bluuebunny/crossref_metadata_2025 huggingface dataset:
CTLA-4 and CD4+CD25+ Regulatory T Cells Inhibit Protective Immunity to Filarial Parasites In Vivo
Authors: Taylor Matthew D. , Harris Anjanette , Babayan Simon A. , Bain Odile , Culshaw Abigail , Allen Judith E. , Maizels Rick M.
Published: April 2014

The T cell coinhibitory receptor CTLA-4 has been implicated in the down-regulation of T cell function that is a quintessential feature of chronic human filarial infections. In a laboratory model of filariasis, Litomosoides sigmodontis infection of susceptible BALB/c mice, we have previously shown that susceptibility is linked both to a CD4+CD25+ regulatory T (Treg) cell response, and to the development of hyporesponsive CD4+ T cells at the infection site, the pleural cavity. We now provide evidence that L. sigmodontis infection drives the proliferation and activation of CD4+Foxp3+ Treg cells in vivo, demonstrated by increased uptake of BrdU and increased expression of CTLA-4, Foxp3, GITR, and CD25 compared with naive controls. The greatest increases in CTLA-4 expression were, however, seen in the CD4+Foxp3− effector T cell population which contained 78% of all CD4+CTLA-4+ cells in the pleural cavity. Depletion of CD25+ cells from the pleural CD4+ T cell population did not increase their Ag-specific proliferative response in vitro, suggesting that their hyporesponsive phenotype is not directly mediated by CD4+CD25+ Treg cells. Once infection had established, killing of adult parasites could be enhanced by neutralization of CTLA-4 in vivo, but only if performed in combination with the depletion of CD25+ Treg cells. This work suggests that during filarial infection CTLA-4 coinhibition and CD4+CD25+ Treg cells form complementary components of immune regulation that inhibit protective immunity in vivo.
NK Dendritic Cells Expanded in IL-15 Exhibit Antitumor Responses In Vivo
Authors: Chaudhry Umer I. , Plitas George , Burt Bryan M. , Kingham T. Peter , Raab Jesse R. , DeMatteo Ronald P.
Published: April 2014

NK dendritic cells (NKDC) are a novel subtype of DC with NK cell properties. IL-15 is a pleiotropic cytokine that plays an obligate role in the proliferation and survival of NK cells. We hypothesized that IL-15 is also essential for NKDC development. NKDC were nearly absent in IL-15−/− mice, but restored by administration of exogenous IL-15. Treatment of wild-type mice with IL-15 caused a 2- to 3-fold expansion of both NK cells and NKDC. After 7 days of culture with IL-15, sorted splenic NKDC expanded 10-fold while NK cells increased 5-fold. NKDC expanded in IL-15 retained their cytolytic capacity but lost the ability to stimulate naive T cells. Meanwhile, NKDC expanded in IL-15 produced 10 times more IFN-γ as fresh NKDC and conferred protection in a tumor prevention model. Thus, IL-15 is essential to the proliferation and survival of NKDC and IL-15 expanded NKDC possess antitumor properties.
iNKT Cells Require CCR4 to Localize to the Airways and to Induce Airway Hyperreactivity
Authors: Meyer Everett H. , Wurbel Marc-André , Staton Tracy L. , Pichavant Muriel , Kan Matthew J. , Savage Paul B. , DeKruyff Rosemarie H. , Butcher Eugene C. , Campbell James J. , Umetsu Dale T.
Published: April 2014

iNKT cells are required for the induction of airway hyperreactivity (AHR), a cardinal feature of asthma, but how iNKT cells traffic to the lungs to induce AHR has not been previously studied. Using several models of asthma, we demonstrated that iNKT cells required the chemokine receptor CCR4 for pulmonary localization and for the induction of AHR. In both allergen-induced and glycolipid-induced models of AHR, wild-type but not CCR4−/− mice developed AHR. Furthermore, adoptive transfer of wild-type but not CCR4−/− iNKT cells reconstituted AHR in iNKT cell-deficient mice. Moreover, we specifically tracked CCR4−/− vs wild-type iNKT cells in CCR4−/−:wild-type mixed BM chimeric mice in the resting state, and when AHR was induced by protein allergen or glycolipid. Using this unique model, we showed that both iNKT cells and conventional T cells required CCR4 for competitive localization into the bronchoalveolar lavage/airways compartment. These results establish for the first time that the pulmonary localization of iNKT cells critical for the induction of AHR requires CCR4 expression by iNKT cells.
CS1 (CRACC, CD319) Induces Proliferation and Autocrine Cytokine Expression on Human B Lymphocytes
Authors: Lee Jae Kyung , Mathew Stephen O. , Vaidya Swapnil V. , Kumaresan Pappanaicken R. , Mathew Porunelloor A.
Published: April 2014

CS1 (CRACC, CD319), a member of the CD2 family of cell surface receptors, is implicated in the activation of NK cell-mediated cytotoxicity. Previous studies showed that CS1 is also expressed on activated B cells. However, the functional role of CS1 in human B-lymphocytes is not known. Two isoforms of CS1, CS1-L and CS1-S, are expressed in human NK cells that differentially regulate NK cell function. CS1-L contains immunoreceptor tyrosine-based switch motifs in its cytoplasmic domain whereas CS1-S lacks immunoreceptor tyrosine-based switch motifs. In this study, we show that human B lymphocytes express only the CS1-L isoform, and its expression is up-regulated upon B cell activation with various stimulators. Moreover, anti-CS1 mAb strongly enhanced proliferation of both freshly isolated as well as activated B cells. The enhanced proliferation effects of CS1 were most prominent on B cells activated by anti-CD40 mAbs and/or hrIL-4. The effects of CS1 on B cell proliferation were shown on both naive and memory B cells. Human cytokine microarray and quantitative real-time PCR results indicated that CS1 activation enhanced mRNA transcripts of flt3 ligand, lymphotoxin A, TNF, and IL-14. Neutralizing Abs against lymphotoxin A, TNF-α, and/or flt3 ligand abolished the ability of CS1 on the B cell proliferation. These results suggest that activation of B lymphocytes, through surface CS1, may be mediated through secretion of autocrine cytokines and CS1 may play a role in the regulation of B lymphocyte proliferation during immune responses.
Krüppel-Like Factor 4 Regulates B Cell Number and Activation-Induced B Cell Proliferation
Authors: Klaewsongkram Jettanong , Yang Yinhua , Golech Susanne , Katz Jonathan , Kaestner Klaus H. , Weng Nan-ping
Published: April 2014

