bluuebunny/crossref_metadata_2025 huggingface dataset:
Cutting Edge: Cbl-b: One of the Key Molecules Tuning CD28- and CTLA-4-Mediated T Cell Costimulation
Authors: Li Dongdong , Gál István , Vermes Csaba , Alegre Maria-Luisa , Chong Anita S. F. , Chen Lieping , Shao Qing , Adarichev Vyacheslava , Xu Xuemei , Koreny Tamas , Mikecz Katalin , Finnegan Alison , Glant Tibor T. , Zhang Jian
Published: April 2014

Cbl-b negatively regulates CD28-dependent T cell activation. In this report, we tested the hypothesis that CD28 and CTLA-4 have opposite roles in tuning T cell activation threshold by controlling the levels of Cbl-b protein expression. We demonstrate that CD28 costimulation potentiates TCR-induced Cbl-b degradation, whereas CTLA-4-B7 interaction is required for Cbl-b re-expression. In support of this finding, Cbl-b expression in CTLA-4 knockout (KO) T cells is significantly reduced, and treating CTLA-4KO mice with human CTLA-4Ig to block CD28-B7 interaction restores Cbl-b expression on T cells. Furthermore, CD28 and CTLA-4 costimulatory effects are compromised in Cbl-bKO T cells. These observations indicate that CD28 and CTLA-4 tightly regulate Cbl-b expression which is critical for establishing the threshold for T cell activation.
Analysis of the Major Patterns of B Cell Gene Expression Changes in Response to Short-Term Stimulation with 33 Single Ligands
Authors: Zhu Xiaocui , Hart Rebecca , Chang Mi Sook , Kim Jong-Woo , Lee Sun Young , Cao Yun Anna , Mock Dennis , Ke Eugene , Saunders Brian , Alexander Angela , Grossoehme Joella , Lin Keng-Mean , Yan Zhen , Hsueh Robert , Lee Jamie , Scheuermann Richard H. , Fruman David A. , Seaman William , Subramaniam Shankar , Sternweis Paul , Simon Melvin I. , Choi Sangdun
Published: April 2014

We examined the major patterns of changes in gene expression in mouse splenic B cells in response to stimulation with 33 single ligands for 0.5, 1, 2, and 4 h. We found that ligands known to directly induce or costimulate proliferation, namely, anti-IgM (anti-Ig), anti-CD40 (CD40L), LPS, and, to a lesser extent, IL-4 and CpG-oligodeoxynucleotide (CpG), induced significant expression changes in a large number of genes. The remaining 28 single ligands produced changes in relatively few genes, even though they elicited measurable elevations in intracellular Ca2+ and cAMP concentration and/or protein phosphorylation, including cytokines, chemokines, and other ligands that interact with G protein-coupled receptors. A detailed comparison of gene expression responses to anti-Ig, CD40L, LPS, IL-4, and CpG indicates that while many genes had similar temporal patterns of change in expression in response to these ligands, subsets of genes showed unique expression patterns in response to IL-4, anti-Ig, and CD40L.
CXCR4-Tropic HIV-1 Envelope Glycoprotein Functions as a Viral Chemokine in Unstimulated Primary CD4+ T Lymphocytes
Authors: Balabanian Karl , Harriague Julie , Décrion Christine , Lagane Bernard , Shorte Spencer , Baleux Françoise , Virelizier Jean-Louis , Arenzana-Seisdedos Fernando , Chakrabarti Lisa A.
Published: April 2014

Interaction of HIV-1 envelope glycoprotein gp120 with the chemokine receptor CXCR4 triggers not only viral entry but also an array of signal transduction cascades. Whether gp120 induces an incomplete or aberrant set of signals, or whether it can function as a full CXCR4 agonist, remains unclear. We report that, in unstimulated human primary CD4+ T cells, the spectrum of signaling responses induced by gp120 through CXCR4 paralleled that induced by the natural ligand stromal cell-derived factor 1/CXCL12. gp120 activated heterotrimeric G proteins and the major G protein-dependent pathways, including calcium mobilization, phosphoinositide-3 kinase, and Erk-1/2 MAPK activation. Interestingly, gp120 caused rapid actin cytoskeleton rearrangements and profuse membrane ruffling, as evidenced by dynamic confocal imaging. This coordinated set of events resulted in a bona fide chemotactic response. Inactivated HIV-1 virions that harbored conformationally intact envelope glycoproteins also caused actin polymerization and chemotaxis, while similar virions devoid of envelope glycoproteins did not. Thus gp120, in monomeric as well as oligomeric, virion-associated form, elicited a complex cellular response that mimicked the effects of a chemokine. HIV-1 has therefore the capacity to dysregulate the vast CD4+ T cell population that expresses CXCR4. In addition, HIV-1 may exploit its chemotactic properties to retain potential target cells and locally perturb their cytoskeleton, thereby facilitating viral transmission.
IL-3 Induces Expression of Lymphatic Markers Prox-1 and Podoplanin in Human Endothelial Cells
Authors: Gröger Marion , Loewe Robert , Holnthoner Wolfgang , Embacher Robert , Pillinger Manuela , Herron G. Scott , Wolff Klaus , Petzelbauer Peter
Published: April 2014

Factors determining lymphatic differentiation in the adult organism are not yet well characterized. We have made the observation that mixed primary cultures of dermal blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) grown under standard conditions change expression of markers during subculture: After passage 6, they uniformly express LEC-specific markers Prox-1 and podoplanin. Using sorted cells, we show that LEC but not BEC constitutively express IL-3, which regulates Prox-1 and podoplanin expression in LEC. The addition of IL-3 to the medium of BEC cultures induces Prox-1 and podoplanin. Blocking IL-3 activity in LEC cultures results in a loss of Prox-1 and podoplanin expression. In conclusion, endogenous IL-3 is required to maintain the LEC phenotype in culture, and the addition of IL-3 to BEC appears to induce transdifferentiation of BEC into LEC.
IL-27 Mediates Complete Regression of Orthotopic Primary and Metastatic Murine Neuroblastoma Tumors: Role for CD8+ T Cells
Authors: Salcedo Rosalba , Stauffer Jimmy K. , Lincoln Erin , Back Timothy C. , Hixon Julie A. , Hahn Cynthia , Shafer-Weaver Kimberly , Malyguine Anatoli , Kastelein Robert , Wigginton Jon M.
Published: April 2014

