bluuebunny/crossref_metadata_2025 huggingface dataset:
The Ontogeny and Fate of NK Cells Marked by Permanent DNA Rearrangements
Authors: Pilbeam Kristy , Basse Per , Brossay Laurent , Vujanovic Nikola , Gerstein Rachel , Vallejo Abbe N. , Borghesi Lisa
Published: April 2014

A subset of NK cells bears incomplete V(D)J rearrangements, but neither the consequence to cell activities nor the precise developmental stages in which recombination occurs is known. These are important issues, as recombination errors cause cancers of the B and T lineages. Using transgenic recombination reporter mice to examine NK cell dynamics in vivo, we show that recombination+ NK cells have distinct developmental patterns in the BM, including reduced homeostatic proliferation and diminished Stat5 phosphorylation. In the periphery, both recombination+ and recombination− NK cells mediate robust functional responses including IFN-γ production, cytolysis, and tumor homing, suggesting that NK cells with distinct developmental histories can be found together in the periphery. We also show that V(D)J rearrangement marks both human cytolytic (CD56dim) and immunoregulatory (CD56bright) populations, demonstrating the distribution of permanent DNA rearrangements across major NK cell subsets in man. Finally, direct quantification of rag transcripts throughout NK cell differentiation in both mouse and man establishes the specific developmental stages that are susceptible to V(D)J rearrangement. Together, these data demonstrate that multipotent progenitors rather than lineage-specified NK progenitors are targets of V(D)J recombination and that NK cells bearing the relics of earlier V(D)J rearrangements have different developmental dynamics but robust biological capabilities in vivo.
Th17 Cells Exhibit a Distinct Calcium Profile from Th1 and Th2 Cells and Have Th1-Like Motility and NF-AT Nuclear Localization
Authors: Weber K. Scott , Miller Mark J. , Allen Paul M.
Published: April 2014

Helper T cell subsets have evolved to respond to different pathogens, and upon activation secrete distinct sets of cytokines. The discovery and identification of Th17 cells, which develop via a unique lineage from Th1 and Th2 cells, have provided new insights into aspects of immune regulation and host defense that were previously unclear. A key early signaling event upon Ag recognition is elevation of intracellular free Ca2+, and cytokine expression can be differentially induced depending on the duration, amplitude, and pattern of Ca2+ signaling. Th1 and Th2 cells can be distinguished by their Ca2+ profiles, and we provide in this study the first report regarding Ca2+ signaling in Th17 cells. Th17 cells have a distinct Ca2+ signaling profile from Th1 and Th2 cells with intermediate sustained Ca2+ levels and increased oscillations compared with Th2 cells. Elevated intracellular Ca2+ has been shown to inhibit T cell motility, and we observed that Th17 cells, like Th1 cells, are less motile than Th2 cells. Analysis of NF-AT nuclear localization revealed that Th1 and Th17 cells have significantly higher levels at later time points compared with Th2 cells. Thus, these findings show that Th17 cells, in addition to their distinct cytokine response from Th1 and Th2 cells, display unique patterns of intracellular Ca2+ signaling and Th1-like motility behavior and nuclear localization of NF-AT.
Ex Vivo Cytokine and Memory T Cell Responses to the 42-kDa Fragment of Plasmodium falciparum Merozoite Surface Protein-1 in Vaccinated Volunteers
Authors: Huaman Maria Cecilia , Martin Laura B. , Malkin Elissa , Narum David L. , Miller Louis H. , Mahanty Siddhartha , Long Carole A.
Published: April 2014

A number of blood-stage malaria Ags are under development as vaccine candidates, but knowledge of the cellular responses to these vaccines in humans is limited. We evaluated the nature and specificity of cellular responses in healthy American volunteers vaccinated with a portion of the major merozoite surface protein-1 (MSP1) of Plasmodium falciparum, MSP142, formulated on Alhydrogel. Volunteers were vaccinated three times with 80 μg of either MSP142-FVO/Alhydrogel or MSP142-3D7/Alhydrogel. Cells collected 2 wk after the third vaccination produced Th1 cytokines, including IFN-γ and IL-2 following Ag stimulation, and greater levels of the Th2 cytokines IL-5 and IL-13; the anti-inflammatory cytokine IL-10 and the molecule CD25 (IL-2Rα) were also detected. The volunteers were evaluated for the MSP142–FVO or MSP142-3D7 specificity of their T cell responses. Comparison of their responses to homologous and heterologous Ags showed ex vivo IFN-γ and IL-5 levels that were significantly higher to homologous rather than to heterologous Ags. The epitopes involved in this stimulation were shown to be present in the dimorphic MSP133 portion of the larger MSP142-3D7 polypeptide, and indirect experiment suggests the same for the MSP142–FVO polypeptide. This contrasts with B cell responses, which were primarily directed to the conserved MSP119 portion. Furthermore, we explored the maturation of memory T cells and found that 46% of vaccinees showed specific memory T cells defined as CD4+CD45RO+CD40L+ after long-term in vitro culture. The identification of human-specific CD4+ memory T cells provides the foundation for future studies of these cells both after vaccination and in field studies.
IFN-γ Arms Human Dendritic Cells to Perform Multiple Effector Functions
Authors: Frasca Loredana , Nasso Maria , Spensieri Fabiana , Fedele Giorgio , Palazzo Raffaella , Malavasi Fabio , Ausiello Clara Maria
Published: April 2014

