bluuebunny/crossref_metadata_2025 huggingface dataset:
TCR Complex-Activated CD8 Adhesion Function by Human T Cells
Authors: Varghese Jay C. , Kane Kevin P.
Published: April 2014

The CD8 receptor plays a central role in the recognition and elimination of virally infected and malignant cells by cytolytic CD8+ T cells. In conjunction with the TCR, the CD8 coreceptor binds Ag-specific class I MHC (MHC-I) molecules expressed by target cells, initiating signaling events that result in T cell activation. Whether CD8 can further function as an adhesion molecule for non-Ag MHC-I is currently unclear in humans. In this study, we show that in human CD8+ T cells, TCR complex signaling activates CD8 adhesion molecule function, resulting in a CD8 interaction with MHC-I that is sufficient to maintain firm T cell adhesion under shear conditions. Secondly, we found that while CD8 adhesive function was triggered by TCR complex activation in differentiated cells, including in vitro generated CTL and ex vivo effector/memory phenotype CD8+ T cells, naive CD8+ T cells were incapable of activated CD8 adhesion. Lastly, we examine the kinetics of, and signaling for, activated CD8 adhesion in humans and identify notable differences from the equivalent CD8 function in mouse. Activated CD8 adhesion induced by TCR signaling may contribute to the more rapid and robust elimination of pathogen-infected cells by differentiated CD8+ T cells.
Calcitonin Gene-Related Peptide Biases Langerhans Cells toward Th2-Type Immunity
Authors: Ding Wanhong , Stohl Lori L. , Wagner John A. , Granstein Richard D.
Published: April 2014

Langerhans cells (LC) are epidermal dendritic cells capable, in several experimental systems, of Ag-presentation for stimulation of cell-mediated immunity. LC have been considered to play a key role in initiation of cutaneous immune responses. Additionally, administration of donor T cells to bone marrow chimeric mice with persistent host LC, but not mice whose LC have been replaced by donor cells, exhibit marked skin graft-vs-host disease, demonstrating that LC can trigger graft-vs-host disease. However, experiments with transgenic mice in which regulatory elements from human langerin were used to drive expression of diphtheria toxin, resulting in absence of LC, suggest that LC may serve to down-regulate cutaneous immunity. LC are associated with nerves containing the neuropeptide calcitonin gene-related peptide (CGRP), and CGRP inhibits LC Ag-presentation in several models including presentation to a Th1 clone. We now report that CGRP enhances LC function for stimulation of Th2 responses. CGRP exposure enhanced LC Ag presentation to a Th2 clone. Upon presentation of chicken OVA by LC to T cells from DO11.10 chicken OVA TCR transgenic mice, pretreatment with CGRP resulted in increased IL-4 production and decreased IFN-γ production. CGRP also inhibited stimulated production of the Th1 chemokines CXCL9 and CXCL10 but induced production of the Th2 chemokines CCL17 and CCL22 by a dendritic cell line and by freshly obtained LC. Changes in production of these chemokines correlated with the effect of CGRP on mRNA levels for these factors. Exposure of LC to nerve-derived CGRP in situ may polarize them toward favoring Th2-type immunity.
Nasal Anti-CD3 Antibody Ameliorates Lupus by Inducing an IL-10-Secreting CD4+CD25−LAP+ Regulatory T Cell and Is Associated with Down-Regulation of IL-17+CD4+ICOS+CXCR5+ Follicular Helper T Cells
Authors: Wu Henry Yim , Quintana Francisco J. , Weiner Howard L.
Published: April 2014

Lupus is an Ab-mediated autoimmune disease. One of the potential contributors to the development of systemic lupus erythematosus is a defect in naturally occurring CD4+CD25+ regulatory T cells. Thus, the generation of inducible regulatory T cells that can control autoantibody responses is a potential avenue for the treatment of systemic lupus erythematosus. We have found that nasal administration of anti-CD3 mAb attenuated lupus development as well as arrested ongoing lupus in two strains of lupus-prone mice. Nasal anti-CD3 induced a CD4+CD25−latency-associated peptide (LAP)+ regulatory T cell that secreted high levels of IL-10 and suppressed disease in vivo via IL-10- and TFG-β-dependent mechanisms. Disease suppression also occurred following adoptive transfer of CD4+CD25−LAP+ regulatory T cells from nasal anti-CD3-treated animals to lupus-prone mice. Animals treated with nasal anti-CD3 had less glomerulonephritis and diminished levels of autoantibodies as measured by both ELISA and autoantigen microarrays. Nasal anti-CD3 affected the function of CD4+ICOS+CXCR5+ follicular helper T cells that are required for autoantibody production. CD4+ICOS+CXCR5+ follicular helper T cells express high levels of IL-17 and IL-21 and these cytokines were down-regulated by nasal anti-CD3. Our results demonstrate that nasal anti-CD3 induces CD4+CD25−LAP+ regulatory T cells that suppress lupus in mice and that it is associated with down-regulation of T cell help for autoantibody production.
Central Tolerance Regulates B Cells Reactive with Goodpasture Antigen α3(IV)NC1 Collagen
Authors: Zhang Ying , Su Susan C. , Hecox Douglas B. , Brady Graham F. , Mackin Katherine M. , Clark Amy G. , Foster Mary H.
Published: April 2014

