bluuebunny/crossref_metadata_2025 huggingface dataset:
Reporter Gene Insertions Reveal a Strictly B Lymphoid-Specific Expression Pattern of Pax5 in Support of Its B Cell Identity Function
Authors: Fuxa Martin , Busslinger Meinrad
Published: April 2014

The transcription factor Pax5 is essential for B cell commitment and development. Although the detailed Pax5 expression pattern within the hemopoietic system is still largely unknown, we previously reported that Pax5 is monoallelically transcribed in pro-B and mature B cells. In this study, we have investigated the expression of Pax5 at single-cell resolution by inserting a GFP or human Cd2 indicator gene under the translational control of an internal ribosomal entry sequence into the 3′ untranslated region of Pax5. These insertions were noninvasive, as B cell development was normal in Pax5ihCd2/ihCd2 and Pax5iGFP/iGFP mice. Transheterozygous Pax5ihCd2/iGFP mice coexpressed GFP and human CD2 at similar levels from pro-B to mature B cells, thus demonstrating biallelic expression of Pax5 at all stages of B cell development. No reporter gene expression could be detected in plasma cells and non-B cells of the hemopoietic system. Moreover, the vast majority of common lymphoid progenitors and pre-pro-B cells in the bone marrow of Pax5iGFP/iGFP mice did not yet express GFP, indicating that Pax5 expression is fully switched on only during the transition from uncommitted pre-pro-B cells to committed pro-B cells. Hence, the transcriptional initiation and B cell-specific expression of Pax5 is entirely consistent with its B cell lineage commitment function.
NF-AT-Mediated Expression of TGF-β1 in Tolerant T Cells
Authors: Nakano Naoko , Hosokawa Hiroyuki , Kohyama Masako , Hozumi Nobumichi
Published: April 2014

During T cell development in the thymus, a certain population of self-reactive thymocytes differentiates into regulatory T cells that suppress otherwise harmful self-reactive T cells. In transgenic mice expressing both TCR that specifically recognizes moth cytochrome c and the moth cytochrome c ligand, a large proportion of CD4+ T cells expresses CD25 and secretes TGF-β1 upon Ag stimulation. Because TGF-β1 expression by these T cells can be decreased by cyclosporin A, a NF-AT inhibitor, NF-AT-mediated TGF-β1 expression in T cells was addressed by characterizing a NF-AT response element in the TGF-β1 promoter. Analysis of the mouse TGF-β1 promoter (−1799 to +793) in transfection experiments in T cell 68-41 hybridoma cells detected NF-AT binding sites at positions +268 and +288 in the proximal promoter region. Binding of NF-AT to this region was detected only in tolerant CD4+ T cells, but not in fully activated CD4+ T cells by chromatin immunoprecipitation assays. Activation of these NF-AT sites was sufficient to induce TGF-β1 promoter activity; however, additional signaling due to full Ag stimulation blocked NF-AT-mediated TGF-β1 expression. This suppression of the TGF-β1 promoter is mediated by the −1079 to −406 region, in which deletion of a GATA-binding motif at position −821 abrogates NF-AT-mediated activation of the TGF-β1 promoter. Therefore, TGF-β1 expression in T cells is controlled by multiple regulatory factors that have distinct functions in response to partial or full TCR activation.
Fragmentation of Two Quantitative Trait Loci Controlling Collagen-Induced Arthritis Reveals a New Set of Interacting Subloci
Authors: Ahlqvist Emma , Bockermann Robert , Holmdahl Rikard
Published: April 2014

Linkage analysis of F2 crosses has led to identification of large numbers of quantitative trait loci (QTL) for complex diseases, but identification of the underlying genes has been more difficult. Reasons for this could be complications that arise from separation of interacting or neighboring loci. We made a partial advanced intercross (PAI) to characterize and fine-map linkage to collagen-induced arthritis in two chromosomal regions derived from the DBA/1 strain and crossed into the B10.Q strain: Cia7 on chromosome 7 and a locus on chromosome 15. Only Cia7 was detected by a previous F2 cross. Linkage analysis of the PAI revealed a different linkage pattern than the F2 cross, adding multiple loci and strong linkage to the previously unlinked chromosome 15 region. Subcongenic strains derived from animals in the PAI confirmed the loci and revealed additional subloci. In total, no less than seven new loci were identified. Several loci interacted and three loci were protective, thus partly balancing the effect of the disease-promoting loci. Our results indicate that F2 crosses do not reveal the full complexity of identified QTLs, and that detection is more dependent on the genetic context of a QTL than the potential effect of the underlying gene.
IL-13 Mediates In Vivo IL-9 Activities on Lung Epithelial Cells but Not on Hematopoietic Cells
Authors: Steenwinckel Valérie , Louahed Jamila , Orabona Ciriana , Huaux François , Warnier Guy , McKenzie Andrew , Lison Dominique , Levitt Roy , Renauld Jean-Christophe
Published: April 2014

