SHP-2 and PD-1-SHP-2 signaling regulate myeloid cell differentiation and antitumor responses.

The inhibitory receptor PD-1 suppresses T cell activation by recruiting the phosphatase SHP-2. However, mice with a T-cell-specific deletion of SHP-2 do not have improved antitumor immunity. Here we showed that mice with conditional targeting of SHP-2 in myeloid cells, but not in T cells, had diminished tumor growth. RNA sequencing (RNA-seq) followed by gene set enrichment analysis indicated the presence of polymorphonuclear myeloid-derived suppressor cells and tumor-associated macrophages (TAMs) with enriched gene expression profiles of enhanced differentiation, activation and expression of immunostimulatory molecules. In mice with conditional targeting of PD-1 in myeloid cells, which also displayed diminished tumor growth, TAMs had gene expression profiles enriched for myeloid differentiation, activation and leukocyte-mediated immunity displaying >50% overlap with enriched profiles of SHP-2-deficient TAMs. In bone marrow, GM-CSF induced the phosphorylation of PD-1 and recruitment of PD-1-SHP-2 to the GM-CSF receptor. Deletion of SHP-2 or PD-1 enhanced GM-CSF-mediated phosphorylation of the transcription factors HOXA10 and IRF8, which regulate myeloid differentiation and monocytic-moDC lineage commitment, respectively. Thus, SHP-2 and PD-1-SHP-2 signaling restrained myelocyte differentiation resulting in a myeloid landscape that suppressed antitumor immunity.

An intrinsic role of IL-33 in Treg cell-mediated tumor immunoevasion.

Regulatory T (Treg) cells accumulate into tumors, hindering the success of cancer immunotherapy. Yet, therapeutic targeting of Treg cells shows limited efficacy or leads to autoimmunity. The molecular mechanisms that guide Treg cell stability in tumors remain elusive. In the present study, we identify a cell-intrinsic role of the alarmin interleukin (IL)-33 in the functional stability of Treg cells. Specifically, IL-33-deficient Treg cells demonstrated attenuated suppressive properties in vivo and facilitated tumor regression in a suppression of tumorigenicity 2 receptor (ST2) (IL-33 receptor)-independent fashion. On activation, Il33-/- Treg cells exhibited epigenetic re-programming with increased chromatin accessibility of the Ifng locus, leading to elevated interferon (IFN)-γ production in a nuclear factor (NF)-κB-T-bet-dependent manner. IFN-γ was essential for Treg cell defective function because its ablation restored Il33-/- Treg cell-suppressive properties. Importantly, genetic ablation of Il33 potentiated the therapeutic effect of immunotherapy. Our findings reveal a new and therapeutically important intrinsic role of IL-33 in Treg cell stability in cancer.

T helper 2 cells control monocyte to tissue-resident macrophage differentiation during nematode infection of the pleural cavity.

The recent revolution in tissue-resident macrophage biology has resulted largely from murine studies performed in C57BL/6 mice. Here, using both C57BL/6 and BALB/c mice, we analyze immune cells in the pleural cavity. Unlike C57BL/6 mice, naive tissue-resident large-cavity macrophages (LCMs) of BALB/c mice failed to fully implement the tissue-residency program. Following infection with a pleural-dwelling nematode, these pre-existing differences were accentuated with LCM expansion occurring in C57BL/6, but not in BALB/c mice. While infection drove monocyte recruitment in both strains, only in C57BL/6 mice were monocytes able to efficiently integrate into the resident pool. Monocyte-to-macrophage conversion required both T cells and interleukin-4 receptor alpha (IL-4Rα) signaling. The transition to tissue residency altered macrophage function, and GATA6+ tissue-resident macrophages were required for host resistance to nematode infection. Therefore, during tissue nematode infection, T helper 2 (Th2) cells control the differentiation pathway of resident macrophages, which determines infection outcome.

Indoleamine 2,3-dioxygenase 1 activation in mature cDC1 promotes tolerogenic education of inflammatory cDC2 via metabolic communication.

