bluuebunny/crossref_metadata_2025 huggingface dataset:
TNF-α Induction of GM2 Expression on Renal Cell Carcinomas Promotes T Cell Dysfunction
Authors: Raval Gira , Biswas Soumika , Rayman Patricia , Biswas Kaushik , Sa Gaurisankar , Ghosh Sankar , Thornton Mark , Hilston Cynthia , Das Tanya , Bukowski Ronald , Finke James , Tannenbaum Charles S.
Published: April 2014

Previous studies from our laboratory demonstrated the role of tumor-derived gangliosides as important mediators of T cell apoptosis, and hence, as one mechanism by which tumors evade immune destruction. In this study, we report that TNF-α secreted by infiltrating inflammatory cells and/or genetically modified tumors augments tumor-associated GM2 levels, which leads to T cell death and immune dysfunction. The conversion of weakly apoptogenic renal cell carcinoma (RCC) clones to lines that can induce T cell death requires 3–5 days of TNF-α pretreatment, a time frame paralleling that needed for TNF-α to stimulate GM2 accumulation by SK-RC-45, SK-RC-54, and SK-RC-13. RCC tumor cell lines permanently transfected with the TNF-α transgene are similarly toxic for T lymphocytes, which correlates with their constitutively elevated levels of GM2. TNF-α increases GM2 ganglioside expression by enhancing the mRNA levels encoding its synthetic enzyme, GM2 synthase, as demonstrated by both RT-PCR and Southern analysis. The contribution of GM2 gangliosides to tumor-induced T cell death was supported by the finding that anti-GM2 Abs significantly blocked T cell apoptosis mediated by TNF-α-treated tumor cells, and by the observation that small interfering RNA directed against TNF-α abrogated GM2 synthase expression by TNF-transfected SK-RC-45, diminished its GM2 accumulation, and inhibited its apoptogenicity for T lymphocytes. Our results indicate that TNF-α signaling promotes RCC-induced killing of T cells by stimulating the acquisition of a distinct ganglioside assembly in RCC tumor cells.
Telomerase in T Lymphocytes: Use It and Lose It?
Authors: Akbar Arne N. , Vukmanovic-Stejic Milica
Published: April 2014

The enzyme telomerase counteracts telomere loss in proliferating cells and extends their capacity for replication. The importance of telomerase is highlighted by the award of the 2006 Albert Lasker Prize for Basic Medical Research for its discovery. Malignant cells subvert telomerase induction to their advantage, and up-regulation of this enzyme confers these populations with unlimited proliferative potential with obvious detrimental consequences. However this enzyme is also essential for the lifelong maintenance of normal cell populations that have a high rate of turnover. Thymic involution in early adulthood dictates that memory T cell populations have to be maintained by continuous proliferation. This highlights the inherent paradox that telomerase down-regulation in T cells may protect against malignancy yet also lead to replicative exhaustion of repeatedly activated memory T cells. In this article, we review the data on telomerase regulation in T lymphocytes and the implications this has for the maintenance of T cell memory.
Cutting Edge: TLR2 Directly Triggers Th1 Effector Functions
Authors: Imanishi Takayuki , Hara Hiromitsu , Suzuki Shinobu , Suzuki Nobutaka , Akira Shizuo , Saito Takashi
Published: April 2014

Toll-like receptors recognize pathogen-associated molecular patterns, activate innate immunity, and consequently modulate adaptive immunity in response to infections. TLRs are also expressed on T cells, and it has been shown that T cell activation is modulated by TLR ligands. However, the functions of TLRs on Th1 and Th2 effector cells and the molecular mechanisms underlying TLR-mediated activation are not fully understood. We analyzed TLR functions and downstream signaling events in both effector T cells. In mouse Th1 cells the stimulation by TLR2 but not by other TLRs directly induced IFN-γ production, cell proliferation, and cell survival without TCR stimulation, and these effects were greatly enhanced by IL-2 or IL-12 through the enhanced activation of MAPKs. In contrast, no TLR affected the function of effector Th2 cells. These results identify TLR2 as a new specific activator of Th1 cell function and imply the involvement in Th1-mediated responses.
Cutting Edge: Regulatory T Cells Induce CD4+CD25−Foxp3− T Cells or Are Self-Induced to Become Th17 Cells in the Absence of Exogenous TGF-β
Authors: Xu LiLi , Kitani Atsushi , Fuss Ivan , Strober Warren
Published: April 2014

Recent studies have shown that TGF-β together with IL-6 induce the differentiation of IL-17-producing T cells (Th17) T cells. We therefore examined whether CD4+CD25+Foxp3+ regulatory T cells, i.e., cells previously shown to produce TGF-β, serve as Th17 inducers. We found that upon activation purified CD25+ T cells (or sorted GFP+ T cells obtained from Foxp3-GFP knockin mice) produce high amounts of soluble TGF-β and when cultured with CD4+CD25−Foxp3− T cells in the presence of IL-6 induce the latter to differentiate into Th17 cells. Perhaps more importantly, upon activation, CD4+CD25+Foxp3+(GFP+) T cells themselves differentiate into Th17 cells in the presence of IL-6 (and in the absence of exogenous TGF-β). These results indicate that CD4+CD25+Foxp3+ regulatory T cells can function as inducers of Th17 cells and can differentiate into Th17 cells. They thus have important implications to our understanding of regulatory T cell function and their possible therapeutic use.
Cutting Edge: Th17 and Regulatory T Cell Dynamics and the Regulation by IL-2 in the Tumor Microenvironment
Authors: Kryczek Ilona , Wei Shuang , Zou Linhua , Altuwaijri Saleh , Szeliga Wojciech , Kolls Jay , Chang Alfred , Zou Weiping
Published: April 2014

