bluuebunny/crossref_metadata_2025 huggingface dataset:
Key Roles of CD4+ T Cells and IFN-γ in the Development of Abdominal Aortic Aneurysms in a Murine Model
Authors: Xiong Wanfen , Zhao Yong , Prall Amy , Greiner Timothy C. , Baxter B. Timothy
Published: April 2014

Abdominal aortic aneurysm (AAA) is one of a number of diseases associated with a prominent inflammatory cell infiltrate and local destruction of structural matrix macromolecules. This inflammatory infiltrate is predominately composed of T lymphocytes and macrophages. Delineating specific contribution of these inflammatory cells and their cytokines in AAA formation is the key to understanding AAA and other chronic inflammatory disease processes. Our previous studies have demonstrated that macrophages are the major source of matrix metalloproteinase-9, which is required for aneurysmal degeneration in the murine AAA model. However, the role of CD4+ T cells, the most abundant infiltrates in aneurysmal aortic tissue, is uncertain. In the present study, we found that in the absence of CD4+ T cells, mice are resistant to aneurysm induction. Previous studies have shown that IFN-γ levels are increased in AAA. IFN-γ is a main product of T cells. Intraperitoneal IFN-γ was able to partially reconstitute aneurysms in CD4−/− mice. Furthermore, mice with a targeted deletion of IFN-γ have attenuation of MMP expression and inhibition of aneurysm development. Aneurysms in IFN-γ−/− mice can be reconstituted by reinfusion of competent splenocytes from the corresponding wild-type mice. This study demonstrates the pivotal role that T cells and the T cell cytokine, IFN-γ, play in orchestrating matrix remodeling in AAA. This study has important implications for other degenerative diseases associated with matrix destruction.
Inhibition of IL-10 Receptor Function in Alveolar Macrophages by Toll-Like Receptor Agonists
Authors: Fernandez Stefan , Jose Purnima , Avdiushko Margarita G. , Kaplan Alan M. , Cohen Donald A.
Published: April 2014

Despite an immunosuppressive lung environment, alveolar macrophages (AM) retain the capacity to respond to microorganisms. This report demonstrates that IL-10, constitutively produced by normal alveolar epithelium, stimulates signal transduction through the IL-10R on AM and that IL-10R function can be inhibited by stimulation of Toll-like receptor (TLR) on AM. IL-10 mRNA and protein were constitutively expressed in normal alveolar epithelium of mice, and IL-10R were constitutively expressed on normal murine AM. Stimulation of AM through TLR2, TLR4, or TLR9 was sufficient to inhibit IL-10R signal transduction, including phosphorylation and nuclear translocation of STAT3 transcription factor. Inhibition of IL-10R function by TLRs was not associated with a decrease in IL-10R expression, but did require expression of the myeloid differentiation factor 88 adaptor protein. Continuous exposure of macrophages to IL-10 caused sustained expression of the chemokine receptors CCR1 and CCR5. However, the addition of TLR ligands inhibited IL-10-induced expression of CCR1 and CCR5. Finally, exposure of macrophages to TLR ligands blocked the ability of IL-10 to inhibit the induction of TNF-α by C2-ceramide. These findings demonstrate a novel regulatory mechanism that may allow AM to overcome inhibitory effects of constitutive IL-10 in the lungs that may permit a more effective response to pulmonary infections.
Respective Roles of Decay-Accelerating Factor and CD59 in Circumventing Glomerular Injury in Acute Nephrotoxic Serum Nephritis
Authors: Lin Feng , Salant David J. , Meyerson Howard , Emancipator Steven , Morgan B. Paul , Medof M. Edward
Published: April 2014

Decay-accelerating factor (DAF or CD55) and CD59 are regulators that protect self cells from C3b deposition and C5b-9 assembly on their surfaces. Their relative roles in protecting glomeruli in immune-mediated renal diseases in vivo are unknown. We induced nephrotoxic serum (NTS) nephritis in Daf1−/−, CD59a−/−, Daf1−/−CD59a−/−, and wild-type (WT) mice by administering NTS IgG. After 18 h, we assessed proteinuria, and performed histological, immunohistochemical, and electron microscopic analyses of kidneys. Twenty-four mice in each group were studied. Baseline albuminuria in the Daf1−/−, CD59a−/−, and Daf1−/−CD59a−/− mice was 82, 83, and 139 as compared with 92 μg/mg creatinine in the WT controls (p > 0.1). After NTS, albuminuria in CD59a−/− and WT mice (186 ± 154 and 183 ± 137 μg/mg creatinine, p > 0.1) was similar. In contrast, Daf1−/− mice developed severe albuminuria (378 ± 520, p < 0.05) that was further exacerbated in Daf1−/−CD59a−/− mice (577 ± 785 μg/mg creatinine, p < 0.05). Glomerular histology showed essentially no infiltrating leukocytes in any group. In contrast, electron microscopy revealed prominent podocyte foot process effacement in Daf1−/− mice with more widespread and severe damage in the double knockouts compared with only mild focal changes in CD59a−/− or WT mice. In all animals, deposition of administered (sheep) NTS Ig was equivalent. This contrasted with marked deposition of both C3 and C9 in Daf1−/−CD59a−/− and Daf1−/− mice, which was evident as early as 2 h post-NTS injection. The results support the proposition that in autoantibody-mediated nephritis, DAF serves as the primary barrier to classical pathway-mediated injury, while CD59 limits consequent C5b-9-mediated cell damage.
A Spontaneous Model for Autoimmune Myocarditis Using the Human MHC Molecule HLA-DQ8
Authors: Taylor Jacqueline A. , Havari Evis , McInerney Marcia F. , Bronson Roderick , Wucherpfennig Kai W. , Lipes Myra A.
Published: April 2014