We have shown previously that IFN-γ-inducing cytokines such as IL-12 can mediate potent antitumor effects against murine solid tumors. IL-27 is a newly described IL-12-related cytokine that potentiates various aspects of T and/or NK cell function. We hypothesized that IL-27 might also mediate potent antitumor activity in vivo. TBJ neuroblastoma cells engineered to overexpress IL-27 demonstrated markedly delayed growth compared with control mice, and complete durable tumor regression was observed in >90% of mice bearing either s.c. or orthotopic intra-adrenal tumors, and 40% of mice bearing induced metastatic disease. The majority of mice cured of their original TBJ-IL-27 tumors were resistant to tumor rechallenge. Furthermore, TBJ-IL-27 tumors were heavily infiltrated by CD8+ T cells, and draining lymph node-derived lymphocytes from mice bearing s.c. TBJ-IL-27 tumors are primed to proliferate more readily when cultured ex vivo with anti-CD3/anti-CD28 compared with lymphocytes from mice bearing control tumors, and to secrete higher levels of IFN-γ. In addition, marked enhancement of local IFN-γ gene expression and potent up-regulation of cell surface MHC class I expression are noted within TBJ-IL-27 tumors compared with control tumors. Functionally, these alterations occur in conjunction with the generation of tumor-specific CTL reactivity in mice bearing TBJ-IL-27 tumors, and the induction of tumor regression via mechanisms that are critically dependent on CD8+, but not CD4+ T cells or NK cells. Collectively, these studies suggest that IL-27 could be used therapeutically to potentiate the host antitumor immune response in patients with malignancy.
Intestinal Cryptopatch Formation in Mice Requires Lymphotoxin α and the Lymphotoxin β Receptor
Authors: Taylor Rebekah T. , Lügering Andreas , Newell Kenneth A. , Williams Ifor R.
Published: April 2014

Interactions between lymphotoxin (LT)α1β2 on inducer cells and the lymphotoxin β receptor (LTβR) on stromal cells initiate development of lymph nodes and Peyer’s patches. In this study, we assessed the contributions of LTα and LTβR to the development of cryptopatches (CP), aggregates of T cell precursors in the mouse small intestine. Mice genetically deficient in LTα or LTβR lacked CP. Bone marrow from LTα-deficient mice was unable to initiate development of CP or isolated lymphoid follicles (ILF) after transfer to CD132-null mice lacking CP and ILF. However, LTα-deficient bone marrow-derived cells contributed to CP formed in CD132-null mice receiving a mixture of wild-type and LTα-deficient bone marrow cells. Transfer of wild-type bone marrow into irradiated LTα-deficient mice resulted in reconstitution of both CP and ILF. However, the LT-dependent formation of CP was distinguished from the LT-dependent formation of ILF and Peyer’s patches by not requiring the presence of an intact NF-κB-inducing kinase gene. CP but not ILF were present in the small intestine from NF-κB-inducing kinase-deficient alymphoplasia mice, indicating that the alternate NF-κB activation pathway required for other types of LTβR-dependent lymphoid organogenesis is dispensable for CP development. In addition, we identified VCAM-1+ cells within both CP and ILF that are candidates for the stromal cells involved in receiving LT-dependent signals from the hemopoietic precursors recruited to CP. These findings demonstrate that interactions between cells expressing LTα1β2 and LTβR are a shared feature in the development of all small intestinal lymphoid aggregates.
Intestinal αβ T Cells Differentiate and Rearrange Antigen Receptor Genes In Situ in the Human Infant
Authors: Williams Amanda M. , Bland Paul W. , Phillips Anne C. , Turner Susan , Brooklyn Trevor , Shaya Gabriel , Spicer Richard D. , Probert Christopher S. J.
Published: April 2014

Intestinal Ag exposure during neonatal life influences appropriate adult immune responses. To define the mechanisms shaping the T cell repertoire during this period, we examined T cell differentiation and receptor diversity in the intestine of human infants. Developmental phenotypes of intraepithelial and lamina propria intestinal T cells from infants aged 1 day to 2 years were assessed ex vivo by flow cytometry and in situ by triple-fluorescent immunohistochemistry. Gene recombination-specific enzymes were assessed by PCR. TCR β-chain V region gene diversity was determined by sequencing. Several different early lineage T cell populations were present neonatally: CD3+4−8− T cells were present at birth and numbers decreased during the neonatal period; CD3+4+8+ T cells were present in low numbers throughout infancy; and CD3+4+8− or CD3+4−8+ T cells increased with age. Very early lineage T cells, CD3−2−7+ and CD3−2+7+, were present neonatally, but were essentially absent at 1 year. Most lamina propria T cells differentiated rapidly after birth, but maturation of intraepithelial T cells took place over 1 year. Intestinal samples from infants less than 6 mo old contained transcripts of T early α and TdT, and 15 of 19 infant samples contained mRNA for RAG-1, some coexpressing RAG-2. TCR β-chain repertoires were polyclonal in infants. Immature T cells, pre-T cells, and genes involved in T cell recombination were found in the intestine during infancy. T cell differentiation occurs within the neonatal human intestine, and the TCR repertoire of these developing immature T cells is likely to be influenced by luminal Ags. Thus, mucosal T cell responsiveness to environmental Ag is shaped in situ during early life.
N-Acetylglucosaminyltransferase V (Mgat5)-Mediated N-Glycosylation Negatively Regulates Th1 Cytokine Production by T Cells
Authors: Morgan Rodney , Gao Guoyan , Pawling Judy , Dennis James W. , Demetriou Michael , Li Baiyong
Published: April 2014