Th17 cells play an active role in inflammation and autoimmune diseases. However, the nature and regulation of Th17 in the context of tumor immunity remain unknown. In this study, we show that parallel to regulatory T (Treg) cells, IL-17+ CD4+ and CD8+ T cells are kinetically induced in multiple tumor microenvironments in mice and humans. Treg cells play a crucial role in tumor immune pathogenesis and temper immune therapeutic efficacy. IL-2 is crucial for the production and function of Treg cells. We now show that IL-2 reduces IL-17+ T cell differentiation in the tumor microenvironment accompanied with an enhanced Treg cell compartment in vitro and in vivo. Altogether, our work demonstrates a dynamic differentiation of IL-17+ T cells in the tumor microenvironment, reveals a novel role for IL-2 in controlling the balance between IL-17+ and Treg cells, and provides new insight of IL-17+ T cells in tumor immune pathology and therapy.
Distinct Roles of Different NF-κB Subunits in Regulating Inflammatory and T Cell Stimulatory Gene Expression in Dendritic Cells
Authors: Wang Junmei , Wang Xingyu , Hussain Sofia , Zheng Ye , Sanjabi Shomyseh , Ouaaz Fatah , Beg Amer A.
Published: April 2014

TLRs play a critical role in inducing inflammatory and immune responses against microbial agents. In this study, we have investigated the role of NF-κB transcription factors in regulating TLR-induced gene expression in dendritic cells, a key APC type. The p50 and cRel NF-κB subunits were found to be crucial for regulating genes important for dendritic cell-induced T cell responses (e.g., CD40, IL-12, and IL-18) but not for genes encoding inflammatory cytokines (e.g., TNF-α, IL-1α, and IL-6). In striking contrast, the RelA subunit was crucial for expression of inflammatory cytokine genes but not T cell stimulatory genes. These novel findings reveal a fundamentally important difference in biological function of genes regulated by different NF-κB subunits. Focusing on RelA target gene specificity mechanisms, we investigated whether the κB site and/or the unique composition of RelA played the most crucial role. Surprisingly, studies of IL-6 expression showed that the κB site is not a primary determinant of RelA target gene specificity. Instead, a major specificity mechanism is the unique ability of RelA to interact with the transcriptional coactivator CREB-binding protein, a function not shared with the closely related cRel subunit. Together, our findings indicate novel and critically important overall roles of NF-κB in TLR-induced gene expression that are mediated by unique functions of distinct subunits.
Vaccinia Virus-Specific CD4+ T Cell Responses Target a Set of Antigens Largely Distinct from Those Targeted by CD8+ T Cell Responses
Authors: Moutaftsi Magdalini , Bui Huynh-Hoa , Peters Bjoern , Sidney John , Salek-Ardakani Shahram , Oseroff Carla , Pasquetto Valerie , Crotty Shane , Croft Michael , Lefkowitz Elliot J. , Grey Howard , Sette Alessandro
Published: April 2014

Recent studies have defined vaccinia virus (VACV)-specific CD8+ T cell epitopes in mice and humans. However, little is known about the epitope specificities of CD4+ T cell responses. In this study, we identified 14 I-Ab-restricted VACV-specific CD4+ T cell epitopes by screening a large set of 2146 different 15-mer peptides in C57BL/6 mice. These epitopes account for ∼20% of the total anti-VACV CD4+ T cell response and are derived from 13 different viral proteins. Surprisingly, none of the CD4+ T cell epitopes identified was derived from VACV virulence factors. Although early Ags were recognized, late Ags predominated as CD4+ T cell targets. These results are in contrast to what was previously found in CD8+ T cells responses, where early Ags, including virulence factors, were prominently recognized. Taken together, these results highlight fundamental differences in immunodominance of CD4+ and CD8+ T cell responses to a complex pathogen.
Regulation of Cell Adhesion by Affinity and Conformational Unbending of α4β1 Integrin
Authors: Chigaev Alexandre , Waller Anna , Zwartz Gordon J. , Buranda Tione , Sklar Larry A.
Published: April 2014

Rapid activation of integrins in response to chemokine-induced signaling serves as a basis for leukocyte arrest on inflamed endothelium. Current models of integrin activation include increased affinity for ligand, molecular extension, and others. In this study, using real-time fluorescence resonance energy transfer to assess α4β1 integrin conformational unbending and fluorescent ligand binding to assess affinity, we report at least four receptor states with independent regulation of affinity and unbending. Moreover, kinetic analysis of chemokine-induced integrin conformational unbending and ligand-binding affinity revealed conditions under which the affinity change was transient whereas the unbending was sustained. In a VLA-4/VCAM-1-specific myeloid cell adhesion model system, changes in the affinity of the VLA-4-binding pocket were reflected in rapid cell aggregation and disaggregation. However, the initial rate of cell aggregation increased 9-fold upon activation, of which only 2.5-fold was attributable to the increased affinity of the binding pocket. These data show that independent regulation of affinity and conformational unbending represents a novel and fundamental mechanism for regulation of integrin-dependent adhesion in which the increased affinity appears to account primarily for the increasing lifetime of the α4β1 integrin/VCAM-1 bond, whereas the unbending accounts for the increased capture efficiency.
Induction of Tolerance in CD8+ T Cells to a Transgenic Autoantigen Expressed in the Liver Does Not Require Cross-Presentation
Authors: Morimoto Junko , Tan Xiaoxio , Teague Ryan M. , Öhlén Claes , Greenberg Philip D.
Published: April 2014