Genome-wide analyses have shown that the MHC class II region is the principal locus that confers susceptibility to a number of human autoimmune diseases. Due to the high degree of linkage disequilibrium across the MHC, it has been difficult to dissect the contribution of individual genes to disease susceptibility. As a result, intensive efforts have been made to generate mice transgenic for human class II molecules as models of autoimmune disease. However, in every case, additional manipulations—such as immunization with Ag in adjuvant, expression of immunostimulants on target tissues, or coexpression of TCR transgenes—have been required to induce disease. In this study, we show that expression of the human HLA-DQ8 (DQA1*0301/DQB1*0302) molecule alone in three lines of transgenic nonobese diabetic murine class II-deficient (mII−/−) mice results in the spontaneous development of autoimmune myocarditis. The disease shares key features of human myocarditis and was characterized by lymphocytic infiltrates in the myocardium and cardiac myocyte destruction, circulating IgG autoantibodies against cardiac myosin heavy chain, and premature death due to heart failure. We demonstrate that myocarditis could be transferred into healthy HLA-DQ8+RAG-1−/−mII−/− nonobese diabetic recipients with lymphocytes, but not sera. It has been widely thought that autoimmune myocarditis is of infectious etiology, with the immune responses arising secondary to cardiac damage from pathogens. These studies provide direct experimental evidence that spontaneous autoimmune myocarditis can occur in the absence of infection and that expression of HLA-DQ8 confers susceptibility to this organ-specific autoimmune disease.
Pulmonary Edema Fluid from Patients with Early Lung Injury Stimulates Fibroblast Proliferation through IL-1β-Induced IL-6 Expression
Authors: Olman Mitchell A. , White Kimberly E. , Ware Lorraine B. , Simmons Warren L. , Benveniste Etty N. , Zhu Sha , Pugin Jerome , Matthay Michael A.
Published: April 2014

Although the fibroproliferative response to lung injury occurs with a high frequency in patients with clinical acute lung injury, the mechanisms that initiate this response are largely unknown. This study was undertaken first to identify fibroblast mitogenic factors in pulmonary edema fluid, and second to examine the human lung fibroblast’s gene expression profile in response to pulmonary edema fluid. The edema fluid obtained from patients with early lung injury has an eightfold higher concentration of IL-1β and a twofold greater IL-1β-dependent mitogenic effect than does fluid obtained from control patients with hydrostatic pulmonary edema. Furthermore, fibroblasts responded to acute lung injury patient-derived edema fluid through production of soluble mediators that possess an autocrine mitogenic effect. Gene array analysis reveals that acute lung injury edema fluid induces several inflammation-modulating and proliferation-related genes in fibroblasts, whose inductions are similarly dependent on bioactive IL-1β. Most notably, the 20-fold induction of IL-6 mRNA and protein was completely blocked by IL-1 receptor antagonist. The combined addition of IL-1β and IL-6 was mitogenic, and the proliferative response to conditioned medium from IL-1β-exposed cells was blocked by antagonistically acting Abs to IL-6 or to gp130. These novel findings indicate that soluble IL-1β bioactivity and autocrine IL-1β-dependent IL-6 up-regulation are critical initiators of fibroblast activation and proliferation and that they likely play a role in the fibroproliferative response seen in human acute lung injury.
CD40 Ligand Dysregulation in HIV Infection: HIV Glycoprotein 120 Inhibits Signaling Cascades Upstream of CD40 Ligand Transcription
Authors: Zhang Rui , Fichtenbaum Carl J. , Hildeman David A. , Lifson Jeffrey D. , Chougnet Claire
Published: April 2014

IL-12 production and up-regulation of CD40 ligand (CD40L) expression are impaired in the PBMC of HIV-infected donors, and exogenous CD40L rescues IL-12 production by such cells. In this study, we implicate dysregulation of CD40L expression in the IL-12 defect associated with HIV by demonstrating that induction of CD40L expression by anti-CD3/CD28 stimulation was directly correlated with the IL-12 productive capacity of PBMC. Further, we demonstrate marked decreases in the induction of CD40L protein and mRNA following anti-CD3/CD28 stimulation in HIV-infected donors compared with uninfected donors, with a tight association between these two levels. Inhibition of CD40L up-regulation was selective, as induction of CD69 or OX40 was not as severely affected. Increased instability of CD40L mRNA did not constitute a major mechanism in CD40L dysregulation, thus suggesting a potential defect in the signaling cascades upstream of transcription. The mechanisms by which HIV infection affects the induction of CD40L expression appear to involve HIV gp120-mediated engagement of CD4. Indeed, anti-CD4 mAb or inactivated HIV virions that harbor a conformationally intact gp120 significantly inhibited CD40L up-regulation at both the protein and mRNA levels. This inhibition was due to the native, virion-associated gp120, as coculture with soluble CD4 or heat treatment of inactivated HIV abolished their effect. These in vitro models mirror the CD40L defect seen in cells from HIV-infected donors and thus provide a suitable model to investigate HIV-induced CD40L dysregulation. Clear elucidation of mechanism(s) may well lead to the development of novel immunotherapeutic approaches to HIV infection.
HIV-1 Does Not Provoke Alteration of Cytokine Gene Expression in Lymphoid Tissue after Acute Infection Ex Vivo
Authors: Audigé Annette , Schlaepfer Erika , Bonanomi Athos , Joller Helene , Knuchel Marlyse C. , Weber Markus , Nadal David , Speck Roberto F.
Published: April 2014