Increased IL-9 expression, either systemically or under the control of lung-specific promoter, induces an asthma-like phenotype, including mucus overproduction, mastocytosis, lung eosinophilia, and airway hyperresponsiveness. These activities correlate with increased production of other Th2 cytokines such as IL-4, IL-5, and IL-13 in IL-9 Tg mice. To determine the exact role of IL-13 in this phenotype, mice overexpressing IL-9 were crossed with IL-13-deficient mice. In these animals, IL-9 could still induce mastocytosis and B lymphocyte infiltration of the lungs. Although IL-9-induced eosinophilia in the peritoneal cavity was not diminished in the absence of IL-13, IL-13 was required for IL-9 to increase eotaxin expression and lung eosinophilia. Mucus production and up-regulation of lung epithelial genes upon IL-9 overexpression were completely abolished in the absence of IL-13. Using hemopoietic cell transfer experiments with recipients that overexpressed IL-9 but were deficient in the IL-9 receptor (IL-9R), we could demonstrate that the effect of IL-9 on lung epithelial cells is indirect and could be fully restored by transfer of hemopoietic cells expressing IL-9R. Mucus production by lung epithelial cells was only up-regulated when hemopoietic cells simultaneously expressed functional IL-9R and IL-13 genes, indicating that IL-13 is not a cofactor but a direct mediator of the effect of IL-9 on lung epithelial cells. Taken together, these data indicate that IL-9 can promote asthma through IL-13-independent pathways via expansion of mast cells, eosinophils, and B cells, and through induction of IL-13 production by hemopoietic cells for mucus production and recruitment of eosinophils by lung epithelial cells.
Immunotherapy of Tumors with Neuroimmune Ligand Capsaicin
Authors: Beltran Jacqueline , Ghosh Amiya K. , Basu Sreyashi
Published: April 2014

Red chili pepper (Capsicum frutescens) is a highly consumed spice throughout the world. Its principal pungent ingredient is the phenol capsaicin (8-methyl-N-vanillyl-6-nonenamide). Capsaicin causes neurogenic inflammation and has analgesic and anti-inflammatory activities. We have observed previously that dendritic cells, a key cell type in immune responses, have the receptor for capsaicin, and engagement of this receptor has powerful immune consequences. In this study, we demonstrate that intratumoral administration of capsaicin into a preexisting tumor results in retarded progression of the injected tumor regardless of whether the tumor is at its early or late stage. Furthermore, it leads to significant inhibition of growth of other, uninjected tumors in the same animal. Capsaicin-elicited immunity is shown to be T cell-mediated and tumor-specific. These results reflect the immunological potency of a neurological ligand in modulating immune response against an established tumor.
Analysis of TCRαβ Combinations used by Simian Immunodeficiency Virus-Specific CD8+ T Cells in Rhesus Monkeys: Implications for CTL Immunodominance
Authors: Hasegawa Atsuhiko , Moriya Chikaya , Liu Huining , Charini William A. , Vinet Heather C. , Subbramanian Ramu A. , Sen Pritha , Letvin Norman L. , Kuroda Marcelo J.
Published: April 2014

Immunodominance is a common feature of Ag-specific CTL responses to infection or vaccines. Understanding the basis of immunodominance is crucial to understanding cellular immunity and viral evasion mechanisms and will provide a rational approach for improving HIV vaccine design. This study was performed comparing CTLs specific for the SIV Gag p11C (dominant) and SIV Pol p68A (subdominant) epitopes that are consistently generated in Mamu-A*01+ rhesus monkeys exposed to SIV proteins. Additionally, vaccinated monkeys were used to prevent any issues of antigenic variation or dynamic changes in CTL responses by continuous Ag exposure. Analysis of the TCR repertoire revealed the usage of higher numbers of TCR clones by the dominant p11C-specific CTL population. Preferential usage of specific TCRs and the in vitro functional TCR-α- and -β-chain-pairing assay suggests that every peptide/MHC complex may only be recognized by a limited number of unique combinations of α- and β-chain pairs. The wider array of TCR clones used by the dominant p11C-specific CTL population might be explained by the higher probability of generating those specific TCR chain pairs. Our data suggest that Ag-specific naive T cell precursor frequency may be predetermined and that this process dictates immunodominance of SIV-specific CD8+ T cell responses. These findings will aid in understanding immunodominance and designing new approaches to modulate CTL responses.
The Activating NKG2D Ligand MHC Class I-Related Chain A Transfers from Target Cells to NK Cells in a Manner That Allows Functional Consequences
Authors: McCann Fiona E. , Eissmann Philipp , Önfelt Björn , Leung Rufina , Davis Daniel M.
Published: April 2014

Recently, it has become apparent that surface proteins commonly transfer between immune cells in contact. Inhibitory receptors and ligands exchange between cells during NK cell surveillance and we report here that NK cells also acquire activating ligands from target cells. Specifically, the stress-inducible activating ligand for NKG2D, MHC class I-related chain A (MICA), transferred to NK cells upon conjugation with MICA-expressing target cells. Acquisition of MICA from target cells was dependent on cell contact and occurred after accumulation of MICA at the immunological synapse. Moreover, transfer of MICA was facilitated by specific molecular recognition via NKG2D and augmented by Src kinase signaling. Importantly, MICA associated with its new host NK cell membrane in an orientation that allowed engagement with NKG2D in trans and indeed could down-regulate NKG2D in subsequent homotypic interactions with other NK cells. MICA captured from target cells could subsequently transfer between NK cells and, more importantly, NK cell degranulation was triggered in such NK cell-NK cell interactions. Thus, NK cells can influence other NK cells with proteins acquired from target cells and our data specifically suggest that NK cells could lyse other NK cells upon recognition of activating ligands acquired from target cells. This mechanism could constitute an important function for immunoregulation of NK cell activity.
Flt3 Ligand Expands Lymphoid Progenitors Prior to Recovery of Thymopoiesis and Accelerates T Cell Reconstitution after Bone Marrow Transplantation
Authors: Wils Evert-Jan , Braakman Eric , Verjans Georges M. G. M. , Rombouts Elwin J. C. , Broers Annoek E. C. , Niesters Hubert G. M. , Wagemaker Gerard , Staal Frank J. T. , Löwenberg Bob , Spits Hergen , Cornelissen Jan J.
Published: April 2014