Krüppel-like factor 4 (Klf4) is a transcription factor and functions in regulating cell differentiation, cell growth, and cell cycle. Although Klf4 is expressed in lymphocytes, its function in lymphocytes is unknown. In this study, we report that the levels of Klf4 expression were low in pro-B cells and continuously increased in pre-B and in mature B cells. Upon activation, Klf4 was rapidly decreased in mature B cells after 2 h of activation. A modest decrease in numbers of pre-B cells in bone marrow and mature B cells in spleen was observed in Klf4-deficient mice. In the absence of Klf4, fewer B cells entered the S phase of the cell cycle and completed cell division in response to the engagement of BCR and/or CD40 in vitro. Furthermore, the delay in entering the cell cycle is associated with decreased expression of cyclin D2 in B cells that lack Klf4 expression. We then demonstrated that Klf4 directly bound to the promoter of cyclin D2 and regulated its expression. These findings demonstrate that Klf4 regulates B cell number and activation-induced B cell proliferation through directly acting on the promoter of cyclin D2.
Autoantigen-Specific TGFβ-Induced Foxp3+ Regulatory T Cells Prevent Autoimmunity by Inhibiting Dendritic Cells from Activating Autoreactive T Cells
Authors: DiPaolo Richard J. , Brinster Carine , Davidson Todd S. , Andersson John , Glass Deborah , Shevach Ethan M.
Published: April 2014

Several strategies are being designed to test the therapeutic potential of Ag-specific regulatory T cells to prevent or treat autoimmune diseases. In this study, we demonstrate that naive CD4+Foxp3− T cells specific for a naturally expressed autoantigen (H+/K+ ATPase) can be converted to Foxp3+ T regulatory cells (Tregs) when stimulated in presence of TGFβ. TGFβ-induced Tregs (iTregs) have all the characteristics of naturally generated regulatory T cells in vitro, and more importantly, are effective at preventing organ-specific autoimmunity in a murine model of autoimmune gastritis. H+/K+ ATPase specific iTregs were able to inhibit the initial priming and proliferation of autoreactive T cells, and appear to do so by acting on H+/K+ ATPase presenting dendritic cells (DC). DC exposed to iTregs in vivo were reduced in their ability to stimulate proliferation and cytokine production by H+/K+ ATPase specific T cells. iTregs specifically reduced CD80 and CD86 expression on the surface of H+/K+ ATPase presenting DC in vitro. These studies reveal the therapeutic potential of Ag specific iTregs to prevent autoimmunity, and provide a mechanism by which this population of regulatory T cells, and perhaps others, mediate their suppressive effects in vivo.
Early Growth Response 1 and NF-ATc1 Act in Concert to Promote Thymocyte Development beyond the β-Selection Checkpoint
Authors: Koltsova Ekaterina K. , Ciofani Maria , Benezra Robert , Miyazaki Toru , Clipstone Neil , Zúñiga-Pflücker Juan Carlos , Wiest David L.
Published: April 2014

Development of immature T cell precursors beyond the β-selection checkpoint is regulated by signals transduced by the pre-TCR complex. The pre-TCR-induced differentiation program is orchestrated by a network of transcription factors that serve to integrate this signaling information. Among these transcription factors are those of the early growth response (Egr) and NF-AT families. In this study, we demonstrate that Egr1 and NF-ATc1 act together to promote development of T cell precursors beyond the β-selection checkpoint to the CD8 immature single-positive and CD4+CD8+ double-positive stages. Moreover, we find that Egr1 and NF-AT cooperatively induce the expression of inhibitor of DNA binding 3 (Id3), a regulatory factor known to play an important role in positive selection of thymocytes, but not previously demonstrated to be required for β-selection. Importantly, we show in this study that Id3 deficiency abrogates the ability of ectopically expressed Egr1 to promote traversal of the β-selection checkpoint. Id3 is presumably essential for traversal of the β-selection checkpoint in this context because of the inability of other inhibitor of DNA binding family members to compensate, since transgenic Egr1 does not induce expression of inhibitor of DNA binding 1 (Id1) or 2 (Id2). Taken together, these data demonstrate that Id3 is a cooperatively induced target that is important for Egr-mediated promotion of development beyond the β-selection checkpoint. Moreover, these data indicate that the ERK and calcium signaling pathways may converge during β-selection through the concerted action of Egr1 and NF-ATc1, respectively.
In Vivo Electroporation Enhances the Immunogenicity of Hepatitis C Virus Nonstructural 3/4A DNA by Increased Local DNA Uptake, Protein Expression, Inflammation, and Infiltration of CD3+ T Cells
Authors: Ahlén Gustaf , Söderholm Jonas , Tjelle Torunn , Kjeken Rune , Frelin Lars , Höglund Urban , Blomberg Pontus , Fons Michael , Mathiesen Iacob , Sällberg Matti
Published: April 2014