Dendritic cells (DCs) are central players in immunity and are used in immune-adoptive vaccine protocols in humans. IFN-γ, mandatory in Th-1 polarization and endowed with regulatory properties, is currently used to condition monocyte-derived DCs (MDDC) in cancer therapy and in clinical trials to treat chronic infectious diseases. We therefore performed a wide analysis of IFN-γ signaling consequences on MDDC multiple effector functions. IFN-γ itself induced IL-27p28 expression and survival but did not promote relevant CCR7-driven migration or activated Th-1 cell recruitment capacity in MDDC. Administered in association with classical maturation stimuli such as CD40 or TLR-4 stimulation, IFN-γ up-regulated IL-27 and IL-12 production, CCR7-driven migration, and activated Th-1 cell recruitment, whereas it decreased IL-10 production and STAT3 phosphorylation. CD38 signaling, which orchestrates migration, survival, and Th-1 polarizing ability of mature MDDC, was involved in IFN-γ-mediated effects. Thus, IFN-γ is a modulator of multiple DC effector functions that can be helpful in MDDC-based vaccination protocols. These data also help understand the dual role exerted by this cytokine as both an inducer and a regulator of inflammation and immune response.
Transcutaneous Anti-Influenza Vaccination Promotes Both CD4 and CD8 T Cell Immune Responses in Humans
Authors: Vogt Annika , Mahé Brice , Costagliola Dominique , Bonduelle Olivia , Hadam Sabrina , Schaefer Gregor , Schaefer Hans , Katlama Christine , Sterry Wolfram , Autran Brigitte , Blume-Peytavi Ulrike , Combadiere Béhazine
Published: April 2014

Induction of T cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. The use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells in CD8 cell cross-priming and suggests that vaccination via the transcutaneous (TC) route may be relevant in the induction of cellular immune responses. We have previously shown that TC application of nanoparticles, on human skin explants, allows targeting of epidermal dendritic cells, possibly via hair follicles. In this study, we have investigated cellular immune responses against an influenza protein-based vaccine by TC vaccination, compared with i.m. vaccination in humans. In this study on 11 healthy volunteers, we found that a newly developed protocol based on cyanocrylate skin surface stripping induced a significant increase in IFNγ-producing T cells specific for influenza vaccine by ELISPOT assays. Interestingly, TC vaccination induced both effector CD4 and CD8 T cell responses, whereas i.m. injection induced strong effector CD4 in the absence of CD8 T cells, as assessed by intracellular cytokine staining and tetramer analyses. This study proposes new perspectives for the development of vaccination strategies that trigger T cell immune responses in humans.
Distinct CD8+ T Cell Repertoires Primed with Agonist and Native Peptides Derived from a Tumor-Associated Antigen
Authors: Hou Yafei , Kavanagh Brian , Fong Lawrence
Published: April 2014

Heteroclitic peptides are used to enhance the immunogenicity of tumor-associated Ags to break T cell tolerance to these self-proteins. One such altered peptide ligand (Cap1–6D) has been derived from an epitope in human carcinoembryonic Ag, CEA605–613 (Cap1). Clinical responses have been seen in colon cancer patients receiving a tumor vaccine comprised of this altered peptide. Whether Cap1–6D serves as a T cell agonist for Cap1-specific T cells or induces different T cells is unknown. We, therefore, examined the T cell repertoires elicited by Cap1–6D and Cap1. Human CTL lines and clones were generated with either Cap1–6D peptide (6D-CTLs) or Cap1 peptide (Cap1-CTLs). The TCR Vβ usage and functional avidity of the T cells induced in parallel against these target peptides were assessed. The predominant CTL repertoire induced by agonist Cap1–6D is limited to TCR Vβ1-J2 with homogenous CDR3 lengths. In contrast, the majority of Cap1-CTLs use different Vβ1 genes and also had diverse CDR3 lengths. 6D-CTLs produce IFN-γ in response to Cap1–6D peptide with high avidity, but respond with lower avidity to the native Cap1 peptide when compared with the Cap1-CTLs. Nevertheless, 6D-CTLs could still lyse targets bearing the native epitope. Consistent with these functional results, 6D-CTLs possess TCRs that bind Cap-1 peptide/MHC tetramer with higher intensity than Cap1-CTLs but form less stable interactions with peptide/MHC as measured by tetramer decay. These results demonstrate that priming with this CEA-derived altered peptide ligand can induce distinct carcinoembryonic Ag-reactive T cells with different functional capacities.
Targeting the Effector Site with IFN-αβ-Inducing TLR Ligands Reactivates Tumor-Resident CD8 T Cell Responses to Eradicate Established Solid Tumors
Authors: Currie Andrew J. , van der Most Robbert G. , Broomfield Steve A. , Prosser Amy C. , Tovey Michael G. , Robinson Bruce W. S.
Published: April 2014

Effective antitumor CD8 T cell responses may be activated by directly targeting the innate immune system within tumors. We investigated this response by injecting a range of TLR agonists into established tumors using a mouse model of malignant mesothelioma stably transduced with the hemagglutinin (HA) gene as a marker Ag (AB1-HA). Persistent delivery of the dsRNA mimetic poly(I:C) into established AB1-HA tumors resulted in complete tumor resolution in 40% of mice, with the remaining mice also showing a significant delay in tumor progression. Experiments in athymic nude mice along with CD8 depletion and IFN-αβ blocking studies revealed that tumor resolution required both CD8 T cells and type I IFN induction, and was associated with local changes in MHC class I expression. Surprisingly, however, tumor resolution was not associated with systemic dissemination or tumor infiltration of effector CD8 T cells. Instead, the antitumor response was critically dependent on the reactivation of tumor-resident CD8 T cell responses. These studies suggest that, once reactivated, pre-existing local CD8 T cell responses are sufficient to resolve established tumors and that in situ type I IFN is a determining factor.
Bone Marrow Stroma Confers Resistance to Apo2 Ligand/TRAIL in Multiple Myeloma in Part by Regulating c-FLIP
Authors: Perez Lia Elena , Parquet Nancy , Shain Kenneth , Nimmanapalli Ramadevi , Alsina Melissa , Anasetti Claudio , Dalton William
Published: April 2014