The cytokine response to invading microorganisms is critical for priming the adaptive immune response. During acute HIV infection, the response is disrupted, but the mechanism is poorly understood. We examined the cytokine response in human lymphoid tissue, acutely infected ex vivo with HIV. Lymphoid tissue was cultured either as blocks or as human lymphocyte aggregate cultures (HLAC) of tonsils and lymph nodes. This approach allowed us to examine the effects of HIV on cytokines using distinct culture techniques. In contrast to HLAC, mock-infected tissue blocks displayed a 50- to 100-fold up-regulation of mRNAs for IL-1β, -6, and -8 in the first 6 days of culture. Parallel increases were also noted at the protein level in the supernatants. Although IL-1β, -6, and -8 are known to synergistically enhance HIV replication, peak HIV replication (measured as p24 Ag) was similar in tissue blocks and HLAC. Surprisingly, vigorous HIV replication of CXCR4- and CCR5-tropic HIV strains did not result in characteristic mRNA profiles for IL-1β, -2, -4, -6, -8, -10, -12, -15, IFN-γ, TNF-α, TGF-β, and β-chemokines in tissue blocks or HLAC. The increased expression of IL-1β, -6, and -8 in tissue blocks may approximate clinical situations with heightened immune activation; neutralization of these cytokines resulted in inhibition of HIV replication, suggesting that these cytokines may contribute to HIV replication in certain clinical settings. These results also indicate that different molecular mechanisms govern HIV replication in tissue blocks and HLAC. Prevention of effective cytokine responses may be an important mechanism that HIV uses during acute infection.
Breakdown of Tolerance to a Self-Peptide of Acetylcholine Receptor α-Subunit Induces Experimental Myasthenia Gravis in Rats
Authors: Baggi Fulvio , Annoni Andrea , Ubiali Federica , Milani Monica , Longhi Renato , Scaioli Widmer , Cornelio Ferdinando , Mantegazza Renato , Antozzi Carlo
Published: April 2014

Experimental autoimmune myasthenia gravis (EAMG), a model for human myasthenia (MG), is routinely induced in susceptible rat strains by a single immunization with Torpedo acetylcholine receptor (TAChR). TAChR immunization induces anti-AChR Abs that cross-react with self AChR, activate the complement cascade, and promote degradation of the postsynaptic membrane of the neuromuscular junction. In parallel, TAChR-specific T cells are induced, and their specific immunodominant epitope has been mapped to the sequence 97–116 of the AChR α subunit. A proliferative T cell response against the corresponding rat sequence (R97–116) was also found in TAChR-immunized rats. To test whether the rat (self) sequence can be pathogenic, we immunized Lewis rats with R97–116 or T97–116 peptides and evaluated clinical, neurophysiological, and immunological parameters. Clinical signs of the disease were noted only in R97–116-immunized animals and were confirmed by electrophysiological signs of impaired neuromuscular transmission. All animals produced Abs against the immunizing peptide, but anti-rat AChR Abs were observed only in animals immunized with the rat peptide. These findings suggested that EAMG in rats can be induced by a single peptide of the self AChR, that this sequence is recognized by T cells and Abs, and that breakdown of tolerance to a self epitope might be an initiating event in the pathogenesis of rat EAMG and MG.
Cysteinyl Leukotrienes and Their Receptors: Cellular Distribution and Function in Immune and Inflammatory Responses
Authors: Kanaoka Yoshihide , Boyce Joshua A.
Published: April 2014

The cysteinyl leukotrienes (cys-LTs) are a family of potent bioactive lipids that act through two structurally divergent G protein-coupled receptors, termed the CysLT1 and CysLT2 receptors. The cloning and characterization of these two receptors has not only reconciled findings of previous pharmacologic profiling studies of contractile tissues, but also has uncovered their expression on a wide array of circulating and tissue-dwelling leukocytes. With the development of receptor-selective reagents, as well as mice lacking critical biosynthetic enzymes, transporter proteins, and the CysLT1 receptor, diverse functions of cys-LTs and their receptors in immune and inflammatory responses have been identified. We review cys-LT biosynthesis; the molecular biology and distribution of the CysLT1 and CysLT2 receptors; the functions of cys-LTs and their receptors in the recruitment and activation of effector leukocytes and induction of adaptive immunity; and the development of fibrosis and airway remodeling in animal models of lung injury and allergic inflammation.
Regulation of IFN Regulatory Factor-7 and IFN-α Production by Enveloped Virus and Lipopolysaccharide in Human Plasmacytoid Dendritic Cells
Authors: Dai Jihong , Megjugorac Nicholas J. , Amrute Sheela B. , Fitzgerald-Bocarsly Patricia
Published: April 2014

Human plasmacytoid dendritic cells (PDC) are a major source of IFN-α upon exposure to enveloped viruses and TLR-7 and TLR-9 ligands. Although IFN regulatory factor-7 (IRF-7) is known to play an essential role in virus-activated transcription of IFN-α genes, the molecular mechanisms of IFN-α production in human PDC remain poorly understood. We and others have recently reported high constitutive levels of IRF-7 expression in PDC as compared with other PBMC. In this study, we demonstrate that both LPS and HSV up-regulate the expression of IRF-7 in PDC, and that this enhancement of IRF-7 is dependent on NF-κB activation. The NF-κB inhibitors MG132 and pyrrolidinedithiocarbamate efficiently inhibited the induction of IRF-7 by HSV or LPS, and also down-regulated the constitutive expression of IRF-7 in PDC and blocked the HSV-induced production of IFN-α. In addition, we found that nuclear translocation of IRF-7 occurred rapidly in response to HSV stimulation, but not in response to LPS, which is consistent with the stimulation of IFN-α production by virus and not by LPS. Although LPS by itself was not able to induce IFN-α production, it led to rapid up-regulation of TLR-4 on PDC and increased the magnitude and accelerated the kinetics of HSV-induced IFN-α production in PDC, providing a mechanism that might be operative in a scenario of mixed infection. In contrast to the current concept of IFN-α regulation established in cell lines, this study strongly supports the immediate availability of high constitutive levels of IRF-7 expression in PDC, and suggests an activation required for IRF-7 that contributes to IFN-α production in virus-stimulated PDC.
Ectopic Expression of HLA-DO in Mouse Dendritic Cells Diminishes MHC Class II Antigen Presentation
Authors: Fallas Jennifer L. , Tobin Helen M. , Lou Olivia , Guo Donglin , Sant’Angelo Derek B. , Denzin Lisa K.
Published: April 2014