The mechanisms by which in vivo electroporation (EP) improves the potency of i.m. DNA vaccination were characterized by using the hepatitis C virus nonstructural (NS) 3/4A gene. Following a standard i.m. injection of DNA with or without in vivo EP, plasmid levels peaked immediately at the site of injection and decreased by 4 logs the first week. In vivo EP did not promote plasmid persistence and, depending on the dose, the plasmid was cleared or almost cleared after 60 days. In vivo imaging and immunohistochemistry revealed that protein expression was restricted to the injection site despite the detection of significant levels of plasmid in adjacent muscle groups. In vivo EP increased and prolonged NS3/4A protein expression levels as well as an increased infiltration of CD3+ T cells at the injection site. These factors most likely additively contributed to the enhanced and broadened priming of NS3/4A-specific Abs, CD4+ T cells, CD8+ T cells, and γ-IFN production. The primed CD8+ responses were functional in vivo, resulting in elimination of hepatitis C virus NS3/4A-expressing liver cells in transiently transgenic mice. Collectively, the enhanced protein expression and inflammation at the injection site following in vivo EP contributed to the priming of in vivo functional immune responses. These localized effects most likely help to insure that the strength and duration of the responses are maintained when the vaccine is tested in larger animals, including rabbits and humans. Thus, the combined effects mediated by in vivo EP serves as a potent adjuvant for the NS3/4A-based DNA vaccine.
Analysis of Eosinophil Turnover In Vivo Reveals Their Active Recruitment to and Prolonged Survival in the Peritoneal Cavity
Authors: Ohnmacht Caspar , Pullner Andrea , van Rooijen Nico , Voehringer David
Published: April 2014

Eosinophils are potent effector cells associated with allergic inflammation and parasite infections. However, limited information exists about their turnover, migration, and survival in vivo. To address these important questions, we determined murine eosinophil turnover under steady state and inflammatory conditions by flow cytometric analysis of BrdU incorporation and analyzed their migration pattern and survival in different tissues after adoptive transfer into recipient mice. In naive mice ∼50% of bone marrow eosinophils were labeled with BrdU during a 15-h pulse, whereas only 10% of splenic eosinophils were labeled within this time frame. Unexpectedly, the rate of eosinophil production did not change during acute infection with the helminth parasite Nippostrongylus brasiliensis despite massive eosinophilia in several tissues. Eosinophils present in lung and peritoneum remained largely BrdU negative, indicating that eosinophilia in end organs was mainly caused by increased survival of already existing eosinophils rather than increased production of new eosinophils in the bone marrow. Adoptive transfer experiments revealed that eosinophils preferentially migrated to the peritoneum in a macrophage-independent and pertussis toxin-sensitive manner, where they survived for several days. Peritoneal eosinophils expressed high levels of the inhibitory receptor Siglec-F, released less eosinophil peroxidase compared with eosinophils from the spleen, and could recirculate to other organs. These results demonstrate that the peritoneum serves as reservoir for eosinophils.
IFN Regulatory Factor 8 Mediates Apoptosis in Nonhemopoietic Tumor Cells via Regulation of Fas Expression
Authors: Yang Dafeng , Thangaraju Muthusamy , Browning Darren D. , Dong Zheng , Korchin Borys , Lev Dina C. , Ganapathy Vadivel , Liu Kebin
Published: April 2014

IFN regulatory factor 8 (IRF8) is a transcription factor that was originally identified in myeloid cells and has been shown to be essential for differentiation and function of hemopoietic cells. Mice with a null mutation of IRF8 exhibit uncontrolled expansion of the granulocytic and monocytic lineages that progress into a phenotype resembling human chronic myelogenous leukemia. In human patients with chronic myelogenous leukemia, IRF8 transcript levels are frequently diminished. Therefore, IRF8 is a key regulator of myeloid tumor development. In this study, we report that IRF8 is a critical regulator of apoptosis in nonhemopoietic tumor cells. Disruption of IRF8 function with IRF8 dominant-negative mutants diminished Fas-mediated apoptosis in sarcoma tumor cells. Both constitutively expressed and IFN-γ-activated IRF8 were involved in regulation of apoptosis. Furthermore, it was found that constitutively expressed IRF8 is associated with the Fas promoter to activate Fas transcription. In addition, disruption of constitutively expressed IRF8 function diminished JAK1 expression and thereby inhibited IFN-γ-initiated induction of STAT1 phosphorylation, which in turn, blocked IFN-γ-induced Fas up-regulation. Interestingly, the constitutively expressed IRF8 was also essential for TNF-α sensitization of Fas-mediated apoptosis because disruption of IRF8 function also inhibited TNF-α-sensitized and Fas-mediated apoptosis. Taken together, our data suggest that IRF8 is an essential mediator of Fas-mediated apoptosis and that IRF8 mediates apoptosis through regulation of Fas expression in nonhemopoietic tumor cells.
The Role of the Prostaglandin D2 Receptor, DP, in Eosinophil Trafficking
Authors: Schratl Petra , Royer Julia F. , Kostenis Evi , Ulven Trond , Sturm Eva M. , Waldhoer Maria , Hoefler Gerald , Schuligoi Rufina , Lippe Irmgard Th. , Peskar Bernhard A. , Heinemann Akos
Published: April 2014

Prostaglandin (PG) D2 is a major mast cell product that acts via two receptors, the D-type prostanoid (DP) and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) receptors. Whereas CRTH2 mediates the chemotaxis of eosinophils, basophils, and Th2 lymphocytes, the role of DP has remained unclear. We report in this study that, in addition to CRTH2, the DP receptor plays an important role in eosinophil trafficking. First, we investigated the release of eosinophils from bone marrow using the in situ perfused guinea pig hind limb preparation. PGD2 induced the rapid release of eosinophils from bone marrow and this effect was inhibited by either the DP receptor antagonist BWA868c or the CRTH2 receptor antagonist ramatroban. In contrast, BWA868c did not inhibit the release of bone marrow eosinophils when this was induced by the CRTH2-selective agonist 13,14-dihydro-15-keto-PGD2. In additional experiments, we isolated bone marrow eosinophils from the femoral cavity and found that these cells migrated toward PGD2. We also observed that BWA868c inhibited this response to a similar extent as ramatroban. Finally, using immunohistochemistry we could demonstrate that eosinophils in human bone marrow specimens expressed DP and CRTH2 receptors at similar levels. Eosinophils isolated from human peripheral blood likewise expressed DP receptor protein but at lower levels than CRTH2. In agreement with this, the chemotaxis of human peripheral blood eosinophils was inhibited both by BWA868c and ramatroban. These findings suggest that DP receptors comediate with CRTH2 the mobilization of eosinophils from bone marrow and their chemotaxis, which might provide the rationale for DP antagonists in the treatment of allergic disease.
Deletion of Secretory Group V Phospholipase A2 Attenuates Cell Migration and Airway Hyperresponsiveness in Immunosensitized Mice
Authors: Muñoz Nilda M. , Meliton Angelo Y. , Arm Jonathan P. , Bonventre Joseph V. , Cho Wonhwa , Leff Alan R.
Published: April 2014