The differentiation of naive CD4+ T cells into either proinflammatory Th1 or proallergic Th2 cells strongly influences autoimmunity, allergy, and tumor immune surveillance. We previously demonstrated that β1,6GlcNAc-branched complex-type (N-acetylglucosaminyltransferase V (Mgat5)) N-glycans on TCR are bound to galectins, an interaction that reduces TCR signaling by opposing agonist-induced TCR clustering at the immune synapse. Mgat5−/− mice display late-onset spontaneous autoimmune disease and enhanced resistance to tumor progression and metastasis. In this study we examined the role of β1,6GlcNAc N-glycan expression in Th1/Th2 cytokine production and differentiation. β1,6GlcNAc N-glycan expression is enhanced by TCR stimulation independent of cell division and declines at the end of the stimulation cycle. Anti-CD3-activated splenocytes and naive T cells from Mgat5−/− mice produce more IFN-γ and less IL-4 compared with wild-type cells, the latter resulting in the loss of IL-4-dependent down-regulation of IL-4Rα. Swainsonine, an inhibitor of Golgi α-mannosidase II, blocked β1,6GlcNAc N-glycan expression and caused a similar increase in IFN-γ production by T cells from humans and mice, but no additional enhancement in Mgat5−/− T cells. Mgat5 deficiency did not alter IFN-γ/IL-4 production by polarized Th1 cells, but caused an ∼10-fold increase in IFN-γ production by polarized Th2 cells. These data indicate that negative regulation of TCR signaling by β1,6GlcNAc N-glycans promotes development of Th2 over Th1 responses, enhances polarization of Th2 cells, and suggests a mechanism for the increased autoimmune disease susceptibility observed in Mgat5−/− mice.
Vaccine-Stimulated, Adoptively Transferred CD8+ T Cells Traffic Indiscriminately and Ubiquitously while Mediating Specific Tumor Destruction
Authors: Palmer Douglas C. , Balasubramaniam Sanjeeve , Hanada Ken-ichi , Wrzesinski Claudia , Yu Zhiya , Farid Shahram , Theoret Marc R. , Hwang Leroy N. , Klebanoff Christopher A. , Gattinoni Luca , Goldstein Allan L. , Yang James C. , Restifo Nicholas P.
Published: April 2014

It has been suggested that antitumor T cells specifically traffic to the tumor site, where they effect tumor destruction. To test whether tumor-reactive CD8+ T cells specifically home to tumor, we assessed the trafficking of gp100-specific pmel-1 cells to large, vascularized tumors that express or do not express the target Ag. Activation of tumor-specific CD8+ pmel-1 T cells with IL-2 and vaccination with an altered peptide ligand caused regression of gp100-positive tumors (B16), but not gp100-negative tumors (methylcholanthrene 205), implanted on opposing flanks of the same mouse. Surprisingly, we found approximately equal and very large numbers of pmel-1 T cells (>25% of all lymphocytes) infiltrating both Ag-positive and Ag-negative tumors. We also found evidence of massive infiltration and proliferation of activated antitumor pmel-1 cells in a variety of peripheral tissues, including lymph nodes, liver, spleen, and lungs, but not peripheral blood. Most importantly, evidence for T cell function, as measured by production of IFN-γ, release of perforin, and activation of caspase-3 in target cells, was confined to Ag-expressing tumor. We thus conclude that CD8+ T cell-mediated destruction of tumor is the result of specific T cell triggering at the tumor site. The ability to induce ubiquitous homing and specific tumor destruction may be important in the case of noninflammatory metastatic tumor foci.
Induction of Central Tolerance by Mature T Cells
Authors: Tian Chaorui , Bagley Jessamyn , Forman Daron , Iacomini John
Published: April 2014

Induction of immunological tolerance is highly desirable for the treatment and prevention of autoimmunity, allergy, and organ transplant rejection. Adoptive transfer of MHC class I disparate mature T cells at the time of reconstitution of mice with syngeneic bone marrow resulted in specific tolerance to allogeneic skin grafts that were matched to the T cell donor strain. Mature allogeneic T cells survived long-term in reconstituted hosts and were able to re-enter the thymus. Analysis of T cell development using transgenic mice expressing an alloantigen-reactive TCR revealed that expression of allogeneic MHC class I on adoptively transferred mature T cells mediated negative selection of developing alloreactive T cells in the thymus. Thus, mature allogeneic T cells are able to mediate central deletion of alloreactive cells and induce transplantation tolerance without the requirement for any other alloantigen-expressing cell type.
Coordinated Expression of Ig-Like Inhibitory MHC Class I Receptors and Acquisition of Cytotoxic Function in Human CD8+ T Cells
Authors: Anfossi Nicolas , Doisne Jean-Marc , Peyrat Marie-Alix , Ugolini Sophie , Bonnaud Olivia , Bossy David , Pitard Vincent , Merville Pierre , Moreau Jean-François , Delfraissy Jean-François , Dechanet-Merville Julie , Bonneville Marc , Venet Alain , Vivier Eric
Published: April 2014