Deficient thymopoiesis and retarded recovery of newly developed CD4+ T cells is one of the most important determinants of impaired immunocompetence after hemopoietic stem cell transplantation. Here we evaluated whether Fms-like tyrosine kinase 3 (Flt3) ligand (FL) alone or combined with IL-7 affects T cell recovery, thymopoiesis, and lymphoid progenitor expansion following bone marrow transplantation in immunodeficient mice. FL strongly accelerated and enhanced the recovery of peripheral T cells after transplantation of a low number of bone marrow cells. An additive effect on T cell recovery was not observed after coadministration of IL-7. Lineage−sca-1+c-kit+flt3+ lymphoid progenitor cell numbers were significantly increased in bone marrow of FL-treated mice before recovery of thymopoiesis. Thymocyte differentiation was advanced to more mature stages after FL treatment. Improved T cell recovery resulted in better immunocompetence against a post-bone marrow transplantation murine CMV infection. Collectively, our data suggest that FL promotes T cell recovery by enhanced thymopoiesis and by expansion of lymphoid progenitors.
Induction of a Th1 Response from Th2-Polarized T Cells by Activated Dendritic Cells: Dependence on TCR:Peptide-MHC Interaction, ICAM-1, IL-12, and IFN-γ
Authors: Radhakrishnan Suresh , Wiehagen Karla R. , Pulko Vesna , Van Keulen Virginia , Faubion William A. , Knutson Keith L. , Pease Larry R.
Published: April 2014

Dendritic cells (DC) are important regulators of T cell immunity. The degree of stimulation, the pattern of costimulatory molecules expressed, and the cytokines secreted by DC dictate the nature of the effector and memory cells generated, particularly with respect to their Th1 or Th2 phenotypes. In this study, we demonstrate that the addition of activated DC to spleen cultures containing established Th2-polarized CD4+ T cells was sufficient to suppress Th2 and induce Th1 cytokines in a recall response, a phenomenon referred to as phenotype reversal. The ability of activated DC to induce phenotype reversal displayed exquisite Ag specificity. The DC activator B7-DC cross-linking Ab (XAb) was >10,000-fold more efficient at inducing phenotype reversal than the TLR agonists CpG-oligodeoxynucleotide and Gardiquimod. Characterization of the mechanisms governing phenotype reversal revealed the requirement for cognate interaction between the TCR:peptide-MHC complex, the expression of the costimulation/adhesion molecule ICAM-1, and secretion of IL-12 and IFN-γ by the activated DC. The requirement for the costimulation/adhesion molecule SLAM (signaling lymphocytic activation molecule) was found to be quantitative. Thus, activation of DC, particularly by crosslinking B7-DC, can modulate well-established Th2 T cell responses in an Ag-specific manner. Because the regulation of mouse and human DC by B7-DC XAb overlaps in several significant ways, immune modulation with B7-DC XAb is a potential strategy for treating Th2-mediated diseases.
Synergistic Effect of Bacillus Calmette Guerin and a Tuberculosis Subunit Vaccine in Cationic Liposomes: Increased Immunogenicity and Protection
Authors: Dietrich Jes , Billeskov Rolf , Doherty T. Mark , Andersen Peter
Published: April 2014

In the present work, we evaluated a new TB vaccine approach based on a combination of the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine and a subunit vaccine consisting of the proteins Ag85B and ESAT-6. We demonstrate that in addition to its vaccine efficacy BCG is an immune modulator that can potentiate a Th1 immune response better than the well-known adjuvant mono phosphoryl lipid A, leading to enhanced recognition of the subunit vaccine Ag85B-ESAT-6. Importantly, adding a vehicle to the vaccine, such as the cationic liposome dimethyl dioctadecyl ammonium bromide (DDA), significantly increased the potentiating effect of BCG. This synergistic effect between BCG and Ag85B-ESAT-6/liposome required drainage to the same lymph node of all vaccine components but did not require direct mixing of the components and was therefore also observed when BCG and Ag85B-ESAT-6/liposome were given as separate injections at sites draining to the same lymph node. The resulting optimized vaccine protocol consisting of BCG and subunit in liposomes (injected side by side) followed by boosting with the subunit in conventional adjuvant resulted in an impressive increase in the protective efficacy of up to 7-fold compared with BCG alone and 3-fold compared with unaugmented BCG boosted by the subunit vaccine. Thus, these studies suggest an immunization strategy where a novel TB subunit vaccine is administered as part of the child vaccination program together with BCG in neonates and followed by subunit boosting.
Overexpression of Suppressor of Cytokine Signaling-3 in T Cells Exacerbates Acetaminophen-Induced Hepatotoxicity
Authors: Numata Kosuke , Kubo Masato , Watanabe Hiroyuki , Takagi Katsumasa , Mizuta Hiroshi , Okada Seiji , Kunkel Steven L. , Ito Takaaki , Matsukawa Akihiro
Published: April 2014

