bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunologic Homeostasis during Infection: Coexistence of Strong Pulmonary Cell-Mediated Immunity to SecondaryCryptococcus neoformansInfection While the Primary Infection Still Persists at Low Levels in the Lungs
Authors: Lindell Dennis M. , Ballinger Megan N. , McDonald Roderick A. , Toews Galen B. , Huffnagle Gary B.
Published: April 2014

Maintenance of immunity to persistent pathogens is poorly understood. In this study, we used a murine model of persistent pulmonary fungal infection to study the ongoing cell-mediated immune response. CBA/J mice with low-level persistent Cryptococcus neoformans infection had CD4+ T cells of effector memory phenotype present in their lungs. Although unable to eliminate the primary infection to sterility, these mice displayed hallmarks of immunologic memory in response to rechallenge with C. neoformans: 1) the secondary cryptococcal challenge was controlled much more rapidly, 2) the inflammatory response developed and resolved more rapidly, 3) CD4+ T and CD8+ T cell responses were higher in magnitude, and 4) effector cytokine production by T cells was greatly enhanced. Depletion of CD4+ T cells at the time of secondary challenge adversely affected clearance of C. neoformans from the lungs. These results demonstrate that persistent low-level infection with C. neoformans does not impair the cell-mediated response to the fungus. Although they are relatively free of overt disease, these mice can respond with a rapid secondary immune response if the burden of C. neoformans increases. These data support the concept that immunologically healthy individuals can maintain low numbers of cryptococci that can become a nidus for re-activation disease during immunodeficient states such as AIDS.
Differential Selection Pressure Exerted on HIV by CTL Targeting Identical Epitopes but Restricted by Distinct HLA Alleles from the Same HLA Supertype
Authors: Leslie Alasdair , Price David A. , Mkhize Pamela , Bishop Karen , Rathod Almas , Day Cheryl , Crawford Hayley , Honeyborne Isobella , Asher Tedi E. , Luzzi Graz , Edwards Anne , Rosseau Christine M. , Mullins James I. , Tudor-Williams Gareth , Novelli Vas , Brander Christian , Douek Daniel C. , Kiepiela Photini , Walker Bruce D. , Goulder Philip J. R.
Published: April 2014

HLA diversity is seen as a major challenge to CTL vaccines against HIV. One current approach focuses on “promiscuous” epitopes, presented by multiple HLA alleles from within the same HLA supertype. However, the effectiveness of such supertype vaccines depends upon the functional equivalence of CTL targeting a particular epitope, irrespective of the restricting HLA. In this study, we describe the promiscuous HIV-specific CTL epitopes presented by alleles within the B7 supertype. Substantial differences were observed in the ability of CTL to select for escape mutation when targeting the same epitope but restricted by different HLA. This observation was common to all six promiscuous B7 epitopes identified. Moreover, with one exception, there were no significant differences in the frequency, magnitude, or immunodominance of the CTL responses restricted by different HLA alleles to explain these discrepancies. This suggests that the unique peptide/MHC complexes generated by even closely related HLA induce CTL responses that are qualitatively different. This hypothesis is supported by additional differences observed between CTL targeting identical epitopes but restricted by different HLA: first, the occurrence of distinct, HLA-specific escape mutation; second, the recruitment of distinct TCR repertoires by particular peptide/MHC complexes; and, third, significant differences in the functional avidity of CTL. Taken together, these data indicate that significant functional differences exist between CTL targeting identical epitopes but restricted by different, albeit closely related HLA. These findings are of relevance to vaccine approaches that seek to exploit HLA supertypes to overcome the problem of HLA diversity.
Galectin-3 Induces Death of Candida Species Expressing Specific β-1,2-Linked Mannans
Authors: Kohatsu Luciana , Hsu Daniel K. , Jegalian Armin G. , Liu Fu-Tong , Baum Linda G.
Published: April 2014

Lectins play a critical role in host protection against infection. The galectin family of lectins recognizes saccharide ligands on a variety of microbial pathogens, including viruses, bacteria, and parasites. Galectin-3, a galectin expressed by macrophages, dendritic cells, and epithelial cells, binds bacterial and parasitic pathogens including Leishmania major, Trypanosoma cruzi, and Neisseria gonorrhoeae. However, there have been no reports of galectins having direct effects on microbial viability. We found that galectin-3 bound only to Candida albicans species that bear β-1,2-linked oligomannans on the cell surface, but did not bind Saccharomyces cerevisiae that lacks β-1,2-linked oligomannans. Surprisingly, binding directly induced death of Candida species containing specific β-1,2-linked oligomannosides. Thus, galectin-3 can act as a pattern recognition receptor that recognizes a unique pathogen-specific oligosaccharide sequence. This is the first description of antimicrobial activity for a member of the galectin family of mammalian lectins; unlike other lectins of the innate immune system that promote opsonization and phagocytosis, galectin-3 has direct fungicidal activity against opportunistic fungal pathogens.
Follistatin-Like Protein-1 Is a Novel Proinflammatory Molecule
Authors: Miyamae Takako , Marinov Anthony D. , Sowders Dawn , Wilson David C. , Devlin Jason , Boudreau Robert , Robbins Paul , Hirsch Raphael
Published: April 2014

While analyzing gene expression in collagen-induced arthritis, we discovered that a poorly characterized gene, follistatin-like protein 1 (FSTL-1), is highly overexpressed in mouse paws during early arthritis, especially at the interface of synovial pannus and eroding bone. In this study, we show that FSTL-1 is a novel proinflammatory molecule with a previously unrecognized role in inflammation. Transfection of FSTL-1 into macrophages and fibroblasts leads to up-regulation of proinflammatory cytokines, including IL-1β, TNF-α, and IL-6. Overexpression of FSTL-1 in mouse paws by gene transfer results in severe paw swelling and arthritis.
Cyclooxygenase-1 Overexpression Decreases Basal Airway Responsiveness but Not Allergic Inflammation
Authors: Card Jeffrey W. , Carey Michelle A. , Bradbury J. Alyce , Graves Joan P. , Lih Fred B. , Moorman Michael P. , Morgan Daniel L. , DeGraff Laura M. , Zhao Yun , Foley Julie F. , Zeldin Darryl C.
Published: April 2014