Cross-presentation of normal self and candidate tumor Ags by bone marrow (BM)-derived APCs that have not been activated has been demonstrated as a major mechanism contributing to acquisition of tolerance by mature T cells that first encounter an Ag in the periphery (cross-tolerance). Following adoptive transfer of naive TCR-transgenic CD8+ T cells into a host expressing a transgenic Ag that is a potentially targetable tumor Ag in normal hepatocytes as a self-Ag, we found that the majority of Ag-specific CD8+ T cells were deleted, with the remaining cells rendered anergic. Studies in BM chimeric mice and with purified cell populations demonstrated that these events were not dependent on cross-presentation by BM-derived APCs including Kupffer cells or liver sinusoidal endothelial cells, and apparently can occur entirely as a consequence of direct recognition of Ag endogenously processed and presented by hepatocytes. Direct recognition of Ag-expressing hepatocytes in vivo induced a proliferative response and up-regulation of activation markers in responding CD8+ T cells, but proliferating cells did not accumulate, with most cells rapidly eliminated, and the persisting T cells lost the capacity to proliferate in response to repeated Ag stimulation. The results suggest that parenchymal tissues may retain the capacity to directly regulate in vivo responses to self-Ags processed and presented in the context of class I MHC molecules.
Type 2 Diabetes Impairs Insulin Receptor Substrate-2-Mediated Phosphatidylinositol 3-Kinase Activity in Primary Macrophages to Induce a State of Cytokine Resistance to IL-4 in Association with Overexpression of Suppressor of Cytokine Signaling-3
Authors: O’Connor Jason C. , Sherry Christina L. , Guest Christopher B. , Freund Gregory G.
Published: April 2014

Chronic elevation of proinflammatory markers in type 2 diabetes (T2D) is well defined, but the role of anti-inflammatory cytokines in T2D is less clear. In this study, we report that normal IL-4-dependent elaboration of IL-1 receptor antagonist (IL-1RA) requires IRS-2-mediated PI3K activity in primary macrophages. We also show that macrophages isolated from obese/diabetic db/db mice have impaired IRS-2-mediated PI3K activity and constitutively overexpress suppressor of cytokine signaling (SOCS)-3, which impairs an important IL-4 anti-inflammatory function. Peritoneal proinflammatory cytokine levels were examined in diabese (db/db) mice, and IL-6 was found to be nearly 7-fold higher than in nondiabese (db/+) control mice. Resident peritoneal macrophages were isolated from db/db mice and were found to constitutively overexpress IL-6 and were unable to elaborate IL-1RA in response to IL-4-like db/+ mouse macrophages. Inhibition of PI3K with wortmannin or blockage of IRS-2/PI3K complex formation with a cell permeable IRS-2-derived tyrosine phosphopeptide inhibited IL-4-dependent IL-1RA production in db/+ macrophages. Examination of IL-4 signaling in db/db macrophages revealed that IL-4-dependent IRS-2/PI3K complex formation and IRS-2 tyrosine phosphorylation was reduced compared with db/+ macrophages. SOCS-3/IL-4 receptor complexes, however, were increased in db/db mouse macrophages compared with db/+ mice macrophages as was db/db mouse macrophage SOCS-3 expression. These results indicate that in the db/db mouse model of T2D, macrophage expression of SOCS-3 is increased, and impaired IL-4-dependent IRS-2/PI3K formation induces a state of IL-4 resistance that disrupts IL-4-dependent production of IL-1RA.
Distinct Subsets of FoxP3+ Regulatory T Cells Participate in the Control of Immune Responses
Authors: Stephens Geoffrey L. , Andersson John , Shevach Ethan M.
Published: April 2014

Expression of the transcription factor FoxP3 is the hallmark of regulatory T cells that play a crucial role in dampening immune responses. A comparison of the development and phenotype of FoxP3+ T cells in relation to the expression of conventional MHC molecules facilitated the identification of several distinct lineages of naive and effector/memory populations of Foxp3+ T cells. One subpopulation of effector/memory Foxp3+ T cells develops in the thymic medulla, whereas the second is thymic independent. Both lineages display a distinct activated phenotype, undergo extensive steady-state proliferation, home to sites of acute inflammation, and are unique in their capacity to mediate Ag-nonspecific suppression of T cell activation directly ex vivo. Effector FoxP3+ T cells may act as a sentinel of tolerance, providing a first line of defense against potentially harmful responses by rapidly suppressing immunity to peripheral self-Ags.
Altered Innate Immune Functioning of Dendritic Cells in Elderly Humans: A Role of Phosphoinositide 3-Kinase-Signaling Pathway
Authors: Agrawal Anshu , Agrawal Sudhanshu , Cao Jia-Ning , Su Houfen , Osann Kathryn , Gupta Sudhir
Published: April 2014

Aging represents a state of paradox where chronic inflammation is associated with declining immune responses. Dendritic cells (DCs) are the major APCs responsible for initiating an immune response. However, DC functions in aging have not been studied in detail. In this study, we have compared the innate immune functions of monocyte-derived myeloid DCs from elderly subjects with DCs from young individuals. We show that although phenotypically comparable, DCs from the aging are functionally different from DCs from the young. In contrast to DCs from the young, DCs from elderly individuals display 1) significantly reduced capacity to phagocytose Ags via macropinocytosis and endocytosis as determined by flow cytometry; 2) impaired capacity to migrate in vitro in response to the chemokines MIP-3β and stromal cell-derived factor-1; and 3) significantly increased LPS and ssRNA-induced secretion of TNF-α and IL-6, as determined by ELISA. Investigations of intracellular signaling revealed reduced phosphorylation of AKT in DCs from the aging, indirectly suggesting decreased activation of the PI3K pathway. Because the PI3K-signaling pathway plays a positive regulatory role in phagocytosis and migration, and also functions as a negative regulator of TLR signaling by inducing activation of p38 MAPK, this may explain the aberrant innate immune functioning of DCs from elderly subjects. Results from real-time PCR and protein expression by flow cytometry demonstrated an increased expression of phosphatase and tensin homolog, a negative regulator of the PI3K-signaling pathway, in DCs from the aging. Increased phosphatase and tensin homolog may thus be responsible for the defect in AKT phosphorylation and, therefore, the altered innate immune response of DCs from elderly humans.
Analysis of the Role of Bleomycin Hydrolase in Antigen Presentation and the Generation of CD8 T Cell Responses
Authors: Towne Charles F. , York Ian A. , Watkin Levi B. , Lazo John S. , Rock Kenneth L.
Published: April 2014

