Liver-resident NK cells confer adaptive immunity in skin-contact inflammation

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Abstract

Liver natural killer (NK) cells were recently reported to possess memory-like properties in contact hypersensitivity (CHS) models. However, the phenotype and origin of these “memory” NK cells cannot be distinguished from other NK cell subpopulations. Here, we define the transcriptional, phenotypic, and functional features of liver NK cell subsets and their roles in mediating CHS. Liver NK cells can be divided into two distinct subsets: CD49a⁺DX5^– and CD49a^–DX5⁺. Substantial transcriptional and phenotypic differences existed between liver CD49a⁺DX5^– NK cells and other NK cell subsets. CD49a⁺DX5^– NK cells possessed memory potential and conferred hapten-specific CHS responses upon hapten challenge. Importantly, CD49a⁺DX5^– NK cells were liver resident and were present in the liver sinusoidal blood, but not the afferent and efferent blood of the liver. Moreover, they appeared to originate from hepatic hematopoietic progenitor/stem cells (HPCs/HSCs) but not from the bone marrow, and maintained their phenotypes in the steady state. Our findings of liver-resident NK cells shed new light on the acquisition of memory-like properties of NK cells.

Authors

Hui Peng, Xiaojun Jiang, Yonglin Chen, Dorothy K. Sojka, Haiming Wei, Xiang Gao, Rui Sun, Wayne M. Yokoyama, Zhigang Tian

Epitope specificity determines pathogenicity and detectability in ANCA-associated vasculitis

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Abstract

Anti-neutrophil cytoplasmic antibody–associated (ANCA-associated) small vessel necrotizing vasculitis is caused by immune-mediated inflammation of the vessel wall and is diagnosed in some cases by the presence of myeloperoxidase-specific antibodies (MPO-ANCA). This multicenter study sought to determine whether differences in ANCA epitope specificity explain why, in some cases, conventional serologic assays do not correlate with disease activity, why naturally occurring anti-MPO autoantibodies can exist in disease-free individuals, and why ANCA are undetected in patients with ANCA-negative disease. Autoantibodies from human and murine samples were epitope mapped using a highly sensitive epitope excision/mass spectrometry approach. Data indicated that MPO autoantibodies from healthy individuals had epitope specificities different from those present in ANCA disease. Importantly, this methodology led to the discovery of MPO-ANCA in ANCA-negative disease that reacted against a sole linear sequence. Autoantibodies against this epitope had pathogenic properties, as demonstrated by their capacity to activate neutrophils in vitro and to induce nephritis in mice. The confounder for serological detection of these autoantibodies was the presence of a fragment of ceruloplasmin in serum, which was eliminated in purified IgG, allowing detection. These findings implicate immunodominant epitopes in the pathology of ANCA-associated vasculitis and suggest that autoantibody diversity may be common to other autoimmune diseases.

Authors

Aleeza J. Roth, Joshua D. Ooi, Jacob J. Hess, Mirjan M. van Timmeren, Elisabeth A. Berg, Caroline E. Poulton, JulieAnne McGregor, Madelyn Burkart, Susan L. Hogan, Yichun Hu, Witold Winnik, Patrick H. Nachman, Coen A. Stegeman, John Niles, Peter Heeringa, A. Richard Kitching, Stephen Holdsworth, J. Charles Jennette, Gloria A. Preston, Ronald J. Falk

Rapamycin-treated human endothelial cells preferentially activate allogeneic regulatory T cells