Pharmacological inhibition or genetic disruption of cyclooxygenase (COX)-1 or COX-2 exacerbates the inflammatory and functional responses of the lung to environmentally relevant stimuli. To further examine the contribution of COX-derived eicosanoids to basal lung function and to allergic lung inflammation, transgenic (Tr) mice were generated in which overexpression of human COX-1 was targeted to airway epithelium. Although no differences in basal respiratory or lung mechanical parameters were observed, COX-1 Tr mice had increased bronchoalveolar lavage fluid PGE2 content compared with wild-type littermates (23.0 ± 3.6 vs 8.4 ± 1.4 pg/ml; p < 0.05) and exhibited decreased airway responsiveness to inhaled methacholine. In an OVA-induced allergic airway inflammation model, comparable up-regulation of COX-2 protein was observed in the lungs of allergic wild-type and COX-1 Tr mice. Furthermore, no genotype differences were observed in allergic mice in total cell number, eosinophil content (70 vs 76% of total cells, respectively), and inflammatory cytokine content of bronchoalveolar lavage fluid, or in airway responsiveness to inhaled methacholine (p > 0.05). To eliminate the presumed confounding effects of COX-2 up-regulation, COX-1 Tr mice were bred into a COX-2 null background. In these mice, the presence of the COX-1 transgene did not alter allergen-induced inflammation but significantly attenuated allergen-induced airway hyperresponsiveness, coincident with reduced airway leukotriene levels. Collectively, these data indicate that COX-1 overexpression attenuates airway responsiveness under basal conditions but does not influence allergic airway inflammation.
Native High-Density Lipoprotein Augments Monocyte Responses to Lipopolysaccharide (LPS) by Suppressing the Inhibitory Activity of LPS-Binding Protein
Authors: Thompson Patricia A. , Kitchens Richard L.
Published: April 2014

High-density lipoprotein (HDL) is an abundant plasma lipoprotein that is generally thought to be anti-inflammatory in both health and infectious disease. It binds and neutralizes the bioactivity of the potent bacterial lipids, LPS and lipoteichoic acid, that stimulate host innate immune responses. LPS-binding protein (LBP) plays an important role in augmenting leukocyte responses to LPS, whereas high concentrations of LBP, in the range of those found in plasma, can be inhibitory. We found that native HDL (nHDL) augmented human monocyte responses to LPS in the presence of inhibitory concentrations of LBP as measured by production of TNF and other cytokines. HDL did not stimulate cells in the absence of LPS, and it did not augment responses that were stimulated by IL-1β or lipoteichoic acid. This activity of HDL was inhibited by trypsin treatment, suggesting that one or more protein constituents of HDL are required. In contrast to nHDL, low-density lipoprotein, and reconstituted HDL did not possess this activity. The total lipoprotein fraction of normal plasma had activity that was similar to that of nHDL, whereas lipoproteins from septic patients with reduced HDL levels had a reduced ability to augment responses to LPS; this activity was restored by adding normal HDL to the patient lipoproteins. Our results demonstrate a novel proinflammatory activity of HDL that may help maintain sensitive host responses to LPS by suppressing the inhibitory activity of LBP. Our findings also raise the possibility that the decline of HDL during sepsis may help control the response to LPS.
Cutting Edge: Treatment of Complement Regulatory Protein Deficiency by Retroviral In Vivo Gene Therapy
Authors: Spitzer Dirk , Wu Xiaobo , Ma Xiucui , Xu Lingfei , Ponder Katherine P. , Atkinson John P.
Published: April 2014

Gene therapy is an attractive means to replace a deficient or defective protein. Using a murine retroviral vector, we provide an example of reconstituting a C regulator by neonatal in vivo gene transfer. The fusion gene containing the mouse C receptor 1-related gene/protein y (Crry) and a single chain Ab fragment with specificity for mouse glycophorin A was placed under transcriptional control of a liver-specific promoter. Shortly after birth, Crry KO mice were injected with the retroviral vectors. Protein expression progressively increased over the next 6–8 wk after which an equilibrium was established. Coating levels on RBCs were obtained that inhibited C activation similar to wild-type cells and remained constant for >1 year. Thus, gene therapy with targeted regulators represents a treatment option to provide a long-term and sustained protein supply for the site-specific blockade of undesirable complement activation.
Cutting Edge: Abortive Proliferation of CD46-Induced Tr1-Like Cells due to a Defective Akt/Survivin Signaling Pathway
Authors: Meiffren Grégory , Flacher Monique , Azocar Olga , Rabourdin-Combe Chantal , Faure Mathias
Published: April 2014

T regulatory cell 1 (Tr1) are low proliferating peripherally induced suppressive T cells. Engaging CD3 and CD46 on human CD4+ T cells induces a Tr1-like phenotype. In this study, we report that human Tr1-like cells do not sustain proliferation over time. The weak proliferation of these cells results first from their inability to sustain expression of various cell cycle-associated proteins, to efficiently degrade the inhibitor of cell cycle progression p27/Kip1 and, as a consequence, in their accumulation in the G0-G1 phase. Also, the reduced proliferation of Tr1-like cells results from their increased sensitivity to death as they divide, through a mechanism that is neither Fas-mediated nor Bcl2/Bcl-xL related. Both properties, impaired cell cycle and death sensitivity, are explained by a specific defective activation of Akt that impairs the expression of Survivin. Thus, our results show that CD3/CD46-induced Tr1-like cells die through a process of abortive proliferation.
Cutting Edge: Inhibition of T Cell Activation by TIM-2
Authors: Knickelbein Jared E. , de Souza Anjali J. , Tosti Richard , Narayan Preeti , Kane Lawrence P.
Published: April 2014