We investigated the role of group V phospholipase A2 (gVPLA2) in OVA-induced inflammatory cell migration and airway hyperresponsiveness (AHR) in C57BL/6 mice. Repeated allergen challenge induced biosynthesis of gVPLA2 in airways. By aerosol, gVPLA2 caused dose-related increase in airway resistance in saline-treated mice; in allergic mice, gVPLA2 caused persistent airway narrowing. Neither group IIa phospholipase A2, a close homolog of gVPLA2, nor W31A, an inactive gVPLA2 mutant with reduced activity, caused airway narrowing in immune-sensitized mice. Pretreatment with MCL-3G1, a blocking Ab against gVPLA2, before OVA challenge blocked fully gVPLA2-induced cell migration and airway narrowing as marked by reduction of migrating leukocytes in bronchoalveolar lavage fluid and decreased airway resistance. We also assessed whether nonspecific AHR caused by methacholine challenge was elicited by gVPLA2 secreted from resident airway cells of immune-sensitized mice. MCL-3G1 also blocked methacholine-induced airway bronchoconstriction in allergic mice. Blockade of bronchoconstriction by MCL-3G1 was replicated in allergic pla2g5−/− mice, which lack the gene encoding gVPLA2. Bronchoconstriction caused by gVPLA2 in pla2g4−/− mice was comparable to that in pla2g4+/+ mice. Our data demonstrate that gVPLA2 is a critical messenger enzyme in the development of AHR and regulation of cell migration during immunosensitization by a pathway that is independent of group IVa phospholipase A2.
A Critical Role for TLR4 in the Pathogenesis of Necrotizing Enterocolitis by Modulating Intestinal Injury and Repair
Authors: Leaphart Cynthia L. , Cavallo Jaime , Gribar Steven C. , Cetin Selma , Li Jun , Branca Maria F. , Dubowski Theresa D. , Sodhi Chhinder P. , Hackam David J.
Published: April 2014

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in preterm infants and is characterized by translocation of LPS across the inflamed intestine. We hypothesized that the LPS receptor (TLR4) plays a critical role in NEC development, and we sought to determine the mechanisms involved. We now demonstrate that NEC in mice and humans is associated with increased expression of TLR4 in the intestinal mucosa and that physiological stressors associated with NEC development, namely, exposure to LPS and hypoxia, sensitize the murine intestinal epithelium to LPS through up-regulation of TLR4. In support of a critical role for TLR4 in NEC development, TLR4-mutant C3H/HeJ mice were protected from the development of NEC compared with wild-type C3H/HeOUJ littermates. TLR4 activation in vitro led to increased enterocyte apoptosis and reduced enterocyte migration and proliferation, suggesting a role for TLR4 in intestinal repair. In support of this possibility, increased NEC severity in C3H/HeOUJ mice resulted from increased enterocyte apoptosis and reduced enterocyte restitution and proliferation after mucosal injury compared with mutant mice. TLR4 signaling also led to increased serine phosphorylation of intestinal focal adhesion kinase (FAK). Remarkably, TLR4 coimmunoprecipitated with FAK, and small interfering RNA-mediated FAK inhibition restored enterocyte migration after TLR4 activation, demonstrating that the FAK-TLR4 association regulates intestinal healing. These findings demonstrate a critical role for TLR4 in the development of NEC through effects on enterocyte injury and repair, identify a novel TLR4-FAK association in regulating enterocyte migration, and suggest TLR4/FAK as a therapeutic target in this disease.
Generation of Th1 and Th2 Chemokines by Human Eosinophils: Evidence for a Critical Role of TNF-α
Authors: Liu Lin Ying , Bates Mary Ellen , Jarjour Nizar N. , Busse William W. , Bertics Paul J. , Kelly Elizabeth A. B.
Published: April 2014

Emerging evidence suggests a role for eosinophils in immune regulation of T cells. Thus, we sought to determine whether human eosinophils may exert their effect via differential generation of Th1 and Th2 chemokines depending on cytokines in their microenvironment and, if so, to establish the conditions under which these chemokines are produced. Eosinophils cultured with TNF-α plus IL-4 had increased mRNA expression and protein secretion of the Th2-type chemokines, CCL17 (thymus and activation-regulated chemokine) and CCL22 (macrophage-derived chemokine). Conversely, the Th1-type chemokines, CXCL9 (monokine induced by IFN-γ) and CXCL10 (IFN-γ-inducible protein-10), were expressed after stimulation with TNF-α plus IFN-γ. Addition of TNF-α appeared to be essential for IFN-γ-induced release of Th1-type chemokines and significantly enhanced IL-4-induced Th2-type chemokines. Inhibition of NF-κB completely blocked the production of both Th1 and Th2 chemokines. Activation of NF-κB, STAT6, and STAT1 was induced in eosinophils by TNF-α, IL-4, and IFN-γ, respectively. However, there was no evidence for enhancement of these signaling events when eosinophils were stimulated with the combination of TNF-α plus IL-4 or TNF-α plus IFN-γ. Thus, independently activated signaling cascades appear to lead to activation of NF-κB, STAT1, and STAT6, which may then cooperate at the promoter level to increase gene transcription. Our data demonstrate that TNF-α is a vital component for eosinophil chemokine generation and that, depending on the cytokines present in their microenvironment, eosinophils can promote either a Th2 or a Th1 immune response, supporting an immunoregulatory role for eosinophils.
A Novel Inflammatory Pathway Involved in Leukocyte Recruitment: Role for the Kinin B1 Receptor and the Chemokine CXCL5
Authors: Duchene Johan , Lecomte Florence , Ahmed Saleh , Cayla Cecile , Pesquero Joao , Bader Michael , Perretti Mauro , Ahluwalia Amrita
Published: April 2014