Conventional dendritic cells (cDCs), cDC1 and cDC2, act both to initiate immunity and maintain self-tolerance. The tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is used by cDCs in maintaining tolerance, but its role in different subsets remains unclear. At homeostasis, only mature CCR7+ cDC1 expressed IDO1 that was dependent on IRF8. Lipopolysaccharide treatment induced maturation and IDO1-dependent tolerogenic activity in isolated immature cDC1, but not isolated cDC2. However, both human and mouse cDC2 could induce IDO1 and acquire tolerogenic function when co-cultured with mature cDC1 through the action of cDC1-derived l-kynurenine. Accordingly, cDC1-specific inactivation of IDO1 in vivo exacerbated disease in experimental autoimmune encephalomyelitis. This study identifies a previously unrecognized metabolic communication in which IDO1-expressing cDC1 cells extend their immunoregulatory capacity to the cDC2 subset through their production of tryptophan metabolite l-kynurenine. This metabolic axis represents a potential therapeutic target in treating autoimmune demyelinating diseases.

Interleukin-17A Serves a Priming Role in Autoimmunity by Recruiting IL-1ß-Producing Myeloid Cells that Promote Pathogenic T Cells.

Interleukin-17A (IL-17A) is a major mediator of tissue inflammation in many autoimmune diseases. Anti-IL-17A is an effective treatment for psoriasis and is showing promise in clinical trials in multiple sclerosis. In this study, we find that IL-17A-defective mice or mice treated with anti-IL-17A at induction of experimental autoimmune encephalomyelitis (EAE) are resistant to disease and have defective priming of IL-17-secreting γδ T (γδT17) cells and Th17 cells. However, T cells from Il17a-/- mice induce EAE in wild-type mice following in vitro culture with autoantigen, IL-1ß, and IL-23. Furthermore, treatment with IL-1ß or IL-17A at induction of EAE restores disease in Il17a-/- mice. Importantly, mobilization of IL-1ß-producing neutrophils and inflammatory monocytes and activation of γδT17 cells is reduced in Il17a-/- mice. Our findings demonstrate that a key function of IL-17A in central nervous system (CNS) autoimmunity is to recruit IL-1ß-secreting myeloid cells that prime pathogenic γδT17 and Th17 cells.

CD8+ T Cells Drive Plaque Smooth Muscle Cell Dedifferentiation in Experimental Atherosclerosis.

BACKGROUND: Atherosclerosis is driven by the infiltration of the arterial intima by diverse immune cells and smooth muscle cells (SMCs). CD8+ T cells promote lesion growth during atherosclerotic lesion development, but their role in advanced atherosclerosis is less clear. Here, we studied the role of CD8+ T cells and their effects on SMCs in established atherosclerosis. METHODS: CD8+ T cells were depleted in (SMC reporter) low-density lipoprotein receptor-deficient (Ldlr-/-) mice with established atherosclerotic lesions. Atherosclerotic lesion formation was examined, and single-cell RNA sequencing of aortic SMCs and their progeny was performed. Additionally, coculture experiments with primary aortic SMCs and CD8+ T cells were conducted. RESULTS: Although we could not detect differences in atherosclerotic lesion size, an increased plaque SMC content was noted in mice after CD8+ T-cell depletion. Single-cell RNA sequencing of aortic lineage-traced SMCs revealed contractile SMCs and a modulated SMC cluster, expressing macrophage- and osteoblast-related genes. CD8+ T-cell depletion was associated with an increased contractile but decreased macrophage and osteoblast-like gene signature in this modulated aortic SMC cluster. Conversely, exposure of isolated aortic SMCs to activated CD8+ T cells decreased the expression of genes indicative of a contractile SMC phenotype and induced a macrophage and osteoblast-like cell state. Notably, CD8+ T cells triggered calcium deposits in SMCs under osteogenic conditions. Mechanistically, we identified transcription factors highly expressed in modulated SMCs, including Runx1, to be induced by CD8+ T cells in cultured SMCs in an IFNγ (interferon-γ)-dependent manner. CONCLUSIONS: We here uncovered CD8+ T cells to control the SMC phenotype in atherosclerosis. CD8+ T cells promote SMC dedifferentiation and drive SMCs to adopt features of an osteoblast-like, procalcifying cell phenotype. Given the critical role of SMCs in atherosclerotic plaque stability, CD8+ T cells could thus be explored as therapeutic target cells during lesion progression.