T cell Ig and mucin domain protein 2 (TIM-2) has been shown to regulate T cell activation in vitro and T cell-mediated disease in vivo. However, it is still not clear whether TIM-2 acts mainly to augment T cell function or to inhibit it. We have directly examined the function of TIM-2 in murine and human T cell lines. Our results indicate that expression of TIM-2 significantly impairs the induction of NFAT and AP-1 transcriptional reporters by not only TCR ligation but also by the pharmacological stimuli PMA and ionomycin. This does not appear to be due to a general effect on cell viability, and the block in NFAT activation can be bypassed by expression of activated alleles of Ras or calcineurin, or MEK kinase, in the case of AP-1. Thus, our data are consistent with a model whereby TIM-2 inhibits T cell activation.
Effect of Lipid Rafts on Cb2 Receptor Signaling and 2-Arachidonoyl-Glycerol Metabolism in Human Immune Cells
Authors: Bari Monica , Spagnuolo Paola , Fezza Filomena , Oddi Sergio , Pasquariello Nicoletta , Finazzi-Agrò Alessandro , Maccarrone Mauro
Published: April 2014

Recently, we have shown that treatment of rat C6 glioma cells with the raft disruptor methyl-β-cyclodextrin (MCD) doubles the binding of anandamide (AEA) to type-1 cannabinoid receptors (CB1R), followed by CB1R-dependent signaling via adenylate cyclase and p42/p44 MAPK activity. In the present study, we investigated whether type-2 cannabinoid receptors (CB2R), widely expressed in immune cells, also are modulated by MCD. We show that treatment of human DAUDI leukemia cells with MCD does not affect AEA binding to CB2R, and that receptor activation triggers similar [35S]guanosine-5′-O-(3-thiotriphosphate) binding in MCD-treated and control cells, similar adenylate cyclase and MAPK activity, and similar MAPK-dependent protection against apoptosis. The other AEA-binding receptor transient receptor potential channel vanilloid receptor subunit 1, the AEA synthetase N-acyl-phosphatidylethanolamine-phospholipase D, and the AEA hydrolase fatty acid amide hydrolase were not affected by MCD, whereas the AEA membrane transporter was inhibited (∼55%) compared with controls. Furthermore, neither diacylglycerol lipase nor monoacylglycerol lipase, which respectively synthesize and degrade 2-arachidonoylglycerol, were affected by MCD in DAUDI or C6 cells, whereas the transport of 2-arachidonoylglycerol was reduced to ∼50%. Instead, membrane cholesterol enrichment almost doubled the uptake of AEA and 2-arachidonoylglycerol in both cell types. Finally, transfection experiments with human U937 immune cells, and the use of primary cells expressing CB1R or CB2R, ruled out that the cellular environment could account per se for the different modulation of CB receptor subtypes by MCD. In conclusion, the present data demonstrate that lipid rafts control CB1R, but not CB2R, and endocannabinoid transport in immune and neuronal cells.
DAP12 Signaling Directly Augments Proproliferative Cytokine Stimulation of NK Cells during Viral Infections
Authors: French Anthony R. , Sjölin Hanna , Kim Sungjin , Koka Rima , Yang Liping , Young Deborah A. , Cerboni Cristina , Tomasello Elena , Ma Averil , Vivier Eric , Kärre Klas , Yokoyama Wayne M.
Published: April 2014

NK cells vigorously proliferate during viral infections. During the course of murine CMV infection, this response becomes dominated by the preferential proliferation of NK cells that express the activation receptor Ly49H. The factors driving such selective NK cell proliferation have not been characterized. In this study, we demonstrate that preferential NK cell proliferation is dependent on DAP12-mediated signaling following the binding of Ly49H to its virally encoded ligand, m157. Ly49H signaling through DAP12 appears to directly augment NK cell sensitivity to low concentrations of proproliferative cytokines such as IL-15. The impact of Ly49H-mediated signaling on NK cell proliferation is masked in the presence of high concentrations of proproliferative cytokines that nonselectively drive all NK cells to proliferate.
Differential Usage of Cellular Niches by Cytomegalovirus versus EBV- and Influenza Virus-Specific CD8+ T Cells
Authors: van Leeuwen Ester M. M. , Koning Jasper J. , Remmerswaal Ester B. M. , van Baarle Debbie , van Lier René A. W. , ten Berge Ineke J. M.
Published: April 2014

Immunological memory provides long-term protection against reinfection or reactivation of pathogens. Murine memory T cell populations may be compressed following infections with new pathogens. Humans have to retain memory T cells directed against a variety of microbes for many decades. Under these circumstances, the effect of pathogens that mount robust T cell reactivity on the pre-existing memory directed against unrelated microbes is unknown. In this study, we studied peripheral blood memory CD8+ T cells directed against different viruses following primary CMV infection in renal transplant recipients. The entrance of CMV-specific CD8+ T cells expanded the Ag-primed CD8+ T cell compartment rather than competing for space with pre-existing memory T cells specific for persistent or cleared viruses. Neither numbers nor phenotype of EBV- or influenza-specific CD8+ T cells was altered by primary CMV infection. CMV-specific CD8+ T cells accumulated over time, resulting in increased total CD8+ T cell numbers. Additionally, they acquired a highly differentiated cytolytic phenotype that was clearly distinct from EBV- or influenza-reactive T cells. Thus, the human immune system appears to be flexible and is able to expand when encountering CMV. In view of the phenotypic differences between virus-specific T cells, this expansion may take place in cellular niches different from those occupied by EBV- or influenza-specific T cells, thereby preserving immunity to these pathogens.
A Preferential Role for STAT5, not Constitutively Active STAT3, in Promoting Survival of a Human Lymphoid Tumor
Authors: Nagy Zsuzsanna S. , Rui Hallgeir , Stepkowski Stanislaw M. , Karras James , Kirken Robert A.
Published: April 2014