The kinin B1 receptor is an inducible receptor not normally expressed but induced by inflammatory stimuli and plays a major role in neutrophil recruitment, particularly in response to the cytokine IL-1β. However, the exact mechanism involved in this response is unclear. The aim of this study was to dissect the molecular mechanism involved, in particular to determine whether specific ELR-CXCL chemokines (specific neutrophil chemoattractants) played a role. Using intravital microscopy, we demonstrated that IL-1β-induced leukocyte rolling, adherence, and emigration in mesenteric venules of wild-type (WT) mice, associated with an increase in B1 receptor mRNA expression, were substantially attenuated (>80%) in B1 receptor knockout mice (B1KO). This effect in B1KO mice was correlated with a selective down-regulation of IL-1β-induced CXCL5 mRNA and protein expression compared with WT mice. Furthermore a selective neutralizing CXCL5 Ab caused profound suppression of leukocyte emigration in IL-1β-treated WT mice. Finally, treatment of human endothelial cells with IL-1β enhanced mRNA expression of the B1 receptor and the human (h) CXCL5 homologues (hCXCL5 and hCXCL6). This response was suppressed by ∼50% when cells were pretreated with the B1 receptor antagonist des-Arg9-[Leu8]-bradykinin while treatment with des-Arg9-bradykinin, the B1 receptor agonist, caused a concentration-dependent increase in hCXCL5 and hCXCL6 mRNA expression. This study unveils a proinflammatory pathway centered on kinin B1 receptor activation of CXCL5 leading to leukocyte trafficking and highlights the B1 receptor as a potential target in the therapeutics of inflammatory disease.
B Cells Amplify IFN-γ Production By T Cells via a TNF-α-Mediated Mechanism
Authors: Menard Laurence C. , Minns Laurie A. , Darche Sylvie , Mielcarz Daniel W. , Foureau David M. , Roos David , Dzierszinski Florence , Kasper Lloyd H. , Buzoni-Gatel Dominique
Published: April 2014

Aside from being the precursors of the Ab-secreting cells, B cells are engaged in other immune functions such as Ag presentation to T cells or cytokine production. These functions may contribute to the pathogenic role of B cells in a wide range of autoimmune diseases. We demonstrate that B cells acquire the capacity to amplify IFN-γ production by CD4 and CD8 T cells during the course of the Th1 inflammatory response to Toxoplasma gondii infection. Using the two following different strategies, we observed that B cells from T. gondii-infected mice, but not from naive mice, induce higher IFN-γ expression by splenic host T cells: 1) reconstitution of B cell-deficient mice with B cells expressing an alloantigen different from the recipients, and 2) adoptive transfer of B and T cells into RAG−/− mice. In vitro assays allowing the physical separation of T and B cells demonstrate that Ag-primed B cells enhance IFN-γ production by T cells in a contact-dependent fashion. Using an OVA-transgenic strain of T. gondii and OVA-specific CD4 T cells, we observed that the proinflammatory effect of B cells is neither Ag specific nor requires MHCII expression. However, TNF-α expressed on the surface of B cells appears to mediate in part the up-regulation of IFN-γ by the effector T cells.
Deficiency of the NF-κB Inhibitor Caspase Activating and Recruitment Domain 8 in Patients with Rheumatoid Arthritis Is Associated with Disease Severity
Authors: Fontalba Ana , Martinez-Taboada Victor , Gutierrez Olga , Pipaon Carlos , Benito Natividad , Balsa Alejandro , Blanco Ricardo , Fernandez-Luna Jose L.
Published: April 2014

Caspase activating and recruitment domain 8 (CARD8) potently inhibits NF-κB signaling, which plays a key role in inflammation, and may contribute to avoid a pathologic activation of NF-κB; however, the transcriptional mechanisms regulating CARD8 expression and the relevance of this protein in inflammatory diseases are poorly understood. We found a NF-κB-binding element within the human CARD8 promoter that was required for transcriptional activity in response to TNF-α and the p65 subunit of NF-κB. Moreover, TNF-α and overexpression of p65 induced the formation of NF-κB-CARD8 promoter complexes. Thus, CARD8 may control NF-κB activation through a regulatory loop. To study the relevance of CARD8 in chronic inflammatory disorders, we functionally characterized a deleterious polymorphism (p.C10X) and studied its association with rheumatoid arthritis (RA). Transfection of cell lines with the allelic variants of CARD8 revealed that full-length (CARD8-L) but not truncated (CARD8-S) protein inhibits NF-κB transcriptional activity, and abrogates the binding of NF-κB to its consensus site. Furthermore, in contrast to the full-length protein, CARD8-S did not modify the expression of NF-κB target genes (cIAP, A1), in response to TNF-α. We analyzed the p.C10X polymorphism in 200 patients with RA, and found that homozygous carriers of the CARD8-S allele have higher disease activity score (p = 0.014), more extra-articular manifestations (p = 0.03), and a lower probability of clinical remission (p = 0.03) than the CARD8-L allele carriers. Overall, our findings provide molecular insight into the expression of CARD8 by NF-κB, and suggest that a deleterious polymorphism of CARD8 may help predict the severity of RA.
Elf-1 Binds to GGAA Elements on the FcRγ Promoter and Represses Its Expression
Authors: Juang Yuang-Taung , Sumibcay Laarni , Tolnay Mate , Wang Ying , Kyttaris Vasileios C. , Tsokos George C.
Published: April 2014