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Abstract

Human graft endothelial cells (ECs) can act as antigen-presenting cells to initiate allograft rejection by host memory T cells. Rapamycin, an mTOR inhibitor used clinically to suppress T cell responses, also acts on DCs, rendering them tolerogenic. Here, we report the effects of rapamycin on EC alloimmunogenicity. Compared with mock-treated cells, rapamycin-pretreated human ECs (rapa-ECs) stimulated less proliferation and cytokine secretion from allogeneic CD4⁺ memory cells, an effect mimicked by shRNA knockdown of mTOR or raptor in ECs. The effects of rapamycin persisted for several days and were linked to upregulation of the inhibitory molecules PD-L1 and PD-L2 on rapa-ECs. Additionally, rapa-ECs produced lower levels of the inflammatory cytokine IL-6. CD4⁺ memory cells activated by allogeneic rapa-ECs became hyporesponsive to restimulation in an alloantigen-specific manner and contained higher percentages of suppressive CD4⁺CD25^hiCD127^loFoxP3⁺ cells that did not produce effector cytokines. In a human-mouse chimeric model of allograft rejection, rapamycin pretreatment of human arterial allografts increased graft EC expression of PD-L1 and PD-L2 and reduced subsequent infiltration of allogeneic effector T cells into the artery intima and intimal expansion. Preoperative conditioning of allograft ECs with rapamycin could potentially reduce immune-mediated rejection.

Authors

Chen Wang, Tai Yi, Lingfeng Qin, Roberto A. Maldonado, Ulrich H. von Andrian, Sanjay Kulkarni, George Tellides, Jordan S. Pober

Blockade of individual Notch ligands and receptors controls graft-versus-host disease

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Abstract

Graft-versus-host disease (GVHD) is the main complication of allogeneic bone marrow transplantation. Current strategies to control GVHD rely on global immunosuppression. These strategies are incompletely effective and decrease the anticancer activity of the allogeneic graft. We previously identified Notch signaling in T cells as a new therapeutic target for preventing GVHD. Notch-deprived T cells showed markedly decreased production of inflammatory cytokines, but normal in vivo proliferation, increased accumulation of regulatory T cells, and preserved anticancer effects. Here, we report that γ-secretase inhibitors can block all Notch signals in alloreactive T cells, but lead to severe on-target intestinal toxicity. Using newly developed humanized antibodies and conditional genetic models, we demonstrate that Notch1/Notch2 receptors and the Notch ligands Delta-like1/4 mediate all the effects of Notch signaling in T cells during GVHD, with dominant roles for Notch1 and Delta-like4. Notch1 inhibition controlled GVHD, but led to treatment-limiting toxicity. In contrast, Delta-like1/4 inhibition blocked GVHD without limiting adverse effects while preserving substantial anticancer activity. Transient blockade in the peritransplant period provided durable protection. These findings open new perspectives for selective and safe targeting of individual Notch pathway components in GVHD and other T cell–mediated human disorders.

Authors

Ivy T. Tran, Ashley R. Sandy, Alexis J. Carulli, Christen Ebens, Jooho Chung, Gloria T. Shan, Vedran Radojcic, Ann Friedman, Thomas Gridley, Amy Shelton, Pavan Reddy, Linda C. Samuelson, Minhong Yan, Christian W. Siebel, Ivan Maillard

IL-6 trans-signaling promotes pancreatitis-associated lung injury and lethality

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Abstract

Acute lung injury (ALI) is an inflammatory disease with a high mortality rate. Although typically seen in individuals with sepsis, ALI is also a major complication in severe acute pancreatitis (SAP). The pathophysiology of SAP-associated ALI is poorly understood, but elevated serum levels of IL-6 is a reliable marker for disease severity. Here, we used a mouse model of acute pancreatitis–associated (AP-associated) ALI to determine the role of IL-6 in ALI lethality. Il6-deficient mice had a lower death rate compared with wild-type mice with AP, while mice injected with IL-6 were more likely to develop lethal ALI. We found that inflammation-associated NF-κB induced myeloid cell secretion of IL-6, and the effects of secreted IL-6 were mediated by complexation with soluble IL-6 receptor, a process known as trans-signaling. IL-6 trans-signaling stimulated phosphorylation of STAT3 and production of the neutrophil attractant CXCL1 in pancreatic acinar cells. Examination of human samples revealed expression of IL-6 in combination with soluble IL-6 receptor was a reliable predictor of ALI in SAP. These results demonstrate that IL-6 trans-signaling is an essential mediator of ALI in SAP across species and suggest that therapeutic inhibition of IL-6 may prevent SAP-associated ALI.