STATs are believed to play key roles in normal and abnormal cell function. In the present work, we investigated the role of STATs in an IL-2-responsive human lymphoblastic lymphoma-derived cell line, YT. Only STAT3 was found constitutively tyrosine phosphorylated, but not other STATs. Hyperactive STAT3 was not attributable to a pre-existing intermediate affinity IL-2R complex and/or hyperactive Jak activity. Depletion of STAT3 protein expression reduced tumor cell viability with protracted kinetics (72–96 h), while TUNEL assays demonstrated cell death occurred via apoptosis. Interestingly, depletion of STAT5 in this same tumor induced more pronounced cell death compared with STAT3 depletion (24 h). Although IL-2 was able to rescue STAT3-depleted cells from death, it could not compensate for the loss of STAT5. To determine the prosurvival function of STAT3 vs STAT5 within the same tumor model, genes were profiled in STAT3- or STAT5-depleted YT cells by apoptosis-specific microarrays. Several differentially expressed genes were identified. Interestingly, those genes involved in NF-κB regulation, such as TNFR-associated factors 2 and 5 and B cell leukemia/lymphoma 10, were readily decreased upon STAT5, but not STAT3, depletion as validated by quantitative RT-PCR. These results suggest that STAT5 and, to a lesser extent, hyperactive STAT3 provide preferential and critical cell survival signals for certain human lymphoid tumors, indicating that nonhyperactive STATs should be considered as therapeutic targets for abrogating tumorigenesis.
The Peroxisome Proliferator-Activated Receptor γ (PPARγ) Ligands 15-Deoxy-Δ12,14-Prostaglandin J2 and Ciglitazone Induce Human B Lymphocyte and B Cell Lymphoma Apoptosis by PPARγ-Independent Mechanisms
Authors: Ray Denise M. , Akbiyik Filiz , Phipps Richard P.
Published: April 2014

Peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor important for adipogenesis and more recently has been shown to be an anticancer target. PPARγ ligands, including the endogenous ligand 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) and synthetic ligands like ciglitazone and troglitazone, all induce apoptosis in normal and malignant human B lymphocytes, but the dependency of PPARγ for apoptosis induction is unknown. In this study, we used a PPARγ dominant-negative approach and a small molecule irreversible PPARγ antagonist and found that these inhibitors prevented PPARγ activation but did not prevent B cell apoptosis induced by 15d-PGJ2 or ciglitazone. In addition, a PPARγ agonist that is a structural analog of 15d-PGJ2, and lacks the electrophilic carbon of the 15d-PGJ2 cyclopentenone ring, activated PPARγ but did not kill B lymphocytes, further supporting a non-PPARγ-mediated mechanism. To further investigate the apoptotic mechanism, the effects of 15d-PGJ2 and ciglitazone on reactive oxygen species were investigated. 15d-PGJ2, but not ciglitazone, potently induced reactive oxygen species in B lymphocytes, implicating the reactive nature of the 15d-PGJ2 structure in the apoptosis mechanism. In addition, 15d-PGJ2 caused an almost complete depletion of intracellular glutathione. Moreover, incubation with glutathione reduced ethyl ester, an antioxidant, prevented apoptosis induced by 15d-PGJ2, but not by ciglitazone. These findings indicate that the expression of PPARγ may not be predictive of whether a normal or malignant B lineage cell is sensitive to PPARγ agonists. Furthermore, these new findings support continued investigation into the use of PPARγ agonists as agents to attenuate normal B cell responses and as anti-B cell lymphoma agents.
Levels of Specific Peptide-HLA Class I Complex Predicts Tumor Cell Susceptibility to CTL Killing
Authors: Weidanz Jon A. , Nguyen Tiffany , Woodburn Tito , Neethling Francisca A. , Chiriva-Internati Maurizio , Hildebrand William H. , Lustgarten Joseph
Published: April 2014

Recognition of tumor-associated Ags (TAAs) on tumor cells by CTLs and the subsequent tumor cell death are assumed to be dependent on TAA protein expression and to correlate directly with the level of peptide displayed in the binding site of the HLA class I molecule. In this study we evaluated whether the levels of Her-2/neu protein expression on human tumor cell lines directly correlate with HLA-A*0201/Her2/neu peptide presentation and CTL recognition. We developed a TCR mimic (TCRm) mAb designated 1B8 that specifically recognizes the HLA-A2.1/Her2/neu peptide (369–377) (Her2(369)-A2) complex. TCRm mAb staining intensity varied for the five human tumor cell lines analyzed, suggesting quantitative differences in levels of the Her2(369)-A2 complex on these cells. Analysis of tumor cell lines pretreated with IFN-γ and TNF-α for Her2/neu protein and HLA-A2 molecule expression did not reveal a direct correlation between the levels of Her2/neu Ag, HLA-A2 molecule, and Her2(369)-A2 complex expression. However, compared with untreated cells, cytokine-treated cell lines showed an increase in Her2(369)-A2 epitope density that directly correlated with enhanced tumor cell death (p = 0.05). Although a trend was observed between tumor cell lysis and the level of the Her2(369)-A2 complex for untreated cells, the association was not significant. These findings suggest that tumor cell susceptibility to CTL-mediated lysis may be predicted based on the level of specific peptide-MHC class I expression rather than on the total level of TAA expression. Further, these studies demonstrate the potential of the TCRm mAb for validation of endogenous HLA-peptide epitopes on tumor cells.
H2-M3-Restricted T Cells Participate in the Priming of Antigen-Specific CD4+ T Cells
Authors: Chow Michael T. , Dhanji Salim , Cross Jennifer , Johnson Pauline , Teh Hung-Sia
Published: April 2014

