficient mice. Btn2a2(-/-) mice exhibited enhanced effector CD4(+) and CD8(+) T cell responses, impaired CD4(+) regulatory T cell induction, potentiated antitumor responses, and exacerbated experimental autoimmune encephalomyelitis. Altered immune responses were attributed to Btn2a2 deficiency in antigen-presenting cells rather than T cells or nonhematopoietic cells. These results provide the first genetic evidence that BTN2A2 is a co-inhibitory molecule that modulates T cell-mediated immunity.
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http://dx.doi.org/10.1084/jem.20150435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4749920PMC
February 2016

Regulation of Immunity by Butyrophilins.

Annu Rev Immunol 2016 05 11;34:151-72. Epub 2016 Jan 11.

Department of Pathology, Immunology Division, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 0XY, United Kingdom; email: ,

Butyrophilin molecules (commonly contracted to BTN), collectively take their name from the eponymous protein in cow's milk. They are considered to be members of the B7 family of costimulatory receptors, which includes B7.1 (CD80), B7.2 (CD86), and related molecules, such as PD-L1 (B7-H1, CD274), ICOS-L (CD275), and B7-H3 (CD276). These coreceptors modulate T cell responses upon antigen presentation by major histocompatibility complex and cognate αβ T cell receptor engagement. Molecules such as BTN3A1 (CD277), myelin oligodendrocyte glycoprotein, and mouse Skint1 and Btnl2, all members of the butyrophilin family, show greater structural and functional diversity than the canonical B7 receptors. Some butyrophilins mediate complex interactions between antigen-presenting cells and conventional αβ T cells, and others regulate the immune responses of specific γδ T cell subsets by mechanisms that have characteristics of both innate and adaptive immunity.
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http://dx.doi.org/10.1146/annurev-immunol-041015-055435DOI Listing
May 2016

Absence of nonhematopoietic MHC class II expression protects mice from experimental autoimmune myocarditis.

Eur J Immunol 2016 Mar 22;46(3):656-64. Epub 2015 Dec 22.

Department of Biochemistry, CIIL, University of Lausanne, Epalinges, Switzerland.

Experimental autoimmune myocarditis (EAM) is a CD4(+) T-cell-mediated model of human inflammatory dilated cardiomyopathies. Heart-specific CD4(+) T-cell activation is dependent on autoantigens presented by MHC class II (MHCII) molecules expressed on professional APCs. In this study, we addressed the role of inflammation-induced MHCII expression by cardiac nonhematopoietic cells on EAM development. EAM was induced in susceptible mice lacking inducible expression of MHCII molecules on all nonhematopoietic cells (pIV-/- K14 class II transactivator (CIITA) transgenic (Tg) mice) by immunization with α-myosin heavy chain peptide in CFA. Lack of inducible nonhematopoietic MHCII expression in pIV-/- K14 CIITA Tg mice conferred EAM resistance. In contrast, cardiac pathology was induced in WT and heterozygous mice, and correlated with elevated cardiac endothelial MHCII expression. Control mice with myocarditis displayed an increase in infiltrating CD4(+) T cells and in expression of IFN-γ, which is the major driver of nonhematopoietic MHCII expression. Mechanistically, IFN-γ neutralization in WT mice shortly before disease onset resulted in reduced cardiac MHCII expression and pathology. These findings reveal a previously overlooked contribution of IFN-γ to induce endothelial MHCII expression in the heart and to progress cardiac pathology during myocarditis.
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http://dx.doi.org/10.1002/eji.201545945DOI Listing
March 2016

RFX transcription factors are essential for hearing in mice.

Nat Commun 2015 Oct 15;6:8549. Epub 2015 Oct 15.

Department of Otorhinolaryngology, School of Medicine, University of Maryland Baltimore, 16 South Eutaw Street Suite 500, Baltimore, Maryland 21201, USA.