Authors

Hong Zhang, Patrick Neuhöfer, Liang Song, Björn Rabe, Marina Lesina, Magdalena U. Kurkowski, Matthias Treiber, Thomas Wartmann, Sara Regnér, Henrik Thorlacius, Dieter Saur, Gregor Weirich, Akihiko Yoshimura, Walter Halangk, Joseph P. Mizgerd, Roland M. Schmid, Stefan Rose-John, Hana Algül

PAR-1 contributes to the innate immune response during viral infection

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Abstract

Coagulation is a host defense system that limits the spread of pathogens. Coagulation proteases, such as thrombin, also activate cells by cleaving PARs. In this study, we analyzed the role of PAR-1 in coxsackievirus B3–induced (CVB3-induced) myocarditis and influenza A infection. CVB3-infected Par1^–/– mice expressed reduced levels of IFN-β and CXCL10 during the early phase of infection compared with Par1^+/+ mice that resulted in higher viral loads and cardiac injury at day 8 after infection. Inhibition of either tissue factor or thrombin in WT mice also significantly increased CVB3 levels in the heart and cardiac injury compared with controls. BM transplantation experiments demonstrated that PAR-1 in nonhematopoietic cells protected mice from CVB3 infection. Transgenic mice overexpressing PAR-1 in cardiomyocytes had reduced CVB3-induced myocarditis. We found that cooperative signaling between PAR-1 and TLR3 in mouse cardiac fibroblasts enhanced activation of p38 and induction of IFN-β and CXCL10 expression. Par1^–/– mice also had decreased CXCL10 expression and increased viral levels in the lung after influenza A infection compared with Par1^+/+ mice. Our results indicate that the tissue factor/thrombin/PAR-1 pathway enhances IFN-β expression and contributes to the innate immune response during single-stranded RNA viral infection.

Authors

Silvio Antoniak, A. Phillip Owens III, Martin Baunacke, Julie C. Williams, Rebecca D. Lee, Alice Weithäuser, Patricia A. Sheridan, Ronny Malz, James P. Luyendyk, Denise A. Esserman, JoAnn Trejo, Daniel Kirchhofer, Burns C. Blaxall, Rafal Pawlinski, Melinda A. Beck, Ursula Rauch, Nigel Mackman

SHP-1 phosphatase activity counteracts increased T cell receptor affinity

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Abstract

Anti-self/tumor T cell function can be improved by increasing TCR-peptide MHC (pMHC) affinity within physiological limits, but paradoxically further increases (K_d < 1 μM) lead to drastic functional declines. Using human CD8⁺ T cells engineered with TCRs of incremental affinity for the tumor antigen HLA-A2/NY-ESO-1, we investigated the molecular mechanisms underlying this high-affinity–associated loss of function. As compared with cells expressing TCR affinities generating optimal function (K_d = 5 to 1 μM), those with supraphysiological affinity (K_d = 1 μM to 15 nM) showed impaired gene expression, signaling, and surface expression of activatory/costimulatory receptors. Preferential expression of the inhibitory receptor programmed cell death-1 (PD-1) was limited to T cells with the highest TCR affinity, correlating with full functional recovery upon PD-1 ligand 1 (PD-L1) blockade. In contrast, upregulation of the Src homology 2 domain-containing phosphatase 1 (SHP-1/PTPN6) was broad, with gradually enhanced expression in CD8⁺ T cells with increasing TCR affinities. Consequently, pharmacological inhibition of SHP-1 with sodium stibogluconate augmented the function of all engineered T cells, and this correlated with the TCR affinity–dependent levels of SHP-1. These data highlight an unexpected and global role of SHP-1 in regulating CD8⁺ T cell activation and responsiveness and support the development of therapies inhibiting protein tyrosine phosphatases to enhance T cell–mediated immunity.

