bluuebunny/crossref_metadata_2025 huggingface dataset:
Contribution of NZB Autoimmunity 2 to Y-Linked Autoimmune Acceleration-Induced Monocytosis in Association with Murine Systemic Lupus
Authors: Kikuchi Shuichi , Santiago-Raber Marie-Laure , Amano Hirofumi , Amano Eri , Fossati-Jimack Liliane , Moll Thomas , Kotzin Brian L. , Izui Shozo
Published: April 2014

The accelerated development of systemic lupus erythematosus (SLE) in BXSB male mice is associated with the presence of the Y-linked autoimmune acceleration (Yaa) mutation, which induces an age-dependent monocytosis. Using a cohort of C57BL/6 (B6) × (NZB × B6)F1 backcross male mice bearing the Yaa mutation, we defined the pathogenic role and genetic basis for Yaa-associated monocytosis. We observed a remarkable correlation of monocytosis with autoantibody production and subsequent development of lethal lupus nephritis, indicating that monocytosis is an additional useful indicator for severe SLE. In addition, we identified an NZB-derived locus on chromosome 1 predisposing to the development of monocytosis, which peaked at Fcgr2b encoding FcγRIIB and directly overlapped with the previously identified NZB autoimmunity 2 (Nba2) locus. The contribution of Nba2 to monocytosis was confirmed by the analysis of Yaa-bearing B6 mice congenic for the NZB-Nba2 locus. Finally, we observed a very low-level expression of FcγRIIB on macrophages bearing the NZB-type Fcgr2b allele, compared with those bearing the B6-type allele, and the development of monocytosis in FcγRIIB haploinsufficient B6 mice carrying the Yaa mutation. These data suggest that the Nba2 locus may play a supplementary role in the pathogenesis of SLE by promoting the development of monocytosis and the activation of effector cells bearing stimulatory FcγR, in addition to its implication in the dysregulated activation of autoreactive B cells.
Evidence to Support the Role of HLA-G5 in Allograft Acceptance through Induction of Immunosuppressive/ Regulatory T Cells
Authors: Le Rond Solène , Azéma Christine , Krawice-Radanne Irène , Durrbach Antoine , Guettier Catherine , Carosella Edgardo D. , Rouas-Freiss Nathalie
Published: April 2014

The soluble HLA-G5 isoform encoded by intron-4 retaining spliced transcript has been previously detected in vivo in sera and grafts from transplanted patients who had significantly better graft acceptance. These findings led us to investigate the role of HLA-G5 in tolerance induction in vitro and its biological relevance in allograft acceptance in vivo. We demonstrated that engagement of Ig-like transcript-2 and Ig-like transcript-4 receptors by HLA-G5 is involved in inhibition of T cell alloproliferative responses. Naive T cells sensitized in vitro with HLA-G5, for as little as 18 h, 1) lost their ability to respond to subsequent allogeneic stimulus, and 2) acquired regulatory properties because they inhibited the reactivity of other T cells. These HLA-G5-induced T cells act in an Ag-nonspecific fashion and through soluble factors. Biological relevance was provided by ex vivo analyzes of samples from liver-kidney cotransplanted patients who had high HLA-G5 serum levels and no graft rejection. We showed that addition of HLA-G5-containing sera from these patients inhibited T cell alloresponses and that serum HLA-G5 was responsible for this inhibition. Notably, PBMC from transplanted patients exposed to high levels of circulating HLA-G5 did not respond to allostimulation and inhibited alloreactivity of other T cells. These results demonstrate that HLA-G5-mediated tolerance involves the induction of immunosuppressive T cells. These findings provide evidence supporting the tolerogenic properties of HLA-G and emphasize its potential application as a relevant therapeutic candidate capable of limiting allograft rejection.
Cutting Edge: Membrane Nanotubes In Vivo: A Feature of MHC Class II+ Cells in the Mouse Cornea
Authors: Chinnery Holly R. , Pearlman Eric , McMenamin Paul G.
Published: April 2014

Membrane nanotubes are a recently discovered form of cellular protrusion between two or more cells whose functions include cell communication, environmental sampling, and protein transfer. Although clearly demonstrated in vitro, evidence of the existence of membrane nanotubes in mammalian tissues in vivo has until now been lacking. Confocal microscopy of whole-mount corneas from wild-type, enhanced GFP chimeric mice, and Cx3cr1gfp transgenic mice revealed long (>300 μm) and fine (<0.8 μm diameter) membrane nanotube-like structures on bone marrow-derived MHC class II+ cells in the corneal stroma, some of which formed distinct intercellular bridges between these putative dendritic cells. The frequency of these nanotubes was significantly increased in corneas subjected to trauma and LPS, which suggests that nanotubes have an important role in vivo in cell-cell communication between widely spaced dendritic cells during inflammation. Identification of these novel cellular processes in the mammalian cornea provides the first evidence of membrane nanotubes in vivo.
Cutting Edge: Foxp3-Mediated Induction of Pim 2 Allows Human T Regulatory Cells to Preferentially Expand in Rapamycin
Authors: Basu Samik , Golovina Tatiana , Mikheeva Tatiana , June Carl H. , Riley James L.
Published: April 2014