Leukocyte-associated immunoglobulin-like receptor-1 blockade in combination with programmed death-ligand 1 targeting therapy mediates increased tumour control in mice.

Collagen expression and structure in the tumour microenvironment are associated with tumour development and therapy response. Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a widely expressed inhibitory collagen receptor. LAIR-2 is a soluble homologue of LAIR-1 that competes for collagen binding. Multiple studies in mice implicate blockade of LAIR-1:collagen interaction in cancer as a promising therapeutic strategy. Here, we investigated the role of LAIR-1 in anti-tumour responses. We show that although LAIR-1 inhibits activation, proliferation, and cytokine production of mouse T cells in vitro, tumour outgrowth in LAIR-1-deficient mice did not differ from wild type mice in several in vivo tumour models. Furthermore, treatment with NC410, a LAIR-2-Fc fusion protein, did not result in increased tumour clearance in tested immunocompetent mice, which contrasts with previous data in humanized mouse models. This discrepancy may be explained by our finding that NC410 blocks human LAIR-1:collagen interaction more effectively than mouse LAIR-1:collagen interaction. Despite the lack of therapeutic impact of NC410 monotherapy, mice treated with a combination of NC410 and anti-programmed death-ligand 1 did show reduced tumour burden and increased survival. Using LAIR-1-deficient mice, we showed that this effect seemed to be dependent on the presence of LAIR-1. Taken together, our data demonstrate that the absence of LAIR-1 signalling alone is not sufficient to control tumour growth in multiple immunocompetent mouse models. However, combined targeting of LAIR-1 and PD-L1 results in increased tumour control. Thus, additional targeting of the LAIR-1:collagen pathway with NC410 is a promising approach to treating tumours where conventional immunotherapy is ineffective.

Interleukin-21-Producing CD4(+) T Cells Promote Type 2 Immunity to House Dust Mites.

Asthma is a T helper 2 (Th2)-cell-mediated disease; however, recent findings implicate Th17 and innate lymphoid cells also in regulating airway inflammation. Herein, we have demonstrated profound interleukin-21 (IL-21) production after house dust mite (HDM)-driven asthma by using T cell receptor (TCR) transgenic mice reactive to Dermatophagoides pteronyssinus 1 and an IL-21GFP reporter mouse. IL-21-producing cells in the mediastinal lymph node (mLN) bore characteristics of T follicular helper (Tfh) cells, whereas IL-21(+) cells in the lung did not express CXCR5 (a chemokine receptor expressed by Tfh cells) and were distinct from effector Th2 or Th17 cells. Il21r(-/-) mice developed reduced type 2 responses and the IL-21 receptor (IL-21R) enhanced Th2 cell function in a cell-intrinsic manner. Finally, administration of recombinant IL-21 and IL-25 synergistically promoted airway eosinophilia primarily via effects on CD4(+) lymphocytes. This highlights an important Th2-cell-amplifying function of IL-21-producing CD4(+) T cells in allergic airway inflammation.

Integration of Th17- and Lymphotoxin-Derived Signals Initiates Meningeal-Resident Stromal Cell Remodeling to Propagate Neuroinflammation.

Tertiary lymphoid tissues (TLTs) have been observed in the meninges of multiple sclerosis (MS) patients, but the stromal cells and molecular signals that support TLTs remain unclear. Here, we show that T helper 17 (Th17) cells induced robust TLTs within the brain meninges that were associated with local demyelination during experimental autoimmune encephalitis (EAE). Th17-cell-induced TLTs were underpinned by a network of stromal cells producing extracellular matrix proteins and chemokines, enabling leukocytes to reside within, rather than simply transit through, the meninges. Within the CNS, interactions between lymphotoxin αß (LTαß) on Th17 cells and LTßR on meningeal radio-resistant cells were necessary for the propagation of de novo interleukin-17 responses, and activated T cells from MS patients expressed elevated levels of LTßR ligands. Therefore, input from both Th17 cells and the lymphotoxin pathway induce the formation of an immune-competent stromal cell niche in the meninges.

Peribronchial Inflammation Resulting from Regulatory T Cell Deficiency Damages the Respiratory Epithelium and Disturbs Barrier Function.