Patients and rodents with Goodpasture’s syndrome (GPS) develop severe autoimmune crescentic glomerulonephritis, kidney failure, and lung hemorrhage due to binding of pathogenic autoantibodies to the NC1 domain of the α3 chain of type IV collagen. Target epitopes are cryptic, normally hidden from circulating Abs by protein-protein interactions and the highly tissue-restricted expression of the α3(IV) collagen chain. Based on this limited Ag exposure, it has been suggested that target epitopes are not available as B cell tolerogens. To determine how pathogenic anti-GPS autoantibody responses are regulated, we generated an Ig transgenic (Tg) mouse model that expresses an Ig that binds α3(IV)NC1 collagen epitopes recognized by serum IgG of patients with GPS. Phenotypic analysis reveals B cell depletion and L chain editing in Tg mice. To determine the default tolerance phenotype in the absence of receptor editing and endogenous lymphocyte populations, we crossed Tg mice two generations with mice deficient in Rag. Resulting Tg Rag-deficient mice have central B cell deletion. Thus, development of Tg anti-α3(IV)NC1 collagen B cells is halted in the bone marrow, at which point the cells are deleted unless rescued by a Rag enzyme-dependent process, such as editing. The central tolerance phenotype implies that tolerizing self-Ag is expressed in bone marrow.
An Epithelial Progenitor Pool Regulates Thymus Growth
Authors: Jenkinson William E. , Bacon Andrea , White Andrea J. , Anderson Graham , Jenkinson Eric J.
Published: April 2014

Thymic epithelium provides an essential cellular substrate for T cell development and selection. Gradual age-associated thymic atrophy leads to a reduction in functional thymic tissue and a decline in de novo T cell generation. Development of strategies tailored toward regeneration of thymic tissue provides an important possibility to improve immune function in elderly individuals and increase the capacity for immune recovery in patients having undergone bone marrow transfer following immunoablative therapies. In this study we show that restriction of the size of the functional thymic epithelial progenitor pool affects the number of mature thymic epithelial cells. Using an embryo fusion chimera-based approach, we demonstrate a reduction in the total number of both embryonic and adult thymic epithelium, which relates to the initial size of the progenitor cell pool. The inability of thymic epithelial progenitor cells to undergo sufficient compensatory proliferation to rescue the deficit in progenitor numbers suggests that in addition to extrinsic regulation of thymus growth by provision of growth factors, intrinsic factors such as a proliferative restriction of thymic epithelial progenitors and availability of progenitor cell niches may limit thymic epithelial recovery. Collectively, our data demonstrate an important level of regulation of thymic growth and recovery at the thymic epithelial progenitor level, providing an important consideration for developing methods targeted toward inducing thymic regeneration.
Expression of CD86 on Human Islet Endothelial Cells Facilitates T Cell Adhesion and Migration
Authors: Lozanoska-Ochser Biliana , Klein Nigel J. , Huang Guo C. , Alvarez Raymond A. , Peakman Mark
Published: April 2014

Pancreatic islet endothelial cells (ECs) form the barrier across which autoreactive T cells transmigrate during the development of islet inflammation in type 1 diabetes. Little is known about the immune phenotype of islet ECs that might shape their molecular interaction with autoreactive T cells before and during the development of islet inflammation. In this study we examined the expression and functional significance of costimulatory molecules by human islet ECs. Freshly isolated human islet ECs constitutively expressed CD86 (B7-2) and ICOS ligand but not CD80 (B7-1) or CD40 costimulatory molecules. The functional activity of islet EC-expressed CD86 was examined by coculture of resting islet ECs with CD4 T cells stimulated by CD3 ligation alone. Marked T cell proliferation in the coculture was completely abrogated by mAb blockade of CD86, confirming that costimulatory properties are conferred on ECs by CD86 expression. In view of its location on the vasculature, we hypothesized a role for CD86 in T cell adhesion/transmigration. In keeping with this, adhesion/transmigration of activated (CD3 ligated) memory (CD45R0+) CD4 T cells across islet ECs was completely inhibited in the presence of CD86 blocking mAb. Identical results were obtained for T cell adhesion using either CTLA-4 blocking mAb or CTLA-4Ig (abatacept), indicating CTLA-4 as the T cell ligand for these CD86-mediated effects. These data suggest a novel role for CD86 expression on the microvasculature, whereby ligation of CTLA-4 on CD4 T cells by CD86 on islet ECs is key to the adhesion of recently activated T cells.
IL-17-Producing Alveolar Macrophages Mediate Allergic Lung Inflammation Related to Asthma
Authors: Song Chuanwang , Luo Liqiong , Lei Zhang , Li Bo , Liang Zhihui , Liu Guanghui , Li Dong , Zhang Guimei , Huang Bo , Feng Zuo-Hua
Published: April 2014

IL-17 is a pivotal proinflammatory molecule in asthmatics. However, the cellular source of IL-17 in asthma has not been identified to date. In this study, we report that macrophages rather than Th17 cells are the main producer of IL-17 in allergic inflammation related to asthma. After OVA challenge in a mouse model mimicking allergic asthma, the increased IL-17+ cells in the lung were mainly CD11b+F4/80+ macrophages, instead of T cells or others. Importantly, IL-17+ alveolar macrophages (AMs), but not IL-17+ interstitial macrophages, were significantly increased after allergen challenge. The increase of IL-17+ AMs was not due to the influx of IL-17+ macrophages from circulation or other tissues, but ascribed to the activation of AMs by mediator(s) secreted by IgE/OVA-activated mast cells. Depleting alveolar macrophages or neutralizing IL-17 prevented the initiation of OVA-induced asthma-related inflammation by inhibiting the increase of inflammatory cells and inflammatory factors in bronchoalveolar lavage fluid. Th2 cytokine IL-10 could down-regulate IL-17 expression in alveolar macrophages. The increased IL-17 and the decreased IL-10 in bronchoalveolar lavage fluid were further confirmed in asthmatic patients. These findings suggest that IL-17 is mainly produced by macrophages but not Th17 cells in allergic inflammation related to asthma. Mast cell-released mediators up-regulate the expression of IL-17 by macrophages, whereas IL-10 down-regulates IL-17 expression.
Critical Role of TLR9 in Acute Graft-versus-Host Disease
Authors: Calcaterra Claudia , Sfondrini Lucia , Rossini Anna , Sommariva Michele , Rumio Cristiano , Ménard Sylvie , Balsari Andrea
Published: April 2014