MHC class I-specific inhibitory receptors are expressed by a subset of memory-phenotype CD8+ T cells. Similar to NK cells, MHC class I-specific inhibitory receptors might subserve on T cells an important negative control that participates to the prevention of autologous damage. We analyzed here human CD8+ T cells that express the Ig-like MHC class I-specific inhibitory receptors: killer cell Ig-like receptor (KIR) and CD85j. The cell surface expression of Ig-like inhibitory MHC class I receptors was found to correlate with an advanced stage of CD8+ T cell maturation as evidenced by the reduced proliferative potential of KIR+ and CD85j+ T cells associated with their high intracytoplasmic perforin content. This concomitant regulation might represent a safety mechanism to control potentially harmful cytolytic CD8+ T cells, by raising their activation threshold. Yet, KIR+ and CD85j+ T cells present distinct features. KIR+CD8+ T cells are poor IFN-γ producers upon TCR engagement. In addition, KIR are barely detectable at the surface of virus-specific T cells during the course of CMV or HIV-1 infection. By contrast, CD85j+CD8+ T cells produce IFN-γ upon TCR triggering, and represent a large fraction of virus-specific T cells. Thus, the cell surface expression of Ig-like inhibitory MHC class I receptors is associated with T cell engagement into various stages of the cytolytic differentiation pathway, and the cell surface expression of CD85j or KIR witnesses to the history of qualitatively and/or quantitatively distinct T cell activation events.
Neuromedin Elicits Cytokine Release in Murine Th2-Type T Cell Clone D10.G4.1
Authors: Johnson Eric N. , Appelbaum Edward R. , Carptenter Donald C. , Cox Richard F. , Disa Jyoti , Foley James J. , Ghosh Sujoy K. , Naselsky Diane P. , Pullen Mark A. , Sarau Henry M. , Scheff Samuel R. , Steplewski Klaudia M. , Zaks-Zilberman Meirav , Aiyar Nambi
Published: April 2014

Neuromedin U (NmU), originally isolated from porcine spinal cord and later from other species, is a novel peptide that potently contracts smooth muscle. NmU interacts with two G protein-coupled receptors designated as NmU-1R and NmU-2R. This study demonstrates a potential proinflammatory role for NmU. In a mouse Th2 cell line (D10.G4.1), a single class of high affinity saturable binding sites for 125I-labeled NmU (KD 364 pM and Bmax 1114 fmol/mg protein) was identified, and mRNA encoding NmU-1R, but not NmU-2R, was present. Competition binding analysis revealed equipotent, high affinity binding of NmU isopeptides to membranes prepared from D10.G4.1 cells. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular Ca2+ concentration (EC50 4.8 nM for human NmU). In addition, NmU also significantly increased the synthesis and release of cytokines including IL-4, IL-5, IL-6, IL-10, and IL-13. Studies using pharmacological inhibitors indicated that maximal NmU-evoked cytokine release required functional phospholipase C, calcineurin, MEK, and PI3K pathways. These data suggest a role for NmU in inflammation by stimulating cytokine production by T cells.
CTLA-4 Engagement Acts as a Brake on CD4+ T Cell Proliferation and Cytokine Production but Is Not Required for Tuning T Cell Reactivity in Adaptive Tolerance
Authors: Inobe Manabu , Schwartz Ronald H.
Published: April 2014

Adaptive tolerance is the physiologic down-regulation of T cell responsiveness in the face of persistent antigenic stimulation. In this study, we examined the role of CTLA-4 in this process using CTLA-4-deficient and wild-type TCR transgenic, Rag2−/−, CD4+ T cells transferred into a T cell-deficient, Ag-expressing host. Surprisingly, we found that the tuning process of adoptively transferred T cells could be induced and the hyporesponsive state maintained in the absence of CTLA-4. Furthermore, movement to a deeper state of anergy following restimulation in vivo in a second Ag-bearing host was also unaffected. In contrast, CTLA-4 profoundly inhibited late T cell expansion in vivo following both primary and secondary transfers, and curtailed IL-2 and IFN-γ production. Removal of this braking function in CTLA-4-deficient mice following Ag stimulation may explain their lymphoproliferative dysregulation.
Regulatory T Cell Suppression and Anergy Are Differentially Regulated by Proinflammatory Cytokines Produced by TLR-Activated Dendritic Cells
Authors: Kubo Takekazu , Hatton Robin D. , Oliver James , Liu Xiaofen , Elson Charles O. , Weaver Casey T.
Published: April 2014

CD25+CD4+ regulatory T cells (Tregs) are required for the maintenance of peripheral tolerance to certain self Ags. In this study, the requirements for murine Treg-suppressive activity and proliferation were examined in the context of the maturation of myeloid dendritic cells (DCs). We find that the suppressive function of Tregs is critically dependent on immature DCs and is readily reversed by the maturation of DCs induced by GM-CSF, but does not require TLR activation of either DCs or Tregs. In contrast, reversal of Treg anergy is dependent on TLR activation of DCs, and involves the potentiation of Treg responsiveness to IL-2 by cooperative effects of IL-6 and IL-1, both of which are produced by TLR-activated, mature DCs. Thus, proinflammatory cytokines produced by TLR-activated, mature DCs are required for reversal of Treg anergy, but are not required to overcome Treg suppression.
CD25− T Cells Generate CD25+Foxp3+ Regulatory T Cells by Peripheral Expansion
Authors: Curotto de Lafaille Maria A. , Lino Andreia C. , Kutchukhidze Nino , Lafaille Juan J.
Published: April 2014

Naturally occurring CD4+ regulatory T cells are generally identified through their expression of CD25. However, in several experimental systems considerable Treg activity has been observed in the CD4+CD25− fraction. Upon adoptive transfer, the expression of CD25 in donor-derived cells is not stable, with CD4+CD25+ cells appearing in CD4+CD25− T cell-injected animals and vice versa. We show in this study that CD25+ cells arising from donor CD25− cells upon homeostatic proliferation in recipient mice express markers of freshly isolated Treg cells, display an anergic state, and suppress the proliferation of other cells in vitro. The maintenance of CD25 expression by CD4+CD25+ cells depends on IL-2 secreted by cotransferred CD4+CD25− or by Ag-stimulated T cells in peripheral lymphoid organs.
Endogenous IFN-α Production by Plasmacytoid Dendritic Cells Exerts an Antiviral Effect on Thymic HIV-1 Infection
Authors: Gurney Kevin B. , Colantonio Arnaud D. , Blom Bianca , Spits Hergen , Uittenbogaart Christel H.
Published: April 2014