Long oligopeptides (>10 residues) are generated during the catabolism of cellular proteins in the cytosol. To be presented to T cells, such peptides must be trimmed by aminopeptidases to the proper size (typically 8–10 residues) to stably bind to MHC class I molecules. Aminopeptidases also destroy epitopes by trimming them to even shorter lengths. Bleomycin hydrolase (BH) is a cytosolic aminopeptidase that has been suggested to play a key role in generating MHC class I-presented peptides. We show that BH-deficient cells from mice are unimpaired in their ability to present epitopes from N-extended precursors or whole Ags and express normal levels of MHC class I molecules. Similarly, BH-deficient mice develop normal CD8+ T cell responses to eight epitopes from three different viruses in vivo. Therefore, BH by itself is not essential for the generation or destruction of MHC class I peptides. In contrast, when BH−/− mice are crossed to mice lacking another cytosolic aminopeptidase, leucine aminopeptidase, the resulting BH−/−leucine aminopeptidase−/− progeny show a selective increase in CD8+ T cell responses to the gp276 epitope from lymphocytic choriomeningitis virus, whereas the ability to present and respond to several other epitopes is unchanged. Therefore, BH does influence presentation of some Ags, although its role is largely redundant with other aminopeptidases.
Human Resting CD4+ T Cells Are Constitutively Inhibited by TGFβ under Steady-State Conditions
Authors: Classen Sabine , Zander Thomas , Eggle Daniela , Chemnitz Jens M. , Brors Benedikt , Büchmann Ingrid , Popov Alexey , Beyer Marc , Eils Roland , Debey Svenja , Schultze Joachim L.
Published: April 2014

Based on studies in knockout mice, several inhibitory factors such as TGFβ, IL-10, or CTLA-4 have been implicated as gate keepers of adaptive immune responses. Lack of these inhibitory molecules leads to massive inflammatory responses mainly mediated by activated T cells. In humans, the integration of these inhibitory signals for keeping T cells at a resting state is less well understood. To elucidate this regulatory network, we assessed early genome-wide transcriptional changes during serum deprivation in human mature CD4+ T cells. The most striking observation was a “TGFβ loss signature” defined by down-regulation of many known TGFβ target genes. Moreover, numerous novel TGFβ target genes were identified that are under the suppressive control of TGFβ. Expression of these genes was up-regulated once TGFβ signaling was lost during serum deprivation and again suppressed upon TGFβ reconstitution. Constitutive TGFβ signaling was corroborated by demonstrating phosphorylated SMAD2/3 in resting human CD4+ T cells in situ, which were dephosphorylated during serum deprivation and rephosphorylated by minute amounts of TGFβ. Loss of TGFβ signaling was particularly important for T cell proliferation induced by low-level TCR and costimulatory signals. We suggest TGFβ to be the most prominent factor actively keeping human CD4+ T cells at a resting state.
p300 Protein Acetyltransferase Activity Suppresses Systemic Lupus Erythematosus-Like Autoimmune Disease in Mice
Authors: Forster Nicole , Gallinat Sven , Jablonska Jadwiga , Weiss Siegfried , Elsässer Hans-Peter , Lutz Werner
Published: April 2014

Conditional knock-in mice expressing a histone acetyltransferase-deficient version of the transcriptional coregulator p300 exclusively in B lymphocytes die prematurely with full penetrance. The mice develop an autoimmune disease similar to systemic lupus erythematosus in its pathological manifestations, such as splenomegaly, glomerulonephritis, vasculitis, deposition of immune complexes, and production of autoantibodies against dsDNA. Aged mice show a severe reduction of transitional and marginal zone B cells and generate aberrant mature B cells. These B cells show diminished proliferation in response to stimulation of the BCR, but respond normally to other stimuli. Yet, the mice mount a normal primary immune response against a T-dependent Ag. In contrast, the memory response is impaired. In addition, serum Ig levels, in particular IgG2b, are increased. We conclude that p300 acetyltransferase activity is essential for maintaining self-tolerance of B lymphocytes. These findings support the concept of treating lupus with inhibitors of protein deacetylases and point to B cells as a critical target of these drugs.
CTL Fail to Accumulate at Sites of HIV-1 Replication in Lymphoid Tissue
Authors: Connick Elizabeth , Mattila Teresa , Folkvord Joy M. , Schlichtemeier Rick , Meditz Amie L. , Ray M. Graham , McCarter Martin D. , MaWhinney Samantha , Hage Aaron , White Cara , Skinner Pamela J.
Published: April 2014

The inability of HIV-1-specific CTL to fully suppress virus replication as well as the failure of administration of exogenous CTL to lower viral loads are not understood. To evaluate the hypothesis that these phenomena are due to a failure of CTL to localize at sites of HIV-1 replication, we assessed the distribution of HIV-1 RNA and HIV-1-specific CTL identified by HIV-1 peptide/HLA class I tetrameric complexes (tetramers) within lymph nodes of 14 HIV-1-infected individuals who were not receiving antiretroviral therapy. A median of 0.04% of follicular compared with 0.001% of extrafollicular CD4+ cells were estimated to be producing HIV-1 RNA, a 40-fold difference (p = 0.0001). Tetramer-stained cells were detected by flow cytometry in disaggregated lymph node cells from 11 subjects and constituted a significantly higher fraction of CD8+ cells in lymph node (mean, 2.15%) than in PBMC (mean, 1.52%; p = 0.02). In situ tetramer staining in three subjects’ lymph nodes, in which high frequencies of tetramer-stained cells were detected, revealed that tetramer-stained cells were primarily concentrated in extrafollicular regions of lymph node and were largely absent within lymphoid follicles. These data confirm that HIV-1-specific CTL are abundant within lymphoid tissues, but fail to accumulate within lymphoid follicles where HIV-1 replication is concentrated, suggesting that lymphoid follicles may be immune-privileged sites. Mechanisms underlying the exclusion of CTL from lymphoid follicles as well as the role of lymphoid follicles in perpetuating other chronic pathogens merit further investigation.
Impaired Mast Cell Maturation and Degranulation and Attenuated Allergic Responses in Ndrg1-Deficient Mice
Authors: Taketomi Yoshitaka , Sunaga Kohei , Tanaka Satoshi , Nakamura Masanori , Arata Satoru , Okuda Tomohiko , Moon Tae-Chul , Chang Hyeun-Wook , Sugimoto Yukihiko , Kokame Koichi , Miyata Toshiyuki , Murakami Makoto , Kudo Ichiro
Published: April 2014