The MHC class II-like molecule HLA-DM (DM) (H-2M in mice) catalyzes the exchange of CLIP for antigenic peptides in the endosomes of APCs. HLA-DO (DO) (H-2O in mice) is another class II-like molecule that is expressed in B cells, but not in other APCs. Studies have shown that DO impairs or modifies the peptide exchange activity of DM. To further evaluate the role of DO in Ag processing and presentation, we generated transgenic mice that expressed the human HLA-DOA and HLA-DOB genes under the control of a dendritic cell (DC)-specific promoter. Our analyses of DCs from these mice showed that as DO levels increased, cell surface levels of Ab-CLIP also increased while class II-peptide levels decreased. The presentation of some, but not all, exogenous Ags to T cells or T hybridomas was significantly inhibited by DO. Surprisingly, H-2M accumulated in DO-expressing DCs and B cells, suggesting that H-2O/DO prolongs the half-life of H-2M. Overall, our studies showed that DO expression impaired H-2M function, resulting in Ag-specific down-modulation of class II Ag processing and presentation.
Ligand Binding to Inhibitory Killer Cell Ig-Like Receptors Induce Colocalization with Src Homology Domain 2-Containing Protein Tyrosine Phosphatase 1 and Interruption of Ongoing Activation Signals
Authors: Vyas Yatin M. , Maniar Hina , Lyddane Clay E. , Sadelain Michel , Dupont Bo
Published: April 2014

Interaction of NK cells with target cells leads to formation of an immunological synapse (IS) at the contact site. NK cells form two distinctly different IS, the inhibitory NK cell IS (NKIS) and the cytolytic NKIS. Cognate ligand binding is sufficient to induce clustering of inhibitory killer cell Ig-like receptors (KIR) and phosphorylation of both the receptor and the phosphatase Src homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1). Recruitment and activation of SHP-1 by a signaling competent inhibitory receptor are essential early events for NK cell inhibition. We have in the present study used three-dimensional immunofluorescence microscopy to analyze distribution of inhibitory KIR, SHP-1, LFA-1, and lipid rafts within the NKIS during cytolytic and noncytolytic interactions. NK clones retrovirally transduced with the inhibitory KIR2DL3 gene fused to GFP demonstrate colocalization of KIR2DL3 with SHP-1 in the center of early inhibitory NKIS. Ligand binding translocates the receptor to the center of the IS where activation signals are accumulating and provides a docking site for SHP-1. SHP-1 and rafts cluster in the center of early inhibitory NKIS and late cytolytic NKIS, and whereas rafts continue to increase in size in cytolytic conjugates, they are rapidly dissolved in inhibitory conjugates. Furthermore, rafts are essential only for cytolytic, not for inhibitory, outcome. These results indicate that the outcome of NK cell-target cell interactions is dictated by early quantitative differences in cumulative activating and inhibitory signals.
IκB Kinase 2 Deficiency in T Cells Leads to Defects in Priming, B Cell Help, Germinal Center Reactions, and Homeostatic Expansion
Authors: Schmidt-Supprian Marc , Tian Jane , Ji Hongbin , Terhorst Cox , Bhan Atul K. , Grant Ethan P. , Pasparakis Manolis , Casola Stefano , Coyle Anthony J. , Rajewsky Klaus
Published: April 2014

Signal transduction from proinflammatory stimuli leading to NF-κB-dependent gene expression is mediated by the IκB kinase 2 (IKK2/IKKβ). Therefore, IKK2 has become an important drug target for treatment of inflammatory conditions. T cells, whose activation depends to a large extent on the activity of NF-κB transcription factors, play important roles in inflammation and autoimmunity. Ablation of IKK2 specifically in T cells in CD4cre/Ikk2FL mice allows their survival and activation by polyclonal stimuli in vitro, suggesting that IKK2 is dispensable for T cell activation. We report in this study that IKK2-deficient T cells expand efficiently in response to superantigen administration in vivo, but are completely deficient in recall responses, most likely due to inefficient priming. IKK2-deficient T cells provide suboptimal B cell help and fail to support germinal center reactions. Finally, IKK2 is essential for homeostatic expansion of naive T cells, reflected by the inability of IKK2-deficient T cells to induce colitis in lymphopenic hosts.
Critical Roles of CXC Chemokine Ligand 16/Scavenger Receptor that Binds Phosphatidylserine and Oxidized Lipoprotein in the Pathogenesis of Both Acute and Adoptive Transfer Experimental Autoimmune Encephalomyelitis
Authors: Fukumoto Noriko , Shimaoka Takeshi , Fujimura Harutoshi , Sakoda Saburo , Tanaka Makoto , Kita Toru , Yonehara Shin
Published: April 2014

The scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SR-PSOX)/CXCL16 is a chemokine expressed on macrophages and dendritic cells, while its receptor expresses on T and NK T cells. We investigated the role of SR-PSOX/CXCL16 on acute and adoptive experimental autoimmune encephalomyelitis (EAE), which is Th1-polarized T cell-mediated autoimmune disease of the CNS. Administration of mAb against SR-PSOX/CXCL16 around the primary immunization decreased disease incidence of acute EAE with associated reduced infiltration of mononuclear cells into the CNS. Its administration was also shown to inhibit elevation of serum IFN-γ level at primary immune response, as well as subsequent generation of Ag-specific T cells. In adoptive transfer EAE, treatment of recipient mice with anti-SR-PSOX/CXCL16 mAb also induced not only decreased clinical disease incidence, but also diminished traffic of mononuclear cells into the CNS. In addition, histopathological analyses showed that clinical development of EAE correlates well with expression of SR-PSOX/CXCL16 in the CNS. All the results show that SR-PSOX/CXCL16 plays important roles in EAE by supporting generation of Ag-specific T cells, as well as recruitment of inflammatory mononuclear cells into the CNS.
In Vivo Generated Th1 Cells Can Migrate to B Cell Follicles to Support B Cell Responses
Authors: Smith Karen M. , Brewer James M. , Rush Catherine M. , Riley Jillian , Garside Paul
Published: April 2014