Graft-vs-host disease (GVHD) is a major complication after allogeneic bone marrow transplantation. Different studies have demonstrated that intestinal bacterial breakdown products and loss of gastrointestinal tract integrity, both induced by conditioning regiments, are critical in the pathogenesis of acute GVHD. Using C57BL/6 knockout mice, we evaluated the role of TLR4 and TLR9, which recognize bacterial LPS and DNA, respectively, in the GVHD associated with allogeneic bone marrow transplantation. When myeloablative-irradiated TLR9 knockout (TLR9−/−) mice were used as graft recipients, survival and clinical score of acute GVHD were improved as compared with the wild-type recipient mice (18/30 vs 1/31 mice still alive at day 70 in a total of four experiments); while no differences were observed using recipient TLR4 knockout (TLR4−/−) mice. The reduced mortality and morbidity in TLR9−/− mice related with reduced stimulatory activity of TLR9−/− spleen APCs after conditioning and reduced proliferation of allogeneic donor T cells. Experiments using TLR9+/+ into TLR9−/− and TLR9−/− into TLR9+/+ chimeric mice as recipients indicated a critical role for nonhematopoietic TLR9+/+ cells interacting with bacterial breakdown products released in myeloablated mice. Altogether these data reveal a novel important role of TLR9 in GVHD, a finding that might provide tools to reduce this complication of allogeneic transplantation.
Unique Receptor Repertoire in Mouse Uterine NK cells
Authors: Yadi Hakim , Burke Shannon , Madeja Zofia , Hemberger Myriam , Moffett Ashley , Colucci Francesco
Published: April 2014

Uterine NK (uNK) cells are a prominent feature of the uterine mucosa and regulate placentation. NK cell activity is regulated by a balance of activating and inhibitory receptors, however the receptor repertoire of mouse uNK cells is unknown. We describe herein two distinct subsets of CD3−CD122+ NK cells in the mouse uterus (comprising decidua and mesometrial lymphoid aggregate of pregnancy) at mid-gestation: a small subset indistinguishable from peripheral NK cells, and a larger subset that expresses NKp46 and Ly49 receptors, but not NK1.1 or DX5. This larger subset reacts with Dolichus biflores agglutinin, a marker of uNK cells in the mouse, and is adjacent to the invading trophoblast. By multiparametric analysis we show that the phenotype of uNK cells is unique and unprecedented in terms of adhesion, activation, and MHC binding potential. Thus, the Ly49 repertoire and the expression of other differentiation markers strikingly distinguish uNK cells from peripheral NK cells, suggesting that a selection process shapes the receptor repertoire of mouse uNK cells.
Immunosurveillance of Lung Melanoma Metastasis in EBI-3-Deficient Mice Mediated by CD8+ T Cells
Authors: Sauer Kerstin A. , Maxeiner Joachim H. , Karwot Roman , Scholtes Petra , Lehr Hans A. , Birkenbach Mark , Blumberg Richard S. , Finotto Susetta
Published: April 2014

EBV-induced gene 3 (EBI-3) codes for a soluble type I receptor homologous to the p40 subunit of IL-12 that is expressed by APCs following activation. In this study, we assessed the role of EBI-3 in a model of lung melanoma metastasis. Intravenous injection of the B16-F10 cell line resulted in a significant reduction of lung tumor metastasis in EBI-3−/− recipient mice compared with wild-type mice. The immunological finding accompanying this effect was the expansion of a newly described cell subset called IFN-γ producing killer dendritic cells associated with CD8+ T cell responses in the lung of EBI-3−/− mice including IFN-γ release and TNF-α-induced programmed tumor cell death. Depletion of CD8+ T cells as well as targeting T-bet abrogated the protective effects of EBI-3 deficiency on lung melanoma metastases. Finally, adoptive transfer of EBI-3−/− CD8+ T cells into tumor bearing wild-type mice inhibited lung metastasis in recipient mice. Taken together, these data demonstrate that targeting EBI-3 leads to a T-bet-mediated antitumor CD8+ T cell responses in the lung.
Cross-Linking of B7-H1 on EBV-Transformed B Cells Induces Apoptosis through Reactive Oxygen Species Production, JNK Signaling Activation, and fasL Expression
Authors: Kim Yeong Seok , Park Ga Bin , Lee Hyun-Kyung , Song Hyunkeun , Choi In-Hak , Lee Wang Jae , Hur Dae Young
Published: April 2014

B7-H1 is a newly identified member of the B7 family with important regulatory functions in cell-mediated immune responses, and it is expressed in human immune cells and several tumors. We first observed that expression of surface B7-H1 on B cells was increased during the immortalization process by EBV, which is strongly related to both inflammation and tumorigenesis. Cross-linking of B7-H1 on EBV-transformed B cells using anti-B7-H1 Ab (clone 130002) induced reactive oxygen species (ROS) generation, mitochondrial disruption, release of apoptotic proteins from mitochondria, and subsequent apoptosis. Inhibition of caspases and ROS generation recovered B7-H1-mediated apoptosis and proteolytic activities of caspase-8, -9, and -3. We observed that B7-H1 stimulation induced both transcription and translation of fasL. ZB4, an antagonistic anti-fas Ab, and NOK-1, an antagonistic anti-fasL Ab, effectively blocked apoptosis without exerting any influence on ROS generation. N-acetylcysteine (NAC) completely blocked the induction of fasL mRNA and protein. We found that B7-H1 stimulation activated the phosphorylation of JNK and c-jun and down-regulated ERK1/2 and p-Akt. NAC blocked the activation of JNK and down-regulation of ERK, but both z-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) and ZB4 did not inhibit JNK activation of B7-H1 stimulation. SP600125 blocked fasL induction and apoptosis but did not affect ROS generation after B7-H1 stimulation. Taken together, we concluded that B7-H1-mediated apoptosis on EBV-transformed B cells may be involved in the induction of fasL, which is evoked by ROS generation and JNK activation after cross-linking of B7-H1. These results provide a new concept for understanding reverse signaling through B7-H1 and another mechanism of tumor immunotherapy using anti-B7-H1.
CX3CR1+c-kit+ Bone Marrow Cells Give Rise to CD103+ and CD103− Dendritic Cells with Distinct Functional Properties
Authors: del Rio Maria-Luisa , Rodriguez-Barbosa Jose-Ignacio , Bölter Jasmin , Ballmaier Matthias , Dittrich-Breiholz Oliver , Kracht Michael , Jung Steffen , Förster Reinhold
Published: April 2014