Plasmacytoid dendritic cells (pDC) are the principal producers of IFN-α in response to viral infection. Because pDC are present in the thymus, we investigated the consequences of HIV-1-induced IFN-α production by thymic pDC. We observed that thymic pDC as well as thymocytes express intracellular IFN-α upon infection with HIV-1. However, only the pDC could suppress HIV-1 replication, because depletion of pDC resulted in enhancement of HIV-1 replication in thymocytes. Thymic pDC could also produce IFN-α in response to CpG oligonucleotides, consistent with the observations of others that peripheral pDC produce IFN-α upon engagement of TLR-9. Importantly, CpG considerably increased IFN-α production induced by HIV-1, and addition of CpG during HIV-1 infection enhanced expression of the IFN response protein MxA in thymocytes and strongly reduced HIV-replication. Our data indicate that thymic pDC modulate HIV-1 replication through secretion of IFN-α. The degree of inhibition depends on the level of IFN-α produced by the thymic pDC.
Papillon-Lefèvre Syndrome: Correlating the Molecular, Cellular, and Clinical Consequences of Cathepsin C/Dipeptidyl Peptidase I Deficiency in Humans
Authors: Pham Christine T. N. , Ivanovich Jennifer L. , Raptis Sofia Z. , Zehnbauer Barbara , Ley Timothy J.
Published: April 2014

A variety of neutral serine proteases are important for the effector functions of immune cells. The neutrophil-derived serine proteases cathepsin G and neutrophil elastase are implicated in the host defense against invading bacterial and fungal pathogens. Likewise, the cytotoxic lymphocyte and NK cell granule-associated granzymes A and B are important for the elimination of virus-infected cells. The activation of many of these serine proteases depends on the N-terminal processing activity of the lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI). Although mice deficient in DPPI have defects in serine protease activation in multiple cellular compartments, the role of DPPI for human serine protease activation is largely undefined. Papillon-Lefèvre syndrome (PLS) is a rare autosomal recessive disease associated with loss-of-function mutations in the DPPI gene locus. In this study, we established that the loss of DPPI activity is associated with severe reduction in the activity and stability of neutrophil-derived serine proteases. Surprisingly, patients with PLS retain significant granzyme activities in a cytotoxic lymphocyte compartment (lymphokine-activated killer) and have normal lymphokine-activated killer-mediated cytotoxicity against K562 cells. Neutrophils from patients with PLS do not uniformly have a defect in their ability to kill Staphylococcus aureus and Escherichia coli, suggesting that serine proteases do not represent the major mechanism used by human neutrophils for killing common bacteria. Therefore, this study defines the consequences of DPPI deficiency for the activation of several immune cell serine proteases in humans, and provides a molecular explanation for the lack of a generalized T cell immunodeficiency phenotype in patients with PLS.
Chemoattractant Signals and β2 Integrin Occupancy at Apical Endothelial Contacts Combine with Shear Stress Signals to Promote Transendothelial Neutrophil Migration
Authors: Cinamon Guy , Shinder Vera , Shamri Revital , Alon Ronen
Published: April 2014

Lymphocyte transendothelial migration (TEM) is promoted by fluid shear signals and apical endothelial chemokines. Studying the role of these signals in neutrophil migration across differently activated HUVEC in a flow chamber apparatus, we gained new insights into how neutrophils integrate multiple endothelial signals to promote TEM. Neutrophils crossed highly activated HUVEC in a β2 integrin-dependent manner but independently of shear. In contrast, neutrophil migration across resting or moderately activated endothelium with low-level β2 integrin ligand activity was dramatically augmented by endothelial-presented chemoattractants, conditional to application of physiological shear stresses and intact β2 integrins. Shear stress signals were found to stimulate extensive neutrophil invaginations into the apical endothelial interface both before and during TEM. A subset of invaginating neutrophils completed transcellular diapedesis through individual endothelial cells within <1 min. Our results suggest that low-level occupancy of β2 integrins by adherent neutrophils can mediate TEM only if properly coupled to stimulatory shear stress and chemoattractant signals transduced at the apical neutrophil-endothelial interface.
Anatomical Heterogeneity of Memory CD4+ T Cells Due to Reversible Adaptation to the Microenvironment
Authors: Kassiotis George , Stockinger Brigitta
Published: April 2014

The memory T cell pool is characterized by a substantial degree of heterogeneity in phenotype and function as well as anatomical distribution, but the underlying mechanisms remain unclear. In this study we confirm that the memory CD4+ T cell pool in wild-type and TCR-transgenic mice consists of heterogeneous subsets, as defined by surface marker expression or cytokine production. Extralymphoid sites contain significant numbers of memory CD4+ T cells, which are phenotypically and functionally distinct from their lymphoid counterparts. However, we show in this study that the phenotype of lymphoid and extralymphoid memory T cells is not stable. Instead, the unique properties of extralymphoid memory T cells are acquired upon migration into extralymphoid sites and are lost when memory T cells migrate back into lymphoid organs. Thus, at least some of the extralymphoid properties may represent a transient activation state that can be adopted by T cells belonging to a single memory T cell pool. Furthermore, such intermittent activation during or after migration into extralymphoid sites could provide an important signal, promoting the survival and functional competence of memory T cells in the absence of Ag.
Differential Activation of ERK, p38, and JNK Required for Th1 and Th2 Deviation in Myelin-Reactive T Cells Induced by Altered Peptide Ligand
Authors: Singh Rana A. K. , Zhang Jingwu Z.
Published: April 2014