The Fc receptor (FcR) γ-chain has been shown to be up-regulated in T cells when the TCR ζ-chain is decreased. We demonstrate that Elf-1, but not other Ets family transcription factors, bind to a cluster of GGAA sites located within the 200 bp upstream from the transcription initiation site of the FcRγ promoter. Forced expression of Elf-1 results in the suppression of FcRγ expression, whereas silencing its expression with small interfering RNA Elf-1 results in increased FcRγ expression. Elf-1 represents the first transcription factor identified to be involved in the transcriptional regulation of FcRγ, and cells that fail to express Elf-1, as is the case with human systemic lupus erythematosus T cells, will express FcRγ-chain.
Defective Expression of Ras Guanyl Nucleotide-Releasing Protein 1 in a Subset of Patients with Systemic Lupus Erythematosus
Authors: Yasuda Shinsuke , Stevens Richard L. , Terada Tomoko , Takeda Masumi , Hashimoto Toko , Fukae Jun , Horita Tetsuya , Kataoka Hiroshi , Atsumi Tatsuya , Koike Takao
Published: April 2014

Dysregulation of Ras guanyl nucleotide-releasing protein 1 (RasGRP1) in mice results in a systemic lupus erythematosus (SLE)-like disorder. We therefore looked for defective isoforms and/or diminished levels of human RasGRP1 in a cohort of SLE patients. PBMCs were collected from twenty healthy individuals and thirty-two patients with SLE. mRNA was isolated and five RasGRP1 cDNAs from each subject were sequenced. T cell lysates from healthy controls and SLE patients also were evaluated for their levels of RasGRP1 protein. The accumulated data led to the identification of 13 new splice variants of the human RasGRP1 gene. Not only did our SLE patients have increased levels and types of these defective transcripts relative to normal individuals, two SLE patients were identified whose PBMCs and T cells contained very little, if any, functional RasGRP1 mRNA and protein. The presence of aberrantly spliced RasGRP1 transcripts also was correlated with lower levels of RasGRP1 protein in the patients’ T cells. The lack of the normal isoform of RasGRP1 in some SLE patients and the increased prevalence of defective isoforms of RasGRP1 in others raise the possibility that dysregulation of this signaling protein contributes to the development of autoimmunity in a subset of SLE patients.
Achieving Donor-Specific Hyporesponsiveness Is Associated with FOXP3+ Regulatory T Cell Recruitment in Human Renal Allograft Infiltrates
Authors: Bestard Oriol , Cruzado Josep M. , Mestre Mariona , Caldés Anna , Bas Jordi , Carrera Marta , Torras Joan , Rama Inés , Moreso Francesc , Serón Daniel , Grinyó Josep M.
Published: April 2014

Exploring new immunosuppressive strategies inducing donor-specific hyporesponsiveness is an important challenge in transplantation. For this purpose, a careful immune monitoring and graft histology assessment is mandatory. Here, we report the results of a pilot study conducted in twenty renal transplant recipients, analyzing the immunomodulatory effects of a protocol based on induction therapy with rabbit anti-thymocyte globulin low doses, sirolimus, and mofetil mycophenolate. Evolution of donor-specific cellular and humoral alloimmune response, peripheral blood lymphocyte subsets and apoptosis was evaluated. Six-month protocol biopsies were performed to assess histological lesions and presence of FOXP3+ regulatory T cells (Tregs) in interstitial infiltrates. After transplantation, there was an early and transient apoptotic effect, mainly within the CD8+HLADR+ T cells, combined with a sustained enhancement of CD4+CD25+high lymphocytes in peripheral blood. The incidence of acute rejection was 35%, all steroid sensitive. Importantly, only pretransplant donor-specific cellular alloreactivity could discriminate patients at risk to develop acute rejection. Two thirds of the patients became donor-specific hyporesponders at 6 and 24 mo, and the achievement of this immunologic state was not abrogated by prior acute rejection episodes. Remarkably, donor-specific hyporesponders had the better renal function and less chronic renal damage. Donor-specific hyporesponsiveness was inhibited by depleting CD4+CD25+high T cells, which showed donor-Ag specificity. FOXP3+CD4+CD25+high Tregs both in peripheral blood and in renal infiltrates were higher in donor-specific hyporesponders than in nonhyporesponders, suggesting that the recruitment of Tregs in the allograft plays an important role for renal acceptance. In conclusion, reaching donor-specific hyporesponsiveness is feasible after renal transplantation and associated with Treg recruitment in the graft.
4-1BB Is Superior to CD28 Costimulation for Generating CD8+ Cytotoxic Lymphocytes for Adoptive Immunotherapy
Authors: Zhang Hua , Snyder Kristen M. , Suhoski Megan M. , Maus Marcela V. , Kapoor Veena , June Carl H. , Mackall Crystal L.
Published: April 2014

Artificial APCs (aAPCs) genetically modified to express selective costimulatory molecules provide a reproducible, cost-effective, and convenient method for polyclonal and Ag-specific expansion of human T cells for adoptive immunotherapy. Among the variety of aAPCs that have been studied, acellular beads expressing anti-CD3/anti-CD28 efficiently expand CD4+ cells, but not CD8+ T cells. Cell-based aAPCs can effectively expand cytolytic CD8+ cells, but optimal costimulatory signals have not been defined. 4-1BB, a costimulatory molecule expressed by a minority of resting CD8+ T cells, is transiently up-regulated by all CD8+ T cells following activation. We compared expansion of human cytolytic CD8+ T cells using cell-based aAPCs providing costimulation via 4-1BB vs CD28. Whereas anti-CD3/anti-CD28 aAPCs mostly expand naive cells, anti-CD3/4-1BBL aAPCs preferentially expand memory cells, resulting in superior enrichment of Ag-reactive T cells which recognize previously primed Ags and efficient expansion of electronically sorted CD8+ populations reactive toward viral or self-Ags. Using HLA-A2-Fc fusion proteins linked to 4-1BBL aAPCs, 3-log expansion of Ag-specific CD8+ CTL was induced over 14 days, whereas similar Ag-specific CD8+ T cell expansion did not occur using HLA-A2-Fc/anti-CD28 aAPCs. Furthermore, when compared with cytolytic T cells expanded using CD28 costimulation, CTL expanded using 4-1BB costimulation mediate enhanced cytolytic capacity due, in part, to NKG2D up-regulation. These results demonstrate that 4-1BB costimulation is essential for expanding memory CD8+ T cells ex vivo and is superior to CD28 costimulation for generating Ag-specific products for adoptive cell therapy.
Administration of a CD25-Directed Immunotoxin, LMB-2, to Patients with Metastatic Melanoma Induces a Selective Partial Reduction in Regulatory T Cells In Vivo
Authors: Powell Daniel J. , Felipe-Silva Aloisio , Merino Maria J. , Ahmadzadeh Mojgan , Allen Tamika , Levy Catherine , White Donald E. , Mavroukakis Sharon , Kreitman Robert J. , Rosenberg Steven A. , Pastan Ira
Published: April 2014