We have previously reported that N-myc downstream regulated gene-1 (NDRG1) is an early inducible protein during the maturation of mouse bone marrow-derived mast cells (BMMCs) toward a connective tissue mast cell-like phenotype. To clarify the function of NDRG1 in mast cells and allergic responses, we herein analyzed mast cell-associated phenotypes of mice lacking the Ndrg1 gene. Allergic responses including IgE-mediated passive systemic and cutaneous anaphylactic reactions were markedly attenuated in Ndrg1-deficient mice as compared with those in wild-type mice. In Ndrg1-deficient mice, dermal and peritoneal mast cells were decreased in number and morphologically abnormal with impaired degranulating ability. Ex vivo, Ndrg1-deficient BMMCs cocultured with Swiss 3T3 fibroblasts in the presence of stem cell factor, a condition that facilitates the maturation of BMMCs toward a CTMC-like phenotype, displayed less exocytosis than replicate wild-type cells after the cross-linking of FcεRI or stimulation with compound 48/80, even though the exocytotic response of IL-3-maintained, immature BMMCs from both genotypes was comparable. Unlike degranulation, the production of leukotriene and cytokines by cocultured BMMCs was unaffected by NDRG1 deficiency. Taken together, the altered phenotypes of Ndrg1-deficient mast cells both in vivo and ex vivo suggest that NDRG1 has roles in the terminal maturation and effector function (degranulation) of mast cells.
The Tick Salivary Protein, Salp15, Inhibits the Development of Experimental Asthma
Authors: Paveglio Sara A. , Allard Jenna , Mayette Jana , Whittaker Laurie A. , Juncadella Ignacio , Anguita Juan , Poynter Matthew E.
Published: April 2014

Activation of Th2 CD4+ T cells is necessary and sufficient to elicit allergic airway disease, a mouse model with many features of human allergic asthma. Effectively controlling the activities of these cells could be a panacea for asthma therapy. Blood-feeding parasites have devised remarkable strategies to effectively evade the immune response. For example, ticks such as Ixodes scapularis, which must remain on the host for up to 7 days to feed to repletion, secrete immunosuppressive proteins. Included among these proteins is the 15-kDa salivary protein Salp15, which inhibits T cell activation and IL-2 production. Our objective for these studies was to evaluate the T cell inhibitory properties of Salp15 in a mouse model of allergic asthma. BALB/cJ mice were Ag sensitized by i.p. injection of OVA in aluminum hydroxide, with or without 50 μg of Salp15, on days 0 and 7. All mice were challenged with aerosolized OVA on days 14–16 and were studied on day 18. Compared with control mice sensitized with Ag, mice sensitized with Ag and Salp15 displayed significantly reduced airway hyperresponsiveness, eosinophilia, Ag-specific IgG1 and IgE, mucus cell metaplasia, and Th2 cytokine secretion in vivo and by CD4+ T cells restimulated with Ag in vitro. Our results demonstrate that Salp15 can effectively prevent the generation of a Th2 immune response and the development of experimental asthma. These studies, and those of others, support the notion that a lack of ectoparasitism may contribute to the increasing prevalence of allergic asthma.
C57BL/6 Mice Genetically Deficient in IL-12/IL-23 and IFN-γ Are Susceptible to Experimental Autoimmune Myasthenia Gravis, Suggesting a Pathogenic Role of Non-Th1 Cells
Authors: Wang Wei , Milani Monica , Ostlie Norma , Okita David , Agarwal Rajeev K. , Caspi Rachel , Conti-Fine Bianca M.
Published: April 2014

Immunization with Torpedo acetylcholine receptor (TAChR) induces experimental autoimmune myasthenia gravis (EAMG) in C57BL/6 (B6) mice. EAMG development needs IL-12, which drives differentiation of Th1 cells. The role of IFN-γ, an important Th1 effector, is not clear and that of IL-17, a proinflammatory cytokine produced by Th17 cells, is unknown. In this study, we examined the effect of simultaneous absence of IL-12 and IFN-γ on EAMG susceptibility, using null mutant B6 mice for the genes of both the IL-12/IL-23 p40 subunit and IFN-γ (dKO mice). Wild-type (WT) B6 mice served as control for EAMG induction. All mice were immunized with TAChR in Freund’s adjuvant. dKO mice developed weaker anti-TAChR CD4+T cells and Ab responses than WT mice. Yet, they developed EAMG symptoms, anti-mouse acetylcholine receptor (AChR) Ab, and CD4+ T cell responses against mouse AChR sequences similar to those of WT mice. dKO and WT mice had similarly reduced AChR content in their muscles, and IgG and complement at the neuromuscular junction. Naive dKO mice had significantly fewer NK, NKT, and CD4+CD25+Foxp3+ T regulatory (Treg) cells than naive WT mice. Treg cells from TAChR-immunized dKO mice had significantly less suppressive activity in vitro than Treg cells from TAChR-immunized WT mice. In contrast, TAChR-specific CD4+ T cells from TAChR-immunized dKO and WT mice secreted comparable amounts of IL-17 after stimulation in vitro with TAChR. The susceptibility of dKO mice to EAMG may be due to reduced Treg function, in the presence of a normal function of pathogenic Th17 cells.
Novel Engineered Trastuzumab Conformational Epitopes Demonstrate In Vitro and In Vivo Antitumor Properties against HER-2/neu
Authors: Garrett Joan T. , Rawale Sharad , Allen Stephanie D. , Phillips Gary , Forni Guido , Morris John C. , Kaumaya Pravin T. P.
Published: April 2014