H2-M3-restricted CD8+ T cells provide early protection against bacterial infections. In this study, we demonstrate that activated H2-M3-restricted T cells provide early signals for efficient CD4+ T cell priming. C57BL/6 mice immunized with dendritic cells coated with the MHC class II-restricted listeriolysin O peptide LLO190–201 (LLO) generated CD4+ T cells capable of responding to Listeria monocytogenes (LM) infection. Inclusion of a H2-M3-restricted formylated peptide fMIGWII (fMIG), but not MHC class Ia-restricted peptides, during immunization with LLO significantly increased IFN-γ-producing CD4+ T cell numbers, which was associated with increased protection against LM infection. Studies with a CD4+ T cell-depleting mAb indicate that the reduction in bacterial load in fMIG plus LLO immunized mice is likely due to augmented numbers of LLO-specific CD4+ T cells, generated with the help of H2-M3-restricted CD8+ T cells. We also found that augmentation of LLO-specific CD4+ T lymphocytes with H2-M3-restricted T cells requires presentation of LLO and fMIG by the same dendritic cells. Interestingly, the augmented CD4+ T cell response generated with fMIG also increased primary LM-specific responses by MHC class Ia-restricted CD8 T cells. Coimmunization with LLO and fMIG also increases the number of memory Ag-specific CD4+ T cells. We also demonstrate that CD8 T cells restricted to another MHC class Ib molecule, Qa-1, whose human equivalent is HLA-E, are also able to enhance Ag-specific CD4+ T cell responses. These results reveal a novel function for H2-M3- and Qa-1-restricted T cells; provision of help to CD4+ Th cells during the primary response.
Tetraspanins CD9 and CD81 Modulate HIV-1-Induced Membrane Fusion
Authors: Gordón-Alonso Mónica , Yañez-Mó María , Barreiro Olga , Álvarez Susana , Muñoz-Fernández M. Ángeles , Valenzuela-Fernández Agustín , Sánchez-Madrid Francisco
Published: April 2014

Protein organization on the membrane of target cells may modulate HIV-1 transmission. Since the tetraspanin CD81 is associated to CD4, the receptor of HIV-1 envelope protein (Env; gp120/gp41), we have explored the possibility that this molecule may modulate the initial steps of HIV-1 infection. On the other hand, CD81 belongs to the tetraspanin family, which has been described as organizers of protein microdomains on the plasma membrane. Therefore, the role of CD81 and other related tetraspanin, CD9, on the cell-to-cell fusion process mediated by HIV-1 was studied. We found that anti-tetraspanin Abs enhanced the syncytia formation induced by HIV-1 envelope proteins and viral entry in human T lymphoblasts. In addition, anti-CD81 Abs triggered its clustering in patches, where CD4 and CXCR4 were included. Moreover, the knocking down of CD81 and CD9 expression resulted in an increase in syncytia formation and viral entry. Accordingly, overexpression of CD81 and CD9 rendered cells less susceptible to Env-mediated syncytia formation. These data indicate that CD9 and CD81 have an important role in membrane fusion induced by HIV-1 envelope.
IL-10- and IL-12-Independent Down-Regulation of Allergic Sensitization by Stimulation of CD40 Signaling
Authors: Hellings Peter W. , Kasran Ahmad , Bullens Dominique , Overbergh Lutgart , Mathieu Chantal , Heremans Hubertine , Matthys Patrick , Boon Louis , Jorissen Mark , Ceuppens Jan L.
Published: April 2014

Interaction between CD154 (CD40 ligand) on activated T lymphocytes and its receptor CD40 has been shown to be critically involved in the generation of cell-mediated as well as humoral immunity. CD40 triggering activates dendritic cells (DC), enhances their cytokine production, up-regulates the expression of costimulatory molecules, and induces their maturation. It is unknown how stimulation of CD40 during sensitization to an airborne allergen may affect the outcome of allergic airway inflammation. We took advantage of a mouse model of allergic asthma and a stimulatory mAb to CD40 (FGK45) to study the effects of CD40-mediated DC activation on sensitization to OVA and subsequent development of OVA-induced airway inflammation. Agonistic anti-CD40 mAb (FGK45) injected during sensitization with OVA abrogated the development of allergic airway inflammation upon repeated airway challenges with OVA. Inhibition of bronchial eosinophilia corresponded with reduced Th2 cytokine production and was independent of IL-12, as evidenced by a similar down-regulatory effect of anti-CD40 mAb in IL-12 p40-deficient mice. In addition, FGK45 equally down-regulated allergic airway inflammation in IL-10-deficient mice, indicating an IL-10-independent mechanism of action of FGK45. In conclusion, our results show that CD40 signaling during sensitization shifts the immune response away from Th2 cytokine production and suppresses allergic airway inflammation in an IL-12- and IL-10-independent way, presumably resulting from enhanced DC activation during sensitization.
Autophagy Is Induced in CD4+ T Cells and Important for the Growth Factor-Withdrawal Cell Death
Authors: Li Changyou , Capan Elizabeth , Zhao Yani , Zhao Jianping , Stolz Donna , Watkins Simon C. , Jin Shengkan , Lu Binfeng
Published: April 2014