The description of Th1 and Th2 T cell subsets rationalized the inverse correlation between humoral and cell-mediated immunity. Although Th1 cells were described to support cell-mediated immune responses, their role in supporting certain B cell responses was firmly established. However, there is now a prevailing preconception that provision of B cell help is entirely the domain of Th2 cells and that Th1 cells lack this capacity. Previous studies demonstrated that immunization using aluminum hydroxide adjuvants induces Ag-specific Th2 responses, whereas incorporation of IL-12 with aluminum hydroxide produces a Th1 inducing adjuvant. By immunizing TCR transgenic recipient mice in this fashion, we have generated Ag-specific, traceable Th1 and Th2 cells in vivo and assessed their follicular migration and ability to support B cell responses. In this study we have shown that in vivo polarized Th1 and Th2 cells clonally expand to similar levels and migrate into B cell follicles in which they support B cell responses to a similar degree. Critically, we present direct evidence that in vivo polarized, IFN-γ secreting Th1 cells migrate into B cell follicles where they can interact with Ag-specific B cells.
Differential Roles for Wiskott-Aldrich Syndrome Protein in Immune Synapse Formation and IL-2 Production
Authors: Cannon Judy L. , Burkhardt Janis K.
Published: April 2014

Wiskott-Aldrich syndrome protein (WASP)-deficient T cells exhibit defects in IL-2 production that are widely believed to stem from primary defects in actin remodeling and immune synapse formation. Surprisingly, however, we find that WASP-deficient T cells responding to Ag-specific APCs polymerize actin and organize talin and PKCθ normally, forming an immune synapse that is stable for at least 3 h. At low doses of peptide, WASP-deficient T cells show less efficient talin and PKCθ polarization. Thus, although WASP may facilitate immune synapse formation at low peptide concentrations, WASP is not required for this process. Defects in IL-2 production are observed even under conditions in which immune synapse formation proceeds normally, suggesting that the role of WASP in regulating IL-2 production is independent of its role in immune synapse formation.
Cross-Reactive TCR Responses to Self Antigens Presented by Different MHC Class II Molecules
Authors: Mycko Marcin P. , Waldner Hanspeter , Anderson David E. , Bourcier Katarzyna D. , Wucherpfennig Kai W. , Kuchroo Vijay K. , Hafler David A.
Published: April 2014

Autoreactive T cells represent a natural repertoire of T cells in both diseased patients and healthy individuals. The mechanisms regulating the function of these autoreactive T cells are still unknown. Ob1A12 is a myelin basic protein (MBP)-reactive Th cell clone derived from a patient with relapsing-remitting multiple sclerosis. Mice transgenic for this human TCR and DRA and DRB1*1501 chains develop spontaneous experimental autoimmune encephalomyelitis. The reactivity of Ob1A12 is reported to be restricted to recognition of MBP peptide 85–99 in the context of DRB1*1501. DRA/DRB1*1501 and the patient’s other restriction element, DRA/DRB1*0401, differ significantly in their amino acid sequences. In this study we describe an altered peptide ligand derived from MBP85–99 with a single amino acid substitution at position 88 (Val to Lys; 88V→K), that could stimulate the Ob1A12.TCR in the context of both DRA/DRB1*1501 and DRA/DRB1*0401. Analysis of a panel of transfected T cell hybridomas expressing Ob1A12.TCR and CD4 indicated that Ob1A12.TCR cross-reactivity in the context of DRA/DRB1*0401 is critically dependent on the presence of the CD4 coreceptor. Furthermore, we found that activation of Ob1A12.TCR with MBP altered peptide ligand 85–99 88V→K presented by DRB1*1501 or DRB1*0401 resulted in significant differences in TCRζ phosphorylation. Our data indicate that injection of altered peptide ligand into patients heterozygous for MHC class II molecules may result in unexpected cross-reactivities, leading to activation of autoreactive T cells.
HLA-B27 Heavy Chain Homodimers Are Expressed in HLA-B27 Transgenic Rodent Models of Spondyloarthritis and Are Ligands for Paired Ig-Like Receptors
Authors: Kollnberger Simon , Bird Lucy A. , Roddis Matthew , Hacquard-Bouder Cecile , Kubagawa Hiromi , Bodmer Helen C. , Breban Maxime , McMichael Andrew J. , Bowness Paul
Published: April 2014

HLA-B27 transgenic rats and strains of HLA-B27-transgenic β2-microglobulin (β2m)-deficient mice develop a multisystem inflammatory disease affecting the joints, skin, and bowel with strong similarity to human spondyloarthritis. We show that HLA-B27 transgenic mice and rats express HC10-reactive, β2m-free HLA-B27 homodimers (B272) and multimers, both intracellularly and at the cell surface of leukocytes, including rat dendritic cells. Fluorescent-labeled tetrameric complexes of HLA-B27 homodimers (B272 tetramers) bind to populations of lymphocytes, monocytes, and dendritic cells. The murine (and probably rat) paired Ig-like receptors (PIRs) are ligands for B272. Thus, B272 tetramers stain RBL cells transfected with murine activating PIR-A4 and inhibitory PIR-B receptors. Murine PIR-A and -B can be immunoprecipitated from the RAW264.7 macrophage cell line, and murine PIR-A can be immunoprecipitated from the J774.A1 line using B272. B272 tetramer staining corresponds to the distribution of PIR expression on lymphoid and myeloid cells and on murine macrophage cell lines. B272 can induce TNF-α release from the J774.A1 macrophage cell line. The binding of B272 to PIR is inhibited by HC10, an mAb that ameliorates arthritis in HLA-B27+ β2m−/− mice. The expression and PIR recognition of B272 could explain the pathogenesis of rodent spondyloarthritis.
The Gads (GrpL) Adaptor Protein Regulates T Cell Homeostasis
Authors: Yankee Thomas M. , Yun Theodore J. , Draves Kevin E. , Ganesh Kolumam , Bevan Michael J. , Murali-Krishna Kaja , Clark Edward A.
Published: April 2014