CD25+CD4+ T regulatory (Treg) cells regulate peripheral self tolerance and possess the ability to suppress antitumor responses, which may in part explain the poor clinical response of cancer patients undergoing active immunization protocols. We have previously shown that in vitro incubation of human PBMC with LMB-2, a CD25-directed immunotoxin, significantly reduced CD25+FOXP3+CD4+ Treg cells without impairing the function of the remaining lymphocytes. In the current study, eight patients with metastatic melanoma were treated with LMB-2 followed by MART-1 and gp100-specific peptide vaccination. LMB-2 administration resulted in a preferential, transient reduction in mean circulating CD25+CD4+ T cell number, from 83 ± 16 cells/μl to a nadir of 17 ± 5 cells/μl, a 79.1% reduction. FOXP3 analysis revealed a less robust depletion with mean FOXP3+CD4+ Treg cell number decreasing from 74 ± 15 cells/μl to 36 ± 8 cells/μl, a 51.4% reduction. FOXP3+CD4+ Treg cells that survived LMB-2-mediated cytotoxicity expressed little or no CD25. Similar to the peripheral blood, immunohistochemical analysis showed a 68.9% mean reduction in FOXP3+CD4+ Treg cell frequency in evaluable lesions. Despite inducing a reduction in Treg cell numbers in vivo, LMB-2 therapy did not augment the immune response to cancer vaccination and no patient experienced an objective response or autoimmunity. These data demonstrate the capacity of a CD25-directed immunotoxin to selectively mediate a transient partial reduction in circulating and tumor-infiltrating Treg cells in vivo, and suggest that more comprehensive Treg cell elimination may be required to bolster antitumor responses in patients with metastatic melanoma.
Autoantibodies in Canine Masticatory Muscle Myositis Recognize a Novel Myosin Binding Protein-C Family Member
Authors: Wu Xiaohua , Li Zhi-fang , Brooks Randolph , Komives Elizabeth A. , Torpey Justin W. , Engvall Eva , Gonias Steven L. , Shelton G. Diane
Published: April 2014

Inflammatory myopathies are a group of autoimmune diseases that affect muscles. In humans, the most common inflammatory myopathies are polymyositis, dermatomyositis, and inclusion body myositis. Autoantibodies may be found in humans with inflammatory myopathies, and these play an important role in diagnosis and disease classification. However, these Abs are typically not muscle specific. Spontaneously occurring canine inflammatory myopathies may be good parallel disorders and provide insights into human myositis. In dogs with inflammatory myopathy, muscle-specific autoantibodies have been found, especially in masticatory muscle myositis. We have identified the major Ag recognized by the autoantibodies in canine masticatory muscle myositis. This Ag is a novel member of the myosin binding protein-C family, which we call masticatory myosin binding protein-C (mMyBP-C). mMyBP-C is localized not only within the masticatory muscle fibers, but also at or near their cell surface, perhaps making it accessible as an immunogen. The gene for mMyBP-C also exists in humans, and mMyBP-C could potentially play a role in certain human inflammatory myopathies. Understanding the role of mMyBP-C in this canine inflammatory myopathy may advance our knowledge of mechanisms of autoimmune inflammatory muscle diseases, not only in dogs, but also in humans.
Cutting Edge: IL-4-Mediated Protection of Primary B Lymphocytes from Apoptosis via Stat6-Dependent Regulation of Glycolytic Metabolism
Authors: Dufort Fay J. , Bleiman Blair F. , Gumina Maria R. , Blair Derek , Wagner Dean J. , Roberts Mary F. , Abu-Amer Yousef , Chiles Thomas C.
Published: April 2014

IL-4 prevents the death of naive B lymphocytes through the up-regulation of antiapoptotic proteins such as Bcl-xL. Despite studies implicating glucose utilization in growth factor-dependent survival of hemopoietic cells, the role of glucose energy metabolism in maintaining B cell viability by IL-4 is unknown. We show that IL-4 triggers glucose uptake, Glut1 expression, and glycolysis in splenic B cells; this is accompanied by increased cellular ATP. Glycolysis inhibition results in apoptosis, even in the presence of IL-4. IL-4-induced glycolysis occurs normally in B cells deficient in insulin receptor substrate-2 or the p85α subunit of PI3K and is not affected by pretreatment with PI3K or MAPK pathway inhibitors. Stat6-deficient B cells exhibit impaired IL-4-induced glycolysis. Cell-permeable, constitutively active Stat6 is effective in restoring IL-4-induced glycolysis in Stat6-deficient B cells. Therefore, besides controlling antiapoptotic proteins, IL-4 mediates B cell survival by regulating glucose energy metabolism via a Stat6-dependent pathway.
Functional Human Antigen-Specific T Cells Produced In Vitro Using Retroviral T Cell Receptor Transfer into Hematopoietic Progenitors
Authors: van Lent Anja U. , Nagasawa Maho , van Loenen Marleen M. , Schotte Remko , Schumacher Ton N. M. , Heemskerk Mirjam H. M. , Spits Hergen , Legrand Nicolas
Published: April 2014