Regulatory T cells (Tregs) that express the transcription factor Foxp3 have a critical role in limiting inflammatory processes and tissue damage. Whether Tregs are functional in maintaining epithelial barriers and in control of tight junction expression has not yet been explored. In this study, we investigated the effect of Treg deficiency on the airway epithelial barrier in an experimental murine model in which diphtheria toxin was repeatedly injected in Foxp3-diphtheria toxin receptor (DTR) mice to deplete Tregs. This resulted in spontaneous peribronchial inflammation and led to a systemic and local increase of IL-4, IL-5, CCL3, IFN-γ, and IL-10 and a local (lung) increase of IL-6 and IL-33 and decreased amphiregulin levels. Moreover, Treg depletion increased airway permeability and decreased epithelial tight junction (protein and mRNA) expression. CTLA4-Ig treatment of Treg-depleted mice almost completely prevented barrier dysfunction together with suppression of lung inflammation and cytokine secretion. Treatment with anti-IL-4 partly reversed the effects of Treg depletion on tight junction expression, whereas neutralization of IL-6 of IFN-γ had either no effect or only a limited effect. We conclude that Tregs are essential to protect the epithelial barrier at the level of tight junctions by restricting spontaneous T cell activation and uncontrolled secretion of cytokines, in particular IL-4, in the bronchi.

Termination of CD40L co-stimulation promotes human B cell differentiation into antibody-secreting cells.

Human naïve B cells are notoriously difficult to differentiate into antibody-secreting cells (ASCs) in vitro while maintaining sufficient cell numbers to evaluate the differentiation process. B cells require T follicular helper (TFH ) cell-derived signals like CD40L and IL-21 during germinal center (GC) responses to undergo differentiation into ASCs. Cognate interactions between B and TFH cells are transient; after TFH contact, B cells cycle between GC light and dark zones where TFH contact is present and absent, respectively. Here, we elucidated that the efficacy of naïve B cells in ACS differentiation is dramatically enhanced by the release of CD40L stimulation. Multiparameter phospho-flow and transcription factor (TF)-flow cytometry revealed that termination of CD40L stimulation downmodulates NF-κB and STAT3 signaling. Furthermore, the termination of CD40 signaling downmodulates C-MYC, while promoting ASC TFs BLIMP1 and XBP-1s. Reduced levels of C-MYC in the differentiating B cells are later associated with crucial downmodulation of the B cell signature TF PAX5 specifically upon the termination of CD40 signaling, resulting in the differentiation of BLIMP1 high expressing cells into ASCs. The data presented here are the first steps to provide further insights how the transient nature of CD40 signaling is in fact needed for efficient human naïve B cell differentiation to ASCs.

Trichuris muris infection drives cell-intrinsic IL4R alpha independent colonic RELMα+ macrophages.

The intestinal nematode parasite Trichuris muris dwells in the caecum and proximal colon driving an acute resolving intestinal inflammation dominated by the presence of macrophages. Notably, these macrophages are characterised by their expression of RELMα during the resolution phase of the infection. The RELMα+ macrophage phenotype associates with the presence of alternatively activated macrophages and work in other model systems has demonstrated that the balance of classically and alternatively activated macrophages is critically important in enabling the resolution of inflammation. Moreover, in the context of type 2 immunity, RELMα+ alternatively activated macrophages are associated with the activation of macrophages via the IL4Rα. Despite a breadth of inflammatory pathologies associated with the large intestine, including those that accompany parasitic infection, it is not known how colonic macrophages are activated towards an alternatively activated phenotype. Here, we address this important knowledge gap by using Trichuris muris infection, in combination with transgenic mice (IL4Rαfl/fl.CX3CR1Cre) and IL4Rα-deficient/wild-type mixed bone marrow chimaeras. We make the unexpected finding that education of colonic macrophages towards a RELMα+, alternatively activated macrophage phenotype during T. muris infection does not require IL4Rα expression on macrophages. Further, this independence is maintained even when the mice are treated with an anti-IFNγ antibody during infection to create a strongly polarised Th2 environment. In contrast to RELMα, PD-L2 expression on macrophages post infection was dependent on IL4Rα signalling in the macrophages. These novel data sets are important, revealing a surprising cell-intrinsic IL4R alpha independence of the colonic RELMα+ alternatively activated macrophage during Trichuris muris infection.