Little is known about the role of the Gads (GrpL) adaptor protein in mature T cell populations. In this study we show that the effects of Gads deficiency on murine CD4+ and CD8+ T cells are markedly different. Gads−/− CD4+ T cells were markedly deficient in the spleen and had an activated phenotype and a rapid turnover rate. When transferred into a wild-type host, Gads−/− CD4+ T cells continued to proliferate at a higher rate than wild-type CD4+ T cells, demonstrating a defect in homeostatic proliferation. Gads−/− CD8+ T cells had a memory-like phenotype, produced IFN-γ in response to ex vivo stimulation, and underwent normal homeostatic proliferation in wild-type hosts. Gads−/− T cells had defective TCR-mediated calcium responses, but had normal activation of ERK. Gads−/− CD4+ T cells, but not CD8+ T cells, had a severe block of TCR-mediated proliferation and a high rate of spontaneous cell death and were highly susceptible to CD95-induced apoptosis. This suggests that the rapid turnover of Gads−/− CD4+ T cells is due to a defect in cell survival. The intracellular signaling pathways that regulate homeostasis in CD4+ and CD8+ T cells are clearly different, and the Gads adaptor protein is critical for homeostasis of CD4+ T cells.
MUC1/sec-Expressing Tumors Are Rejected In Vivo by a T Cell-Dependent Mechanism and Secrete High Levels of CCL2
Authors: Grosso Joseph F. , Herbert Lynn M. , Owen Jennifer L. , Lopez Diana M.
Published: April 2014

MUC1/sec is a secreted form of the glycoprotein mucin 1 (MUC1). To characterize the role that MUC1 and MUC1/sec have in tumor progression, these genes were expressed in DA-3 mammary tumor cells. DA-3 cells and DA-3 cells expressing the transmembrane MUC1 gene (DA-3/TM) grow with similar kinetics in BALB/c mice. Surprisingly, DA-3 cells expressing and secreting MUC1/sec (DA-3/sec) fail to form tumors in vivo. The mechanism of rejection was evaluated using mice deficient in constituents of the immune system. All mice lacking IFN-γ, NK, NKT, or macrophages formed DA-3/sec tumors that regressed shortly after implantation. However, progressively growing DA-3/sec tumors developed in mice devoid of T lymphocytes. The importance of T lymphocytes in the rejection of DA-3/sec tumors was further supported by detection of DA-3-specific CTL in mice challenged with the DA-3/sec tumor. Recruitment of appropriate APC and effector cells is an important first step in the tumor clearance. Indeed, DA-3/sec cells or cell supernatants recruited 3–4 times as many macrophages as DA-3/TM cells in vivo, suggesting that a secreted chemotactic product is produced from DA-3/sec cells. RNA and protein analysis of DA-3/sec cells revealed that several genes are up-regulated by MUC1/sec expression, including MCP-1 (CCL-2). These results suggest DA-3/sec cells are capable of recruiting immune cells, and that rejection of DA-3/sec tumors, although aided by cells of the innate immune response, is ultimately due to T cell-mediated events.
Antigen Presentation to Celiac Lesion-Derived T Cells of a 33-Mer Gliadin Peptide Naturally Formed by Gastrointestinal Digestion
Authors: Qiao Shuo-Wang , Bergseng Elin , Molberg Øyvind , Xia Jiang , Fleckenstein Burkhard , Khosla Chaitan , Sollid Ludvig M.
Published: April 2014

Celiac disease is an HLA-DQ2-associated disorder characterized by intestinal T cell responses to ingested wheat gluten proteins. A peptide fragment of 33 residues (α2-gliadin 56–88) produced by normal gastrointestinal proteolysis contains six partly overlapping copies of three T cell epitopes and is a remarkably potent T cell stimulator after deamidation by tissue transglutaminase (TG2). This 33-mer is rich in proline residues and adopts the type II polyproline helical conformation in solution. In this study we report that after deamidation, the 33-mer bound with higher affinity to DQ2 compared with other monovalent peptides harboring gliadin epitopes. We found that the TG2-treated 33-mer was presented equally effectively by live and glutaraldehyde-fixed, EBV-transformed B cells. The TG2-treated 33-mer was also effectively presented by glutaraldehyde-fixed dendritic cells, albeit live dendritic cells were the most effective APCs. A strikingly increased T cell stimulatory potency of the 33-mer compared with a 12-mer peptide was also seen with fixed APCs. The 33-mer showed binding maximum to DQ2 at pH 6.3, higher than maxima found for other high affinity DQ2 binders. The 33-mer is thus a potent T cell stimulator that does not require further processing within APC for T cell presentation and that binds to DQ2 with a pH profile that promotes extracellular binding.
α-Fetoprotein Impairs APC Function and Induces Their Apoptosis
Authors: Um Soon Ho , Mulhall Catherine , Alisa Akeel , Ives Annette Robyn , Karani John , Williams Roger , Bertoletti Antonio , Behboudi Shahriar
Published: April 2014

α-Fetoprotein (AFP) is a tumor-associated Ag, and its serum level is elevated in patients with hepatocellular carcinoma (HCC). In vitro, AFP induces functional impairment of dendritic cells (DCs). This was demonstrated by the down-regulation of CD40 and CD86 molecules and the impairment of allostimulatory function. Also, AFP was found to induce significant apoptosis of DCs, and AFP-treated DCs produced low levels of IL-12 and TNF-α, a cytokine pattern that could hamper an efficient antitumor immune response. Ex vivo, APCs of patients with HCC and high levels of AFP produced lower levels of TNF-α than that of healthy individuals. In conclusion, these results illustrate that AFP induces dysfunction and apoptosis of APCs, thereby offering a mechanism by which HCC escapes immunological control.
Inactivation of c-Cbl or Cbl-b Differentially Affects Signaling from the High Affinity IgE Receptor
Authors: Zhang Juan , Chiang Yungping J. , Hodes Richard J. , Siraganian Reuben P.
Published: April 2014