Dendritic cells (DC) represent a rather heterogeneous cell population with regard to morphology, phenotype, and function and, like most cells of the immune system, are subjected to a continuous renewal process. CD103+ (integrin αE) DC have been identified as a major mucosal DC subset involved in the induction of tissue-specific homing molecules on T cells, but little is known about progenitors able to replenish this DC subset. Herein we report that lineage (lin)−CX3CR1+c-kit+ (GFP+c-kit+) bone marrow cells can differentiate to either CD11c+CD103− or CD11c+CD103+ DC in vitro and in vivo. Gene expression as well as functional assays reveal distinct phenotypical and functional properties of both subsets generated in vitro. CD103− DC exhibit enhanced phagocytosis and respond to LPS stimulation by secreting proinflammatory cytokines, whereas CD103+ DC express high levels of costimulatory molecules and efficiently induce allogeneic T cell proliferation. Following adoptive transfer of GFP+c-kit+ bone marrow cells to irradiated recipients undergoing allergic lung inflammation, we identified donor-derived CD103+ DC in lung and the lung-draining bronchial lymph node. Collectively, these data indicate that GFP+c-kit+ cells contribute to the replenishment of CD103+ DC in lymphoid and nonlymphoid organs.
Organizer-Like Reticular Stromal Cell Layer Common to Adult Secondary Lymphoid Organs
Authors: Katakai Tomoya , Suto Hidenori , Sugai Manabu , Gonda Hiroyuki , Togawa Atsushi , Suematsu Sachiko , Ebisuno Yukihiko , Katagiri Koko , Kinashi Tatsuo , Shimizu Akira
Published: April 2014

Mesenchymal stromal cells are crucial components of secondary lymphoid organs (SLOs). Organogenesis of SLOs involves specialized stromal cells, designated lymphoid tissue organizer (LTo) in the embryonic anlagen; in the adult, several distinct stromal lineages construct elaborate tissue architecture and regulate lymphocyte compartmentalization. The relationship between the LTo and adult stromal cells, however, remains unclear, as does the precise number of stromal cell types that constitute mature SLOs are unclear. From mouse lymph nodes, we established a VCAM-1+ICAM-1+MAdCAM-1+ reticular cell line that can produce CXCL13 upon LTβR stimulation and support primary B cell adhesion and migration in vitro. A similar stromal population sharing many characteristics with the LTo, designated marginal reticular cells (MRCs), was found in the outer follicular region immediately underneath the subcapsular sinus of lymph nodes. Moreover, MRCs were commonly observed at particular sites in various SLOs even in Rag2−/− mice, but were not found in ectopic lymphoid tissues, suggesting that MRCs are a developmentally determined element. These findings lead to a comprehensive view of the stromal composition and architecture of SLOs.
Automated Identification of Complementarity Determining Regions (CDRs) Reveals Peculiar Characteristics of CDRs and B Cell Epitopes
Authors: Ofran Yanay , Schlessinger Avner , Rost Burkhard
Published: April 2014

Exact identification of complementarity determining regions (CDRs) is crucial for understanding and manipulating antigenic interactions. One way to do this is by marking residues on the antibody that interact with B cell epitopes on the antigen. This, of course, requires identification of B cell epitopes, which could be done by marking residues on the antigen that bind to CDRs, thus requiring identification of CDRs. To circumvent this vicious circle, existing tools for identifying CDRs are based on sequence analysis or general biophysical principles. Often, these tools, which are based on partial data, fail to agree on the boundaries of the CDRs. Herein we present an automated procedure for identifying CDRs and B cell epitopes using consensus structural regions that interact with the antigens in all known antibody-protein complexes. Consequently, we provide the first comprehensive analysis of all CDR-epitope complexes of known three-dimensional structure. The CDRs we identify only partially overlap with the regions suggested by existing methods. We found that the general physicochemical properties of both CDRs and B cell epitopes are rather peculiar. In particular, only four amino acids account for most of the sequence of CDRs, and several types of amino acids almost never appear in them. The secondary structure content and the conservation of B cell epitopes are found to be different than previously thought. These characteristics of CDRs and epitopes may be instrumental in choosing which residues to mutate in experimental search for epitopes. They may also assist in computational design of antibodies and in predicting B cell epitopes.
n-3 Polyunsaturated Fatty Acids Suppress the Localization and Activation of Signaling Proteins at the Immunological Synapse in Murine CD4+ T Cells by Affecting Lipid Raft Formation
Authors: Kim Wooki , Fan Yang-Yi , Barhoumi Rola , Smith Roger , McMurray David N. , Chapkin Robert S.
Published: April 2014

The molecular properties of immunosuppressive n-3 polyunsaturated fatty acids (PUFA) have not been fully elucidated. Using CD4+ T cells from wild-type control and fat-1 transgenic mice (enriched in n-3 PUFA), we show that membrane raft accumulation assessed by Laurdan (6-dodecanoyl-2-dimethyl aminonaphthalene) labeling was enhanced in fat-1 cells following immunological synapse (IS) formation by CD3-specific Ab expressing hybridoma cells. However, the localization of protein kinase Cθ, phospholipase Cγ-1, and F-actin into the IS was suppressed. In addition, both the phosphorylation status of phospholipase Cγ-1 at the IS and cell proliferation as assessed by CFSE labeling and [3H]thymidine incorporation were suppressed in fat-1 cells. These data imply that lipid rafts may be targets for the development of dietary agents for the treatment of autoimmune and chronic inflammatory diseases.
Loss of Protein Kinase Cθ, Bcl10, or Malt1 Selectively Impairs Proliferation and NF-κB Activation in the CD4+ T Cell Subset
Authors: Kingeter Lara M. , Schaefer Brian C.
Published: April 2014