Addition of rapamycin to cultures of expanding natural CD4+CD25+Foxp3+ T regulatory cells (Tregs) helps maintain their suppressive activity, but the underlying mechanism is unclear. Pim 2 is a serine/threonine kinase that can confer rapamycin resistance. Unexpectedly, pim 2 was found to be constitutively expressed in freshly isolated, resting Tregs, but not in CD4+CD25− T effector cells. Introduction of Foxp3, but not Foxp3Δ2, into effector T cells induced pim 2 expression and conferred preferential expansion in the presence of rapamycin, indicating that Foxp3 can regulate pim 2 expression. Finally, we determined there is a positive correlation between Treg expansion and Foxp3 expression in the presence of rapamycin. Together, these results indicate that Tregs are programmed to be resistant to rapamycin, providing further rationale for why this immunosuppressive drug should be used in conjunction with expanded Tregs.
Cutting Edge: Overlapping Functions of TLR7 and TLR9 for Innate Defense against a Herpesvirus Infection
Authors: Zucchini Nicolas , Bessou Gilles , Traub Stephanie , Robbins Scott H. , Uematsu Satoshi , Akira Shizuo , Alexopoulou Lena , Dalod Marc
Published: April 2014

As initially demonstrated with murine cytomegalovirus (MCMV), plasmacytoid dendritic cells (pDCs) are the major source of IFN-α/β in response to a variety of viruses in vivo. However, contradictory results have been obtained pertaining to the mechanisms promoting IFN-α/β production by pDCs in response to MCMV. In this study we show that TLR7 and TLR9 exert redundant functions for IFN-α/β, IL-12p40, and TNF-α production by pDCs in vivo during MCMV infection. In contrast, we confirm that systemic production of IL-12p70 strictly depends on TLR9. The combined loss of TLR7 and TLR9 recapitulates critical features of the phenotype of MyD88-deficient mice, including a dramatic decrease in systemic IFN-α/β levels, an increase in viral load, and increased susceptibility to MCMV-induced mortality. This is the first demonstration of the implication of TLR7 in the recognition of a DNA virus.
Lck-Dependent Tyrosine Phosphorylation of Diacylglycerol Kinase α Regulates Its Membrane Association in T Cells
Authors: Merino Ernesto , Ávila-Flores Antonia , Shirai Yasuhito , Moraga Ignacio , Saito Naoaki , Mérida Isabel
Published: April 2014

TCR engagement triggers phospholipase Cγ1 activation through the Lck-ZAP70-linker of activated T cell adaptor protein pathway. This leads to generation of diacylglycerol (DAG) and mobilization of intracellular Ca2+, both essential for TCR-dependent transcriptional responses. TCR ligation also elicits transient recruitment of DAG kinase α (DGKα) to the lymphocyte plasma membrane to phosphorylate DAG, facilitating termination of DAG-regulated signals. The precise mechanisms governing dynamic recruitment of DGKα to the membrane have not been fully elucidated, although Ca2+ influx and tyrosine kinase activation were proposed to be required. We show that DGKα is tyrosine phosphorylated, and identify tyrosine 335 (Y335), at the hinge between the atypical C1 domains and the catalytic region, as essential for membrane localization. Generation of an Ab that recognizes phosphorylated Y335 demonstrates Lck-dependent phosphorylation of endogenous DGKα during TCR activation and shows that pY335DGKα is a minor pool located exclusively at the plasma membrane. Our results identify Y335 as a residue critical for DGKα function and suggest a mechanism by which Lck-dependent phosphorylation and Ca2+ elevation regulate DGKα membrane localization. The concerted action of these two signals results in transient, receptor-regulated DGKα relocalization to the site at which it exerts its function as a negative modulator of DAG-dependent signals.
Systemic Control of Plasmacytoid Dendritic Cells by CD8+ T Cells and Commensal Microbiota
Authors: Fujiwara Daisuke , Wei Bo , Presley Laura L. , Brewer Sarah , McPherson Michael , Lewinski Michael A. , Borneman James , Braun Jonathan
Published: April 2014

The composition of the intestinal microbial community is a distinctive individual trait that may divergently influence host biology. Because dendritic cells (DC) regulate the quality of the host response to microbiota, we evaluated DC in mice bearing distinct enteric microbial communities divergent for colitis susceptibility. Surprisingly, a selective, systemic reduction of plasmacytoid dendritic cells (pDC) was observed in isogenic mice with different microbiota: restricted flora (RF) vs specific pathogen free (SPF). This reduction was not observed in germfree mice, suggesting that the pDC deficiency was not simply due to a lack of intestinal microbial products. The microbial action was linked to cytotoxic CD8+ T cells, since pDC in RF mice were preserved in the CD8−/− and perforin−/− genotypes, partially restored by anti-CD8β Ab, and augmented in SPF mice bearing the TAP−/− genotype. Direct evidence for pDC cytolysis was obtained by rapid and selective pDC depletion in SPF mice transferred with RF CD8+ T cells. These data indicate that commensal microbiota, via CTL activation, functionally shape systemic immune regulation that may modify risk of inflammatory disease.
Pathogen Proliferation Governs the Magnitude but Compromises the Function of CD8 T Cells
Authors: Sad Subash , Dudani Renu , Gurnani Komal , Russell Marsha , van Faassen Henk , Finlay Brett , Krishnan Lakshmi
Published: April 2014

CD8+ T cell memory is critical for protection against many intracellular pathogens. However, it is not clear how pathogen virulence influences the development and function of CD8+ T cells. Salmonella typhimurium (ST) is an intracellular bacterium that causes rapid fatality in susceptible mice and chronic infection in resistant strains. We have constructed recombinant mutants of ST, expressing the same immunodominant Ag OVA, but defective in various key virulence genes. We show that the magnitude of CD8+ T cell response correlates directly to the intracellular proliferation of ST. Wild-type ST displayed efficient intracellular proliferation and induced increased numbers of OVA-specific CD8+ T cells upon infection in mice. In contrast, mutants with defective Salmonella pathogenicity island II genes displayed poor intracellular proliferation and induced reduced numbers of OVA-specific CD8+ T cells. However, when functionality of the CD8+ T cell response was measured, mutants of ST induced a more functional response compared with the wild-type ST. Infection with wild-type ST, in contrast to mutants defective in pathogenicity island II genes, induced the generation of mainly effector-memory CD8+ T cells that expressed little IL-2, failed to mediate efficient cytotoxicity, and proliferated poorly in response to Ag challenge in vivo. Taken together, these results indicate that pathogens that proliferate rapidly and chronically in vivo may evoke functionally inferior memory CD8+ T cells which may promote the survival of the pathogen.
IFN-γ and IL-17 Production in Experimental Autoimmune Encephalomyelitis Depends on Local APC–T Cell Complement Production
Authors: Liu Jinbo , Lin Feng , Strainic Michael G. , An Fengqi , Miller Robert H. , Altuntas Cengiz Z. , Heeger Peter S. , Tuohy Vincent K. , Medof M. Edward
Published: April 2014