Sensorineural hearing loss is a common and currently irreversible disorder, because mammalian hair cells (HCs) do not regenerate and current stem cell and gene delivery protocols result only in immature HC-like cells. Importantly, although the transcriptional regulators of embryonic HC development have been described, little is known about the postnatal regulators of maturating HCs. Here we apply a cell type-specific functional genomic analysis to the transcriptomes of auditory and vestibular sensory epithelia from early postnatal mice. We identify RFX transcription factors as essential and evolutionarily conserved regulators of the HC-specific transcriptomes, and detect Rfx1,2,3,5 and 7 in the developing HCs. To understand the role of RFX in hearing, we generate Rfx1/3 conditional knockout mice. We show that these mice are deaf secondary to rapid loss of initially well-formed outer HCs. These data identify an essential role for RFX in hearing and survival of the terminally differentiating outer HCs.
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http://dx.doi.org/10.1038/ncomms9549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4634137PMC
October 2015

RFX2 Is a Major Transcriptional Regulator of Spermiogenesis.

PLoS Genet 2015 Jul 10;11(7):e1005368. Epub 2015 Jul 10.

Department of Pathology and Immunology, University of Geneva Medical School, CMU, Geneva, Switzerland.

Spermatogenesis consists broadly of three phases: proliferation of diploid germ cells, meiosis, and finally extensive differentiation of the haploid cells into effective delivery vehicles for the paternal genome. Despite detailed characterization of many haploid developmental steps leading to sperm, only fragmentary information exists on the control of gene expression underlying these processes. Here we report that the RFX2 transcription factor is a master regulator of genes required for the haploid phase. A targeted mutation of Rfx2 was created in mice. Rfx2-/- mice are perfectly viable but show complete male sterility. Spermatogenesis appears to progress unperturbed through meiosis. However, haploid cells undergo a complete arrest in spermatid development just prior to spermatid elongation. Arrested cells show altered Golgi apparatus organization, leading to a deficit in the generation of a spreading acrosomal cap from proacrosomal vesicles. Arrested cells ultimately merge to form giant multinucleated cells released to the epididymis. Spermatids also completely fail to form the flagellar axoneme. RNA-Seq analysis and ChIP-Seq analysis identified 139 genes directly controlled by RFX2 during spermiogenesis. Gene ontology analysis revealed that genes required for cilium function are specifically enriched in down- and upregulated genes showing that RFX2 allows precise temporal expression of ciliary genes. Several genes required for cell adhesion and cytoskeleton remodeling are also downregulated. Comparison of RFX2-regulated genes with those controlled by other major transcriptional regulators of spermiogenesis showed that each controls independent gene sets. Altogether, these observations show that RFX2 plays a major and specific function in spermiogenesis.
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http://dx.doi.org/10.1371/journal.pgen.1005368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498915PMC
July 2015

TLR3-Mediated CD8+ Dendritic Cell Activation Is Coupled with Establishment of a Cell-Intrinsic Antiviral State.

J Immunol 2015 Aug 22;195(3):1025-33. Epub 2015 Jun 22.

Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, CH-1211 Geneva, Switzerland;

Because of their unique capacity to cross-present Ags to CD8(+) T cells, mouse lymphoid tissue-resident CD8(+) dendritic cells (DCs) and their migratory counterparts are critical for priming antiviral T cell responses. High expression of the dsRNA sensor TLR3 is a distinctive feature of these cross-presenting DC subsets. TLR3 engagement in CD8(+) DCs promotes cross-presentation and the acquisition of effector functions required for driving antiviral T cell responses. In this study, we performed a comprehensive analysis of the TLR3-induced antiviral program and cell-autonomous immunity in CD8(+) DC lines and primary CD8(+) DCs. We found that TLR3-ligand polyinosinic-polycytidylic acid and human rhinovirus infection induced a potent antiviral protection against Sendai and vesicular stomatitis virus in a TLR3 and type I IFN receptor-dependent manner. Polyinosinic-polycytidylic acid-induced antiviral genes were identified by mass spectrometry-based proteomics and transcriptomics in the CD8(+) DC line. Nanostring nCounter experiments confirmed that these antiviral genes were induced by TLR3 engagement in primary CD8(+) DCs, and indicated that many are secondary TLR3-response genes requiring autocrine IFN-β stimulation. TLR3-activation thus establishes a type I IFN-dependent antiviral program in a DC subtype playing crucial roles in priming adaptive antiviral immune responses. This mechanism is likely to shield the priming of antiviral responses against inhibition or abrogation by the viral infection. It could be particularly relevant for viruses detected mainly by TLR3, which may not trigger type I IFN production by DCs that lack TLR3, such as plasmacytoid DCs or CD8(-) DCs.
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http://dx.doi.org/10.4049/jimmunol.1402033DOI Listing
August 2015

Immune tolerance. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4⁺ T cells.