Autophagy is a tightly regulated catabolic mechanism that degrades proteins and organelles. Autophagy mediates programmed cell death under certain conditions. To determine the role of autophagy in T cells, we examined, in mouse CD4+ T cells, conditions under which autophagy is induced and alterations of the cell fate when autophagy is blocked. We have found that resting naive CD4+ T cells do not contain detectable autophagosomes. Autophagy can be observed in activated CD4+ T cells upon TCR stimulation, cytokine culturing, and prolonged serum starvation. Induction of autophagy in T cells requires JNK and the class III PI3K. Autophagy is inhibited by caspases and mammalian target of rapamycin in T cells. Interestingly, more Th2 cells than Th1 cells undergo autophagy. Th2 cells become more resistant to growth factor-withdrawal cell death when autophagy is blocked using either chemical inhibitors 3-methyladenine, or by RNA interference knockdown of beclin 1 and Atg7. Therefore, autophagy is an important mechanism that controls homeostasis of CD4+ T cells.
The Cyclin-Dependent Kinase Inhibitor p27kip1 Is Required for Transplantation Tolerance Induced by Costimulatory Blockade
Authors: Rowell Emily A. , Wang Liqing , Hancock Wayne W. , Wells Andrew D.
Published: April 2014

The cyclin-dependent kinase (CDK) inhibitor p27kip1 is an important negative regulator of the cell cycle that sets a threshold for mitogenic signals in T lymphocytes, and is required for T cell anergy in vitro. To determine whether p27kip1 is required for tolerance in vivo, we performed cardiac allograft transplantation under conditions of combined CD28/CD40L costimulatory blockade. Although this treatment induced long-term allograft survival in wild-type recipients, costimulatory blockade was no longer sufficient to induce tolerance in mice lacking p27kip1. Rejected allografts from p27kip1−/− mice contained more CD4+ T lymphocytes and exhibited more tissue damage than allografts from tolerant, wild-type mice. Infiltrating p27kip1-deficient T cells, but not wild-type T cells, exhibited nuclear expression of cyclins E and A, indicating uncontrolled T cell cycle progression in the graft. The failure of tolerance in p27kip1−/− mice was also accompanied by markedly increased numbers of allospecific, IFN-γ-producing cells in the periphery, and occurred despite apparently normal regulatory T cell activity. These data demonstrate that the CDK inhibitor p27kip1 enforces the costimulatory requirement for the expansion and differentiation of alloimmune effector T lymphocytes in vivo, and point to CDKs as novel targets for immunosuppressive or tolerance-inducing therapies.
Distinct Characteristics of Murine STAT4 Activation in Response to IL-12 and IFN-α
Authors: Berenson Lisa S. , Gavrieli Maya , Farrar J. David , Murphy Theresa L. , Murphy Kenneth M.
Published: April 2014

The role of type I IFN in Th1 development, STAT4 activation, and IFN-γ production in murine T cells has remained unresolved despite extensive examination. Initial studies indicated that IFN-α induced Th1 development and IFN-γ production in human, but not murine, T cells, suggesting species-specific differences in signaling. Later studies suggested that IFN-α also induced Th1 development in mice, similar to IL-12. More recent studies have questioned whether IFN-α actually induces Th1 development even in the human system. In the present study, we compared the capacity of IL-12 and IFN-α to induce Th1 differentiation, STAT4 phosphorylation, and IFN-γ production in murine T cells. First, we show that IFN-α, in contrast to IL-12, cannot induce Th1 development. However, in differentiated Th1 cells, IFN-α can induce transient, but not sustained, STAT4 phosphorylation and, in synergy with IL-18, can induce transient, but not sustained, IFN-γ production in Th1 cells, in contrast to the sustained actions of IL-12. Furthermore, loss of STAT1 increases IFN-α-induced STAT4 phosphorylation, but does not generate levels of STAT4 activation or IFN-γ production achieved by IL-12 or convert transient STAT4 activation into a sustained response. Our findings agree with recent observations in human T cells that IFN-α-induced STAT4 activation is transient and unable to induce Th1 development, and indicate that IFN-α may act similarly in human and murine T cells.
IL-4-Secreting NKT Cells Prevent Hypersensitivity Pneumonitis by Suppressing IFN-γ-Producing Neutrophils
Authors: Hwang Su Jin , Kim Sanghee , Park Weon Seo , Chung Doo Hyun
Published: April 2014

Hypersensitivity pneumonitis (HP) is mediated by Th1 immune response. NKT cells regulate immune responses by modulating the Th1/Th2 balance. Therefore, we postulated that NKT cells play a critical role in the development of the HP by modulating the Th1/Th2 response. To address this issue, we explored the functional roles of NKT cells in Saccharopolyspora rectivirgula (SR)-induced HP. In CD1d−/− mice, the HP was worse in terms of histological changes, hydroxyproline levels, the CD4:CD8 ratio in bronchoalveolar lavage fluid, and SR-specific immune responses than in control mice. CD1d−/− mice showed elevated IFN-γ production in the lung during the HP, and this was produced mainly by Gr-1+ neutrophils. The blockade of IFN-γ in CD1d−/− mice attenuated the HP, whereas the injection of rIFN-γ aggravated it. Moreover, the depletion of Gr-1+ neutrophils reduced CD8+ T cell numbers in bronchoalveolar lavage fluid during the HP. The adoptive transfer of IL-4−/− mouse NKT cells did not attenuate the HP, whereas wild-type or IFN-γ−/− mouse NKT cells suppressed the HP. In conclusion, NKT cells producing IL-4 play a protective role in SR-induced HP by suppressing IFN-γ-producing neutrophils, which induce the activation and proliferation of CD8+ T cells in the lung.
Innate Response to Focal Necrotic Injury Inside the Blood-Brain Barrier
Authors: Kim Jiyun V. , Dustin Michael L.
Published: April 2014