IFN-γ- and IL-17-producing T cells autoreactive across myelin components are central to the pathogenesis of multiple sclerosis. Using direct in vivo, adoptive transfer, and in vitro systems, we show in this study that the generation of these effectors in myelin oligodendrocyte glycoprotein35–55-induced experimental autoimmune encephalomyelitis depends on interactions of locally produced C3a/C5a with APC and T cell C3aR/C5aR. In the absence of the cell surface C3/C5 convertase inhibitor decay-accelerating factor (DAF), but not the combined absence of DAF and C5aR and/or C3aR on APC and T cells, a heightened local autoimmune response occurs in which myelin destruction is markedly augmented in concert with markedly more IFN-γ+ and IL-17+ T cell generation. The augmented T cell response is due to increased IL-12 and IL-23 elaboration by APCs together with increased T cell expression of the receptors for each cytokine. The results apply to initial generation of the IL-17 phenotype because naive CD62Lhigh Daf1−/− T cells produce 3-fold more IL-17 in response to TGF-β and IL-6, whereas CD62Lhigh Daf1−/−C5aR−/−C3aR−/− T cells produce 4-fold less.
CD83 Expression in CD4+ T Cells Modulates Inflammation and Autoimmunity
Authors: Reinwald Simone , Wiethe Carsten , Westendorf Astrid M. , Breloer Minka , Probst-Kepper Michael , Fleischer Bernhard , Steinkasserer Alexander , Buer Jan , Hansen Wiebke
Published: April 2014

The transmembrane protein CD83 has been initially described as a maturation marker for dendritic cells. Moreover, there is increasing evidence that CD83 also regulates B cell function, thymic T cell maturation, and peripheral T cell activation. Herein, we show that CD83 expression confers immunosuppressive function to CD4+ T cells. CD83 mRNA is differentially expressed in naturally occurring CD4+CD25+ regulatory T cells, and upon activation these cells rapidly express large amounts of surface CD83. Transduction of naive CD4+CD25− T cells with CD83 encoding retroviruses induces a regulatory phenotype in vitro, which is accompanied by the induction of Foxp3. Functional analysis of CD83-transduced T cells in vivo demonstrates that these CD83+Foxp3+ T cells are able to interfere with the effector phase of severe contact hypersensitivity reaction of the skin. Moreover, adoptive transfer of these cells prevents the paralysis associated with experimental autoimmune encephalomyelitis, suppresses proinflammatory cytokines IFN-γ and IL-17, and increases antiinflammatory IL-10 in recipient mice. Taken together, our data provide the first evidence that CD83 expression can contribute to the immunosuppressive function of CD4+ T cells in vivo.
Deacetylase Activity Is Required for STAT5-Dependent GM-CSF Functional Activity in Macrophages and Differentiation to Dendritic Cells
Authors: Sebastián Carlos , Serra Maria , Yeramian Andrée , Serrat Neus , Lloberas Jorge , Celada Antonio
Published: April 2014

After interaction with its receptor, GM-CSF induces phosphorylation of the β-chain in two distinct domains in macrophages. One induces activation of mitogen-activated protein kinases and the PI3K/Akt pathway, and the other induces JAK2-STAT5. In this study we describe how trichostatin A (TSA), which inhibits deacetylase activity, blocks JAK2-STAT5-dependent gene expression but not the expression of genes that depend on the signal transduction induced by the other domain of the receptor. TSA treatment inhibited the GM-CSF-dependent proliferation of macrophages by interfering with c-myc and cyclin D1 expression. However, M-CSF-dependent proliferation, which requires ERK1/2, was unaffected. Protection from apoptosis, which involves Akt phosphorylation and p21waf-1 expression, was not modified by TSA. GM-CSF-dependent expression of MHC class II molecules was inhibited because CIITA was not induced. The generation of dendritic cells was also impaired by TSA treatment because of the inhibition of IRF4, IRF2, and RelB expression. TSA mediates its effects by preventing the recruitment of RNA polymerase II to the promoter of STAT5 target genes and by inhibiting their expression. However, this drug did not affect STAT5A or STAT5B phosphorylation or DNA binding. These results in GM-CSF-treated macrophages reveal a relationship between histone deacetylase complexes and STAT5 in the regulation of gene expression.
Regulatory T Cells Inhibit Dendritic Cells by Lymphocyte Activation Gene-3 Engagement of MHC Class II
Authors: Liang Bitao , Workman Craig , Lee Janine , Chew Claude , Dale Benjamin M. , Colonna Lucrezia , Flores Marcella , Li Nianyu , Schweighoffer Edina , Greenberg Steven , Tybulewicz Victor , Vignali Dario , Clynes Raphael
Published: April 2014