Apo2 ligand (Apo2L)/TRAIL induces apoptosis of cancer cells that express the specific receptors while sparing normal cells. Because the tumor microenvironment protects myeloma from chemotherapy, we investigated whether hemopoietic stroma induces resistance to Apo2L/TRAIL apoptosis in this disease. Apo2L/TRAIL-induced death was diminished in myeloma cell lines (RPMI 8226, U266, and MM1s) directly adhered to a human immortalized HS5 stroma cell line but not adhered to fibronectin. In a Transwell assay, with myeloma in the upper well and HS5 cells in the lower well, Apo2L/TRAIL apoptosis was reduced when compared with cells exposed to medium in the lower well. Using HS5 and myeloma patients’ stroma-conditioned medium, we determined that soluble factor(s) produced by stroma–myeloma interactions are responsible for a reversible Apo2/TRAIL apoptosis resistance. Soluble factor(s) attenuated procaspase-8, procaspase-3, and poly(ADP-ribose) polymerase cleavage and diminished mitochondrial membrane potential changes without affecting Bcl-2 family proteins and/or Apo2L/TRAIL receptors. Soluble factor(s) increased the baseline levels of the anti-apoptotic protein c-FLIP in all cell lines tested. Inhibition of c-FLIP by means of RNA interference increased Apo2/TRAIL sensitivity in RPMI 8226 cells. Unlike direct adhesion to fibronectin, soluble factor(s) have no impact on c-FLIP redistribution within cellular compartments. Cyclohexamide restored Apo2L/TRAIL sensitivity in association with down-regulation of c-FLIP, suggesting that c-FLIP synthesis, not intracellular traffic, is essential for soluble factor(s) to regulate c-FLIP. Additionally, IL-6 conferred resistance to Apo2L/TRAIL-mediated apoptosis in association with increased c-FLIP levels. In conclusion, the immune cytotoxic effect of Apo2L/TRAIL can be restored at least in part by c-FLIP pathway inhibitors.
Colonic Patches Direct the Cross-Talk Between Systemic Compartments and Large Intestine Independently of Innate Immunity
Authors: Chang Sun-Young , Cha Hye-Ran , Uematsu Satoshi , Akira Shizuo , Igarashi Osamu , Kiyono Hiroshi , Kweon Mi-Na
Published: April 2014

Although the mucosal and the systemic immune compartments are structurally and functionally independent, they engage in cross-talk under specific conditions. To investigate this cross-talk, we vaccinated mice with tetanus toxoid together with cholera toxin with s.c. priming followed by intrarectal (IR) boosting. Interestingly, higher numbers of Ag-specific IgA and IgG Ab-secreting cells (ASCs) were detected in the lamina propria of the large intestine of mice vaccinated s.c.-IR. Ag-specific ASCs from the colon migrated to SDF-1α/CXCL12 and mucosae-associated epithelial chemokine/CCL28, suggesting that CXCR4+ and/or CCR10+ IgA ASCs found in the large intestine after s.c.-IR are of systemic origin. In the colonic patches-null mice, IgA ASCs in the large intestine were completely depleted. Furthermore, the accumulation of IgA ASCs in the colonic patches by inhibition of their migration with FTY720 revealed that colonic patches are the IgA class-switching site after s.c.-IR. Most interestingly, s.c.-IR induced numbers of Ag-specific IgA ASCs in the large intestine of TLR2−/−, TLR4−/−, MyD88−/−, and TRIF−/− mice that were comparable with those of wild-type mice. Taken together, our results suggest the possibility that cross-talk could occur between the large intestine and the systemic immune compartments via the colonic patches without the assistance of innate immunity.
Suppression of Th2 Cell Development by Notch Ligands Delta1 and Delta4
Authors: Sun Jie , Krawczyk Connie J. , Pearce Edward J.
Published: April 2014

Notch signaling plays important roles in Th cell activation. We show that in response to TLR ligation, dendritic cells up-regulate expression of Notch ligands Delta1 and Delta4 via a MyD88-dependent pathway. Expression of Delta1 or Delta4 by dendritic cells enhanced their ability to activate naive Th cells and promote Th1 cell development, and allowed them to strongly inhibit Th2 cell development. Promotion of Th1 cell development was dependent on IFN-γ and T-bet expression by responding Th cells. However, the inhibition of Th2 cell development occurred independently of IFN-γ or T-bet, and resulted from a block in IL-4-initiated commitment to the Th2 lineage. The promotion of Th1 cell development by Delta is not a reflection of the delivery of pro-Th1 instructional signal, but rather it is the result of a block in the downstream effects initiated by IL-4 signaling.
The Receptor Tyrosine Kinase RON Represses HIV-1 Transcription by Targeting RNA Polymerase II Processivity
Authors: Klatt Alicia , Zhang Zhiqiang , Kalantari Parisa , Hankey Pamela A. , Gilmour David S. , Henderson Andrew J.
Published: April 2014

Efficient HIV-1 transcription requires the induction of cellular transcription factors, such as NF-κB, and the viral factor Tat, which through the recruitment of P-TEFb enhances processive transcription. However, whether cellular signals repress HIV-1 transcription to establish proviral latency has not been well studied. Previously, it has been shown that the receptor tyrosine kinase RON inhibits HIV transcription. To gain insights into the biochemical mechanisms by which RON inhibits transcription we examined the binding of transcription factors to the HIV provirus long terminal repeat using chromatin immunoprecipitation. RON expression decreased basal levels of NF-κB and RNA polymerase II (Pol II) binding to the HIV provirus long terminal repeat but did not prevent the induction of these complexes following treatment with cytokines. However, RON did decrease efficient transcription elongation because reduced RNA Pol II was associated with HIV-1 genomic sequences downstream of the transcriptional start site. There was a correlation between RON expression and increased binding of factors that negatively regulate transcription elongation, NELF, Spt5, and Pcf11. Furthermore, the ability of RON to inhibit HIV-1 transcription was sensitive to a histone deacetylase inhibitor and was associated with nucleosome remodeling. These results indicate that RON represses HIV transcription at multiple transcriptional check points including initiation, elongation and chromatin organization and are the first studies to show that cellular signaling pathways target Pol II pausing to repress gene expression.
Differential Roles for the E2A Activation Domains in B Lymphocytes and Macrophages
Authors: Bhalla Savita , Spaulding Christina , Brumbaugh Rachel L. , Zagort Derek E. , Massari Mark E. , Murre Cornelis , Kee Barbara L.
Published: April 2014