Trastuzumab is a growth-inhibitory humanized Ab targeting the oncogenic protein HER-2/neu. Although trastuzumab is approved for treatment of advanced breast cancer, a number of concerns exist with passive immunotherapy. Treatment is expensive and has a limited duration of action, necessitating repeated administrations of the mAb. Active immunotherapy with conformational B cell epitopes affords the possibility of generating an enduring immune response, eliciting protein-reactive high-affinity anti-peptide Abs. The three-dimensional structure of human HER-2 in complex with trastuzumab reveals that the Ag-binding region of HER-2 spans residues 563–626 that comprises an extensive disulfide-bonding pattern. To delineate the binding region of HER-2, we have designed four synthetic peptides with different levels of conformational flexibility. Chimeric peptides incorporating the measles virus fusion “promiscuous” T cell epitope via a four-residue linker sequence were synthesized, purified, and characterized. All conformational peptides were recognized by trastuzumab and prevented the function of trastuzumab inhibiting tumor cell proliferation, with 563–598 and 597–626 showing greater reactivity. All epitopes were immunogenic in FVB/N mice with Abs against 597–626 and 613–626 recognizing HER-2. The 597–626 epitope was immunogenic in outbred rabbits eliciting Abs which recognized HER-2, competed with trastuzumab for the same epitope, inhibited proliferation of HER-2-expressing breast cancer cells in vitro and caused their Ab-dependent cell-mediated cytotoxicity. Moreover, immunization with the 597–626 epitope significantly reduced tumor burden in transgenic BALB-neuT mice. These results suggest the peptide B cell immunogen is appropriate as a vaccine for HER-2-overexpressing cancers because the resulting Abs show analogous biological properties to trastuzumab.
STAT1 Expression in Dendritic Cells, but Not T Cells, Is Required for Immunity toLeishmania major
Authors: Johnson Leanne M. , Scott Phillip
Published: April 2014

The generation of Th1 responses is important for resistance to intracellular pathogens, including the parasite, Leishmania major. Although IFN-γR/STAT1 signaling promotes a Th1 response via the up-regulation of T-bet, the requirement for STAT1 in Th1 cell differentiation remains controversial. Although in some cases Th1 cells develop independently of STAT1, STAT1−/− mice fail to develop a Th1 response during L. major infection. However, the interpretation of this result is complicated by the role STAT1 plays in Ag presentation and, more importantly, in elimination of parasites by macrophages, because both defective Ag presentation and increased parasite burden can influence Th cell development. To resolve this issue, we assessed the ability of STAT1−/− T cells to become Th1 cells and protect mice against L. major following adoptive transfer into STAT1-sufficient mice. We found that whereas T-bet is critical for the differentiation of protective Th1 cells during L. major infection, IFN-γR and STAT1 are dispensable. Given that a STAT1-independent Th1 cell response was generated by STAT1-sufficient APCs, but not by STAT1−/− cells, we next addressed whether dendritic cells (DCs) require STAT1 signaling to effectively present Ag. We found that STAT1−/− DCs had impaired up-regulation of MHC and costimulatory molecules, and, as a consequence, the absence of STAT1 resulted in reduced Th1 cell priming. Taken together, these results demonstrate that T cell expression of STAT1 is not required for the development of Th1 cells protective against L. major and instead stress the importance of STAT1 signaling in DCs for the optimal induction of Th1 responses.
Early Virus-Associated Bystander Events Affect the Fitness of the CD8 T Cell Response to Persistent Virus Infection
Authors: Andrews Nicolas P. , Pack Christopher D. , Vezys Vaiva , Barber Glen N. , Lukacher Aron E.
Published: April 2014

Chronic Ag exposure during persistent viral infection erodes virus-specific CD8 T cell numbers and effector function, with a concomitant loss of pathogen control. Less clear are the respective contributions of Ag-specific and Ag-nonspecific (bystander) events on the quantity, quality, and maintenance of antiviral CD8 T cells responding to persistent virus infection. In this study, we show that low-dose inoculation with mouse polyomavirus (PyV) elicits a delayed, but numerically equivalent, antiviral CD8 T cell response compared with high-dose inoculation. Low-dose infection generated virus-specific CD8 T cells endowed with multicytokine functionality and a superior per cell capacity to produce IFN-γ. PyV-specific CD8 T cells primed by low-dose inoculation also expressed higher levels of IL-7Rα and bcl-2 and possessed enhanced Ag-independent survival. Importantly, the quantity and quality of the antiviral CD8 T cell response elicited by dendritic cell-mediated immunization were mitigated by infection with a mutant PyV lacking the dominant CD8 T cell viral epitope. These findings suggest that the fitness of the CD8 T cell response to persistent virus infection is programmed in large part by early virus-associated Ag-nonspecific factors, and imply that limiting bystander inflammation at the time of inoculation, independent of Ag load, may optimize adaptive immunity to persistent viral infection.
Dual Binding Specificity of a Borrelia hermsii-Associated Complement Regulator-Acquiring Surface Protein for Factor H and Plasminogen Discloses a Putative Virulence Factor of Relapsing Fever Spirochetes
Authors: Rossmann Evelyn , Kraiczy Peter , Herzberger Pia , Skerka Christine , Kirschfink Michael , Simon Markus M. , Zipfel Peter F. , Wallich Reinhard
Published: April 2014