Lymphocyte activation gene-3 (LAG-3) is a CD4-related transmembrane protein expressed by regulatory T cells that binds MHC II on APCs. It is shown in this study that during Treg:DC interactions, LAG-3 engagement with MHC class II inhibits DC activation. MHC II cross-linking by agonistic Abs induces an ITAM-mediated inhibitory signaling pathway, involving FcγRγ and ERK-mediated recruitment of SHP-1 that suppresses dendritic cell maturation and immunostimulatory capacity. These data reveal a novel ITAM-mediated inhibitory signaling pathway in DCs triggered by MHC II engagement of LAG-3, providing a molecular mechanism in which regulatory T cells may suppress via modulating DC function.
Disruption of Mutually Negative Regulatory Feedback Loop between Interferon-Inducible p202 Protein and the E2F Family of Transcription Factors in Lupus-Prone Mice
Authors: Panchanathan Ravichandran , Xin Hong , Choubey Divaker
Published: April 2014

Studies have identified IFN-inducible Ifi202 gene as a lupus susceptibility gene (encoding p202 protein) in mouse models of lupus disease. However, signaling pathways that regulate the Ifi202 expression in cells remain to be elucidated. We found that steady-state levels of Ifi202 mRNA and protein were high in mouse embryonic fibroblasts (MEFs) from E2F1 knockout (E2F1−/−) and E2F1 and E2F2 double knockout (E2F1−/−E2F2−/−) mice than isogenic wild-type MEFs. Moreover, overexpression of E2F1 in mouse fibroblasts decreased expression of p202. Furthermore, expression of E2F1, but not E2F4, transcription factor in mouse fibroblasts repressed the activity of 202-luc-reporter in promoter-reporter assays. Interestingly, the E2F1-mediated transcriptional repression of the 202-luc-reporter was independent of p53 and pRb expression. However, the repression was dependent on the ability of E2F1 to bind DNA. We have identified a potential E2F DNA-binding site in the 5′-regulatory region of the Ifi202 gene, and mutations in this E2F DNA-binding site reduced the E2F1-mediated transcriptional repression of 202-luc-reporter. Because p202 inhibits the E2F1-mediated transcriptional activation of genes, we compared the expression of E2F1 and its target genes in splenic cells from lupus-prone B6.Nba2 congenic mice, which express increased levels of p202, with age-matched C57BL/6 mice. We found that increased expression of Ifi202 in the congenic mice was associated with inhibition of E2F1-mediated transcription and decreased expression of E2F1 and its target genes that encode proapoptotic proteins. Our observations support the idea that increased Ifi202 expression in certain strains of mice contributes to lupus susceptibility in part by inhibiting E2F1-mediated functions.
IL-12 Signaling Drives CD8+ T Cell IFN-γ Production and Differentiation of KLRG1+ Effector Subpopulations during Toxoplasma gondii Infection
Authors: Wilson Douglas C. , Matthews Suzanne , Yap George S.
Published: April 2014

IFN-γ-producing CD8+ T lymphocytes are essential effector cells that mediate protective immunity during murine toxoplasmosis, and yet their effector development remains poorly characterized. Vaccination with the carbamoyl phosphate synthase (CPS) mutant strain of Toxoplasma gondii was used to examine the CD8+ T cell response in the peritoneal effector site. Four CTL subpopulations with varying effector potentials were defined based on the expression of effector molecules and the cell surface activation markers CD62L and killer cell lectin-like receptor G1 (KLRG1). Further phenotypic analysis revealed that the acquisition of KLRG1 among effector subpopulations correlated with the down-regulation of both IL-7R and CD27, suggesting that KLRG1 marks dominant, end-stage effector cells. Using gene-targeted mice, we tested the in vivo requirements of key IL-12 signaling components for effector CTL differentiation. Contrary to established models of viral and bacterial infection, CD8+ T cell-intrinsic IL-12 signaling was required for the generation of IFN-γ-producing CTLs in response to T. gondii. Importantly, the development of the KLRG1+ effector subpopulations, but not the memory precursor-containing KLRG1− effector subset, was critically reliant on IL-12. Furthermore, IL-12 signaling-dependent T-bet expression was also found to be important for differentiation of KLRG1+ effectors. Our results underscore a vital role for IL-12 in not only the induction of IFN-γ expression but also in the development of heterogeneous subpopulations of effector CD8+ T cells generated in response to the intracellular parasite T. gondii.
An Interaction between CD200 and Monoclonal Antibody Agonists to CD200R2 in Development of Dendritic Cells That Preferentially Induce Populations of CD4+CD25+ T Regulatory Cells
Authors: Gorczynski Reg , Khatri Ismat , Lee Lydia , Boudakov Ivo
Published: April 2014

In previous studies we reported that while interaction between the relatively ubiquitously expressed molecule CD200 and one of its receptors, CD200R1, resulted in direct suppression of alloreactivity, engagement of alternate receptors led instead to altered differentiation of dendritic cells (DCs) from marrow precursors, which could in turn foster development of Foxp3+ regulatory T cells. We have explored this effect of engagement of alternate receptors by using a monoclonal agonist Ab to CD200R2 and investigating expression of TLRs on DCs induced in vivo and in vitro after CD200 stimulation in mice in which the gene encoding CD200R1 was deleted. CD200 stimulation was achieved by using either a soluble form of CD200 (CD200Fc) or overexpression of CD200 as a doxycycline-inducible transgene. Although broadly similar effects were seen, consistent with the hypothesis that triggering of CD200R2 does produce DCs with a characteristic TLR repertoire, there are subtle differences in suppression of alloreactivity achieved by CD200 delivered in these two manners, which is consistent with a complexity of CD200:CD200R engagement not previously appreciated.
Conversion of Tyrosine to the Inflammation-Associated Analog 3′-Nitrotyrosine at Either TCR- or MHC-Contact Positions Can Profoundly Affect Recognition of the MHC Class I-Restricted Epitope of Lymphocytic Choriomeningitis Virus Glycoprotein 33 by CD8 T Cells
Authors: Hardy Lani L. , Wick Darin A. , Webb John R.
Published: April 2014