The E2A gene encodes two E protein/class I basic helix-loop-helix transcription factors, E12 and E47, that are essential for B lymphopoiesis. In addition to the DNA-binding and protein dimerization domain, the E proteins share two highly conserved transcription activation domains. In this study, we show that both activation domains are required for optimal E2A-dependent transcription. Surprisingly, however, neither activation domain is required for E2A to rescue B lymphopoiesis from E2A−/− hemopoietic progenitors, although the N terminus of E2A, which harbors some transcription capacity, is required. Therefore, the E protein activation domains function redundantly in promoting B cell development. In contrast, the N-terminal activation domain, AD1, is required for a newly described ability of E2A to suppress macrophage development in vitro. Our findings demonstrate distinct functionalities for the E protein activation domains in B lymphocytes and macrophages.
Role of the β-Subunit Arginine/Lysine Finger in Integrin Heterodimer Formation and Function
Authors: Gupta Vineet , Alonso José Luis , Sugimori Takashi , Issafi Makram , Xiong Jiang-Ping , Arnaout M. Amin
Published: April 2014

Formation of the integrin αβ heterodimer is essential for cell surface expression and function. At the core of the αβ interface is a conserved Arg/Lys “finger” from the β-subunit that inserts into a cup-like “cage” formed of two layers of aromatic residues in the α-subunit. We evaluated the role of this residue in heterodimer formation in an αA-lacking and an αA-containing integrin αVβ3 and αMβ2 (CD11b/CD18), respectively. Arg261 of β3 was mutated to Ala or Glu; the corresponding Lys252 of β2 was mutated to Ala, Arg, Glu, Asp, or Phe; and the effects on heterodimer formation in each integrin examined by ELISA and immunoprecipitation in HEK 293 cells cotransfected with plasmids encoding the α- and β-subunits. The Arg261Glu (but not Arg261Ala) substitution significantly impaired cell surface expression and heterodimer formation of αVβ3. Although Lys252Arg, and to a lesser extent Lys252Ala, were well tolerated, each of the remaining substitutions markedly reduced cell surface expression and heterodimer formation of CD11b/CD18. Lys252Arg and Lys252Ala integrin heterodimers displayed a significant increase in binding to the physiologic ligand iC3b. These data demonstrate an important role of the Arg/Lys finger in formation of a stable integrin heterodimer, and suggest that subtle changes at this residue affect the activation state of the integrin.
NK Cells Lyse T Regulatory Cells That Expand in Response to an Intracellular Pathogen
Authors: Roy Sugata , Barnes Peter F. , Garg Ankita , Wu Shiping , Cosman David , Vankayalapati Ramakrishna
Published: April 2014

We evaluated the capacity of NK cells to influence expansion of CD4+CD25+FoxP3+ regulatory T cells (Tregs) in response to microbial Ags, using Mycobacterium tuberculosis as a model. We previously found that Tregs expand when CD4+ cells and monocytes are exposed to M. tuberculosis. Addition of NK cells that were activated by monokines (IL-12, IL-15, and IL-18) or by exposure to M. tuberculosis-stimulated monocytes reduced Treg expansion in response to M. tuberculosis. NK cell inhibition of Treg expansion was not mediated through IFN-γ. Activated NK cells lysed expanded, but not freshly isolated Tregs. Although monokines increased NK cell expression of the activating receptors NKp46, NKG2D, 2B4, CD16, and DNAM-1, only anti-NKG2D and anti-NKp46 inhibited NK cell lysis of expanded Tregs. Of five NKG2D ligands, only UL16-binding protein 1 (ULBP1) was up-regulated on M. tuberculosis-expanded Tregs, and anti-ULBP1 inhibited NK cell lysis of expanded Tregs. M. tuberculosis-stimulated monocytes activated NK cells to lyse expanded Tregs, and this was also inhibited by anti-NKG2D and anti-ULBP1, confirming the physiological relevance of this effect. Our study identifies a potential new role for NK cells in maintaining the delicate balance between the regulatory and effector arms of the immune response.
Cytosolic Antiviral RNA Recognition Pathway Activates Caspases 1 and 3
Authors: Rintahaka Johanna , Wiik Daniel , Kovanen Panu E. , Alenius Harri , Matikainen Sampsa
Published: April 2014