Science 2015 May 23;348(6238):1031-5. Epub 2015 Apr 23.

Jill Roberts Institute for Research in Inflammatory Bowel Disease, Joan and Sanford I. Weill Department of Medicine, Gastroenterology Division, and Department of Microbiology and Immunology, Weill Cornell Medical College, Cornell University, New York, NY, USA.

Inflammatory CD4(+) T cell responses to self or commensal bacteria underlie the pathogenesis of autoimmunity and inflammatory bowel disease (IBD), respectively. Although selection of self-specific T cells in the thymus limits responses to mammalian tissue antigens, the mechanisms that control selection of commensal bacteria-specific T cells remain poorly understood. Here, we demonstrate that group 3 innate lymphoid cell (ILC3)-intrinsic expression of major histocompatibility complex class II (MHCII) is regulated similarly to thymic epithelial cells and that MHCII(+) ILC3s directly induce cell death of activated commensal bacteria-specific T cells. Further, MHCII on colonic ILC3s was reduced in pediatric IBD patients. Collectively, these results define a selection pathway for commensal bacteria-specific CD4(+) T cells in the intestine and suggest that this process is dysregulated in human IBD.
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http://dx.doi.org/10.1126/science.aaa4812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4449822PMC
May 2015

NLRC5 exclusively transactivates MHC class I and related genes through a distinctive SXY module.

PLoS Genet 2015 Mar 26;11(3):e1005088. Epub 2015 Mar 26.

Department of Biochemistry, University of Lausanne, Epalinges, Switzerland.

MHC class II (MHCII) genes are transactivated by the NOD-like receptor (NLR) family member CIITA, which is recruited to SXY enhancers of MHCII promoters via a DNA-binding "enhanceosome" complex. NLRC5, another NLR protein, was recently found to control transcription of MHC class I (MHCI) genes. However, detailed understanding of NLRC5's target gene specificity and mechanism of action remained lacking. We performed ChIP-sequencing experiments to gain comprehensive information on NLRC5-regulated genes. In addition to classical MHCI genes, we exclusively identified novel targets encoding non-classical MHCI molecules having important functions in immunity and tolerance. ChIP-sequencing performed with Rfx5(-/-) cells, which lack the pivotal enhanceosome factor RFX5, demonstrated its strict requirement for NLRC5 recruitment. Accordingly, Rfx5-knockout mice phenocopy Nlrc5 deficiency with respect to defective MHCI expression. Analysis of B cell lines lacking RFX5, RFXAP, or RFXANK further corroborated the importance of the enhanceosome for MHCI expression. Although recruited by common DNA-binding factors, CIITA and NLRC5 exhibit non-redundant functions, shown here using double-deficient Nlrc5(-/-)CIIta(-/-) mice. These paradoxical findings were resolved by using a "de novo" motif-discovery approach showing that the SXY consensus sequence occupied by NLRC5 in vivo diverges significantly from that occupied by CIITA. These sequence differences were sufficient to determine preferential occupation and transactivation by NLRC5 or CIITA, respectively, and the S box was found to be the essential feature conferring NLRC5 specificity. These results broaden our knowledge on the transcriptional activities of NLRC5 and CIITA, revealing their dependence on shared enhanceosome factors but their recruitment to distinct enhancer motifs in vivo. Furthermore, we demonstrated selectivity of NLRC5 for genes encoding MHCI or related proteins, rendering it an attractive target for therapeutic intervention. NLRC5 and CIITA thus emerge as paradigms for a novel class of transcriptional regulators dedicated for transactivating extremely few, phylogenetically related genes.
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http://dx.doi.org/10.1371/journal.pgen.1005088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374748PMC
March 2015

Ag-presenting CpG-activated pDCs prime Th17 cells that induce tumor regression.