Immunohistochemical detection of increased levels of protein-associated nitrotyrosine has become widely used as a surrogate marker of in situ inflammation. However, the potential consequences of protein-associated nitrotyrosine formation in terms of cellular immune recognition has received surprisingly little attention. Using a well-defined I-EK-restricted epitope of pigeon cytochrome c, we previously demonstrated that conversion of a single tyrosine residue to nitrotyrosine can have a profound effect on recognition by CD4 T cells. In this study, we used the MHC class I-restricted epitope of lymphocytic choriomeningitis virus glycoprotein (gp33) to demonstrate that conversion of tyrosine to nitrotyrosine can also profoundly affect recognition of MHC class I-restricted epitopes. Conversion of the Y4 residue of the gp33 epitope to nitrotyrosine completely abrogated recognition by gp33-specific T cells from P14 TCR-transgenic mice. In contrast, CD8+ T cells specific for “nitrated gp33” (NY-gp33) can be readily elicited in C57BL/6 mice after immunization with NY-gp33 peptide. Interestingly, T-T hybridomas specific for NY-gp33 peptide were found to fall into two distinct subsets, being specific for NY-gp33 presented in the context of either H-2Db or H-2Kb. This latter result is surprising in light of previous structural studies showing that Y4 comprises a critical TCR-contact residue when presented by H-2Db but that the same residue points downward into the peptide-binding groove of the MHC when presented by H-2Kb. Together, these results indicate that nitrotyrosine formation can impact T cell recognition both directly, through alteration of TCR-contact residues, or indirectly, through alterations in MHC-contact positions.
Preferential Development of CD4 and CD8 T Regulatory Cells in RasGRP1-Deficient Mice
Authors: Chen Xiaoxi , Priatel John J. , Chow Michael T. , Teh Hung-Sia
Published: April 2014

RasGRP1 and Sos are two Ras-guanyl-nucleotide exchange factors that link TCR signal transduction to Ras and MAPK activation. Recent studies demonstrate positive selection of developing thymocytes is crucially dependent on RasGRP1, whereas negative selection of autoreactive thymocytes appears to be RasGRP1 independent. However, the role of RasGRP1 in T regulatory (Treg) cell development and function is unknown. In this study, we characterized the development and function of CD4+CD25+Foxp3+ and CD8+CD44highCD122+ Treg lineages in RasGRP1−/− mice. Despite impaired CD4 Treg cell development in the thymus, the periphery of RasGRP1−/− mice contained significantly increased frequencies of CD4+Foxp3+ Treg cells that possessed a more activated cell surface phenotype. Furthermore, on a per cell basis, CD4+Foxp3+ Treg cells from mutant mice are more suppressive than their wild-type counterparts. Our data also suggest that the lymphopenic environment in the mutant mice plays a dominant role of favored peripheral development of CD4 Treg cells. These studies suggest that whereas RasGRP1 is crucial for the intrathymic development of CD4 Treg cells, it is not required for their peripheral expansion and function. By contrast to CD4+CD25+Foxp3+ T cells, intrathymic development of CD8+CD44highCD122+ Treg cells is unaffected by the RasGRP1−/− mutation. Moreover, RasGRP1−/− mice contained greater numbers of CD8+CD44highCD122+ T cells in the spleen, relative to wild-type mice. Activated CD8 Treg cells from RasGRP1−/− mice retained their ability to synthesize IL-10 and suppress the proliferation of wild-type CD8+CD122− T cells, albeit at a much lower efficiency than wild-type CD8 Treg cells.
Prevention of Allograft Tolerance by Bacterial Infection with Listeria monocytogenes
Authors: Wang Tongmin , Chen Luqiu , Ahmed Emily , Ma Lianli , Yin Dengping , Zhou Ping , Shen Jikun , Xu Honglin , Wang Chyung-Ru , Alegre Maria-Luisa , Chong Anita S.
Published: April 2014

Exposure to certain viruses and parasites has been shown to prevent the induction of transplantation tolerance in mice via the generation of cross-reactive memory T cell responses or the induction of bystander activation. Bacterial infections are common in the perioperative period of solid organ allograft recipients in the clinic, and correlations between bacterial infections and acute allograft rejection have been reported. However, whether bacterial infections at the time of transplantation have any effect on the generation of transplantation tolerance remains to be established. We used the Gram-positive intracellular bacterium Listeria monocytogenes (LM) as a model pathogen because its effects on immune responses are well described. Perioperative LM infection prevented cardiac and skin allograft acceptance induced by anti-CD154 and donor-specific transfusion in mice. LM-mediated rejection was not due to the generation of cross-reactive T cells and was largely independent of signaling via MyD88, an adaptor for most TLRs, IL-1, and IL-18. Instead, transplant rejection following LM infection was dependent on the expression of the phagosome-lysing pore former listeriolysin O and on type I IFN receptor signaling. Our results indicate that bacterial exposure at the time of transplantation can antagonize tolerogenic regimens by enhancing alloantigen-specific immune responses independently of the generation of cross-reactive memory T cells.
Fungal Proteases Induce Th2 Polarization through Limited Dendritic Cell Maturation and Reduced Production of IL-12
Authors: Lamhamedi-Cherradi Salah-Eddine , Martin Rachel Elizabeth , Ito Tomoki , Kheradmand Farrah , Corry David Brian , Liu Yong-Jun , Moyle Matthew
Published: April 2014