Cytokines have been implicated in the progression of acetaminophen (APAP)-induced acute liver injury. Suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine signaling by inhibiting the JAK-STAT pathway, but their role in APAP hepatotoxicity is unknown. In this present study, we attempted to explore the role of SOCS3 in T cells in APAP-induced liver injury. Mice with a cell-specific overexpression of SOCS3 in T cells (SOCS3Tg, in which Tg is transgenic) exhibited exaggerated hepatic injury after APAP challenge, as evidenced by increased serum alanine aminotransferase levels, augmented hepatic necrosis, and decreased survival relative to the wild-type mice. Adaptive transfer of SOCS3Tg-CD4+ T cells into T and B cell-deficient RAG-2−/− mice resulted in an exacerbated liver injury relative to the control. In SOCS3Tg mice, hepatocyte apoptosis was enhanced with decreased expression of antiapoptotic protein bcl-2, whereas hepatocyte proliferation was reduced with altered cell cycle-regulatory proteins. Levels of IFN-γ and TNF-α in the circulation were augmented in SOCS3Tg mice relative to the control. Studies using neutralizing Abs indicated that elevated IFN-γ and TNF-α were responsible for the exacerbated hepatotoxicity in SOCS3Tg mice. Activation of STAT1 that is harmful in liver injury was augmented in SOCS3Tg hepatocytes. Alternatively, hepatoprotective STAT3 activation was decreased in SOCS3Tg hepatocytes, an event that was associated with augmented SOCS3 expression in the hepatocytes. Altogether, these results suggest that forced expression of SOCS3 in T cells is deleterious in APAP hepatotoxicity by increasing STAT1 activation while decreasing STAT3 activation in hepatocytes, possibly through elevated IFN-γ and TNF-α.
IL-21 Has a Pathogenic Role in a Lupus-Prone Mouse Model and Its Blockade with IL-21R.Fc Reduces Disease Progression
Authors: Herber Deborah , Brown Thomas P. , Liang Spencer , Young Deborah A. , Collins Mary , Dunussi-Joannopoulos Kyri
Published: April 2014

Systemic lupus erythematosus is a complex autoimmune disease characterized by dysregulated interactions between autoreactive T and B lymphocytes and the development of anti-nuclear Abs. The recently described pleiotropic cytokine IL-21 has been shown to regulate B cell differentiation and function. IL-21 is produced by activated T lymphocytes and its interactions with IL-21R are required for isotype switching and differentiation of B cells into Ab-secreting cells. In this report, we studied the impact of blocking IL-21 on disease in the lupus-prone MRL-Faslpr mouse model. Mice treated for 10 wk with IL-21R.Fc fusion protein had reduced proteinuria, fewer IgG glomerular deposits, no glomerular basement membrane thickening, reduced levels of circulating dsDNA autoantibodies and total sera IgG1 and IgG2a, and reduced skin lesions and lymphadenopathy, compared with control mice. Also, treatment with IL-21R.Fc resulted in a reduced number of splenic T lymphocytes and altered splenic B lymphocyte ex vivo function. Our data show for the first time that IL-21 has a pathogenic role in the MRL-Faslpr lupus model by impacting B cell function and regulating the production of pathogenic autoantibodies. From a clinical standpoint, these results suggest that blocking IL-21 in systemic lupus erythematosus patients may represent a promising novel therapeutic approach.
Immobilized Stromal Cell-Derived Factor-1α Triggers Rapid VLA-4 Affinity Increases to Stabilize Lymphocyte Tethers on VCAM-1 and Subsequently Initiate Firm Adhesion
Authors: DiVietro Jeffrey A. , Brown David C. , Sklar Larry A. , Larson Richard S. , Lawrence Michael B.
Published: April 2014

The integrin VLA-4 (α4β1) mediates tethering and rolling events as well as firm adhesion of leukocytes to VCAM-1. Unlike selectins, VLA-4 integrin-mediated lymphocyte adhesiveness can be modulated by chemokines through intracellular signaling pathways. To investigate the effects of the chemokine stromal cell-derived factor-1α (SDF-1α) on VLA-4-mediated lymphocyte adhesion, human PBL were flowed over VCAM-1 substrates in a parallel plate flow chamber with surface-immobilized SDF-1α, a potent activator of firm adhesion. The initial tethering interactions had a median lifetime of 200 ms, consistent with the half-life of low-affinity VLA-4-VCAM-1 bonds. Immobilized SDF-1α acted within the lifetime of a primary tether to stabilize initial tethering interactions, increasing the likelihood a PBL would remain interacting with the surface. As expected, the immobilized SDF-1α also increased the ratio of PBL firm adhesion to rolling. An LDV peptide-based small molecule that preferentially binds high-affinity VLA-4 reduced PBL firm adhesion to VCAM-1 by 90%. The reduction in firm adhesion due to blockage of high-affinity VLA-4 was paralleled by a 4-fold increase in the fraction of rolling PBL. Chemokine activation of PBL firm adhesion on VCAM-1 depended on induction of high-affinity VLA-4 rather than recruitment of a pre-existing pool of high-affinity VLA-4 as previously thought.
Costimulation Blockade Inhibits Allergic Sensitization but Does Not Affect Established Allergy in a Murine Model of Grass Pollen Allergy
Authors: Linhart Birgit , Bigenzahn Sinda , Hartl Arnulf , Lupinek Christian , Thalhamer Josef , Valenta Rudolf , Wekerle Thomas
Published: April 2014

Type I allergy is characterized by the development of an initial Th2-dependent allergen-specific IgE response, which is boosted upon a subsequent allergen encounter. Although the immediate symptoms of allergy are mainly IgE-mediated, allergen-specific T cell responses contribute to the late phase as well as to the chronic manifestations of allergy. This study investigates the potential of costimulation blockade with CTLA4Ig and an anti-CD154 mAb for modifying the allergic immune response to the major timothy grass pollen allergen Phl p 5 in a mouse model. BALB/c mice were treated with the costimulation blockers at the time of primary sensitization to the Phl p 5 allergen or at the time of a secondary allergen challenge. Costimulation blockade (CTLA4Ig plus anti-CD154 or anti-CD154 alone) at the time of sensitization prevented the development of allergen-specific IgE, IgM, IgG, and IgA responses compared with untreated but sensitized mice. However, costimulation blockade had no influence on established IgE responses in sensitized mice. Immediate-type reactions as analyzed by a rat basophil leukemia cell mediator release assay were only suppressed by early treatment but not by a costimulation blockade after sensitization. CTLA4Ig given alone failed to suppress both the primary and the secondary allergen-specific Ab responses. Allergen-specific T cell activation was suppressed in mice by early as well as by a late costimulation blockade, suggesting that IgE responses in sensitized mice are independent of T cell help. Our results indicate that T cell suppression alone without active immune regulation or a shifting of the Th2/Th1 balance is not sufficient for the treatment of established IgE responses in an allergy.
Unravelling the Complexity of T Cell Abnormalities in Common Variable Immunodeficiency
Authors: Giovannetti Antonello , Pierdominici Marina , Mazzetta Francesca , Marziali Marco , Renzi Cristina , Mileo Anna Maria , De Felice Marco , Mora Barbara , Esposito Antonella , Carello Rossella , Pizzuti Antonio , Paggi Marco G. , Paganelli Roberto , Malorni Walter , Aiuti Fernando
Published: April 2014