The cytosolic proteins protein kinase Cθ (PKCθ), Bcl10, and Malt1 play critical roles in TCR signaling to the transcription factor NF-κB. Our data confirm that CD4+ T cells from PKCθ, Bcl10, and Malt1 knockout mice show severe impairment of proliferation in response to TCR stimulation. Unexpectedly, we find that knockout CD8+ T cells proliferate to a similar extent as wild-type cells in response to strong TCR signals, although a survival defect prevents their accumulation. Both CD4+ and CD8+ knockout T cells express activation markers, including CD25, following TCR stimulation. Addition of exogenous IL-2 rescues survival of knockout CD4+ and CD8+ T cells, but fails to overcome the proliferation defect of CD4+ T cells. CD4+ T cells from knockout mice are extremely deficient in TCR-induced NF-κB activation, whereas NF-κB activation is only partially impaired in CD8+ T cells. Overall, our results suggest that defects in TCR signaling through PKCθ, Bcl10, and Malt1 predominantly impair NF-κB activation and downstream functional responses of CD4+ T cells. In contrast, CD8+ T cells maintain substantial NF-κB signaling, implying the existence of a significant TCR-regulated NF-κB activation pathway in CD8+ T cells that is independent of PKCθ, Bcl10, and Malt1.
Ikaros Regulates Notch Target Gene Expression in Developing Thymocytes
Authors: Chari Sheila , Winandy Susan
Published: April 2014

Both Ikaros and Notch are essential for normal T cell development. Collaborative mutations causing a reduction in Ikaros activity and an increase in Notch activation promote T cell leukemogenesis. Although the molecular mechanisms of this cooperation have been studied, its consequences in thymocyte development remain unexplored. In this study, we show that Ikaros regulates expression of a subset of Notch target genes, including Hes1, Deltex1, pTa, Gata3, and Runx1, in both Ikaros null T cell leukemia lines and Ikaros null primary thymocytes. In Ikaros null leukemia cells, Notch deregulation occurs at both the level of Notch receptor cleavage and expression of Notch target genes, because re-expression of Ikaros in these cells down-regulates Notch target gene expression without affecting levels of intracellular cleaved Notch. In addition, abnormal expression of Notch target genes is observed in Ikaros null double-positive thymocytes, in the absence of detectable intracellular cleaved Notch. Finally, we show that this role of Ikaros is specific to double-positive and single-positive thymocytes because derepression of Notch target gene expression is not observed in Ikaros null double-negative thymocytes or lineage-depleted bone marrow. Thus, in this study, we provide evidence that Ikaros and Notch play opposing roles in regulation of a subset of Notch target genes and that this role is restricted to developing thymocytes where Ikaros is required to appropriately regulate the Notch program as they progress through T cell development.
Endoplasmic Reticulum Aminopeptidase Associated with Antigen Processing Regulates Quality of Processed Peptides Presented by MHC Class I Molecules
Authors: Kanaseki Takayuki , Shastri Nilabh
Published: April 2014

Effective immune surveillance by CD8 T cells depends on the presentation of diverse peptides by MHC class I (pMHC I) molecules on the cell surface. The pMHC I repertoire is shaped in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with Ag processing (ERAAP). The ERAAP activity is required for producing peptides of appropriate length for generating optimal pMHC I. Paradoxically, ERAAP also inhibits generation of certain peptides such as the SVL9 (SSVVGVWYL) peptide encoded by the H13a histocompatibility gene and presented by Db MHC by an unknown mechanism. In this study, we show that the presentation of the SVL9-Db complex is inhibited when other peptides compete for binding Db. Conversely, improving the binding of SVL9 peptide to Db suppresses the inhibition. Interestingly, the inhibitory effect of competitor peptides is observed only when ERAAP is expressed in the same cells. Thus, ERAAP, in concert with MHC I molecules, regulates the quality of processed peptides presented on the cell surface.
Impact of Protective IL-2 Allelic Variants on CD4+Foxp3+ Regulatory T Cell Function In Situ and Resistance to Autoimmune Diabetes in NOD Mice
Authors: Sgouroudis Evridiki , Albanese Alexandre , Piccirillo Ciriaco A.
Published: April 2014

Type I diabetes (T1D) susceptibility is inherited through multiple insulin-dependent diabetes (Idd) genes. NOD.B6 Idd3 congenic mice, introgressed with an Idd3 allele from T1D-resistant C57BL/6 mice (Idd3B6), show a marked resistance to T1D compared with control NOD mice. The protective function of the Idd3 locus is confined to the Il2 gene, whose expression is critical for naturally occurring CD4+Foxp3+ regulatory T (nTreg) cell development and function. In this study, we asked whether Idd3B6 protective alleles in the NOD mouse model confer T1D resistance by promoting the cellular frequency, function, or homeostasis of nTreg cells in vivo. We show that resistance to T1D in NOD.B6 Idd3 congenic mice correlates with increased levels of IL-2 mRNA and protein production in Ag-activated diabetogenic CD4+ T cells. We also observe that protective IL2 allelic variants (Idd3B6 resistance allele) also favor the expansion and suppressive functions of CD4+Foxp3+ nTreg cells in vitro, as well as restrain the proliferation, IL-17 production, and pathogenicity of diabetogenic CD4+ T cells in vivo more efficiently than control do nTreg cells. Lastly, the resistance to T1D in Idd3 congenic mice does not correlate with an augmented systemic frequency of CD4+Foxp3+ nTreg cells but more so with the ability of protective IL2 allelic variants to promote the expansion of CD4+Foxp3+ nTreg cells directly in the target organ undergoing autoimmune attack. Thus, protective, IL2 allelic variants impinge the development of organ-specific autoimmunity by bolstering the IL-2 producing capacity of self-reactive CD4+ T cells and, in turn, favor the function and homeostasis of CD4+Foxp3+ nTreg cells in vivo.
Complete Characterization of Killer Ig-Like Receptor (KIR) Haplotypes in Mauritian Cynomolgus Macaques: Novel Insights into Nonhuman Primate KIR Gene Content and Organization
Authors: Bimber Benjamin N. , Moreland Anna J. , Wiseman Roger W. , Hughes Austin L. , O'Connor David H.
Published: April 2014