Allergens are capable of polarizing the T cell immune response toward a Th2 cytokine profile in a process that is mediated by dendritic cells (DCs). Proteases derived from Aspergillus species (Aspergillus proteases; AP) have been shown to induce a Th2-like immune response when administered directly to the airway and without adjuvant or prior priming immunizations at sites remote from the lung in models of allergic airway disease. To explore mechanisms that underlie the Th2 immune response, we have investigated the effect of AP on DC function. We found that human DCs derived from CD14+ monocytes from healthy donors underwent partial maturation when incubated with AP. Naive allogeneic T cells primed with AP-activated DCs proliferated and displayed enhanced production of IL-4 and reduced expression of IFN-γ as compared with naive T cells primed with LPS-activated DCs. Global gene expression analysis of DCs revealed relatively low expression of IL-12p40 in AP-activated DCs as compared with those activated by LPS, and this was confirmed at the protein level by ELISA. Exogenous IL-12p70 added to cocultures of DCs and T cells resulted in reduced IL-4 and increased IFN-γ expression when DCs were activated with AP. When the proteolytic activity of AP was neutralized by chemical inactivation it failed to up-regulate costimulatory molecules on DCs, and these DCs did not prime a Th2 response in naive T cells. These findings provide a mechanism for explaining how proteolytically active allergens could preferentially induce Th2 responses through limited maturation of DCs with reduced production of IL-12.
Inorganic Arsenic Activates Reduced NADPH Oxidase in Human Primary Macrophages through a Rho Kinase/p38 Kinase Pathway
Authors: Lemarie Anthony , Bourdonnay Emilie , Morzadec Claudie , Fardel Olivier , Vernhet Laurent
Published: April 2014

Inorganic arsenic is an immunotoxic environmental contaminant to which millions of humans are chronically exposed. We recently demonstrated that human primary macrophages constituted a critical target for arsenic trioxide (As2O3), an inorganic trivalent form. To specify the effects of arsenic on macrophage phenotype, we investigated in the present study whether As2O3 could regulate the activity of NADPH oxidase, a major superoxide-generating enzymatic system in human phagocytes. Our results show that superoxide levels were significantly increased in a time-dependent manner in blood monocyte-derived macrophages treated with 1 μM As2O3 for 72 h. Concomitantly, As2O3 induced phosphorylation and membrane translocation of the NADPH oxidase subunit p47phox and it also increased translocation of Rac1 and p67phox. Apocynin, a selective inhibitor of NADPH oxidases, prevented both p47phox translocation and superoxide production. NADPH oxidase activation was preceded by phosphorylation of p38-kinase in As2O3-treated macrophages. The p38-kinase inhibitor SB-203580 prevented phosphorylation and translocation of p47phox and subsequent superoxide production. Pretreatment of macrophages with the Rho-kinase inhibitor Y-27632 was found to mimic inhibitory effects of SB-203580 and to prevent As2O3-induced phosphorylation of p38 kinase. Treatment with As2O3 also resulted in an increased secretion of the proinflammatory chemokine CCL18 that was fully inhibited by both apocynin and SB-203580. Taken together, our results demonstrate that As2O3 induced a marked activation of NADPH oxidase in human macrophages, likely through stimulation of a Rho-kinase/p38-kinase pathway, and which may contribute to some of the deleterious effects of inorganic arsenic on macrophage phenotype.
Characterization of IL-10-Secreting T Cells Derived from Regulatory CD4+CD25+ Cells by the TIRC7 Surface Marker
Authors: Wakkach Abdelilah , Augier Séverine , Breittmayer Jean-Philippe , Blin-Wakkach Claudine , Carle Georges F.
Published: April 2014

Natural CD25+CD4+ regulatory T cells (Treg) are essential for self-tolerance and for the control of T cell-mediated immune pathologies. However, the identification of Tregs in an ongoing immune response or in inflamed tissues remains elusive. Our experiments indicate that TIRC7, T cell immune response cDNA 7, a novel membrane molecule involved in the regulation of T lymphocyte activation, identifies two Treg subsets (CD25lowTIRC7+ and CD25highTIRC7−) that are characterized by the expression of Foxp3 and a suppressive activity in vitro and in vivo. We also showed that the CD25lowTIRC7+ subset represents IL-10-secreting Tregs in steady state, which is accumulated intratumorally in a tumor-bearing mice model. Blockade of the effect of IL-10 reversed the suppression imposed by the CD25lowTIRC7+ subset. Interestingly, these IL-10-secreting cells derived from the CD25highTIRC7− subset, both in vitro and in vivo, in response to tumoral Ags. Our present results strongly support the notion that, in the pool of natural Tregs, some cells can recognize foreign Ags and that this recognition is an essential step in their expansion and suppressive activity in vivo.
Suppression of Normal and Malignant Kit Signaling by a Bispecific Antibody Linking Kit with CD300a
Authors: Bachelet Ido , Munitz Ariel , Berent-Maoz Beata , Mankuta David , Levi-Schaffer Francesca
Published: April 2014

Through its receptor Kit (CD117), stem cell factor (SCF) critically regulates human mast cell (MC) differentiation, survival, priming, and activation. The dominance of SCF in setting these parameters compels stringent contra-regulation to maintain a balanced MC phenotype. We have synthesized a library of bispecific Ab fragments to examine the effect of linking Kit with CD300a. In this study, we report that CD300a exerts a strong inhibitory effect on Kit-mediated SCF-induced signaling, consequently impairing MC differentiation, survival, and activation in vitro. This effect derives from Kit-mediated tyrosine phosphorylation of CD300a and recruitment of the SHIP-1 but not of SH2-containing protein phosphatase 1. CD300a inhibits the constitutive activation of the human leukemic HMC-1 cells but not their survival. Finally, CD300a abrogates the allergic reaction induced by SCF in a murine model of cutaneous anaphylaxis. Our findings highlight CD300a as a novel regulator of Kit in human MC and suggest roles for this receptor as a suppressor of Kit signaling in MC-related disorders.
Loss of STAT3 in CD4+ T Cells Prevents Development of Experimental Autoimmune Diseases
Authors: Liu Xuebin , Lee Yun Sang , Yu Cheng-Rong , Egwuagu Charles E.
Published: April 2014