Cancer Res 2014 Nov 24;74(22):6430-40. Epub 2014 Sep 24.

Department of Pathology and Immunology, University of Geneva Medical School, Geneva, Switzerland.

Plasmacytoid dendritic cells (pDC) rapidly and massively produce type I IFN and other inflammatory cytokines in response to foreign nucleic acids, thereby indirectly influencing T-cell responses. Moreover, antigen (Ag)-presenting pDCs directly regulate T-cell differentiation. Depending on the immune environment, pDCs exhibit either tolerogenic or immunogenic properties. Here, we show that CpG-activated pDCs promote efficient Th17 differentiation. Indeed, Th17 responses are defective in mice selectively lacking MHCII on pDCs upon antigenic challenge. Importantly, in those mice, the frequency of Th17 cells infiltrating solid tumors is impaired. As a result, the recruitment of infiltrating leukocytes in tumors, including tumor-specific cytotoxic T lymphocytes (CTL), is altered and results in increased tumor growth. Importantly, following immunization with tumor Ag and CpG-B, MHCII-restricted Ag presentation by pDCs promotes the differentiation of antitumor Th17 cells that induce intratumor CTL recruitment and subsequent regression of established tumors. Our results highlight a new role for Ag presenting activated pDCs in promoting the development of Th17 cells and impacting on antitumor immunity.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-1149DOI Listing
November 2014

HEATR2 plays a conserved role in assembly of the ciliary motile apparatus.

PLoS Genet 2014 Sep 18;10(9):e1004577. Epub 2014 Sep 18.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine at The University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom.

Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme.
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http://dx.doi.org/10.1371/journal.pgen.1004577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4168999PMC
September 2014

MHC Class II-restricted antigen presentation by plasmacytoid dendritic cells drives proatherogenic T cell immunity.

Circulation 2014 Oct 15;130(16):1363-73. Epub 2014 Sep 15.

From the Division of Cardiovascular Medicine, Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom (A.P.S., D.M., L.M.M., L.L.B., A.J.F., J.E.H., Z.M.); Centre for Immunobiology, Institute of Infection, Immunity, and Inflammation, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow, United Kingdom (P.M., S.R.S., H.C., G.G.); Institut National de la Santé et de la Recherche Médicale, Unit 970, Paris Cardiovascular Research Center, Paris, France (H.A., Z.M.); the Department of Pharmacy, University of Naples Federico II, Naples, Italy (P.M., G.G.); Institute of Immunobiology, Kantonal Hospital St. Gallen, CH-9007 St. Gallen, Switzerland (B.L.); the Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland (W.R.); Center for Molecular Medicine, Department of Medicine, Karolinska University Hospital, Karolinska Institute, Stockholm, Sweden (G.K.H.); the Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY (B.R.); and the Department of Pathology, University of Geneva Medical School, CH-1211 Geneva, Switzerland (S.H.).

Background: Plasmacytoid dendritic cells (pDCs) bridge innate and adaptive immune responses and are important regulators of immuno-inflammatory diseases. However, their role in atherosclerosis remains elusive.

Methods And Results: Here, we used genetic approaches to investigate the role of pDCs in atherosclerosis. Selective pDC deficiency in vivo was achieved using CD11c-Cre × Tcf4(-/flox) bone marrow transplanted into Ldlr(-/-) mice. Compared with control Ldlr(-/-) chimeric mice, CD11c-Cre × Tcf4(-/flox) mice had reduced atherosclerosis levels. To begin to understand the mechanisms by which pDCs regulate atherosclerosis, we studied chimeric Ldlr(-/-) mice with selective MHCII deficiency on pDCs. Significantly, these mice also developed reduced atherosclerosis compared with controls without reductions in pDC numbers or changes in conventional DCs. MHCII-deficient pDCs showed defective stimulation of apolipoprotein B100-specific CD4(+) T cells in response to native low-density lipoprotein, whereas production of interferon-α was not affected. Finally, the atheroprotective effect of selective MHCII deficiency in pDCs was associated with significant reductions of proatherogenic T cell-derived interferon-γ and lesional T cell infiltration, and was abrogated in CD4(+) T cell-depleted animals.