The Cbl family of proteins negatively regulate signaling from tyrosine kinase-coupled receptors. Among the three members of this family, only c-Cbl and Cbl-b are expressed in hemopoietic cells. To examine the role of c-Cbl and Cbl-b in FcεRI signaling, mast cell cultures from wild-type, c-Cbl−/−, and Cbl-b−/− mice were generated. Cell growth rates and cell surface expression of FcεRI were similar in the different cell populations. Compared with control cells, Cbl-b inactivation resulted in increases in FcεRI-induced Ca2+ response and histamine release. FcεRI-induced tyrosine phosphorylation of total cellular proteins, Syk, and phospholipase C-γ was also enhanced by Cbl-b deficiency, whereas receptor-initiated phosphorylation of Vav, JNK, and p38 kinases was not changed in these cells. In contrast to Cbl-b, c-Cbl deficiency had no detectable effect on FcεRI-induced histamine release or on the phosphorylation of total cellular proteins or Syk. The absence of c-Cbl increased the phosphorylation of ERK after receptor stimulation, but resulted in slightly reduced p38 phosphorylation and Ca2+ response. These results suggest that Cbl-b and c-Cbl have divergent effects on FcεRI signal transduction and that Cbl-b, but not c-Cbl, functions as a negative regulator of FcεRI-induced degranulation.
Characterization of Distinct Conventional and Plasmacytoid Dendritic Cell-Committed Precursors in Murine Bone Marrow
Authors: Diao Jun , Winter Erin , Chen Wenhao , Cantin Claude , Cattral Mark S.
Published: April 2014

The developmental pathways and differentiation relationship of dendritic cell (DC) subsets remain unclear. We report that murine CD11c+MHC II− bone marrow cells, which are immediate DC precursors of CD8α+, CD8α−, and B220+ DC in vivo, can be separated into B220+ and B220− DC precursor subpopulations. Purified B220− DC precursors expand, and generate exclusively mature CD11c+CD11b+B220− DC in vitro and after adoptive transfer. B220+ DC precursors, which resemble plasmacytoid pre-DC, have a lower proliferative potential than B220− DC precursors and generate both CD11b− B220+ and CD11b+B220− DC populations. Both DC precursor populations can give rise to CD8α+ and CD8α− DC subtypes. Our findings indicate that CD11c+MHC II−B220+ and CD11c+MHC II−B220− bone marrow cells are distinct DC lineage-restricted precursors.
Transcription-Dependent Somatic Hypermutation Occurs at Similar Levels on Functional and Nonfunctional Rearranged IgH Alleles
Authors: Delpy Laurent , Sirac Christophe , Le Morvan Caroline , Cogné Michel
Published: April 2014

Allelic exclusion of IgH chain expression is stringently established before or during early B cell maturation. It likely relies both on cellular mechanisms, selecting those cells in which a single receptor allows the best possible Ag response, and on molecular restrictions of gene accessibility to rearrangement. The extent to which transcriptional control may be involved is unclear. Transcripts arising from nonfunctional alleles would undergo nonsense-mediated degradation and their virtual absence in mature cells cannot ensure that transcription per se is down-regulated. By contrast, somatic hypermutation may provide an estimate of primary transcription in Ag-activated cells since both processes are directly correlated. For coding regions, the rate and nature of mutations also depend upon Ag binding constraints. By sequencing intronic sequence downstream mouse VDJ genes, we could show in the absence of such constraints that somatic hypermutation intrinsically targets nonfunctional rearranged alleles at a frequency approaching that of functional alleles, suggesting that transcription also proceeds on both alleles at a similar rate. By contrast and confirming the strong dependency of somatic hypermutation upon transcription, we show that artificial blockade of transcription on the nonfunctional allele by a knock-in neomycin resistance cassette keeps the VDJ region unmutated even when its promoter is intact and when it is fully rearranged.
Histidines Are Critical for Heparin-Dependent Activation of Mast Cell Tryptase
Authors: Hallgren Jenny , Bäckström Stefan , Estrada Sergio , Thuveson Maria , Pejler Gunnar
Published: April 2014

Mast cell tryptase is a tetrameric serine protease that is stored in complex with negatively charged heparin proteoglycans in the secretory granule. Tryptase has potent proinflammatory properties and has been implicated in diverse pathological conditions such as asthma and fibrosis. Previous studies have shown that tryptase binds tightly to heparin, and that heparin is required in the assembly of the tryptase tetramer as well as for stabilization of the active tetramer. Because the interaction of tryptase with heparin is optimal at acidic pH, we investigated in this study whether His residues are of importance for the heparin binding, tetramerization, and activation of the tryptase mouse mast cell protease 6. Molecular modeling of mouse mast cell protease 6 identified four His residues, H35, H106, H108, and H238, that are conserved among pH-dependent tryptases and are exposed on the molecular surface, and these four His residues were mutated to Ala. In addition, combinations of different mutations were prepared. Generally, the single His-Ala mutations did not cause any major defects in heparin binding, activation, or tetramerization, although some effect of the H106A mutation was observed. However, when several mutations were combined, large defects in all of these parameters were observed. Of the mutants, the triple mutant H106A/H108A/H238A was the most affected with an almost complete inability to bind to heparin and to form active tryptase tetramers. Taken together, this study shows that surface-exposed histidines mediate the interaction of mast cell tryptase with heparin and are of critical importance in the formation of active tryptase tetramers.
B Cell Lipid Rafts Regulate Both Peptide-Dependent and Peptide-Independent APC-T Cell Interaction
Authors: Setterblad Niclas , Bécart Stéphane , Charron Dominique , Mooney Nuala
Published: April 2014