Th17 cells are implicated in CNS autoimmune diseases. We show that mice with targeted-deletion of Stat3 in CD4+ T cells (CD4Stat3−/−) do not develop experimental autoimmune uveoretinitis (EAU) or experimental autoimmune encephalomyelitis. Defective Th17 differentiation noted in CD4Stat3−/− mice is compensated by exaggerated increases in Foxp3-, IL-10-, IL-4-, and IFN-γ-expressing T cells, suggesting critical roles of STAT3 in shaping Ag-specific CD4+ T cell repertoire. In mice with EAU, a high percentage of IL-17-expressing T cells in their peripheral lymphoid organs also secrete IFN-γ while these double-expressors are absent in CD4Stat3−/− and wild-type mice without EAU, raising the intriguing possibility that uveitis maybe mediated by Th17 and IL-17-expressing Th1 cells. Resistance of Stat3-deficient mice to EAU derives in part from an inability of uveitogenic Th17 and Th1 cells to enter eyes or brain of the CD4Stat3−/− mouse because of the reduction in the expression of activated α4/β1 integrins on CD4Stat3−/− T cells. Adoptive transfer of activated interphotoreceptor retinoid-binding protein-specific uveitogenic T cells induced in CD4Stat3−/− mice a severe EAU characterized by development of retinal folds, infiltration of inflammatory cells into the retina, and destruction of retinal architecture, underscoring our contention that the loss of STAT3 in CD4+ T cells results in an intrinsic developmental defect that renders CD4Stat3−/− resistant to CNS inflammatory diseases. STAT3 requirement for IL-17 production by Th17, generation of double positive T cells expressing IL-17 and IFN-γ, and for T cell trafficking into CNS tissues suggests that STAT3 may be a therapeutic target for modulating uveitis, sceritis, or multiple sclerosis.
Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Inhibits Proximal TCR Signaling by Targeting ZAP-70
Authors: Chen Zhangguo , Chen Lanfen , Qiao Shuo-Wang , Nagaishi Takashi , Blumberg Richard S.
Published: April 2014

The long cytoplasmic tail (CT) isoforms of carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1) are expressed on activated human T cells and possess two ITIM motifs in the CT. These isoforms of CEACAM1 are inhibitory for T cell responses initiated by the TCR/CD3 complex with the inhibition dependent upon the ITIMs of CEACAM1 and Src homology 2 domain-containing phosphatase 1 (SHP-1). However, the mechanism by which this inhibition occurs in T cells is unknown. We demonstrate here that the Src family kinase, Lck, and the ability of CEACAM1 to bind homophilically are required for the ITIM phosphorylation of CEACAM1 that is a prerequisite for CEACAM1 association with SHP-1. We further show that CEACAM1 associates with and recruits SHP-1 to the TCR/CD3 complex leading to decreased phosphorylation of CD3-ζ and ZAP-70 and consequently decreased activation of the elements downstream of ZAP-70. This is physiologically relevant because extinction of SHP-1 expression or blockade of homophilic binding by CEACAM1 using a Fab that specifically recognizes the homophilic binding region of human CEACAM1 increases the cytolytic function initiated by the TCR/CD3 complex. These studies show that long CT isoforms of CEACAM1 orchestrate an inhibitory program that abrogates extremely proximal events downstream of the TCR/CD3 complex by focusing on the activation of ZAP-70.
Functional Activity of MD-2 Polymorphic Variant Is Significantly Different in Soluble and TLR4-Bound Forms: Decreased Endotoxin Binding by G56R MD-2 and Its Rescue by TLR4 Ectodomain
Authors: Vašl Jožica , Prohinar Polonca , Gioannini Theresa L. , Weiss Jerrold P. , Jerala Roman
Published: April 2014

MD-2 is an essential component of endotoxin (LPS) sensing, binding LPS independently and when bound to the ectodomain of the membrane receptor TLR4. Natural variation of proteins involved in the LPS-recognition cascade such as the LPS-binding protein, CD14, and TLR4, as well as proteins involved in intracellular signaling downstream of LPS binding, affect the cellular response to endotoxin and host defense against bacterial infections. We now describe the functional properties of two nonsynonymous coding polymorphisms of MD-2, G56R and P157S, documented in HapMap. As predicted from the MD-2 structure, the P157S mutation had little or no effect on MD-2 function. In contrast, the G56R mutation, located close to the LPS-binding pocket, significantly decreased cellular responsiveness to LPS. Soluble G56R MD-2 showed markedly reduced LPS binding that was to a large degree rescued by TLR4 coexpression or presence of TLR4 ectodomain. Thus, cells that express TLR4 without MD-2 and whose response to LPS depends on ectopically produced MD-2 were most affected by expression of the G56R variant of MD-2. Coexpression of wild-type and G56R MD-2 yielded an intermediate phenotype with responses to LPS diminished to a greater extent than that resulting from expression of the D299G TLR4 polymorphic variant.
Single and Dual Amino Acid Substitutions in TCR CDRs Can Enhance Antigen-Specific T Cell Functions
Authors: Robbins Paul F. , Li Yong F. , El-Gamil Mona , Zhao Yangbing , Wargo Jennifer A. , Zheng Zhili , Xu Hui , Morgan Richard A. , Feldman Steven A. , Johnson Laura A. , Bennett Alan D. , Dunn Steven M. , Mahon Tara M. , Jakobsen Bent K. , Rosenberg Steven A.
Published: April 2014