Tick-borne relapsing fever in North America is primarily caused by the spirochete Borrelia hermsii. The pathogen employs multiple strategies, including the acquisition of complement regulators and antigenic variation, to escape innate and humoral immunity. In this study we identified in B. hermsii a novel member of the complement regulator-acquiring surface protein (CRASP) family, designated BhCRASP-1, that binds the complement regulators factor H (FH) and FH-related protein 1 (FHR-1) but not FH-like protein 1 (FHL-1). BhCRASP-1 specifically interacts with the short consensus repeat 20 of FH, thereby maintaining FH-associated cofactor activity for factor I-mediated C3b inactivation. Furthermore, ectopic expression of BhCRASP- 1 converted the serum-sensitive Borrelia burgdorferi B313 strain into an intermediate complement-resistant strain. Finally, we report for the first time that BhCRASP-1 binds plasminogen/plasmin in addition to FH via, however, distinct nonoverlapping domains. The fact that surface-bound plasmin retains its proteolytic activity suggest that the dual binding specificity of BhCRASP-1 for FH and plasminogen/plasmin contributes to both the dissemination/invasion of B. hermsii and its resistance to innate immunity.
Inhibition of Allergen-Induced Airway Remodeling in Smad 3-Deficient Mice
Authors: Le Annie V. , Cho Jae Youn , Miller Marina , McElwain Shauna , Golgotiu Kirsti , Broide David H.
Published: April 2014

Intracellular signaling pathways that converge on Smad 3 are used by both TGF-β and activin A, key cytokines implicated in the process of fibrogenesis. To determine the role of Smad 3 in allergen-induced airway remodeling, Smad 3-deficient and wild-type (WT) mice were sensitized to OVA and challenged by repetitive administration of OVA for 1 mo. Increased levels of activin A and increased numbers of peribronchial TGF-β1+ cells were detected in WT and Smad 3-deficient mice following repetitive OVA challenge. Smad 3-deficient mice challenged with OVA had significantly less peribronchial fibrosis (total lung collagen content and trichrome staining), reduced thickness of the peribronchial smooth muscle layer, and reduced epithelial mucus production compared with WT mice. As TGF-β and Smad 3 signaling are hypothesized to mediate differentiation of fibroblasts to myofibroblasts in vivo, we determined the number of peribronchial myofibroblasts (Col-1+ and α-smooth muscle actin+) as assessed by double-label immunofluorescence microscopy. Although the number of peribronchial myofibroblasts increased significantly in WT mice following OVA challenge, there was a significant reduction in the number of peribronchial myofibroblasts in OVA-challenged Smad 3-deficient mice. There was no difference in levels of eosinophilic airway inflammation or airway responsiveness in Smad 3-deficient compared with WT mice. These results suggest that Smad 3 signaling is required for allergen-induced airway remodeling, as well as allergen-induced accumulation of myofibroblasts in the airway. However, Smad 3 signaling does not contribute significantly to airway responsiveness.
Soluble TNF-Like Cytokine (TL1A) Production by Immune Complexes Stimulated Monocytes in Rheumatoid Arthritis
Authors: Cassatella Marco A. , da Silva Gabriela Pereira , Tinazzi Ilaria , Facchetti Fabio , Scapini Patrizia , Calzetti Federica , Tamassia Nicola , Wei Ping , Nardelli Bernardetta , Roschke Viktor , Vecchi Annunciata , Mantovani Alberto , Bambara Lisa M. , Edwards Steven W. , Carletto Antonio
Published: April 2014

TNF-like cytokine (TL1A) is a newly identified member of the TNF superfamily of ligands that is important for T cell costimulation and Th1 polarization. However, despite increasing information about its functions, very little is known about expression of TL1A in normal or pathological states. In this study, we report that mononuclear phagocytes appear to be a major source of TL1A in rheumatoid arthritis (RA), as revealed by their strong TL1A expression in either synovial fluids or synovial tissue of rheumatoid factor (RF)-seropositive RA patients, but not RF−/RA patients. Accordingly, in vitro experiments revealed that human monocytes express and release significant amounts of soluble TL1A when stimulated with insoluble immune complexes (IC), polyethylene glycol precipitates from the serum of RF+/RA patients, or with insoluble ICs purified from RA synovial fluids. Monocyte-derived soluble TL1A was biologically active as determined by its capacity to induce apoptosis of the human erythroleukemic cell line TF-1, as well as to cooperate with IL-12 and IL-18 in inducing the production of IFN-γ by CD4+ T cells. Because RA is a chronic inflammatory disease with autoimmune etiology, in which ICs, autoantibodies (including RF), and various cytokines contribute to its pathology, our data suggest that TL1A could be involved in its pathogenesis and contribute to the severity of RA disease that is typical of RF+/RA patients.
Inactivation of Cdc42 Is Necessary for Depolymerization of Phagosomal F-Actin and Subsequent Phagosomal Maturation
Authors: Lerm Maria , Brodin Veronika Patcha , Ruishalme Iida , Stendahl Olle , Särndahl Eva
Published: April 2014

Phagocytosis is a complex process involving the activation of various signaling pathways, such as the Rho GTPases, and the subsequent reorganization of the actin cytoskeleton. In neutrophils, Rac and Cdc42 are activated during phagocytosis but less is known about the involvement of these GTPases during the different stages of the phagocytic process. The aim of this study was to elucidate the role of Cdc42 in phagocytosis and the subsequent phagosomal maturation. Using a TAT-based protein transduction technique, we introduced dominant negative and constitutively active forms of Cdc42 into neutrophil-like HL60 (human leukemia) cells that were allowed to phagocytose IgG-opsonized yeast particles. Staining of cellular F-actin in cells transduced with constitutively active Cdc42 revealed that the activation of Cdc42 induced sustained accumulation of periphagosomal actin. Moreover, the fusion of azurophilic granules with the phagosomal membrane was prevented by the accumulated F-actin. In contrast, introducing dominant negative Cdc42 impaired the translocation per se of azurophilic granules to the periphagosomal area. These results show that efficient phagosomal maturation and the subsequent eradication of ingested microbes in human neutrophils is dependent on a strictly regulated Cdc42. To induce granule translocation, Cdc42 must be in its active state but has to be inactivated to allow depolymerization of the F-actin cage around the phagosome, a process essential for phagolysosome formation.
Corticosteroid Inhibition of Growth-Related Oncogene Protein-α via Mitogen-Activated Kinase Phosphatase-1 in Airway Smooth Muscle Cells
Authors: Issa Razao , Xie Shaoping , Khorasani Nadia , Sukkar Maria , Adcock Ian M. , Lee Kang-Yun , Chung Kian Fan
Published: April 2014