Lipocalin 2 modulates dendritic cell activity and shapes immunity to influenza in a microbiome dependent manner.

Lipocalin 2 (LCN2) is a secreted glycoprotein with roles in multiple biological processes. It contributes to host defense by interference with bacterial iron uptake and exerts immunomodulatory functions in various diseases. Here, we aimed to characterize the function of LCN2 in lung macrophages and dendritic cells (DCs) using Lcn2-/- mice. Transcriptome analysis revealed strong LCN2-related effects in CD103+ DCs during homeostasis, with differential regulation of antigen processing and presentation and antiviral immunity pathways. We next validated the relevance of LCN2 in a mouse model of influenza infection, wherein LCN2 protected from excessive weight loss and improved survival. LCN2-deficiency was associated with enlarged mediastinal lymph nodes and increased lung T cell numbers, indicating a dysregulated immune response to influenza infection. Depletion of CD8+ T cells equalized weight loss between WT and Lcn2-/- mice, proving that LCN2 protects from excessive disease morbidity by dampening CD8+ T cell responses. In vivo T cell chimerism and in vitro T cell proliferation assays indicated that improved antigen processing by CD103+ DCs, rather than T cell intrinsic effects of LCN2, contribute to the exacerbated T cell response. Considering the antibacterial potential of LCN2 and that commensal microbes can modulate antiviral immune responses, we speculated that LCN2 might cause the observed influenza phenotype via the microbiome. Comparing the lung and gut microbiome of WT and Lcn2-/- mice by 16S rRNA gene sequencing, we observed profound effects of LCN2 on gut microbial composition. Interestingly, antibiotic treatment or co-housing of WT and Lcn2-/- mice prior to influenza infection equalized lung CD8+ T cell counts, suggesting that the LCN2-related effects are mediated by the microbiome. In summary, our results highlight a novel regulatory function of LCN2 in the modulation of antiviral immunity.

Indoleamine 2,3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells.

Regulation of tryptophan metabolism by indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) is a highly versatile modulator of immunity. In inflammation, interferon-γ is the main inducer of IDO for the prevention of hyperinflammatory responses, yet IDO is also responsible for self-tolerance effects in the longer term. Here we show that treatment of mouse plasmacytoid DCs (pDCs) with transforming growth factor-ß (TGF-ß) conferred regulatory effects on IDO that were mechanistically separable from its enzymic activity. We found that IDO was involved in intracellular signaling events responsible for the self-amplification and maintenance of a stably regulatory phenotype in pDCs. Thus, IDO has a tonic, nonenzymic function that contributes to TGF-ß-driven tolerance in noninflammatory contexts.

Interleukin-33 improves local immunity during Gram-negative pneumonia by a combined effect on neutrophils and inflammatory monocytes.

Pneumonia represents a major health care burden and Gram-negative bacteria provide an increasing therapeutic challenge at least in part through the emergence of multidrug-resistant strains. IL-33 is a multifunctional cytokine belonging to the IL-1 family that can affect many different cell types. We sought here to determine the effect of recombinant IL-33 on the host response during murine pneumonia caused by the common Gram-negative pathogen Klebsiella pneumoniae. IL-33 pretreatment prolonged survival for more than 1 day during lethal airway infection and decreased bacterial loads at the primary site of infection and distant organs. Postponed treatment with IL-33 (3 h) also reduced bacterial growth and dissemination. IL-33-mediated protection was not observed in mice deficient for the IL-33 receptor component IL-1 receptor-like 1. IL-33 induced a brisk type 2 response, characterized by recruitment of type 2 innate lymphoid cells to the lungs and enhanced release of IL-5 and IL-13. However, neither absence of innate lymphoid cells or IL-13, nor blocking of IL-5 impacted on IL-33 effects in mice infected with Klebsiella. Likewise, IL-33 remained effective in reducing bacterial loads in mice lacking B, T, and natural killer T cells. Experiments using antibody-mediated cell depletion indicated that neutrophils and inflammatory monocytes were of importance for antibacterial defense. The capacity of IL-33 to restrict bacterial growth in the lungs was strongly reduced in mice depleted of both neutrophils and inflammatory monocytes, but not in mice selectively depleted of either one of these cell types. These results suggest that IL-33 boosts host defense during bacterial pneumonia by a combined effect on neutrophils and inflammatory monocytes. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

ARGX-117, a therapeutic complement inhibiting antibody targeting C2.