We investigated several phenotypic and functional parameters of T cell-mediated immunity in a large series of common variable immunodeficiency (CVID) patients. We demonstrated that the vast majority of CVID patients presented multiple T cell abnormalities intimately related among them, the severity of which was reflected in a parallel loss of CD4+ naive T cells. A strong correlation between the number of CD4+ naive T cells and clinical features was observed, supporting the subgrouping of patients according to their number of naive CD4+ T lymphocytes. A reduced thymic output and disrupted CD4+ and CD8+ TCR repertoires paralleled the contraction of CD4+ naive T cell pools. The evaluation of activation markers and cytokine production indicated a strong T cell activation that was significantly related to the increased levels of T cell turnover and apoptosis. Finally, discrete genetic profiles could be demonstrated in groups of patients showing extremely diverse T cell subset composition and function. Naive CD4+ T cell levels were significantly associated with the switched memory B cell-based classification, although the concordance between the respective subgroups did not exceed 58.8%. In conclusion, our data highlight the key role played by the T cell compartment in the pathogenesis of CVID, pointing to the need to consider this aspect for classification of this disease.
NK Cells and Cancer
Authors: Zamai Loris , Ponti Cristina , Mirandola Prisco , Gobbi Giuliana , Papa Stefano , Galeotti Laura , Cocco Lucio , Vitale Marco
Published: April 2014

In this review, we overview the main features and functions of NK cells, focusing on their role in cell-mediated immune response to tumor cells. In parallel, we discuss the information available in the field of NK cell receptors and offer a wide general overview of functional aspects of cell targeting and killing, focusing on the recent acknowledgments on the efficacy of NK cells after cytokine and mAb administration in cancer therapy. Since efficacy of NK cell-based immunotherapy has been proven in KIR-mismatch regimens or in TRAIL-dependent apoptosis, the ability to manipulate the balance of activating and inhibitory receptors on NK cells and of their cognate ligands, as well as the sensitivity of tumor cells to apoptosis, opens new perspectives for NK cell-based immunotherapy.
Cutting Edge: Immature Human Dendritic Cells Express Latency-Associated Peptide and Inhibit T Cell Activation in a TGF-β-Dependent Manner
Authors: Gandhi Roopali , Anderson David E. , Weiner Howard L.
Published: April 2014

Dendritic cells (DCs) play a critical role in both initiating immune responses and in maintaining peripheral tolerance. However, the exact mechanism by which DCs instruct/influence the generation of effector vs regulatory T cells is not clear. In this study, we present evidence that TGF-β, an important immunoregulatory molecule, is present on the surface of ex vivo immature human DCs bound by latency-associated peptide (LAP). Maturation of DCs upon stimulation with LPS results in loss of membrane-bound LAP and up-regulation of HLA class II and costimulatory molecules. The presence of LAP on immature DCs selectively inhibits Th1 cell but not Th17 cell differentiation and is required for differentiation and/or survival of Foxp3-positive regulatory T cells. Taken together, our results indicate that surface expression of TGF-β on DCs in association with LAP is one of the mechanisms by which immature DCs limit T cell activation and thus prevent autoimmune responses.
Cutting Edge: Limiting Amounts of IL-7 Do Not Control Contraction of CD4+ T Cell Responses
Authors: Tripathi Pulak , Mitchell Thomas C. , Finkelman Fred , Hildeman David A.
Published: April 2014

During the acute T cell response most effector T cells die while some survive and become memory T cells. Selective expression of CD127 (IL-7Rα) on effector T cells has been proposed to engender their survival into the memory pool. We assessed the role of IL-7 in effector T cell survival using MHC class II tetramers to track a CD4+ T cell response following infection with a recombinant vaccinia virus (rVV-2W1S). Exogenous IL-7 prevented the contraction of the 2W1S-specific CD4+ T cell response after rVV-2W1S infection. IL-7 increased proliferation of, and Bcl-2 expression within, 2W1S-specific T cells; the latter was required for IL-7-driven prevention of contraction. Conversely, in vivo neutralization of IL-7 or Bcl-2 did not exacerbate the contraction of 2W1S-specific CD4+ T cells. These data suggest that IL-7 administration may enhance the survival of effector T cells but that IL-7 is not the limiting factor during normal contraction of the response.
The T Cell Costimulator TL1A Is Induced by FcγR Signaling in Human Monocytes and Dendritic Cells
Authors: Prehn John L. , Thomas Lisa S. , Landers Carol J. , Yu Qi T. , Michelsen Kathrin S. , Targan Stephan R.
Published: April 2014