Single and dual amino acid substitution variants were generated in the TCR CDRs of three TCRs that recognize tumor-associated Ags. Substitutions that enhance the reactivity of TCR gene-modified T cells to the cognate Ag complex were identified using a rapid RNA-based transfection system. The screening of a panel of variants of the 1G4 TCR, that recognizes a peptide corresponding to amino acid residues 157–165 of the human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of the HLA-A*02 class I allele, resulted in the identification of single and dual CDR3α and CDR2β amino acid substitutions that dramatically enhanced the specific recognition of NY-ESO-1+/HLA-A*02+ tumor cell lines by TCR gene-modified CD4+ T cells. Within this group of improved TCRs, a dual substitution in the 1G4 TCR CDR3α chain was identified that enhanced Ag-specific reactivity in gene-modified CD4+ and CD8+ T cells. Separate experiments on two distinct TCRs that recognize the MART-1 27–35 (AAGIGILTV) peptide/HLA-A*02 Ag complex characterized single amino acid substitutions in both TCRs that enhanced CD4+ T cell Ag-specific reactivity. These results indicate that simple TCR substitution variants that enhance T cell function can be identified by rapid transfection and assay techniques, providing the means for generating potent Ag complex-specific TCR genes for use in the study of T cell interactions and in T cell adoptive immunotherapy.
Mannose-Binding Lectin (MBL)-Associated Serine Protease (MASP)-1 Contributes to Activation of the Lectin Complement Pathway
Authors: Takahashi Minoru , Iwaki Daisuke , Kanno Kazuko , Ishida Yumi , Xiong Jie , Matsushita Misao , Endo Yuichi , Miura Shigeto , Ishii Naoto , Sugamura Kazuo , Fujita Teizo
Published: April 2014

The complement system plays an important role in innate immunity. In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme. It has been suggested that MASP-2 is responsible for the activation of C4. Other serine proteases (MASP-1 and MASP-3) are also associated with MBL or ficolins; however, their functions are still controversial. In this study, a MASP-1- and MASP-3-deficient mouse model (MASP1/3−/−) was generated by a gene targeting strategy to investigate the roles of MASP-1 and MASP-3 in the lectin pathway. Serum derived from MASP1/3−/− mice showed significantly lower activity of both C4 and C3 deposition on mannan-agarose, and this low activity was restored by the addition of recombinant MASP-1. MASP-1/3-deficient serum showed a significant delay for activation of MASP-2 compared with normal serum. Reconstitution of recombinant MASP-1 in MASP-1/3-deficient serum was able to promote the activation of MASP-2. From these results, we propose that MASP-1 contributes to the activation of the lectin pathway, probably through the activation of MASP-2.
Lipoproteins Are Critical TLR2 Activating Toxins in Group B Streptococcal Sepsis
Authors: Henneke Philipp , Dramsi Shaynoor , Mancuso Giuseppe , Chraibi Kamila , Pellegrini Elisabeth , Theilacker Christian , Hübner Johannes , Santos-Sierra Sandra , Teti Giuseppe , Golenbock Douglas T. , Poyart Claire , Trieu-Cuot Patrick
Published: April 2014

Group B streptococcus (GBS) is the most important cause of neonatal sepsis, which is mediated in part by TLR2. However, GBS components that potently induce cytokines via TLR2 are largely unknown. We found that GBS strains of the same serotype differ in released factors that activate TLR2. Several lines of genetic and biochemical evidence indicated that lipoteichoic acid (LTA), the most widely studied TLR2 agonist in Gram-positive bacteria, was not essential for TLR2 activation. We thus examined the role of GBS lipoproteins in this process by inactivating two genes essential for bacterial lipoprotein (BLP) maturation: the prolipoprotein diacylglyceryl transferase gene (lgt) and the lipoprotein signal peptidase gene (lsp). We found that Lgt modification of the N-terminal sequence called lipobox was not critical for Lsp cleavage of BLPs. In the absence of lgt and lsp, lipoprotein signal peptides were processed by the type I signal peptidase. Importantly, both the Δlgt and the Δlsp mutant were impaired in TLR2 activation. In contrast to released factors, fixed Δlgt and Δlsp GBS cells exhibited normal inflammatory activity indicating that extracellular toxins and cell wall components activate phagocytes through independent pathways. In addition, the Δlgt mutant exhibited increased lethality in a model of neonatal GBS sepsis. Notably, LTA comprised little, if any, inflammatory potency when extracted from Δlgt GBS. In conclusion, mature BLPs, and not LTA, are the major TLR2 activating factors from GBS and significantly contribute to GBS sepsis.
Tick Saliva Inhibits Dendritic Cell Migration, Maturation, and Function while Promoting Development of Th2 Responses
Authors: Skallová Anna , Iezzi Giandomenica , Ampenberger Franziska , Kopf Manfred , Kopecký Jan
Published: April 2014

Similarly to other blood-feeding arthropods, ticks have evolved immunosuppressive mechanisms enabling them to overcome the host immune system. Although the immunomodulatory effect of tick saliva on several cell populations of the immune system has been extensively studied, little is known about its impact on dendritic cells (DCs). We have examined the effect of Ixodes ricinus tick saliva on DC function in vitro and in vivo. Exposure of DCs to tick saliva in vitro resulted in impaired maturation, upon CD40 or TLR9, TLR3 and TLR7 ligation, as well as reduced Ag presentation capacity. Administration of tick saliva in vivo significantly inhibited maturation and early migration of DCs from inflamed skin to draining lymph nodes, and decreased the capacity of lymph node DCs to present soluble Ag to specific T cells. Moreover, saliva-exposed DCs failed to induce efficient Th1 and Th17 polarization and promoted development of Th2 responses. Our data reveal a complex inhibitory effect exerted by tick saliva on DC function. Given the role of DCs as the key instigators of adaptive immune responses, alteration of their function might represent a major mechanism of tick-mediated immune evasion.