During an innate immune response, macrophages recognize viruses by their pattern recognition receptors. In this study, we have studied the role of membrane-associated TLRs and cytoplasmic retinoic acid inducible gene-I (RIG-I)-like receptors (RLR) in regulation of IFN-β, IL-29, IL-1β, and IL-18 production and caspases 1 and 3 activation in human macrophages. We provide evidence that TLRs are mainly involved in transcriptional up-regulation of IL-1β gene expression, whereas cytosolic dsRNA recognition pathway stimulates powerful IFN-β and IL-29 gene transcription. However, robust IL-1β secretion occurred only if two TLRs were triggered simultaneously or if a single TLR was activated in conjunction with the RLR pathway. Markedly, TLR activation did not stimulate IL-18 processing or secretion. In contrast, triggering of cytosolic RNA recognition pathway with poly(I:C) transfection or influenza A virus infection resulted in caspase-1- and -3-mediated proteolytic processing of pro-IL-18 and secretion of biologically active IL-18. Furthermore, caspase 3-dependent processing of pro-IL-18 was also observed in human HaCaT keratinocytes, and forced expression of RIG-I and its downstream effector, mitochondrial antiviral signaling protein, activated proteolytic processing of pro-IL-18, caspase-3, and apoptosis in these cells. The present results indicate that in addition to robust IFN-β, IL-29, IL-1β, and IL-18 generation, RIG-I/mitochondrial antiviral signaling protein pathway activates caspase-3, suggesting a role for these RIG-I-like receptors beyond the innate cytokine response, hence, in the induction of apoptosis of the virus-infected cell.
Helminth Infection withLitomosoides sigmodontisInduces Regulatory T Cells and Inhibits Allergic Sensitization, Airway Inflammation, and Hyperreactivity in a Murine Asthma Model
Authors: Dittrich Anna M. , Erbacher Annika , Specht Sabine , Diesner Felix , Krokowski Martin , Avagyan Angela , Stock Philippe , Ahrens Birgit , Hoffmann Wolfgang H. , Hoerauf Achim , Hamelmann Eckard
Published: April 2014

Numerous epidemiological studies have shown an inverse correlation between helminth infections and the manifestation of atopic diseases, yet the immunological mechanisms governing this phenomenon are indistinct. We therefore investigated the effects of infection with the filarial parasite Litomosoides sigmodontis on allergen-induced immune reactions and airway disease in a murine model of asthma. Infection with L. sigmodontis suppressed all aspects of the asthmatic phenotype: Ag-specific Ig production, airway reactivity to inhaled methacholine, and pulmonary eosinophilia. Similarly, Ag-specific recall proliferation and overall Th2 cytokine (IL-4, IL-5, and IL-3) production were significantly reduced after L. sigmodontis infection. Analysis of splenic mononuclear cells and mediastinal lymph nodes revealed a significant increase in the numbers of T cells with a regulatory phenotype in infected and sensitized mice compared with sensitized controls. Additionally, surface and intracellular staining for TGF-β on splenic CD4+ T cells as well as Ag-specific TGF-β secretion by splenic mononuclear cells was increased in infected and sensitized animals. Administration of Abs blocking TGF-β or depleting regulatory T cells in infected animals before allergen sensitization and challenges reversed the suppressive effect with regard to airway hyperreactivity, but did not affect airway inflammation. Despite the dissociate results of the blocking experiments, these data point toward an induction of regulatory T cells and enhanced secretion of the immunomodulatory cytokine TGF-β as one principle mechanism. In conclusion, our data support the epidemiological evidence and enhance the immunological understanding concerning the impact of helminth infections on atopic diseases thus providing new insights for the development of future studies.
Autocrine Regulation of IL-21 Production in Human T Lymphocytes
Authors: Caprioli Flavio , Sarra Massimiliano , Caruso Roberta , Stolfi Carmine , Fina Daniele , Sica Giuseppe , MacDonald Thomas T. , Pallone Francesco , Monteleone Giovanni
Published: April 2014

IL-21 has pathologic function in immune-inflammatory diseases. IL-21 mediates its functions through a heterodimeric receptor, composed of a specific subunit, termed IL-21R, and the common γ-chain. IL-21 is mostly produced by CD4+ T cells, but molecular mechanisms that regulate IL-21 synthesis are not fully understood. The fact that CD4+ T cells express high levels of IL-21R and are capable of functionally responding to IL-21 raises the possibility that IL-21 may regulate its own production. We here show that IL-21 enhances IL-21 RNA and protein expression in human peripheral blood CD3+ T cells in a dose- and time-dependent fashion. Additionally, both IL-7 and IL-15, but not IL-4, induce IL-21, thus suggesting that common γ-chain signals are not sufficient to promote IL-21 synthesis. Analysis of molecular mechanisms underlying IL-21 induction reveals that IL-21 activates Stat3 and enhances its recruitment to IL-21 gene promoter. Pharmacologic inhibition and knockdown of Stat3 by small interference RNA largely prevent IL-21 induction in IL-21-treated cells. Consistently, IL-21 is inducible in T cells by IL-6, another cytokine that activates Stat3. Finally, we show that IL-21 positively regulates its own expression in human intestinal CD3+ lamina propria lymphocytes, and blockade of endogenous IL-21 in cultures of CD3+ lamina propria lymphocytes isolated from patients with Crohn’s disease, a chronic inflammatory bowel disease characterized by high IL-21, down-regulates Stat3 activation and IL-21 expression. These data suggest the existence of a positive autocrine loop that could help to amplify and stabilize IL-21-driven, T cell-mediated responses.
Cyclin-Dependent Kinase Inhibitors Block Leukocyte Adhesion and Migration
Authors: Liu Li , Schwartz Barbara , Tsubota Yoshiaki , Raines Elaine , Kiyokawa Hiroaki , Yonekawa Karyn , Harlan John M. , Schnapp Lynn M.
Published: April 2014

Leukocyte trafficking is a tightly regulated process essential for an appropriate inflammatory response. We now report a new adhesion pathway that allows unstimulated leukocytes to adhere to and migrate through exposed endothelial matrix or high-density ligand, a process we have termed ligand-induced adhesion. This ligand-induced adhesion is integrin mediated, but in contrast to phorbol ester-stimulated adhesion, it is not dependent on the small GTPase Rap-1 activity. Instead, we show a critical role for cyclin-dependent kinase (Cdk) 4 in ligand-induced adhesion by three independent lines of evidence: inhibition by pharmacological inhibitors of Cdk, inhibition by dominant-negative construct of Cdk4, and inhibition by Cdk4 small interfering RNA. The major substrate of Cdk4, Rb, is not required for ligand-induced adhesion, suggesting the involvement of a novel Cdk4 substrate. We also demonstrate that Cdk4−/− mice have impaired recruitment of lymphocytes to the lung following injury. The finding that Cdk inhibitors can block leukocyte adhesion and migration may expand the clinical indications for this emerging class of therapeutics.
Sphingosine 1-Phosphate 1 and TLR4 Mediate IFN-β Expression in Human Gingival Epithelial Cells
Authors: Eskan Mehmet A. , Rose Beate G. , Benakanakere Manjunatha R. , Lee Menq-Jer , Kinane Denis F.
Published: April 2014