In vitro production of human T cells with known Ag specificity is of major clinical interest for immunotherapy against tumors and infections. We have performed TCRαβ gene transfer into human hemopoietic progenitors from postnatal thymus or umbilical cord blood, and subsequently cultured these precursors on OP9 stromal cells expressing the Notch human ligand Delta-like1. We report here that fully mature, functional T cells with controlled Ag specificity are obtained from such cultures. Using vectors encoding TCRαβ-chains directed against melanoma (MART-1), viral (CMV), and minor histocompatibility (HA-2) Ags, we show that the obtained Ag-specific T cells exert cytolytic activity against their cognate Ag and expand in vitro upon specific TCR stimulation. Therapeutic applications may arise from these results because they provide a way to produce large numbers of autologous mature Ag-specific T cells in vitro from undifferentiated hemopoietic progenitors.
Differential Requirements for OX40 Signals on Generation of Effector and Central Memory CD4+ T Cells
Authors: Soroosh Pejman , Ine Shouji , Sugamura Kazuo , Ishii Naoto
Published: April 2014

Memory T cells can be divided into effector memory (TEM) and central memory (TCM) subsets based on their effector function and homing characteristics. Although previous studies have demonstrated that TCR and cytokine signals mediate the generation of the two memory subsets of CD8+ T cells, the mechanisms for generation of the CD4+ TEM and TCM cell subsets are unknown. We found that OX40-deficient mice showed a marked reduction in the number of CD4+ TEM cells, whereas the number of CD4+ TCM cells was normal. Adoptive transfer experiments using Ag-specific CD4+ T cells revealed that OX40 signals during the priming phase were indispensable for the optimal generation of the CD4+ TEM, but not the CD4+ TCM population. In a different transfer experiment with in vitro established CD4+CD44highCD62Llow (TEM precursor) and CD4+CD44highCD62Lhigh (TCM precursor) subpopulations, OX40-KO TEM precursor cells could not survive in the recipient mice, whereas wild-type TEM precursor cells differentiated into both TEM and TCM cells. In contrast, TCM precursor cells mainly produced TCM cells regardless of OX40 signals, implying the dispensability of OX40 for generation of TCM cells. Nevertheless, survival of OX40-KO TEM cells was partially rescued in lymphopenic mice. During in vitro recall responses, the OX40-KO TEM cells that were generated in lymphopenic recipient mice showed impaired cytokine production, suggesting an essential role for OX40 not only on generation but also on effector function of CD4+ TEM cells. Collectively, the present results indicate differential requirements for OX40 signals on generation of CD4+ TEM and TCM cells.
The Importance of Myeloid-Derived Suppressor Cells in the Regulation of Autoimmune Effector Cells by a Chronic Contact Eczema
Authors: Marhaba Rachid , Vitacolonna Mario , Hildebrand Dagmar , Baniyash Michal , Freyschmidt-Paul Pia , Zöller Margot
Published: April 2014

Induction of a chronic eczema is a most efficient therapy for alopecia areata (AA). We had noted a reduction in regulatory T cells during AA induction and wondered whether regulatory T cells may become recruited or expanded during repeated skin sensitization or whether additional regulatory cells account for hair regrowth. AA could not be cured by the transfer of CD4+CD25high lymph node cells from mice repeatedly treated with a contact sensitizer. This obviously is a consequence of a dominance of freshly activated cells as compared with regulatory CD4+CD25+ T cells. Instead, a population of Gr-1+CD11b+ cells was significantly increased in skin and spleen of AA mice repeatedly treated with a contact sensitizer. Gr-1+CD11b+ spleen cells mostly expressed CD31. Expression of several proinflammatory cytokines as well as of the IFN-γ receptor and the TNF receptor I were increased. Particularly in the skin, Gr-1+ cells expressed several chemokines and CCR8 at high levels. Gr-1+CD11b+ cells most potently suppressed AA effector cell proliferation in vitro and promoted partial hair regrowth in vivo. When cocultured with CD4+ or CD8+ cells from AA mice, the Gr-1+CD11b+ cells secreted high levels of NO. However, possibly due to high level Bcl-2 protein expression in AA T cells, apoptosis induction remained unaltered. Instead, ζ-chain expression was strongly down-regulated, which was accompanied by a decrease in ZAP70 and ERK1/2 phosphorylation. Thus, a chronic eczema supports the expansion and activation of myeloid suppressor cells that, via ζ-chain down-regulation, contribute to autoreactive T cell silencing in vitro and in vivo.
CD8 T Cell Responses to Myelin Oligodendrocyte Glycoprotein-Derived Peptides in Humanized HLA-A*0201-Transgenic Mice
Authors: Mars Lennart T. , Bauer Jan , Gross David A. , Bucciarelli Florence , Firat Huseyin , Hudrisier Denis , Lemonnier François , Kosmatopoulos Kostas , Liblau Roland S.
Published: April 2014

Multiple sclerosis (MS) is a demyelinating inflammatory disease of the CNS. Though originally believed to be CD4-mediated, additional immune effector mechanisms, including myelin-specific CD8+ T cells, are now proposed to participate in the pathophysiology of MS. To study the immunologic and encephalitogenic behavior of HLA-A*0201-binding myelin-derived epitopes in vivo, we used a humanized HLA-A*0201-transgenic mouse model. Eight HLA-A*0201-binding peptides derived from myelin oligodendrocyte glycoprotein (MOG), an immunodominant myelin self-Ag, were identified in silico. After establishing their relative affinity for HLA-A*0201 and their capacity to form stable complexes with HLA-A*0201 in vitro, their immunological characteristics were studied in HLA-A*0201-transgenic mice. Five MOG peptides, which bound stably to HLA-A*0201 exhibited strong immunogenicity by inducing a sizeable MOG-specific HLA-A*0201-restricted CD8+ T cell response in vivo. Of these five candidate epitopes, four were processed by MOG-transfected RMA target cells and two peptides proved immunodominant in vivo in response to a plasmid-encoding native full-length MOG. One of the immunodominant MOG peptides (MOG181) generated a cytotoxic CD8+ T cell response able to aggravate CD4+-mediated EAE. Therefore, this detailed in vivo characterization provides a hierarchy of candidate epitopes for MOG-specific CD8+ T cell responses in HLA-A*0201 MS patients identifying the encephalitogenic MOG181 epitope as a primary candidate.