Autoreactive T cells can be induced by altered peptide ligands to switch Th1 and Th2 phenotypes. The underlying molecular mechanism is critical for understanding of activation of autoreactive T cells and development of novel therapeutic strategies for autoimmune conditions. In this study, we demonstrated that analog peptides of an immunodominant epitope of myelin basic protein (residues 83–99) with alanine substitution at Val86 and His88 had a unique partial agonistic property in the induction of Th1 or Th2 deviation in MBP83–99-reactive T cell clones typical of Th0 phenotype. The observed phenotypic switch involved differential activation of ERK, p38, and JNK MAPKs. More specifically, Th1 deviation induced by peptide 86V→A (86A) correlated with enhanced p38 and JNK activities, while Th2 deviation by peptide 88H→A (88A) was associated with up-regulated ERK activity and a basal level of p38 and JNK activity. Further characterization revealed that a specific inhibitor for ERK selectively prevented Th2 deviation of MBP83–99-specific T cells. Conversely, specific inhibitors for p38 and JNK blocked Th1 deviation in the same T cell preparations induced by peptide 86A. The findings have important implications in our understanding of regulation of ERK, p38, and JNK by altered peptide ligands and their role in cytokine regulation and phenotype switch of autoreactive T cells.
A Sudden Decline in Active Membrane-Bound TGF-β Impairs Both T Regulatory Cell Function and Protection against Autoimmune Diabetes
Authors: Gregg Randal K. , Jain Renu , Schoenleber Scott J. , Divekar Rohit , Bell J. Jeremiah , Lee Hyun-Hee , Yu Ping , Zaghouani Habib
Published: April 2014

Autoimmunity presumably manifests as a consequence of a shortfall in the maintenance of peripheral tolerance by CD4+CD25+ T regulatory cells (Tregs). However, the mechanism underlying the functional impairment of Tregs remains largely undefined. In this study a glutamic acid decarboxylase (GAD) diabetogenic epitope was expressed on an Ig to enhance tolerogenic function, and the resulting Ig-GAD expanded Tregs in both young and older insulitis-positive, nonobese diabetic (NOD) mice, but delayed autoimmune diabetes only in the former. Interestingly, Tregs induced at 4 wk of age had significant active membrane-bound TGF-β (mTGF-β) and sustained protection against diabetes, whereas Tregs expanded during insulitis had minimal mTGF-β and could not protect against diabetes. The Tregs probably operate suppressive function through mTGF-β, because Ab blockade of mTGF-β nullifies protection against diabetes. Surprisingly, young Tregs that modulated pathogenic T cells maintained stable frequency over time in the protected animals, but decreased their mTGF-β at the age of 8 wk. More strikingly, these 8-wk-old mTGF-β-negative Tregs, which were previously protective, became unable to confer resistance against diabetes. Thus, a developmental decline in active mTGF-β nullifies Treg function, leading to a break in tolerance and the onset of diabetes.
Plasmablast and Plasma Cell Production and Distribution in Trout Immune Tissues
Authors: Bromage Erin S. , Kaattari Ilsa M. , Zwollo Patty , Kaattari Stephen L.
Published: April 2014

These studies describe the in vitro and ex vivo generation of plasmablasts and plasma cells in trout (Oncorhynchus mykiss) peripheral blood and splenic and anterior kidney tissues. Cells were derived either from naive trout and cultured with the polyclonal activator, Escherichia coli LPS, or from trout that had been immunized with trinitrophenyl-keyhole limpet hemocyanin. Hydroxyurea was used to resolve populations of replicating (plasmablast) and nonreplicating (plasma cell) Ab-secreting cells (ASC). Complete inhibition of Ig secretion was only observed within the PBL. Both anterior kidney and splenic lymphocytes possessed a subset of ASCs that were hydroxyurea resistant. Thus, in vitro production of plasma cells appears to be restricted to the latter two tissues, whereas peripheral blood is exclusively restricted to the production of plasmablasts. After immunization with trinitrophenyl-keyhole limpet hemocyanin, specific ASC could be isolated from all immune organs; however, the anterior kidney contained 98% of all ASC. Late in the response (>10 wk), anterior kidney ASC secreted specific Ab for at least 15 days in culture, indicating that they were long-lived plasma cells. Cells from spleen and peripheral blood lost all capacity to secrete specific Ab in the absence of Ag. Late in the Ab response, high serum titer levels are solely the result of Ig secretion from anterior kidney plasma cells.
Expansion of Myeloid Suppressor Cells in SHIP-Deficient Mice Represses Allogeneic T Cell Responses
Authors: Ghansah Tomar , Paraiso Kim H. T. , Highfill Steven , Desponts Caroline , May Sarah , McIntosh Joseph K. , Wang Jia-Wang , Ninos John , Brayer Jason , Cheng Fengdong , Sotomayor Eduardo , Kerr William G.
Published: April 2014

Previously we demonstrated that SHIP−/− mice accept allogeneic bone marrow transplants (BMT) without significant acute graft-vs-host disease (GvHD). In this study we show that SHIP−/− splenocytes and lymph node cells are poor stimulators of allogeneic T cell responses that cause GvHD. Intriguingly, SHIP−/− splenocytes prime naive T cell responses to peptide epitopes, but, conversely, are partially impaired for priming T cell responses to whole Ag. However, dendritic cells (DC) purified from SHIP−/− splenocytes prime T cell responses to allogeneic targets, peptide epitopes, and whole Ag as effectively as SHIP+/+ DC. These findings point to an extrinsic effect on SHIP−/− DC that impairs priming of allogeneic T cell responses. Consistent with this extrinsic effect, we found that a dramatic expansion of myeloid suppressor cells in SHIP−/− mice impairs priming of allogeneic T cells. These findings suggest that SHIP expression or its activity could be targeted to selectively compromise T cell responses that mediate GvHD and graft rejection.
Early Growth Response Gene-2, a Zinc-Finger Transcription Factor, Is Required for Full Induction of Clonal Anergy in CD4+ T Cells
Authors: Harris John E. , Bishop Kenneth D. , Phillips Nancy E. , Mordes John P. , Greiner Dale L. , Rossini Aldo A. , Czech Michael P.
Published: April 2014