IFN-β production is a critical step in human innate immune responses and is primarily controlled at the transcription level by highly ordered mechanisms. IFN-β can be induced by pattern-recognition receptors such as the TLR4. S1P1 is a G protein-coupled receptor, which has a high affinity for sphingosine 1-phosphate (S1P). Although many of the receptors and signaling pathways leading to the expression of IFN-β have been identified and characterized, it is still unclear how IFN-β is regulated in primary human gingival epithelial cells (HGECs). In this study, we demonstrate that S1P1 and TLR4, acting in unison, play an important role in IFN-β expression at the protein and mRNA level in HGECs. We demonstrate that the expression of both IFN-β and IFN-inducible protein-10 (CXCL-10) is significantly up-regulated by LPS and S1P or LPS and a specific S1P1 agonist. This enhanced innate immune response is attenuated in HGECs by small interfering RNA knockdown of either TLR4 or S1P1. Moreover, we show that triggering of TLR4 results in the increased expression of S1P1 receptors. Furthermore, we found that IFN-regulatory factor 3 activation was maximized by LPS and S1P through PI3K. Our data show that triggering TLR4 increases S1P1, such that both TLR4 and S1P1 acting through PI3K enhancement of IFN-regulatory factor 3 activation increase IFN-β expression in epithelial cells. The functional association between TLR4 and the S1P1 receptor demonstrates a novel mechanism in the regulation of IFN-β and CXCL-10 in human primary gingival epithelial cells.
Cyclooxygenase-2 Deficiency Enhances Th2 Immune Responses and Impairs Neutrophil Recruitment in Hepatic Ischemia/Reperfusion Injury
Authors: Hamada Takashi , Tsuchihashi Seiichiro , Avanesyan Armine , Duarte Sergio , Moore Carolina , Busuttil Ronald W. , Coito Ana J.
Published: April 2014

Cyclooxygenase-2 (COX-2) is a prostanoid-synthesizing enzyme that is critically implicated in a variety of pathophysiological processes. Using a COX-2-deficient mouse model, we present data that suggest that COX-2 has an active role in liver ischemia/reperfusion (I/R) injury. We demonstrate that COX-2-deficient mice had a significant reduction in liver damage after I/R insult. The inability of COX-2−/− to elaborate COX-2 products favored a Th2-type response in these mice. COX-2−/− livers after I/R injury showed significantly decreased levels of IL-2, as well as IL-12, a cytokine known to have a central role in Th1 effector cell differentiation. Moreover, such livers expressed enhanced levels of the anti-inflammatory cytokine IL-10, shifting the balance in favor of a Th2 response in COX-2-deficient mice. The lack of COX-2 expression resulted in decreased levels of CXCL2, a neutrophil-activating chemokine, reduced infiltration of MMP-9-positive neutrophils, and impaired late macrophage activation in livers after I/R injury. Additionally, Bcl-2 and Bcl-xL were normally expressed in COX-2−/− livers after injury, whereas respective wild-type controls were almost depleted of these two inhibitors of cell death. In contrast, caspase-3 activation and TUNEL-positive cells were depressed in COX-2−/− livers. Therefore, our data support the concept that COX-2 is involved in the pathogenic events occurring in liver I/R injury. The data also suggest that potential valuable therapeutic approaches in liver I/R injury may result from further studies aimed at identifying specific COX-2-derived prostanoid pathways.
Polarized Localization of Epithelial CXCL11 in Chronic Obstructive Pulmonary Disease and Mechanisms of T Cell Egression
Authors: Porter Joanna C. , Falzon Mary , Hall Alan
Published: April 2014

The exit of lymphocytes from the interstitium of the lung, across the bronchial epithelium and into the airway lumen, is known as egression, or luminal clearance. Egression is important for immune surveillance and the resolution of inflammation, but the mechanisms involved are unknown. We show that egression of human T cells across the bronchial epithelium is a multistep process, driven in part by a polarized transepithelial gradient of CXCL11 that is up-regulated in patients with chronic obstructive airways disease. Previous studies have shown that T cells can migrate across a disrupted bronchial epithelium, but we provide evidence that egression does not require epithelial injury, and can take place across an intact epithelial barrier. After negotiating the extracellular matrix, the T cell adheres to the basal surface of the bronchial epithelial cell using α4 and leukocyte function associated-1 integrins before crossing the epithelium in an leukocyte function associated-1-dependent way. We demonstrate an egression-dependent decrease in transepithelial resistance across the epithelium without gross alteration in tight-junction proteins. The process of egression has been relatively overlooked when considering the control of leukocyte trafficking in the lung and other epithelial organs. This study highlights the role of the respiratory epithelium in the trafficking of T lymphocytes from the pulmonary interstitium and into the large airways, during the onset and resolution of pulmonary inflammation.
Histone Deimination As a Response to Inflammatory Stimuli in Neutrophils
Authors: Neeli Indira , Khan Salar N. , Radic Marko
Published: April 2014