BACKGROUND: Activation of the classical and lectin pathway of complement may contribute to tissue damage and organ dysfunction of antibody-mediated diseases and ischemia-reperfusion conditions. Complement factors are being considered as targets for therapeutic intervention. OBJECTIVE: We sought to characterize ARGX-117, a humanized inhibitory monoclonal antibody against complement C2. METHODS: The mode-of-action and binding characteristics of ARGX-117 were investigated in detail. Furthermore, its efficacy was analyzed in in vitro complement cytotoxicity assays. Finally, a pharmacokinetic/pharmacodynamic study was conducted in cynomolgus monkeys. RESULTS: Through binding to the Sushi-2 domain of C2, ARGX-117 prevents the formation of the C3 proconvertase and inhibits classical and lectin pathway activation upstream of C3 activation. As ARGX-117 does not inhibit the alternative pathway, it is expected not to affect the antimicrobial activity of this complement pathway. ARGX-117 prevents complement-mediated cytotoxicity in in vitro models for autoimmune hemolytic anemia and antibody-mediated rejection of organ transplants. ARGX-117 exhibits pH- and calcium-dependent target binding and is Fc-engineered to increase affinity at acidic pH to the neonatal Fc receptor, and to reduce effector functions. In cynomolgus monkeys, ARGX-117 dose-dependently reduces free C2 levels and classical pathway activity. A 2-dose regimen of 80 and 20 mg/kg separated by a week, resulted in profound reduction of classical pathway activity lasting for at least 7 weeks. CONCLUSIONS: ARGX-117 is a promising new complement inhibitor that is uniquely positioned to target both the classical and lectin pathways while leaving the alternative pathway intact.

Regulatory Role for NK Cells in a Mouse Model of Systemic Juvenile Idiopathic Arthritis.

Mice deficient in IFN-γ (IFN-γ knockout [KO] mice) develop a systemic inflammatory syndrome in response to CFA, in contrast to CFA-challenged wild-type (WT) mice who only develop a mild inflammation. Symptoms in CFA-challenged IFN-γ KO resemble systemic juvenile idiopathic arthritis (sJIA), a childhood immune disorder of unknown cause. Dysregulation of innate immune cells is considered to be important in the disease pathogenesis. In this study, we used this murine model to investigate the role of NK cells in the pathogenesis of sJIA. NK cells of CFA-challenged IFN-γ KO mice displayed an aberrant balance of activating and inhibitory NK cell receptors, lower expression of cytotoxic proteins, and a defective NK cell cytotoxicity. Depletion of NK cells (via anti-IL-2Rß and anti-Asialo-GM1 Abs) or blockade of the NK cell activating receptor NKG2D in CFA-challenged WT mice resulted in increased severity of systemic inflammation and appearance of sJIA-like symptoms. NK cells of CFA-challenged IFN-γ KO mice and from anti-NKG2D-treated mice showed defective degranulation capacities toward autologous activated immune cells, predominantly monocytes. This is in line with the increased numbers of activated inflammatory monocytes in these mice which was particularly reflected in the expression of CCR2, a chemokine receptor, and in the expression of Rae-1, a ligand for NKG2D. In conclusion, NK cells are defective in a mouse model of sJIA and impede disease development in CFA-challenged WT mice. Our findings point toward a regulatory role for NK cells in CFA-induced systemic inflammation via a NKG2D-dependent control of activated immune cells.

CD62L Is a Functional and Phenotypic Marker for Circulating Innate Lymphoid Cell Precursors.

Innate lymphoid cells (ILCs) guard epithelial tissue integrity during homeostasis, but can be potent immune effector cells during inflammation. Precursors to all ILC subsets (ILC precursors [ILCP]) have been identified in human peripheral blood (PB). We found that during homeostasis, ILCP in PB of mouse and human expressed homing receptors for secondary lymphoid organs, mainly CD62L. These ILCP entered mouse lymph nodes in a CD62L-dependent way and relied on S1P receptors for their exit. Importantly, CD62L expression was absent on human ILCs expressing NKp44 in tonsils and PB of Crohn disease patients, and relatively fewer CD62L+ ILCP were present in PB of Crohn disease patients. These data are in agreement with selective expression of CD62L on nonactivated ILCP. As such, we conclude that CD62L not only serves as a functional marker of ILCP, but has potential to be used in the clinic as a diagnostic marker in inflammatory disorders.