Ag-specific immune tolerance results from the induction of cellular mechanisms that limit T cell responses to selective Ags. One of these mechanisms is characterized by attenuated proliferation and decreased IL-2 production in fully stimulated CD4+ Th cells and is denoted T cell anergy. We report the identification of the early growth response gene (Egr-2; Krox-20), a zinc-finger transcription factor, as a key protein required for induction of anergy in cultured T cells. Gene array screening revealed high Egr-2 expression distinctly persists in anergized but not proliferating murine A.E7 T cells. In contrast, Egr-1, a related family member induced upon costimulation, displays little or no expression in the anergic state. IL-2-mediated abrogation of anergy causes rapid depletion of Egr-2 protein. Full stimulation of anergic A.E7 T cells fails to enhance IL-2 and Egr-1 expression, whereas Egr-2 expression is greatly increased. Silencing Egr-2 gene expression by small interfering RNA treatment of cultured A.E7 T cells before incubation with anti-CD3 alone prevents full induction of anergy. However, small interfering RNA-mediated depletion of Egr-2 5 days after anergy induction does not appear to abrogate hyporesponsiveness to stimulation. These data indicate that sustained Egr-2 expression is necessary to induce a full anergic state through the actions of genes regulated by this transcription factor.
Peptidoglycan Molecular Requirements Allowing Detection by the Drosophila Immune Deficiency Pathway
Authors: Stenbak Carolyn R. , Ryu Ji-Hwan , Leulier François , Pili-Floury Sebastien , Parquet Claudine , Hervé Mireille , Chaput Catherine , Boneca Ivo G. , Lee Won-Jae , Lemaitre Bruno , Mengin-Lecreulx Dominique
Published: April 2014

Innate immune recognition of microbes is a complex process that can be influenced by both the host and the microbe. Drosophila uses two distinct immune signaling pathways, the Toll and immune deficiency (Imd) pathways, to respond to different classes of microbes. The Toll pathway is predominantly activated by Gram-positive bacteria and fungi, while the Imd pathway is primarily activated by Gram-negative bacteria. Recent work has suggested that this differential activation is achieved through peptidoglycan recognition protein (PGRP)-mediated recognition of specific forms of peptidoglycan (PG). In this study, we have further analyzed the specific PG molecular requirements for Imd activation through the pattern recognition receptor PGRP-LC in both cultured cell line and in flies. We found that two signatures of Gram-negative PG, the presence of diaminopimelic acid in the peptide bridge and a 1,6-anhydro form of N-acetylmuramic acid in the glycan chain, allow discrimination between Gram-negative and Gram-positive bacteria. Our results also point to a role for PG oligomerization in Imd activation, and we demonstrate that elements of both the sugar backbone and the peptide bridge of PG are required for optimum recognition. Altogether, these results indicate multiple requirements for efficient PG-mediated activation of the Imd pathway and demonstrate that PG is a complex immune elicitor.
Site-Specific Inhibitors of NADPH Oxidase Activity and Structural Probes of Flavocytochrome b: Characterization of Six Monoclonal Antibodies to the p22phox Subunit
Authors: Taylor Ross M. , Burritt James B. , Baniulis Danas , Foubert Thomas R. , Lord Connie I. , Dinauer Mary C. , Parkos Charles A. , Jesaitis Algirdas J.
Published: April 2014

The integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the human phagocyte NADPH oxidase, an enzyme complex that initiates a cascade of reactive oxygen species important in the elimination of infectious agents. This study reports the generation and characterization of six mAbs (NS1, NS2, NS5, CS6, CS8, and CS9) that recognize the p22phox subunit of the Cyt b heterodimer. Each of the mAbs specifically detected p22phox by Western blot analysis but did not react with intact neutrophils in FACS studies. Phage display mapping identified core epitope regions recognized by mAbs NS2, NS5, CS6, CS8, and CS9. Fluorescence resonance energy transfer experiments indicated that mAbs CS6 and CS8 efficiently compete with Cascade Blue-labeled mAb 44.1 (a previously characterized, p22phox-specific mAb) for binding to Cyt b, supporting phage display results suggesting that all three Abs recognize a common region of p22phox. Energy transfer experiments also suggested the spatial proximity of the mAb CS9 and mAb NS1 binding sites to the mAb 44.1 epitope, while indicating a more distant proximity between the mAb NS5 and mAb 44.1 epitopes. Cell-free oxidase assays demonstrated the ability of mAb CS9 to markedly inhibit superoxide production in a concentration-dependent manner, with more moderate levels of inhibition observed for mAbs NS1, NS5, CS6, and CS8. A combination of computational predictions, available experimental data, and results obtained with the mAbs reported in this study was used to generate a novel topology model of p22phox.
Plasticity within the Antigen-Combining Site May Manifest as Molecular Mimicry in the Humoral Immune Response
Authors: Goel Manisha , Krishnan Lavanya , Kaur Surinder , Kaur Kanwal J. , Salunke Dinakar M.
Published: April 2014