Posttranslational modifications, such as the deimination of arginine to citrulline by peptidyl arginine deiminase (PAD4), change protein structure and function. For autoantigens, covalent modifications represent a mechanism to sidestep tolerance and stimulate autoimmunity. To examine conditions leading to histone deimination in neutrophils, we used Abs that detect citrullines in the N terminus of histone H3. Deimination was investigated in human neutrophils and HL-60 cells differentiated into granulocytes. We observed rapid and robust H3 deimination in HL-60 cells exposed to LPS, TNF, lipoteichoic acid, f-MLP, or hydrogen peroxide, which are stimuli that activate neutrophils. Importantly, we also observed H3 deimination in human neutrophils exposed to these stimuli. Citrullinated histones were identified as components of extracellular chromatin traps (NETs) produced by degranulating neutrophils. In contrast, apoptosis proceeded without detectable H3 deimination in HL-60 cells exposed to staurosporine or camptothecin. We conclude that histone deimination in neutrophils is induced in response to inflammatory stimuli and not by treatments that induce apoptosis. Our results further suggest that deiminated histone H3, a covalently modified form of a prominent nuclear autoantigen, is released to the extracellular space as part of the neutrophil response to infections. The possible association of a modified autoantigen with microbial components could, in predisposed individuals, increase the risk of autoimmunity.
Role of Sphingosine 1-Phosphate in the Pathogenesis of Sjögren’s Syndrome
Authors: Sekiguchi Masahiro , Iwasaki Tsuyoshi , Kitano Masayasu , Kuno Hideki , Hashimoto Naoaki , Kawahito Yutaka , Azuma Masayuki , Hla Timothy , Sano Hajime
Published: April 2014

Primary Sjögren’s syndrome (SS) is an autoimmune disease characterized by inflammatory mononuclear cell infiltration and destruction of epithelial cells of lacrimal and salivary glands. Sphingosine 1-phosphate (S1P) and signaling through its receptor S1P1 have been implicated in many critical cellular events including inflammation, cancer, and angiogenesis. This study was undertaken to examine the role of S1P1 signaling in the pathogenesis of primary SS. S1P1 and sphingosine kinase 1, which converts sphingosine to S1P, were detected in the cytoplasm of inflammatory mononuclear cells, vascular endothelial cells, and epithelial cells in all labial salivary glands by immunohistochemistry. The expression of S1P1 in inflammatory mononuclear cells was enhanced in advanced stages of primary SS. S1P enhanced proliferation and IFN-γ production by CD4+ T cells. The enhancing effect of S1P on IFN-γ production by CD4+ T cells was stronger in patients with primary SS than in healthy controls. S1P also enhanced Fas expression and Fas-mediated caspase-3 induction in salivary gland epithelial cells. IL-6 expression was detected in the cytoplasm of inflammatory mononuclear cells and ductal epithelial cells and was enhanced in advanced stages of primary SS. Furthermore, both IFN-γ and S1P augmented IL-6 secretion by salivary gland epithelial cells. These effects of S1P were inhibited by pretreatment of pertussis toxin. Our data reveal that S1P1 signaling may modulate the autoimmune phenotype of primary SS by the action of immune as well as epithelial cells.
Distinct, Specific IL-17- and IL-22-Producing CD4+ T Cell Subsets Contribute to the Human Anti-Mycobacterial Immune Response
Authors: Scriba Thomas J. , Kalsdorf Barbara , Abrahams Deborah-Ann , Isaacs Fatima , Hofmeister Jessica , Black Gillian , Hassan Hisham Y. , Wilkinson Robert J. , Walzl Gerhard , Gelderbloem Sebastian J. , Mahomed Hassan , Hussey Gregory D. , Hanekom Willem A.
Published: April 2014

We investigated whether the proinflammatory T cell cytokines IL-17 and IL-22 are induced by human mycobacterial infection. Remarkably, >20% of specific cytokine-producing CD4+ T cells in peripheral blood of healthy, mycobacteria-exposed adults expressed IL-17 or IL-22. Specific IL-17- and IL-22-producing CD4+ T cells were distinct from each other and from Th1 cytokine-producing cells. These cells had phenotypic characteristics of long-lived central memory cells. In patients with tuberculosis disease, peripheral blood frequencies of these cells were reduced, whereas bronchoalveolar lavage fluid contained higher levels of IL-22 protein compared with healthy controls. IL-17 was not detected in this fluid, which may be due to suppression by Th1 cytokines, as PBMC IL-17 production was inhibited by IFN-γ in vitro. However, Th1 cytokines had no effect on IL-22 production in vitro. Our results imply that the magnitude and complexity of the anti-mycobacterial immune response have historically been underestimated. IL-17- and IL-22-producing CD4+ T cells may play important roles in the human immune response to mycobacteria.
Plasticity of Macrophage Function during Tumor Progression: Regulation by Distinct Molecular Mechanisms
Authors: Biswas Subhra K. , Sica Antonio , Lewis Claire E.
Published: April 2014

Recent studies have shown that macrophages play an important part in both tumor initiation and various key steps in growth and metastasis. These cells show a remarkable degree of plasticity during tumor development with a “switch” in macrophage phenotypes occurring during the course of tumor progression. During chronic inflammation they appear to predispose a given tissue to tumor initiation by the release of factors that promote neoplastic transformation. Following this, their phenotype shifts more toward one that is immunosuppressive and supports tumor growth, angiogenesis, and metastasis. In this review, we discuss the evidence for this plasticity of macrophage functions, the specific signaling mechanisms that may be regulating it, and the new targets for anticancer therapies highlighted by these findings.
Cutting Edge: Activation by Innate Cytokines or Microbial Antigens Can Cause Arrest of Natural Killer T Cell Patrolling of Liver Sinusoids
Authors: Velázquez Peter , Cameron Thomas O. , Kinjo Yuki , Nagarajan Niranjana , Kronenberg Mitchell , Dustin Michael L.
Published: April 2014