Authors

Michael Hebeisen, Lukas Baitsch, Danilo Presotto, Petra Baumgaertner, Pedro Romero, Olivier Michielin, Daniel E. Speiser, Nathalie Rufer

2-photon imaging of phagocyte-mediated T cell activation in the CNS

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Abstract

Autoreactive T cells can infiltrate the CNS to cause disorders such as multiple sclerosis. In order to visualize T cell activation in the CNS, we introduced a truncated fluorescent derivative of nuclear factor of activated T cells (NFAT) as a real-time T cell activation indicator. In experimental autoimmune encephalomyelitis, a rat model of multiple sclerosis, we tracked T cells interacting with structures of the vascular blood-brain barrier (BBB). 2-photon imaging documented the cytoplasmic-nuclear translocation of fluorescent NFAT, indicative of calcium-dependent activation of the T cells in the perivascular space, but not within the vascular lumen. The activation was related to contacts with the local antigen-presenting phagocytes and was noted only in T cells with a high pathogenic potential. T cell activation implied the presentation of an autoantigen, as the weakly pathogenic T cells, which remained silent in the untreated hosts, were activated upon instillation of exogenous autoantigen. Activation did not cogently signal long-lasting arrest, as individual T cells were able to sequentially contact fresh APCs. We propose that the presentation of local autoantigen by BBB-associated APCs provides stimuli that guide autoimmune T cells to the CNS destination, enabling them to attack the target tissue.

Authors

Marija Pesic, Ingo Bartholomäus, Nikolaos I. Kyratsous, Vigo Heissmeyer, Hartmut Wekerle, Naoto Kawakami

Specialized role of migratory dendritic cells in peripheral tolerance induction

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Abstract

Harnessing DCs for immunotherapies in vivo requires the elucidation of the physiological role of distinct DC populations. Migratory DCs traffic from peripheral tissues to draining lymph nodes charged with tissue self antigens. We hypothesized that these DC populations have a specialized role in the maintenance of peripheral tolerance, specifically, to generate suppressive Foxp3⁺ Tregs. To examine the differential capacity of migratory DCs versus blood-derived lymphoid-resident DCs for Treg generation in vivo, we targeted a self antigen, myelin oligodendrocyte glycoprotein, using antibodies against cell surface receptors differentially expressed in these DC populations. Using this approach together with mouse models that lack specific DC populations, we found that migratory DCs have a superior ability to generate Tregs in vivo, which in turn drastically improve the outcome of experimental autoimmune encephalomyelitis. These results provide a rationale for the development of novel therapies targeting migratory DCs for the treatment of autoimmune diseases.

Authors

Juliana Idoyaga, Christopher Fiorese, Lori Zbytnuik, Ashira Lubkin, Jennifer Miller, Bernard Malissen, Daniel Mucida, Miriam Merad, Ralph M. Steinman

An obligate cell-intrinsic function for CD28 in Tregs

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Abstract

Tregs expressing the transcription factor FOXP3 are critical for immune homeostasis. The costimulatory molecule CD28 is required for optimal activation and function of naive T cells; however, its role in Treg function has been difficult to dissect, as CD28 is required for thymic Treg development, and blockade of CD28-ligand interactions has confounding effects in trans on nonregulatory cells. To address this question, we created Treg-specific Cd28 conditional knockout mice. Despite the presence of normal numbers of FOXP3⁺ cells, these animals accumulated large numbers of activated T cells, developed severe autoimmunity that primarily affected the skin and lungs, and failed to appropriately resolve induced experimental allergic encephalomyelitis. This in vivo functional impairment was accompanied by dampened expression of CTLA-4, PD-1, and CCR6. Disease occurrence was not due to subversion of Cd28-deficient Tregs into pathogenic cells, as complementation with normal Tregs prevented disease occurrence. Interestingly, in these “competitive” environments, Cd28-deficient Tregs exhibited a pronounced proliferative/survival disadvantage. These data demonstrate clear postmaturational roles for CD28 in FOXP3⁺ Tregs and provide mechanisms which we believe to be novel to explain how interruption of CD28-ligand interactions may enhance immune responses independent of effects on thymic development or on other cell types.

Authors

Ruan Zhang, Alexandria Huynh, Gregory Whitcher, JiHoon Chang, Jonathan S. Maltzman, Laurence A. Turka