Conclusions: This study supports a proatherogenic role for pDCs in murine atherosclerosis and identifies a critical role for MHCII-restricted antigen presentation by pDCs in driving proatherogenic T cell immunity.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.114.011090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4428652PMC
October 2014

Hepatocyte growth factor limits autoimmune neuroinflammation via glucocorticoid-induced leucine zipper expression in dendritic cells.

J Immunol 2014 Sep 11;193(6):2743-52. Epub 2014 Aug 11.

Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland; Unit of Neuroimmunology and Multiple Sclerosis, Division of Neurology, Department of Clinical Neurosciences, Geneva University Hospital, 1211 Geneva, Switzerland; Division of Laboratory Medicine, Department of Genetic and Laboratory Medicine, Geneva University Hospital, 1211 Geneva, Switzerland

Autoimmune neuroinflammation, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), a prototype for T cell-mediated autoimmunity, is believed to result from immune tolerance dysfunction leading to demyelination and substantial neurodegeneration. We previously showed that CNS-restricted expression of hepatocyte growth factor (HGF), a potent neuroprotective factor, reduced CNS inflammation and clinical deficits associated with EAE. In this study, we demonstrate that systemic HGF treatment ameliorates EAE through the development of tolerogenic dendritic cells (DCs) with high expression levels of glucocorticoid-induced leucine zipper (GILZ), a transcriptional repressor of gene expression and a key endogenous regulator of the inflammatory response. RNA interference-directed neutralization of GILZ expression by DCs suppressed the induction of tolerance caused by HGF. Finally, adoptive transfer of HGF-treated DCs from wild-type but not GILZ gene-deficient mice potently mediated functional recovery in recipient mice with established EAE through effective modulation of autoaggressive T cell responses. Altogether, these results show that by inducing GILZ in DCs, HGF reproduces the mechanism of immune regulation induced by potent immunomodulatory factors such as IL-10, TGF-β1, and glucocorticoids and therefore that HGF therapy may have potential in the treatment of autoimmune dysfunctions.
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http://dx.doi.org/10.4049/jimmunol.1302338DOI Listing
September 2014

Extensive remodeling of DC function by rapid maturation-induced transcriptional silencing.

Nucleic Acids Res 2014 Sep 7;42(15):9641-55. Epub 2014 Aug 7.

Department of Pathology and Immunology, University of Geneva Medical School, CH-1211 Geneva, Switzerland

The activation, or maturation, of dendritic cells (DCs) is crucial for the initiation of adaptive T-cell mediated immune responses. Research on the molecular mechanisms implicated in DC maturation has focused primarily on inducible gene-expression events promoting the acquisition of new functions, such as cytokine production and enhanced T-cell-stimulatory capacity. In contrast, mechanisms that modulate DC function by inducing widespread gene-silencing remain poorly understood. Yet the termination of key functions is known to be critical for the function of activated DCs. Genome-wide analysis of activation-induced histone deacetylation, combined with genome-wide quantification of activation-induced silencing of nascent transcription, led us to identify a novel inducible transcriptional-repression pathway that makes major contributions to the DC-maturation process. This silencing response is a rapid primary event distinct from repression mechanisms known to operate at later stages of DC maturation. The repressed genes function in pivotal processes--including antigen-presentation, extracellular signal detection, intracellular signal transduction and lipid-mediator biosynthesis--underscoring the central contribution of the silencing mechanism to rapid reshaping of DC function. Interestingly, promoters of the repressed genes exhibit a surprisingly high frequency of PU.1-occupied sites, suggesting a novel role for this lineage-specific transcription factor in marking genes poised for inducible repression.
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http://dx.doi.org/10.1093/nar/gku674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4150779PMC
September 2014

Repression of arginase-2 expression in dendritic cells by microRNA-155 is critical for promoting T cell proliferation.

J Immunol 2014 Aug 9;193(4):1690-700. Epub 2014 Jul 9.

Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, CH-1211 Geneva, Switzerland; and

Arginine, a semiessential amino acid implicated in diverse cellular processes, is a substrate for two arginases-Arg1 and Arg2-having different expression patterns and functions. Although appropriately regulated Arg1 expression is critical for immune responses, this has not been documented for Arg2. We show that Arg2 is the dominant enzyme in dendritic cells (DCs) and is repressed by microRNA-155 (miR155) during their maturation. miR155 is known to be strongly induced in various mouse and human DC subsets in response to diverse maturation signals, and miR155-deficient DCs exhibit an impaired ability to induce Ag-specific T cell responses. By means of expression profiling studies, we identified Arg2 mRNA as a novel miR155 target in mouse DCs. Abnormally elevated levels of Arg2 expression and activity were observed in activated miR155-deficient DCs. Conversely, overexpression of miR155 inhibited Arg2 expression. Bioinformatic and functional analyses confirmed that Arg2 mRNA is a direct target of miR155. Finally, in vitro and in vivo functional assays using DCs exhibiting deregulated Arg2 expression indicated that Arg2-mediated arginine depletion in the extracellular milieu impairs T cell proliferation. These results indicate that miR155-induced repression of Arg2 expression is critical for the ability of DCs to drive T cell activation by controlling arginine availability in the extracellular environment.
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http://dx.doi.org/10.4049/jimmunol.1301913DOI Listing
August 2014

Lymph node stromal cells acquire peptide-MHCII complexes from dendritic cells and induce antigen-specific CD4⁺ T cell tolerance.

J Exp Med 2014 Jun 19;211(6):1153-66. Epub 2014 May 19.

Department of Pathology and Immunology, University of Geneva Medical School, 1211 Geneva, Switzerland

Dendritic cells (DCs), and more recently lymph node stromal cells (LNSCs), have been described to tolerize self-reactive CD8(+) T cells in LNs. Although LNSCs express MHCII, it is unknown whether they can also impact CD4(+) T cell functions. We show that the promoter IV (pIV) of class II transactivator (CIITA), the master regulator of MHCII expression, controls endogenous MHCII expression by LNSCs. Unexpectedly, LNSCs also acquire peptide-MHCII complexes from DCs and induce CD4(+) T cell dysfunction by presenting transferred complexes to naive CD4(+) T cells and preventing their proliferation and survival. Our data reveals a novel, alternative mechanism where LN-resident stromal cells tolerize CD4(+) T cells through the presentation of self-antigens via transferred peptide-MHCII complexes of DC origin.
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http://dx.doi.org/10.1084/jem.20132000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4042642PMC
June 2014

Selective antibody intervention of Toll-like receptor 4 activation through Fc γ receptor tethering.

J Biol Chem 2014 May 15;289(22):15309-18. Epub 2014 Apr 15.

From the NovImmune SA, 14 Chemin des Aulx, 1228 Plan les Ouates, Switzerland.

Inflammation is mediated mainly by leukocytes that express both Toll-like receptor 4 (TLR4) and Fc γ receptors (FcγR). Dysregulated activation of leukocytes via exogenous and endogenous ligands of TLR4 results in a large number of inflammatory disorders that underlie a variety of human diseases. Thus, differentially blocking inflammatory cells while sparing structural cells, which are FcγR-negative, represents an elegant strategy when targeting the underlying causes of human diseases. Here, we report a novel tethering mechanism of the Fv and Fc portions of anti-TLR4 blocking antibodies that achieves increased potency on inflammatory cells. In the presence of ligand (e.g. lipopolysaccharide (LPS)), TLR4 traffics into glycolipoprotein microdomains, forming concentrated protein platforms that include FcγRs. This clustering produces a microenvironment allowing anti-TLR4 antibodies to co-engage TLR4 and FcγRs, increasing their avidity and thus substantially increasing their inhibitory potency. Tethering of antibodies to both TLR4 and FcγRs proves valuable in ameliorating inflammation in vivo. This novel mechanism of action therefore has the potential to enable selective intervention of relevant cell types in TLR4-driven diseases.
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http://dx.doi.org/10.1074/jbc.M113.537936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140888PMC
May 2014