Structural and physiological facets of carbohydrate-peptide mimicry were addressed by analyzing the Ab response to α-d-mannopyranoside. mAbs against α-d-mannopyranoside were generated and screened with the carbohydrate-mimicking 12 mer (DVFYPYPYASGS) peptide. Three mAbs, 2D10, 1H11, and 1H7, which were subjected to detailed analysis, exhibit diverse V gene usage, indicating their independent germline origins. Although the mAb 1H7 was specific in binding only to the immunizing Ag, the Abs 2D10 and 1H11 recognize the 12 mer peptide as well as the immunogen, α-d-mannopyranoside. The Abs that recognize mimicry appear to bind to a common epitope on the peptide and do not share the mode of peptide binding with Con A. Binding kinetics and thermodynamics of Ag recognition suggest that the Ab that does not recognize peptide-carbohydrate mimicry probably has a predesigned mannopyranoside-complementing site. In contrast, the mimicry-recognizing Abs adopt the Ag-combining site only on exposure to the sugar, exploiting the conformational flexibility in the CDRs. Although the mAb 1H7 showed unique specificity toward mannopyranoside, the mimicry-recognizing Abs 2D10 and 1H11 exhibited degenerate specificities with regard to other sugar moieties. It is proposed that the degeneracy of specificity arising from the plasticity at the Ag-combining site in a subset of the Ab clones may be responsible for exhibiting molecular mimicry in the context of Ab response.
Genetic Dissection of Lupus Pathogenesis: Sle3/5 Impacts IgH CDR3 Sequences, Somatic Mutations, and Receptor Editing
Authors: Wakui Masatoshi , Kim Jinho , Butfiloski Edward J. , Morel Laurence , Sobel Eric S.
Published: April 2014

Sle3/5 is a lupus susceptibility locus identified on mouse chromosome 7 of the New Zealand Black/New Zealand White (NZB/NZW)-derived NZM2410 strain. Based on previous observations, this locus appears to contribute to lupus pathogenesis through its impact on diversification of immune responses. To understand how Sle3/5 affects somatic diversification of humoral responses, we analyzed IgH rearrangements preferentially encoding hapten-reactive IgG1 repertoires after immunization and assessed peripheral IgH VDJ recombination activities in C57BL/6 (B6) mice congenic for Sle3/5 (B6.Sle3/5). In addition to altered somatic VH mutation profiles, sequences from B6.Sle3/5 mice exhibited atypical IgH CDR3 structures characteristic of autoreactive B cells and consistent with peripheral B cells bearing putatively edited receptors. Significant expression of Rag genes and circular VHD gene excision products were detected in splenic mature B cells of B6.Sle3/5 but not B6 mice, showing that peripheral IgH rearrangements occurred beyond allelic exclusion. Taken together, on the nonautoimmune background, Sle3/5 affected VHDJH junctional diversity and VH mutational diversity and led to recombinational activation of allelically excluded IgH genes in the periphery. Such impact on somatic IgH diversification may contribute to the development of autoreactive B cell repertoires. This is the first report to present evidence for significant association of a lupus susceptibility locus, which has been mapped to a chromosomal region in which no Ig genes have been identified, with somatic IgH sequence diversity and peripheral H chain receptor editing or revision without relying upon Ig transgene strategies.
A Distinct Region of the Murine IFN-γ Promoter Is Hypomethylated from Early T Cell Development through Mature Naive and Th1 Cell Differentiation, but Is Hypermethylated in Th2 Cells
Authors: Winders Benjamin R. , Schwartz Ronald H. , Bruniquel Denis
Published: April 2014

Reports on the status of DNA methylation of the IFN-γ gene during T cell development in human and mouse have presented somewhat contradictory results. In this study we demonstrate in the mouse that methylation of the IFN-γ promoter inhibits its transcriptional activity, and define a small hypomethylated region in T cells that correlates with transcription. The IFN-γ promoter was also hypomethylated in NK cells, but not in B cells or nonhemopoietic tissues. Surprisingly, unlike the promoters of the IL-2 and IL-4 genes, the IFN-γ promoter was hypomethylated in naive CD4+ and CD8+ T cells, and in this form from very early in T cell development. A population of non-B, non-T, non-NK cells containing the hypomethylated promoter was also found in the bone marrow. The hypomethylated state appears stable until peripheral CD4+ T cells differentiate in response to Ag and APC. After T cell stimulation in vitro under Th2 conditions, but far less so under Th1 conditions, CD4+ cells display a more methylated IFN-γ promoter, which may contribute to the lack of expression of IFN-γ in these preactivated cells. Our experiments support a new model of IFN-γ chromatin structural changes in murine T cell development that differs from what has been previously published for human T cells.
Chicken Ig-Like Receptor B2, a Member of a Multigene Family, Is Mainly Expressed on B Lymphocytes, Recruits Both Src Homology 2 Domain Containing Protein Tyrosine Phosphatase (SHP)-1 and SHP-2, and Inhibits Proliferation
Authors: Viertlboeck Birgit C. , Crooijmans Richard P. M. A. , Groenen Martien A. M. , Göbel Thomas W. F.
Published: April 2014

Ig-like inhibitory receptors have been the focus of intensive research particularly in mouse and human. We report the cloning and characterization of three novel inhibitory chicken Ig-like receptors (CHIR) that display a two Ig-domain extracellular structure, a transmembrane region lacking charged residues and a cytoplasmic domain containing two ITIM. The localization of all receptors to a small genomic region and the hybridization pattern indicated that they belong to a multigene family. The genomic structure of the extracellular domain with two exons encoding the signal peptide and single exons for each Ig domain resembled that of all human leukocyte Ig-like receptors and killer cell Ig-like receptors, whereas the exons encoding the C terminus displayed a structure closely resembling killer cell Ig-like receptor genes. A mAb generated against one receptor designated CHIR-B2 reacted with all B cells and a small T cell subset, but not with monocytes, thrombocytes, or various leukocyte-derived cell lines. The mAb immunoprecipitated a 46-kDa protein from bursal cells and transfected cells. The Src homology 2 domain containing protein tyrosine phosphatase (SHP)-2 bound to CHIR-B2 even in unstimulated cells, whereas pervanadate treatment induced the tyrosine phosphorylation and recruitment of several CHIR-B2-associated proteins including SHP-1 and increased levels of SHP-2. Moreover, mAb cross-linking of CHIR-B2 reduced the proliferation of a stable transfected cell line. Together, we have identified a multigene family containing multiple CHIR including one receptor designated CHIR-B2 that is mainly expressed on B lymphocytes and inhibits cellular proliferation by recruitment of SHP-1 and SHP-2.