The recently described TL1A/DR3 ligand/receptor pair mediates strong costimulation of Th1 cells. Activation of T and NK cells induces DR3 expression, permitting soluble recombinant TL1A to increase IFN-γ production and proliferation of these cells. Gut T cells and macrophages express TL1A, especially in Crohn’s disease (CD), and there is a strong association between CD and tl1a single nucleotide polymorphisms. Murine studies implicate TL1A in gut inflammation. To determine whether professional T cell-activating cells can express TL1A, fresh blood monocytes and monocyte-derived dendritic cells were stimulated with various activating ligands, including TLR agonists, IFN-γ, and immune complexes. FcγR stimulation strongly induced TL1A mRNA in both cell types, which correlated with the detection of TL1A on the cell surface and in cell culture medium. TLR agonists capable of inducing IL-6 and TNF-α in monocytes and dendritic cells did not induce surface nor soluble TL1A. Furthermore, we demonstrate that TL1A production in monocytes leads to enhancement of T cell responses. The induction of TL1A on APCs via specific pathway stimulation suggests a role for TL1A in Th1 responses to pathogens, and in CD.
15-Deoxy-Δ12,14-Prostaglandin J2 Negatively Regulates rankl Gene Expression in Activated T Lymphocytes: Role of NF-κB and Early Growth Response Transcription Factors
Authors: Fionda Cinzia , Nappi Filomena , Piccoli Mario , Frati Luigi , Santoni Angela , Cippitelli Marco
Published: April 2014

Receptor activator of NF-κB ligand (RANKL) and its receptor RANK are cell surface proteins abundantly expressed in bone and lymphoid tissues, whose interaction triggers different signaling pathways leading to activation and differentiation of osteoclasts, pivotal actors of the normal bone remodeling cycle. Moreover, RANKL may act as an immunomodulator, representing an important dendritic cell survival factor produced by activated T cells. A large body of research has shown that not only does the RANKL/RANK system regulate the physiology of bone development but also plays an important pathological role in bone destruction mediated by inflammatory disorders or bone metastatic tumors. 15-Deoxy-Δ12,14-PGJ2 (15d-PGJ2) is a cyclopentenone-type PG endowed with anti-inflammatory properties and produced by different cells, including those of the immune system. Although 15d-PGJ2 has been studied as a natural ligand of the peroxisome proliferator-activated receptor-γ nuclear receptor, relevant peroxisome proliferator-activated receptor-γ-independent actions mediated by this prostanoid have been described. In this study, we describe the effect of 15d-PGJ2 on the expression of the rankl gene in T lymphocytes. We show that 15d-PGJ2 inhibits rankl mRNA expression, protein, and rankl promoter activity by mechanisms mediated by its chemically reactive cyclopentenone moiety. Our data also indicate that 15d-PGJ2 represses rankl activation by interfering with the expression and/or activity of the transcription factors NF-κB, early growth response-2, and early growth response-3, whose altered balancing and transactivation may contribute for the repression of this gene. These results place rankl as a novel molecular target for the different immunoregulatory activities mediated by 15d-PGJ2. The physiological and pharmacological implications of these observations are discussed.
Infiltration of a Mesothelioma by IFN-γ-Producing Cells and Tumor Rejection after Depletion of Regulatory T Cells
Authors: Rudge Geordie , Barrett Simon P. , Scott Bernadette , van Driel Ian R.
Published: April 2014

Depletion of CD4+CD25+Foxp3+ regulatory T cells (CD25+ Treg) with an anti-CD25 Ab results in immune-mediated rejection of tolerogenic solid tumors. In this study, we have examined the immune response to a mesothelioma tumor in mice after depletion of CD25+ cells to elucidate the cellular mechanisms of CD25+ Treg, a subject over which there is currently much conjecture. Tumor rejection was found to be primarily due to the action of CD8+ T cells, although CD4+ cells appeared to play some role. Depletion of CD25+ cells resulted in an accumulation in tumor tissue of CD4+ and CD8+ T cells and NK cells that were producing the potent antitumor cytokine IFN-γ. Invasion of tumors by CD8+ T cells was partially dependent on the presence of CD4+ T cells. Although a significant increase in the proliferation and number of tumor-specific CD8+ T cells was observed in lymph nodes draining the tumor of anti-CD25-treated mice, this effect was relatively modest compared with the large increase in IFN-γ-producing T cells found in tumor tissue, which suggests that the migration of T cells into tumor tissue may also have been altered. Depletion of CD25+ cells did not appear to modulate antitumor CTL activity on a per cell basis. Our data suggests that CD25+ Treg limit the accumulation of activated T cells producing IFN-γ in the tumor tissue and, to a lesser extent, activation and/or rate of mitosis of tumor-specific T cells in lymph nodes.
The Constitutive Tyrosine Phosphorylation of CD3ζ Results from TCR-MHC Interactions That Are Independent of Thymic Selection
Authors: Becker Amy M. , DeFord-Watts Laura M. , Wuelfing Christoph , van Oers Nicolai S. C.
Published: April 2014

The TCR complex, when isolated from thymocytes and peripheral T cells, contains a constitutively tyrosine-phosphorylated CD3ζ molecule termed p21. Previous investigations have shown that the constitutive phosphorylation of CD3ζ results from TCR interactions with MHC molecules occurring in both the thymus and the periphery. To determine what contribution the selection environment had on this constitutive phosphorylation, we analyzed CD3ζ from several distinct class I- and II-restricted TCR-transgenic mice where thymocyte development occurred in either a selecting or a nonselecting MHC environment. Herein, we report that constitutively phosphorylated CD3ζ (p21) was present in thymocytes that developed under nonselecting peptide-MHC conditions. These findings strongly support the model that the TCR has an inherent avidity for MHC molecules before repertoire selection. Biochemical analyses of the TCR complex before and after TCR stimulation suggested that the constitutively phosphorylated CD3ζ subunit did not contribute to de novo TCR signals. These findings may have important implications for T cell functions during self-MHC recognition under normal and autoimmune circumstances.
Incomplete Depletion and Rapid Regeneration of Foxp3+ Regulatory T Cells Following Anti-CD25 Treatment in Malaria-Infected Mice
Authors: Couper Kevin N. , Blount Daniel G. , de Souza J. Brian , Suffia Isabelle , Belkaid Yasmine , Riley Eleanor M.
Published: April 2014