Killer Ig-like receptors (KIRs) are implicated in protection from multiple pathogens including HIV, human papillomavirus, and malaria. Nonhuman primates such as rhesus and cynomolgus macaques are important models for the study of human pathogens; however, KIR genetics in nonhuman primates are poorly defined. Understanding KIR allelic diversity and genomic organization are essential prerequisites to evaluate NK cell responses in macaques. In this study, we present a complete characterization of KIRs in Mauritian cynomolgus macaques, a geographically isolated population. In this study we demonstrate that only eight KIR haplotypes are present in the entire population and characterize the gene content of each. Using the simplified genetics of this population, we construct a model for macaque KIR genomic organization, defining four putative KIR3DL loci, one KIR3DH, two KIR2DL, and one KIR1D. We further demonstrate that loci defined in Mauritian cynomolgus macaques can be applied to rhesus macaques. The findings from this study fundamentally advance our understanding of KIR genetics in nonhuman primates and establish a foundation from which to study KIR signaling in disease pathogenesis.
An Indispensable Role for the Chemokine Receptor CCR10 in IgA Antibody-Secreting Cell Accumulation
Authors: Morteau Olivier , Gerard Craig , Lu Bao , Ghiran Sorina , Rits Miriam , Fujiwara Yuko , Law Yuetching , Distelhorst Kathryn , Nielsen Elizabeth M. , Hill Erica D. , Kwan Raymond , Lazarus Nicole H. , Butcher Eugene C. , Wilson Eric
Published: April 2014

The differential expression of chemokines and chemokine receptors, by tissues and leukocytes, respectively, contributes to the specific accumulation of leukocyte subsets to different tissues. CCR10/CCL28 interactions are thought to contribute to the accumulation of IgA Ab-secreting cells (ASC) to mucosal surfaces, such as the gastrointestinal tract and the lactating mammary gland. Although the role of CCL28 in lymphocyte homing is well established, direct in vivo evidence for CCR10 involvement in this process has not been previously shown. In this study, we describe the generation of a CCR10-deficient mouse model. Using this model, we demonstrate that CCR10 is critical for efficient localization and accumulation of IgA ASC to the lactating mammary gland. Surprisingly, IgA ASC accumulation to the gastrointestinal tract is minimally impacted in CCR10-deficient mice. These results provide the first direct evidence of CCR10 involvement in lymphocyte homing and accumulation in vivo, and demonstrate that reliance on CCR10-mediated recruitment of IgA ASC varies dramatically within mucosal tissues.
Prior Immunization with Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus (SARS-CoV) Nucleocapsid Protein Causes Severe Pneumonia in Mice Infected with SARS-CoV
Authors: Yasui Fumihiko , Kai Chieko , Kitabatake Masahiro , Inoue Shingo , Yoneda Misako , Yokochi Shoji , Kase Ryoichi , Sekiguchi Satoshi , Morita Kouichi , Hishima Tsunekazu , Suzuki Hidenori , Karamatsu Katsuo , Yasutomi Yasuhiro , Shida Hisatoshi , Kidokoro Minoru , Mizuno Kyosuke , Matsushima Kouji , Kohara Michinori
Published: April 2014

The details of the mechanism by which severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia are unclear. We investigated the immune responses and pathologies of SARS-CoV-infected BALB/c mice that were immunized intradermally with recombinant vaccinia virus (VV) that expressed either the SARS-CoV spike (S) protein (LC16m8rVV-S) or simultaneously all the structural proteins, including the nucleocapsid (N), membrane (M), envelope (E), and S proteins (LC16m8rVV-NMES) 7–8 wk before intranasal SARS-CoV infection. The LC16m8rVV-NMES-immunized group exhibited as severe pneumonia as the control groups, although LC16m8rVV-NMES significantly decreased the pulmonary SARS-CoV titer to the same extent as LC16m8rVV-S. To identify the cause of the exacerbated pneumonia, BALB/c mice were immunized with recombinant VV that expressed the individual structural proteins of SARS-CoV (LC16mOrVV-N, -M, -E, -S) with or without LC16mOrVV-S (i.e., LC16mOrVV-N, LC16mOrVV-M, LC16mOrVV-E, or LC16mOrVV-S alone or LC16mOrVV-N + LC16mOrVV-S, LC16mOrVV-M + LC16mOrVV-S, or LC16mOrVV-E + LC16mOrVV-S), and infected with SARS-CoV more than 4 wk later. Both LC16mOrVV-N-immunized mice and LC16mOrVV-N + LC16mOrVV-S-immunized mice exhibited severe pneumonia. Furthermore, LC16mOrVV-N-immunized mice upon infection exhibited significant up-regulation of both Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5) cytokines and down-regulation of anti-inflammatory cytokines (IL-10, TGF-β), resulting in robust infiltration of neutrophils, eosinophils, and lymphocytes into the lung, as well as thickening of the alveolar epithelium. These results suggest that an excessive host immune response against the nucleocapsid protein of SARS-CoV is involved in severe pneumonia caused by SARS-CoV infection. These findings increase our understanding of the pathogenesis of SARS.
Long-Term Immunity against Actual Poxviral HLA Ligands as Identified by Differential Stable Isotope Labeling
Authors: Meyer Verena S. , Kastenmuller Wolfgang , Gasteiger Georg , Franz-Wachtel Mirita , Lamkemeyer Tobias , Rammensee Hans-Georg , Stevanovic Stefan , Sigurdardottir Dagmar , Drexler Ingo
Published: April 2014