Formation of an immunological synapse (IS) between APCs and T CD4+ lymphocytes is a key event in the initiation and the termination of the cognate immune response. We have analyzed the contribution of the APC to IS formation and report the implication of the actin cytoskeleton, the signaling proteins and the lipid rafts of B lymphocytes. Recruitment of MHC class II molecules to the IS is concomitant with actin cytoskeleton-dependent B cell raft recruitment. B cell actin cytoskeleton disruption abrogates both IS formation and T cell activation, whereas protein kinase C inhibition only impairs T cell activation. Pharmacological B cell lipid raft disruption inhibited peptide-dependent T lymphocyte activation and induced peptide-independent but HLA-DR-restricted APC-T cell conjugate formation. Such peptide-independent conjugates did not retain the ability to activate T cells. Thus, B cell lipid rafts are bifunctional by regulating T cell activation and imposing peptide stringency.
IL-23 Provides a Limited Mechanism of Resistance to Acute Toxoplasmosis in the Absence of IL-12
Authors: Lieberman Linda A. , Cardillo Fabiola , Owyang Alexander M. , Rennick Donna M. , Cua Daniel J. , Kastelein Robert A. , Hunter Christopher A.
Published: April 2014

IL-23 and IL-12 are heterodimeric cytokines which share the p40 subunit, but which have unique second subunits, IL-23p19 and IL-12p35. Since p40 is required for the development of the Th1 type response necessary for resistance to Toxoplasma gondii, studies were performed to assess the role of IL-23 in resistance to this pathogen. Increased levels of IL-23 were detected in mice infected with T. gondii and in vitro stimulation of dendritic cells with this pathogen resulted in increased levels of mRNA for this cytokine. To address the role of IL-23 in resistance to T. gondii, mice lacking the p40 subunit (common to IL-12 and IL-23) and mice that lack IL-12 p35 (specific for IL-12) were infected and their responses were compared. These studies revealed that p40−/− mice rapidly succumbed to toxoplasmosis, while p35−/− mice displayed enhanced resistance though they eventually succumbed to this infection. In addition, the administration of IL-23 to p40−/− mice infected with T. gondii resulted in a decreased parasite burden and enhanced resistance. However, the enhanced resistance of p35−/− mice or p40−/− mice treated with IL-23 was not associated with increased production of IFN-γ. When IL-23p19−/− mice were infected with T. gondii these mice developed normal T cell responses and controlled parasite replication to the same extent as wild-type mice. Together, these studies indicate that IL-12, not IL-23, plays a dominant role in resistance to toxoplasmosis but, in the absence of IL-12, IL-23 can provide a limited mechanism of resistance to this infection.
Innate BALB/c Enteric Epithelial Responses to Trichinella spiralis: Inducible Expression of a Novel Goblet Cell Lectin, Intelectin-2, and Its Natural Deletion in C57BL/10 Mice
Authors: Pemberton Alan D. , Knight Pamela A. , Gamble John , Colledge William H. , Lee Jin-Kyu , Pierce Michael , Miller Hugh R. P.
Published: April 2014

Infection of mice with the nematode parasite Trichinella spiralis induces changes in the proteome of the jejunal epithelium, including substantial up-regulation of a novel variant of interlectin. In this study we sequence this novel lectin, termed intelectin-2, and compare expression levels during T. spiralis infection of resistant (BALB/c) with susceptible (C57BL/10) mouse strains. Intelectin-2 was cloned and sequenced from BALB/c mRNA extracted on day 14 of infection, and was found to have 91% amino acid identity with intelectin (within our study termed intelectin-1). Intelectin-2 transcripts were up-regulated early (day 3) during infection with T. spiralis in BALB/c mice, suggesting an innate response, and levels remained high through to day 14 (time of parasite rejection). Immunohistochemistry of jejunal sections with a rabbit polyclonal Ab to Xenopus laevis 35-kDa cortical granule lectin (XL35; 68% identity with intelectin-2) followed a similar pattern, with intense labeling of goblet and Paneth cells at day 14. However, intelectin-2 transcripts and protein were absent, and immunohistochemistry negative when C57BL/10 mice were infected with T. spiralis. Genomic PCR and Southern blotting confirmed that the intelectin-2 gene is absent from the C57BL/10 genome. The presence of intelectin-2 in resistant BALB/c mice, its absence from the susceptible C57BL/10 strain and the kinetics of its up-regulation during T. spiralis infection suggest that this novel lectin may serve a protective role in the innate immune response to parasite infection.
Bordetella Type III Secretion and Adenylate Cyclase Toxin Synergize to Drive Dendritic Cells into a Semimature State
Authors: Skinner Jason A. , Reissinger Annette , Shen Hao , Yuk Ming H.
Published: April 2014

Bordetella bronchiseptica establishes persistent infection of the murine respiratory tract. We hypothesize that long-term colonization is mediated in part by bacteria-driven modulation of dendritic cells (DCs) leading to altered adaptive immune responses. Bone marrow-derived DCs (BMDCs) from C57BL/6 mice infected with live B. bronchiseptica exhibited high surface expression of MHCII, CD86, and CD80. However, B. bronchiseptica-infected BMDCs did not exhibit significant increases in CD40 surface expression and IL-12 secretion compared with BMDCs treated with heat-killed B. bronchiseptica. The B. bronchiseptica type III secretion system (TTSS) mediated the increase in MHCII, CD86, and CD80 surface expression, while the inhibition of CD40 and IL-12 expression was mediated by adenylate cyclase toxin (ACT). IL-6 secretion was independent of the TTSS and ACT. These phenotypic changes may result from differential regulation of MAPK signaling in DCs. Wild-type B. bronchiseptica activated the ERK 1/2 signaling pathway in a TTSS-dependent manner. Additionally, ACT was found to inhibit p38 signaling. These data suggest that B. bronchiseptica drive DC into a semimature phenotype by altering MAPK signaling. These semimature DCs may induce tolerogenic immune responses that allow the persistent colonization of B. bronchiseptica in the host respiratory tract.