Investigation of the role of regulatory T cells (Treg) in model systems is facilitated by their depletion using anti-CD25 Abs, but there has been considerable debate about the effectiveness of this strategy. In this study, we have compared the depletion and repopulation of CD4+CD25+Foxp3+ Treg in uninfected and malaria-infected mice using 7D4 and/or PC61 anti-CD25 Abs. We find that numbers and percentages of CD25high cells, but not Foxp3+ cells, are transiently reduced after 7D4 treatment, whereas treatment with PC61 alone or in combination with 7D4 (7D4 plus PC61) reduces but does not eliminate Foxp3+ cells for up to 2 wk. Importantly, all protocols fail to eliminate significant populations of CD25−Foxp3+ or CD25lowFoxp3+ cells, which retain potent regulatory capacity. By adoptive transfer we show that repopulation of the spleen by CD25highFoxp3+ cells results from the re-expression of CD25 on peripheral populations of CD25−Foxp3+ but not from the conversion of peripheral Foxp3− cells. CD25highFoxp3+ repopulation occurs more rapidly in 7D4-treated mice than in 7D4 plus PC61-treated mice, reflecting ongoing clearance of emergent CD25+Foxp3+ cells by persistent PC61 Ab. However, in 7D4 plus PC61-treated mice undergoing acute malaria infection, repopulation of the spleen by CD25+Foxp3+ cells occurs extremely rapidly, with malaria infection driving proliferation and CD25 expression in peripheral CD4+CD25−Foxp3+ cells and/or conversion of CD4+CD25−Foxp3− cells. Finally, we reveal an essential role for IL-2 for the re-expression of CD25 by Foxp3+ cells after anti-CD25 treatment and observe that TGF-β is required, in the absence of CD25 and IL-2, to maintain splenic Foxp3+ cell numbers and a normal ratio of Treg:non-Treg cells.
Gadd45α Regulates p38-Dependent Dendritic Cell Cytokine Production and Th1 Differentiation
Authors: Jirmanova Ludmila , Janković Dragana , Fornace Albert J. , Ashwell Jonathan D.
Published: April 2014

Gadd45α inhibits the activation of p38 by the T cell alternative pathway involving phosphorylation of p38 Tyr323. Given that T cell p38 may play a role in Th1 development, the response to Th-skewing Ags was analyzed in Gadd45α−/− mice. Despite constitutively increased p38 activity in Gadd45α−/− T cells, the Th1 immune response to Toxoplasma gondii Ag (STAg), was diminished. In contrast to T cells, dendritic cells (DC) lacked the alternative p38 activation pathway. Gadd45α−/− DCs responded to STAg with low levels of MAP kinase cascade-dependent p38 activation, IL-12 production, and CD40 expression. Wild-type T cells transferred into Gadd45α−/− recipients had a diminished Th1 response to STAg, whereas Gadd45α−/− T cells transferred into wild-type hosts behaved normally. Therefore, Gadd45α has tissue-specific and opposing functions on p38 activity, and Gadd45α-regulated p38 activation in DCs is a critical event in Th1 polarization in vivo.
FcεRI Aggregation Promotes Survival of Connective Tissue-Like Mast Cells but Not Mucosal-Like Mast Cells
Authors: Ekoff Maria , Strasser Andreas , Nilsson Gunnar
Published: April 2014

Mast cells play a critical role in IgE-dependent immediate hypersensitivity reactions. This is facilitated by their capacity to release inflammatory mediators and to undergo activation-induced survival upon cross-linking of the high-affinity IgE-receptor (FcεRI). Due to their heterogeneity, mast cells can be divided into two major groups: the connective tissue mast cells and the mucosal mast cells. We have previously shown that IL-3-dependent bone marrow-derived mast cells can undergo activation-induced survival that is dependent on the prosurvival gene A1. In this study, we have used two different protocols to develop murine connective tissue-like mast cells (CTLMC) and mucosal-like mast cells (MLMC) to investigate their capacity to survive an allergic reaction in vitro. In this study, we demonstrate that FcεRI stimulation promotes survival of CTLMC but not MLMC. Similarly, a prominent induction of A1 is observed only in CTLMC but not MLMC. MLMC have a higher basal level of the proapoptotic protein Bim compared with CTLMC. These findings demonstrate a difference among mast cell populations in their ability to undergo activation-induced survival after FcεRI stimulation, which might explain the slower turnover of CTMC in IgE-dependent reactions.
Human Dendritic Cells Acquire a Semimature Phenotype and Lymph Node Homing Potential through Interaction with CD4+CD25+ Regulatory T Cells
Authors: Bayry Jagadeesh , Triebel Frédéric , Kaveri Srini V. , Tough David F.
Published: April 2014