Viral peptides are presented by HLA class I on infected cells to activate CD8+ T cells. Several immunogenic peptides have been identified indirectly by epitope prediction and screening of T cell responses to poxviral vectors, including modified vaccinia virus Ankara (MVA) currently being tested as recombinant or smallpox vaccines. However, for the development of optimal vaccination and immunomonitoring strategies, it is essential to characterize the actual viral HLA ligand repertoire of infected cells. We used an innovative approach to identify naturally processed MVA HLA ligands by differential HPLC-coupled mass spectrometry. We describe 12 viral peptides presented by HLA-A*0201 and 3 by HLA-B*0702. All HLA-A*0201 ligands participated in the memory response of MVA-immune donors, and several were immunogenic in Dryvax vaccinees. Eight epitopes were novel. Viral HLA ligand presentation and viral protein abundance did not correlate. All ligands were expressed early during the viral life cycle, and a pool of three of these mediated stronger protection against a lethal challenge in mice as compared with late epitopes. This highlights the reliability of the comparative mass spectrometry-based technique to identify relevant viral CD8+ T cell epitopes for optimizing the monitoring of protective immune responses and the development of effective peptide-based vaccines.
CCL3-CCR5 Axis Regulates Intratumoral Accumulation of Leukocytes and Fibroblasts and Promotes Angiogenesis in Murine Lung Metastasis Process
Authors: Wu Yu , Li Ying-Yi , Matsushima Kouji , Baba Tomohisa , Mukaida Naofumi
Published: April 2014

Metastasis proceeds through interaction between cancer cells and resident cells such as leukocytes and fibroblasts. An i.v. injection of a mouse renal cell carcinoma, Renca, into wild-type mice resulted in multiple metastasis foci in lungs and was associated with intratumoral accumulation of macrophages, granulocytes, and fibroblasts. A chemokine, CCL3, was detected in infiltrating cells and, to a lesser degree, tumor cells, together with an infiltration of leukocytes expressing CCR5, a specific receptor for CCL3. A deficiency of the CCL3 or CCR5 gene markedly reduced the number of metastasis foci in the lung, and the analysis using bone marrow chimeric mice revealed that both bone marrow- and non-bone marrow-derived cells contributed to metastasis formation. CCL3- and CCR5-deficient mice exhibited a reduction in intratumoral accumulation of macrophages, granulocytes, and fibroblasts. Moreover, intratumoral neovascularization, an indispensable process for metastasis, was attenuated in these gene-deficient mice. Intrapulmonary expression of matrix metalloproteinase (MMP)-9 and hepatocyte growth factor (HGF) was enhanced in wild-type mice, and the increases were markedly diminished in CCL3- and CCR5-deficient mice. Furthermore, MMP-9 protein was detected in macrophages and granulocytes, the cells that also express CCR5 and in vitro stimulation by CCL3-induced macrophages to express MMP-9. Intratumoral fibroblasts expressed CCR5 and HGF protein. In vitro CCL3 stimulated fibroblasts to express HGF. Collectively, the CCL3-CCR5 axis appears to regulate intratumoral trafficking of leukocytes and fibroblasts, as well as MMP-9 and HGF expression, and as a consequence to accelerate neovascularization and subsequent metastasis formation.
Prolonged Microglial Cell Activation and Lymphocyte Infiltration following Experimental Herpes Encephalitis
Authors: Marques Cristina P. , Cheeran Maxim C-J. , Palmquist Joseph M. , Hu Shuxian , Urban Stina L. , Lokensgard James R.
Published: April 2014

Experimental murine herpes simplex virus (HSV)-1 brain infection stimulates microglial cell-driven proinflammatory chemokine production which precedes the presence of brain-infiltrating systemic immune cells. In the present study, we investigated the phenotypes and infiltration kinetics of leukocyte trafficking into HSV-infected murine brains. Using real-time bioluminescence imaging, the infiltration of luciferase-positive splenocytes, transferred via tail vein injection into the brains of HSV-infected animals, was followed over an 18-day time course. Flow cytometric analysis of brain-infiltrating leukocytes at 5, 8, 14, and 30 days postinfection (d.p.i.), was performed to assess their phenotype. A predominantly macrophage (CD45highCD11b+Ly6Chigh) and neutrophil (CD45highCD11b+Ly6G+) infiltration was seen early during infection, with elevated levels of TNF-α mRNA expression. By 14 d.p.i., the phenotypic profile shifted to a predominantly lymphocytic (CD45highCD3+) infiltrate. This lymphocyte infiltrate was detected until 30 d.p.i., when infectious virus could not be recovered, with CD8+ and CD4+ T cells present at a 3:1 ratio, respectively. This T lymphocyte infiltration paralleled increased IFN-γ mRNA expression in the brain. Activation of resident microglia (CD45intCD11b+) was also detected until 30 d.p.i., as assessed by MHC class II expression. Activated microglial cells were further identified as the predominant source of IL-1β. In addition, infected mice given primed immunocytes at 4 d.p.i. showed a significant increase in mortality. Taken together, these results demonstrate that intranasal infection results in early macrophage and neutrophil infiltration into the brain followed by prolonged microglial activation and T lymphocyte retention. Similar prolonged neuroimmune activation may contribute to the neuropathological sequelae observed in herpes encephalitis patients.
Receptor-Interacting Protein Homotypic Interaction Motif-Dependent Control of NF-κB Activation via the DNA-Dependent Activator of IFN Regulatory Factors
Authors: Kaiser William J. , Upton Jason W. , Mocarski Edward S.
Published: April 2014