Involvement of Dendritic Cells and Th17 Cells in Induced Tertiary Lymphoid Structures in a Chronic Beryllium Disease Mouse Model.

OBJECTIVE: To study airway pathophysiology and the role of dendritic cells (DCs) and IL-17 receptor (IL-17R) signals in a mouse model for CBD. METHODS: Here, we present a CBD mouse model in which mice were exposed to beryllium during three weeks. We also exposed IL-17R-deficient mice and mice in which DCs were depleted. RESULTS: Eight weeks after the initial beryllium exposure, an inflammatory response was detected in the lungs. Mice displayed inflammation of the lower airways that included focal dense infiltrates, granuloma-like foci, and tertiary lymphoid structure (TLS) containing T cells, B cells, and germinal centers. Alveolar cell analysis showed significantly increased numbers of CD4+ T cells expressing IFNγ, IL-17, or both cytokines. The pathogenic role of IL-17R signals was demonstrated in IL-17R-deficient mice, which had strongly reduced lung inflammation and TLS development following beryllium exposure. In CBD mice, pulmonary DC subsets including CD103+ conventional DCs (cDCs), CD11b+ cDCs, and monocyte-derived DCs (moDCs) were also prominently increased. We used diphtheria toxin receptor-mediated targeted cell ablation to conditionally deplete DCs and found that DCs are essential for the maintenance of TLS in CBD. Furthermore, the presence of antinuclear autoantibodies in the serum of CBD mice showed that CBD had characteristics of autoimmune disease. CONCLUSIONS: We generated a translational model of sarcoidosis driven by beryllium and show that DCs and IL-17R signals play a pathophysiological role in CBD development as well as in established CBD in vivo.

DNGR1-Cre-mediated Deletion of Tnfaip3/A20 in Conventional Dendritic Cells Induces Pulmonary Hypertension in Mice.

Chronic perivascular inflammation is a prominent feature in the lungs of idiopathic pulmonary arterial hypertension. Although the proportions of conventional dendritic cells (cDCs) and plasmacytoid DCs are increased in idiopathic pulmonary arterial hypertension lungs, it remains unknown whether activated cDCs play a pathogenic role. The Tnfaip3 gene encodes the ubiquitin-binding protein A20, which is a negative regulator of NF-κB, critically involved in DC activation. Targeting of Tnfaip3/A20 in cDCs was achieved by Clec9a (DNGR1)-Cre-mediated excision of the Tnfaip3 gene in Tnfaip3DNGR1-KO mice. Mice were evaluated for signs of pulmonary hypertension (PH) using right heart catheterization, echocardiography, and measurement of the Fulton index. Inflammation was assessed by immunohistochemistry and flow cytometry. Pulmonary cDCs and monocyte-derived DCs from 31-week-old Tnfaip3DNGR1-KO mice showed modulated expression of cell surface activation markers compared with Tnfaip3DNGR1-WT mice. Tnfaip3DNGR1-KO mice developed elevated right ventricular systolic pressure and right ventricular hypertrophy. The lungs of these mice displayed increased vascular remodeling and perivascular and peribronchial immune cell infiltration resembling tertiary lymphoid organs. Proportions of activated T cells and expression of IL-1ß, IL-6, and IL-10 were enhanced in the lungs of Tnfaip3DNGR1-KO mice. Autoreactive IgA and IgG1 was detected in BAL and autoreactive IgA recognizing pulmonary endothelial antigens was present in the serum of Tnfaip3DNGR1-KO mice. All signs of PH were ameliorated in Tnfaip3DNGR1-KO mice by anti-IL-6 antibody treatment. These results indicate that activation of the NF-κB pathway in DCs, through deletion of A20/Tnfaip3, leads to experimental PH with accompanied pulmonary inflammation in an IL-6-dependent fashion.