Natural killer T (NKT) cells are innate-like lymphocytes that rapidly secrete large amounts of effector cytokines upon activation. Recognition of α-linked glycolipids presented by CD1d leads to the production of IL-4, IFN-γ, or both, while direct activation by the synergistic action of IL-12 and IL-18 leads to IFN-γ production only. We previously reported that in vitro cultured dendritic cells can modulate NKT cell activation and, using intravital fluorescence laser scanning microscopy, we reported that the potent stimulation of NKT cells results in arrest within hepatic sinusoids. In this study, we examine the relationship between murine NKT cell patrolling and activation. We report that NKT cell arrest results from activation driven by limiting doses of a bacteria-derived weak agonist, galacturonic acid-containing glycosphingolipid, or a synthetic agonist, α-galactosyl ceramide. Interestingly, NKT cell arrest also results from IL-12 and IL-18 synergistic activation. Thus, innate cytokines and natural microbial TCR agonists trigger sinusoidal NKT cell arrest and an effector response.
Cutting Edge: Progesterone Regulates IFN-α Production by Plasmacytoid Dendritic Cells
Authors: Hughes Grant C. , Thomas Sunil , Li Chang , Kaja Murali-Krishna , Clark Edward A.
Published: April 2014

Use of the progesterone (Pg) birth control depot medroxyprogesterone acetate (DMPA) increases a woman’s risk for sexually transmitted infection with HIV or HSV-2 via unknown mechanisms. Plasmacytoid dendritic cells (pDCs) are circulating and tissue-resident sentinels capable of making large quantities of IFN-α upon recognizing viruses through TLRs 7 and 9. In this study, we show that Pg inhibits TLR9-induced IFN-α production by human and mouse pDCs and that DMPA impairs TLR9- and virus-induced IFN-α production by pDCs in mice, providing a potential explanation for how DMPA impairs innate antiviral immunity in women. Pg failed to inhibit the Mda-5 pathway of IFN-α induction in dendritic cells, suggesting that Pg regulates select antiviral DC programs. This may occur through selective blockade of IFN regulatory factor-7 activation, a novel steroid action. Thus, through inhibition of TLR-mediated IFN-α production by pDCs, Pg may regulate antiviral immunity.
4-1BB Engagement Costimulates NKT Cell Activation and Exacerbates NKT Cell Ligand-Induced Airway Hyperresponsiveness and Inflammation
Authors: Kim Dong-Hyeon , Chang Woo-Sung , Lee Yoon-Sook , Lee Kyoo-A , Kim Yoon-Keun , Kwon Byoung S. , Kang Chang-Yuil
Published: April 2014

Multiple studies have demonstrated that 4-1BB (CD137), a member of the TNF receptor superfamily, is expressed on several immune cells including activated T cells. However, the expression and the role of 4-1BB on natural killer T (NKT) cells have not been fully characterized. In this study, it was shown that 4-1BB was not expressed on naive NKT cells but was rapidly induced on activated NKT cells by TCR engagement with α-galactosylceramide (α-GalCer). Also, 4-1BB signaling provided by 3H3, an agonistic anti-4-1BB mAb, promoted NKT cell activation resulting in enhanced cytokine production of NKT cells driven by α-GalCer. When NKT cell-driven airway immune responses were evaluated by intranasal administration of α-GalCer, airway hyperresponsiveness (AHR) and lung inflammation were significantly more aggravated in mice treated with 3H3 and α-GalCer than in mice treated with α-GalCer alone. These aggravations were accompanied by up-regulation of IL-4, IL-13, and IFN-γ production. Interestingly, AHR was not developed in IL-4Rα-deficient mice treated with α-GalCer with or without 3H3 but was exacerbated in IFN-γ-deficient mice. Our study suggests that 4-1BB on NKT cells functions as a costimulatory molecule and exacerbates the induction of NKT cell-mediated AHR, which is dependent on the IL-4Rα-mediated pathway.
Inhibition of STAT3 Promotes the Efficacy of Adoptive Transfer Therapy Using Type-1 CTLs by Modulation of the Immunological Microenvironment in a Murine Intracranial Glioma
Authors: Fujita Mitsugu , Zhu Xinmei , Sasaki Kotaro , Ueda Ryo , Low Keri L. , Pollack Ian F. , Okada Hideho
Published: April 2014

A variety of cancers, including malignant gliomas, show aberrant activation of STAT3, which plays a pivotal role in negative regulation of antitumor immunity. We hypothesized that inhibition of STAT3 signals would improve the efficacy of T cell adoptive transfer therapy by reversal of STAT3-induced immunosuppression in a murine GL261 intracranial glioma model. In vitro treatment of GL261 cells with JSI-124, a STAT3 inhibitor, reversed highly phosphorylated status of STAT3. Systemic i.p. administration of JSI-124 in glioma-bearing immunocompetent mice, but not athymic mice, resulted in prolonged survival, suggesting a role of adaptive immunity in the antitumor effect. Furthermore, JSI-124 promoted maturation of tumor-infiltrating CD11c+ dendritic cells and activation of tumor-conditioned cytotoxic T cells, enhanced dendritic cells and GL261 production of CXCL-10, a critical chemokine for attraction of Tc1 cells. When i.p. JSI-124 administration was combined with i.v. transfer of Pmel-I mouse-derived type-1 CTLs (Tc1), glioma-bearing mice exhibited prolonged survival compared with i.p. JSI-124 or i.v. Tc1 therapy alone. Flow cytometric analyses of brain infiltrating lymphocytes revealed that JSI-124-treatment enhanced the tumor-homing of i.v. transferred Tc1 cells in a CXCL-10-dependent fashion. Systemic JSI-124 administration also up-regulated serum IL-15 levels, and promoted the persistence of transferred Tc1 in the host. These data suggest that systemic inhibition of STAT3 signaling can reverse the suppressive immunological environment of intracranial tumor bearing mice both systemically and locally, thereby promoting the efficacy of adoptive transfer therapy with Tc1.