DNA-dependent activator of IFN regulatory factors (IRF; DAI, also known as ZBP1 or DLM-1) is a cytosolic DNA sensor that initiates IRF3 and NF-κB pathways leading to activation of type I IFNs (IFNα, IFNβ) and other cytokines. In this study, induction of NF-κB is shown to depend on the adaptor receptor-interacting protein kinase (RIP)1, acting via a RIP homotypic interaction motif (RHIM)-dependent interaction with DAI. DAI binds to and colocalizes with endogenous RIP1 at characteristic cytoplasmic granules. Suppression of RIP1 expression by RNAi abrogates NF-κB activation as well as IFNβ induction by immunostimulatory DNA. DAI also interacts with RIP3 and this interaction potentiates DAI-mediated activation of NF-κB, implicating RIP3 in regulating this RHIM-dependent pathway. The role of DAI in activation of NF-κB in response to immunostimulatory DNA appears to be analogous to sensing of dsRNA by TLR3 in that both pathways involve RHIM-dependent signaling that is mediated via RIP1, reinforcing a central role for this adaptor in innate sensing of intracellular microbes.
Hepatitis C Virus Immune Escape via Exploitation of a Hole in the T Cell Repertoire
Authors: Wölfl Matthias , Rutebemberwa Alleluiah , Mosbruger Timothy , Mao Qing , Li Hong-mei , Netski Dale , Ray Stuart C. , Pardoll Drew , Sidney John , Sette Alessandro , Allen Todd , Kuntzen Thomas , Kavanagh Daniel G. , Kuball Jürgen , Greenberg Philip D. , Cox Andrea L.
Published: April 2014

Hepatitis C virus (HCV) infection frequently persists despite eliciting substantial virus-specific immune responses. Thus, HCV infection provides a setting in which to investigate mechanisms of immune escape that allow for viral persistence. Viral amino acid substitutions resulting in decreased MHC binding or impaired Ag processing of T cell epitopes reduce Ag density on the cell surface, permitting evasion of T cell responses in chronic viral infection. Substitutions in viral epitopes that alter TCR contact residues frequently result in escape, but via unclear mechanisms because such substitutions do not reduce surface presentation of peptide-MHC complexes and would be expected to prime T cells with new specificities. We demonstrate that a known in vivo HCV mutation involving a TCR contact residue significantly diminishes T cell recognition and, in contrast to the original sequence, fails to effectively prime naive T cells. This mutant epitope thus escapes de novo immune recognition because there are few highly specific cognate TCR among the primary human T cell repertoire. This example is the first on viral immune escape via exploitation of a “hole” in the T cell repertoire, and may represent an important general mechanism of viral persistence.
MyD88 Signaling Is Not Essential for Induction of Antigen-Specific B Cell Responses but Is Indispensable for Protection against Streptococcus pneumoniae Infection following Oral Vaccination with Attenuated Salmonella Expressing PspA Antigen
Authors: Park Sung-Moo , Ko Hyun-Jeong , Shim Doo-Hee , Yang Jin-Young , Park Yong-Ho , Curtiss Roy , Kweon Mi-Na
Published: April 2014

TLRs directly induce innate host defense responses, but the mechanisms of TLR-mediated adaptive immunity remain subject to debate. In this study, we clarified a role of TLR-mediated innate immunity for induction of adaptive immunity by oral vaccination with a live recombinant attenuated Salmonella enteric serovar Typhimurium vaccine (RASV) strain expressing Streptococcus pneumoniae surface protein A (PspA) Ag. Of note, oral or intranasal vaccination with RASV expressing PspA resulted in identical or even significantly higher levels of PspA-specific IgG and IgA responses in the systemic and mucosal compartments of MyD88−/− mice of either BALB/c or C57BL/6 background when compared with those of wild-type mice. Although PspA-specific CD4+ T cell proliferation in the MyD88−/− mice was minimal, depletion of CD4+ T cells abolished PspA-specific IgG and IgA responses in the MyD88−/− mice of BALB/c background. Of the greatest interest, MyD88−/− mice that possessed high levels of PspA-specific IgG and IgA responses but minimal levels of CD4+ T cell responses died earlier than nonvaccinated and vaccinated wild-type mice following i.v. or intranasal challenge with virulent S. pneumoniae. Taken together, these results suggest that innate immunity activated by MyD88 signals might not be necessary for Ag-specific Ab induction in both systemic and mucosal sites but is critical for protection following oral vaccination with attenuated Salmonella expressing PspA.
Functional Dichotomy of Dendritic Cells following Interaction withLeishmania braziliensis: Infected Cells Produce High Levels of TNF-α, whereas Bystander Dendritic Cells Are Activated to Promote T Cell Responses
Authors: Carvalho Lucas P. , Pearce Edward J. , Scott Phillip
Published: April 2014

Leishmania braziliensis infections are often associated with exaggerated immune responses that can sometimes lead to severe disease associated with high levels of IFN-γ and TNF-α. To explore the role played by dendritic cells (DCs) in these responses, we characterized DCs that were exposed to L. braziliensis. We found that DCs cultured with L. braziliensis parasites up-regulated DC activation markers and produced IL-12 and TNF-α. However, not all DCs in the culture became infected, and an analysis of infected and uninfected DCs demonstrated that the up-regulation of activation markers and IL-12 production was primarily confined to the uninfected (bystander) DCs. Further studies with Transwell chambers and parasite fractions indicated that the activation of bystander DCs was mediated by a soluble parasite product, in a type 1 IFN- and MyD88-independent, but TNF-α-dependent fashion, and that the activated DCs were more efficient at presenting Ag than control DCs. In contrast, L. braziliensis-infected DCs failed to up-regulate activation markers, but exhibited a dramatic enhancement in their ability to produce TNF-α in response to LPS as compared with uninfected DCs. These findings uncover a dual role for DCs in L. braziliensis infection: T cell activation by bystander DCs due to enhanced Ag-presenting capacity following exposure to soluble parasite products, and increased production of TNF-α by infected cells that may contribute to the local control of the parasites, but concomitantly induce immunopathology.