Expression of the inflammatory chemokine, growth-related oncogene protein-α (GRO-α), from airway smooth muscle cells (ASMC) is regulated by pathways involving NF-κB and MAPK activation. We determined the effects of dexamethasone on GRO-α induced by IL-1β or TNF-α with respect to the role of MAPK pathways and of MAPK phosphatase-1 (MKP-1). Human ASMC were studied in primary culture at confluence. Dexamethasone (10−8–10−5 M) partially inhibited GRO-α expression and release induced by IL-1β and TNF-α; this was associated with an inhibition of JNK, but not of p38 or ERK phosphorylation. Together with IL-1β or TNF-α, dexamethasone rapidly induced mRNA and protein expression of MKP-1, which dephosphorylates MAPKs. Using MKP-1 small interfering RNA (siRNA) to block the expression of IL-1β- and dexamethasone-induced MKP-1 by 50%, JNK phosphorylation was doubled. The inhibitory effect of dexamethasone on GRO-α release was partially reversed in ASMC treated with MKP-1 siRNA compared with those treated with scrambled siRNA. In contrast, overexpression of MKP-1 led to a reduction in IL-1β-induced release of GRO-α, but the inhibitory effects of dexamethasone were preserved. Nuclear translocation of the glucocorticoid receptor was increased in ASMC exposed to dexamethasone and IL-1β. Using chromatin immunoprecipitation assay, glucocorticoid receptor binding to the MKP-1 promoter was increased by IL-1β and dexamethasone compared with either alone. Glucocorticoids and IL-1β or TNF-α modulate GRO-α release partly through the inhibition of JNK pathway, resulting from an up-regulation of MKP-1 expression.
Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release
Authors: Tang Daolin , Kang Rui , Xiao Weimin , Jiang Lei , Liu Meidong , Shi Yongzhong , Wang Kangkai , Wang Haichao , Xiao Xianzhong
Published: April 2014

In response to inflammatory stimuli (e.g., endotoxin, proinflammatory cytokines) or oxidative stress, macrophages actively release a ubiquitous nuclear protein, high-mobility group box 1 (HMGB1), to sustain an inflammatory response to infection or injury. In this study, we demonstrated mild heat shock (e.g., 42.5°C, 1 h), or enhanced expression of heat shock protein (Hsp) 72 (by gene transfection) similarly rendered macrophages resistant to oxidative stress-induced HMGB1 cytoplasmic translocation and release. In response to oxidative stress, cytoplasmic Hsp72 translocated to the nucleus, where it interacted with nuclear proteins including HMGB1. Genetic deletion of the nuclear localization sequence (NLS) or the peptide binding domain (PBD) from Hsp72 abolished oxidative stress-induced nuclear translocation of Hsp72-ΔNLS (but not Hsp72-ΔPBD), and prevented oxidative stress-induced Hsp72-ΔPBD-HMGB1 interaction in the nucleus. Furthermore, impairment of Hsp72-ΔNLS nuclear translocation, or Hsp72-ΔPBD-HMGB1 interaction in the nucleus, abrogated Hsp72-mediated suppression of HMGB1 cytoplasmic translocation and release. Taken together, these experimental data support a novel role for nuclear Hsp72 as a negative regulator of oxidative stress-induced HMGB1 cytoplasmic translocation and release.
IFN-γ Protects Cerulein-Induced Acute Pancreatitis by Repressing NF-κB Activation
Authors: Hayashi Takahito , Ishida Yuko , Kimura Akihiko , Iwakura Yoichiro , Mukaida Naofumi , Kondo Toshikazu
Published: April 2014

We explored the pathophysiological roles of IFN-γ in cerulein-induced acute pancreatitis. In wild-type (WT) mice, cerulein injection caused acute pancreatitis as evidenced by increased serum amylase levels and pathological changes such as interstitial edema, vacuolization, acinar cell necrosis, and neutrophil infiltration in pancreas. Concomitantly, cerulein treatment augmented intrapancreatic gene expression of TNF-α, KC/CXCL1, MIP-2/CXCL2, cyclooxygenase-2 (COX-2), and IFN-γ in WT mice. In situ hybridization combined with immunofluorescence analyses demonstrated that infiltrating neutrophils expressed IFN-γ mRNA. Unexpectedly, IFN-γ−/− mice exhibited exacerbated cerulein-induced pancreatic injury, with enhanced neutrophil recruitment. Moreover, intrapancreatic gene expression of TNF-α, KC/CXCL1, MIP-2/CXCL2, and COX-2 were significantly exaggerated in IFN-γ−/− mice, compared with WT mice. Cerulein activated NF-κB, an indispensable transcription factor for gene transcription of TNF-α, KC/CXCL1, MIP-2/CXCL2, and COX-2, in pancreas of cerulein-treated WT mice as evidenced by the increases in nuclear amount and DNA-binding activity of NF-κB p65. In comparison with WT mice, IFN-γ−/− mice exhibited exaggerated and prolonged NF-κB activation, probably due to reduced acetylation of Stat1, a main signal transducer of IFN-γ, because acetylated Stat1 can inhibit NF-κB activation. Indeed, IFN-γ acetylated Stat1 and reciprocally reduced NF-κB activation and COX-2 expression in neutrophils. Finally, even when administered 4 h after the first cerulein injection, IFN-γ remarkably attenuated acute pancreatitis in both WT and IFN-γ−/− mice, with reduced NF-κB activation and COX-2 expression. Thus, IFN-γ can have anti-inflammatory effects on acute pancreatitis by depressing the proinflammatory consequences of NF-κB activation.