We have studied the initial innate immune response to focal necrotic injury on different sides of the mouse blood-brain barrier by two-photon intravital microscopy. Transgenic mice in which the promoter of the myeloid isoform of lysozyme drives GFP were used to track granulocytes and monocytes. Necrotic injury in the meninges, but not the brain parenchyma, recruited GFP+ cells within minutes that fully surrounded the necrotic site within a day. Recently, it has been suggested that microglial cells and astrocytes cooperate to mount a distinct response to laser injury behind the blood-brain barrier. We followed the microglial response in heterozygous knockin mice in which GFP replaces CX3CR1 coding sequence. Prior to injury, microglial cell bodies were immobile over days, but moved to the laser injury site within 1 day. We followed astrocytes, which have been proposed to cooperate with microglial cells in response to focal injury, using transgenic mice in which glial fibrillary acidic protein promoter drives GFP expression. Before injury fine astrocyte processes permeate the parenchyma. Astrocytes polarized toward the injury in an ATP, connexin hemichannels, and intracellular Ca2+-dependent process. The astrocytes network established a cytoplasmic Ca2+ gradient that preceded the microglial response. This is consistent with astrocyte-microglial collaboration to mount this innate response that excludes blood leukocytes.
CXCR5 Identifies a Subset of Vγ9Vδ2 T Cells which Secrete IL-4 and IL-10 and Help B Cells for Antibody Production
Authors: Caccamo Nadia , Battistini Luca , Bonneville Marc , Poccia Fabrizio , Fournié Jean Jacques , Meraviglia Serena , Borsellino Giovanna , Kroczek Richard A. , La Mendola Carmela , Scotet Emmanuel , Dieli Francesco , Salerno Alfredo
Published: April 2014

Vγ9Vδ2 T lymphocytes recognize nonpeptidic Ags and mount effector functions in cellular immune responses against microorganisms and tumors, but little is known about their role in Ab-mediated immune responses. We show here that expression of CXCR5 identifies a unique subset of Vγ9Vδ2 T cells which express the costimulatory molecules ICOS and CD40L, secrete IL-2, IL-4, and IL-10 and help B cells for Ab production. These properties portray CXCR5+Vγ9Vδ2 T cells as a distinct memory T cell subset with B cell helper function.
Suppression of Experimental Autoimmune Myasthenia Gravis by Granulocyte-Macrophage Colony-Stimulating Factor Is Associated with an Expansion of FoxP3+ Regulatory T Cells
Authors: Sheng Jian Rong , Li Liangcheng , Ganesh Balaji B. , Vasu Chenthamarakshan , Prabhakar Bellur S. , Meriggioli Matthew N.
Published: April 2014

Dendritic cells (DCs) have the potential to activate or tolerize T cells in an Ag-specific manner. Although the precise mechanism that determines whether DCs exhibit tolerogenic or immunogenic functions has not been precisely elucidated, growing evidence suggests that DC function is largely dependent on differentiation status, which can be manipulated using various growth factors. In this study, we investigated the effects of mobilization of specific DC subsets—using GM-CSF and fms-like tyrosine kinase receptor 3-ligand (Flt3-L)—on the susceptibility to induction of experimental autoimmune myasthenia gravis (EAMG). We administered GM-CSF or Flt3-L to C57BL/6 mice before immunization with acetylcholine receptor (AChR) and observed the effect on the frequency and severity of EAMG development. Compared with AChR-immunized controls, mice treated with Flt3-L before immunization developed EAMG at an accelerated pace initially, but disease frequency and severity was comparable at the end of the observation period. In contrast, GM-CSF administered before immunization exerted a sustained suppressive effect against the induction of EAMG. This suppression was associated with lowered serum autoantibody levels, reduced T cell proliferative responses to AChR, and an expansion in the population of FoxP3+ regulatory T cells. These results highlight the potential of manipulating DCs to expand regulatory T cells for the control of autoimmune diseases such as MG.
Absence of Innate MyD88 Signaling Promotes Inducible Allograft Acceptance
Authors: Walker Wendy E. , Nasr Isam W. , Camirand Geoffrey , Tesar Bethany M. , Booth Carmen J. , Goldstein Daniel R.
Published: April 2014

Prior experimental strategies to induce transplantation tolerance have focused largely on modifying adaptive immunity. However, less is known concerning the role of innate immune signaling in the induction of transplantation tolerance. Using a highly immunogenic murine skin transplant model that resists transplantation tolerance induction when innate immunity is preserved, we show that absence of MyD88, a key innate Toll like receptor signal adaptor, abrogates this resistance and facilitates inducible allograft acceptance. In our model, absence of MyD88 impairs inflammatory dendritic cell responses that reduce T cell activation. This effect increases T cell susceptibility to suppression mediated by CD4+CD25+ regulatory T cells. Therefore, this study provides evidence that absence of MyD88 promotes inducible allograft acceptance and implies that inhibiting innate immunity may be a potential, clinically relevant strategy to facilitate transplantation tolerance.
SAP Is Required for Th Cell Function and for Immunity to Influenza
Authors: Kamperschroer Cris , Dibble John P. , Meents Dana L. , Schwartzberg Pamela L. , Swain Susan L.
Published: April 2014

Ab is a crucial component of protective immunity to infection, but Ab responses do not proceed normally when defects occur in a protein called signaling lymphocytic activation molecule-associated protein (SAP). To explain this Ab defect, we analyzed B cell and plasma cell responses under conditions of SAP deficiency. Our results demonstrate that SAP-deficient (SAP knockout (KO)) mice have a profound CD4 T cell-intrinsic defect in generating Ag-specific plasma cells following challenge with model Ags or influenza virus, resulting in low Ag-specific Ab titers. We also show that SAP is required in CD4 T cells for normal division and expansion of B cells. These B cell and plasma cell defects were observed during the expansion phase of the primary immune response, indicating early defects in Th cell activity. In fact, additional experiments revealed a nearly complete lack of T cell help for B cells in SAP KO mice. Our work suggests that the ability of SAP to promote T-dependent humoral immune responses is important for antiviral immunity because mice lacking SAP are unable to prevent high dose secondary influenza infection, and because passive transfer of IgG in immune serum from wild-type, but not SAP KO mice can protect mice from an otherwise lethal influenza infection. Overall, our results demonstrate that SAP is required in CD4 T cells for their ability to help B cell responses and promote influenza-specific immunity.
Galectin-1 Binds Different CD43 Glycoforms to Cluster CD43 and Regulate T Cell Death
Authors: Hernandez Joseph D. , Nguyen Julie T. , He Jiale , Wang Wei , Ardman Blair , Green Jonathan M. , Fukuda Minoru , Baum Linda G.
Published: April 2014

Galectin-1 kills immature thymocytes and activated peripheral T cells by binding to glycans on T cell glycoproteins including CD7, CD45, and CD43. Although roles for CD7 and CD45 in regulating galectin-1-induced death have been described, the requirement for CD43 remains unknown. We describe a novel role for CD43 in galectin-1-induced death, and the effects of O-glycan modification on galectin-1 binding to CD43. Loss of CD43 expression reduced galectin-1 death of murine thymocytes and human T lymphoblastoid cells, indicating that CD43 is required for maximal T cell susceptibility to galectin-1. CD43, which is heavily O-glycosylated, contributes a significant fraction of galectin-1 binding sites on T cells, as T cells lacking CD43 bound ∼50% less galectin-1 than T cells expressing CD43. Although core 2 modification of O-glycans on other glycoprotein receptors is critical for galectin-1-induced cross-linking and T cell death, galectin-1 bound to CD43 fusion proteins modified with either unbranched core 1 or branched core 2 O-glycans and expression of core 2 O-glycans did not enhance galectin-1 binding to CD43 on T cells. Moreover, galectin-1 binding clustered CD43 modified with either core 1 or core 2 O-glycans on the T cell surface. Thus, CD43 bearing either core 1 or core 2 O-glycans can positively regulate T cell susceptibility to galectin-1, identifying a novel function for CD43 in controlling cell death. In addition, these studies demonstrate that different T cell glycoproteins on the same cell have distinct requirements for glycan modifications that allow recognition and cross-linking by galectin-1.
A Single Polymorphism Disrupts the Killer Ig-Like Receptor 2DL2/2DL3 D1 Domain
Authors: VandenBussche Christopher J. , Dakshanamurthy Sivanesan , Posch Phillip E. , Hurley Carolyn Katovich
Published: April 2014

Genetic polymorphisms found in the killer Ig-like receptor (KIR), two domains, long cytoplasmic tail 2/3 (KIR2DL2/3) locus are responsible for the differential binding of KIR2DL2/3 allelic products with their HLA-C ligands and have been associated with the resolution of hepatitis C infection. In our study, a KIR CD3ζ fusion-binding assay did not detect any interaction between the KIR2DL2*004 extracellular domain and several putative KIR2DL2/3 ligands. To determine the amino acid polymorphism(s) responsible for the KIR2DL2*004 phenotype, we mutated the polymorphic residues of full-length KIR and expressed them in human Jurkat cells. Flow cytometry analysis failed to detect the surface expression of receptors containing a threonine at position 41 (T41), a polymorphism specific to KIR2DL2*004. Confocal microscopy showed that receptors containing T41 were retained inside the cell and had a perinuclear localization, possibly indicating that their extracellular domain was misfolded. Most KIR2DL2/3 alleles possess an arginine at position 41 (R41), and we predicted through molecular modeling and demonstrated by mutagenesis that R41 most likely interacts with the nearby residues Y77 and D47. Interaction between these residues would maintain C strand contact with the C′ and F strands of the D1 domain β-sheet. Furthermore, R41 and Y77 are conserved in the C and F strand amino acid alignments of Ig-like superfamily members, and may therefore be necessary for the structural integrity of other immune response proteins. Our data indicate that the extracellular T41 polymorphism encoded by the KIR2DL2*004 allele most likely results in misfolding of the D1 domain and complete intracellular retention of the receptor.
Differential and Nonredundant Roles of Phospholipase Cγ2 and Phospholipase Cγ1 in the Terminal Maturation of NK Cells
Authors: Regunathan Jeyarani , Chen Yuhong , Kutlesa Snjezana , Dai Xuezhi , Bai Li , Wen Renren , Wang Demin , Malarkannan Subramaniam
Published: April 2014

NK cells play a central role in mediating innate immune responses. Activation of NK cells results in cytotoxicity, cytokine, and chemokine secretions. In this study, we show that in mice with targeted deletion of phospholipase Cγ (PLCγ)2, one of the key signal transducers, there are profound effects on the development and terminal maturation of NK cells. Lack of PLCγ2 significantly impaired the ability of lineage-committed NK precursor cells to acquire subset-specific Ly49 receptors and thereby terminal maturation of NK cells. Overexpression of isozyme, PLCγ1, in PLCγ2-deficient NK cells resulted in the successful Ly49 acquisition and terminal maturation of the NK cells; however, it could only partially rescue NKG2D-mediated cytotoxicity with no cytokine production. Furthermore, PLCγ2-deficient NK cells failed to mediate antitumor cytotoxicity and inflammatory cytokine production, displaying a generalized hyporesponsiveness. Our results strongly demonstrate that PLCγ1 and PLCγ2 play nonredundant and obligatory roles in NK cell ontogeny and in its effector functions.