Interactions between dendritic cells (DC) and T cells are known to involve the delivery of signals in both directions. We sought to characterize the effects on human DC of contact with different subsets of activated CD4+ T cells. The results showed that interaction with CD25highCD4+ regulatory T cells (Tregs) caused DC to take on very different properties than contact with naive or memory phenotype T cells. Whereas non-Tregs stimulated DC maturation, culture with Tregs produced DC with a mixed phenotype. By many criteria, Tregs inhibited DC maturation, inducing down-regulation of costimulatory molecules and T cell stimulatory activity. However, DC exposed to Tregs also showed some changes typically associated with DC maturation, namely, increased expression of CCR7 and MHC class II molecules, and gained the ability to migrate in response to the CCR7 ligand CCL19. Both soluble factors and cell-associated molecules were shown to be involved in Treg modulation of DC, with lymphocyte activation gene 3 (LAG-3) playing a predominant role in driving maturation-associated changes. The data show that Tregs induce the generation of semimature DC with the potential to migrate into lymphoid organs, suggesting a possible mechanism by which Tregs down-modulate immune responses.
Paired Ig-Like Receptors Bind to Bacteria and Shape TLR-Mediated Cytokine Production
Authors: Nakayama Masafumi , Underhill David M. , Petersen Timothy W. , Li Bin , Kitamura Toshio , Takai Toshiyuki , Aderem Alan
Published: April 2014

The innate immune system uses a wide variety of pattern recognition receptors including TLRs, scavenger receptors, and lectins to identify potential pathogens. A carefully regulated balance between activation and inhibition must be kept to avoid detrimental and inappropriate inflammatory responses. In this study, we identify murine-paired Ig-like receptor (PIR)-B, and its human orthologs Ig-like transcript 2 and Ig-like transcript 5 as novel receptors for Staphylococcus aureus. PIR-B contains four ITIM motifs and is thought to be an inhibitory receptor. Expression of these receptors enables NIH3T3 cells to bind S. aureus. In mouse bone marrow-derived macrophages, masking of PIR-B by anti-PIR mAb or genetic deletion of PIR-B shows significantly impaired recognition of S. aureus and enhanced TLR-mediated inflammatory responses to the bacteria. These data suggest a novel mechanism for innate immune regulation by paired Ig-like receptor family members.
TGF-β Suppresses IFN-γ-STAT1-Dependent Gene Transcription by Enhancing STAT1-PIAS1 Interactions in Epithelia but Not Monocytes/Macrophages
Authors: Reardon Colin , McKay Derek M.
Published: April 2014

IFN-γ and TGF-β are important regulators of mucosal immunity, typically functioning in opposition to each other. In this study, we assessed whether TGF-β could modulate IFN-γ-induced STAT1 signaling. Model epithelial cell lines (HEp-2, HT-29, and T84) or monocytes/macrophages (THP-1 cell line, human blood mononuclear cells) were pretreated with TGF-β (1 ng/ml; 5–60 min), followed by IFN-γ exposure (20 ng/ml; 30 min), and then STAT1 transcriptional activity, DNA-binding activity, phosphorylation, and methylation were assessed. Some epithelia were transfected with an expression plasmid encoding SMAD7 to block TGF-β-SMAD signaling. Epithelia, but not macrophages, pretreated with TGF-β were hyporesponsive to IFN-γ stimulation as indicated by reduced expression of four STAT1-regulated genes and reduced STAT1 DNA binding on EMSA. However, STAT1 Tyr701-, Ser727 phosphorylation, and nuclear recruitment of STAT1 were not significantly different in IFN-γ with or without TGF-β-treated cells, indicating that the effects of TGF-β are downstream of IFN-γR-JAK-STAT1 interaction. The TGF-β effect was not dependent on ERK1/2, p38, or JNK activation but was prevented by overexpression of the inhibitory SMAD7 protein. Additional studies suggest that TGF-β blockade of IFN-γ activity in epithelia is via enhanced sequestering of STAT1 by pre-existing protein inhibitor of activated STAT1. These results demonstrate that TGF-β rapidly suppresses IFN-γ-driven STAT1 signaling by reducing DNA binding via promotion of STAT1-protein inhibitor of activated STAT1 interactions and not inhibition of STAT1 activation; an event that may be specific to epithelia and represent a novel mode of action of TGF-β.
Analysis of 6912 Unselected Somatic Hypermutations in Human VDJ Rearrangements Reveals Lack of Strand Specificity and Correlation between Phase II Substitution Rates and Distance to the Nearest 3′ Activation-Induced Cytidine Deaminase Target
Authors: Ohm-Laursen Line , Barington Torben
Published: April 2014

The initial event of somatic hypermutation (SHM) is the deamination of cytidine residues by activation-induced cytidine deaminase (AID). Deamination is followed by the replication over uracil and/or different error-prone repair events. We sequenced 659 nonproductive human IgH rearrangements (IGHV3-23*01) from blood B lymphocytes enriched for CD27-positive memory cells. Analyses of 6,912 unique, unselected substitutions showed that in vivo hot and cold spots for the SHM of C and G residues corresponded closely to the target preferences reported for AID in vitro. A detailed analysis of all possible four-nucleotide motifs present on both strands of the VH gene showed significant correlations between the substitution frequencies in reverse complementary motifs, suggesting that the SHM machinery targets both strands equally well. An analysis of individual JH and D gene segments showed that the substitution frequencies in the individual motifs were comparable to the frequencies found in the VH gene. Interestingly, JH6-carrying sequences were less likely to undergo SHM (average 15.2 substitutions per VH region) than sequences using JH4 (18.1 substitutions, p = 0.03). We also found that the substitution rates in G and T residues correlated inversely with the distance to the nearest 3′ WRC AID hot spot motif on both the nontranscribed and transcribed strands. This suggests that phase II SHM takes place 5′ of the initial AID deamination target and primarily targets T and G residues or, alternatively, the corresponding A and C residues on the opposite strand.
Extracellular Isoforms of CD6 Generated by Alternative Splicing Regulate Targeting of CD6 to the Immunological Synapse
Authors: Castro Mónica A. A. , Oliveira Marta I. , Nunes Raquel J. , Fabre Stéphanie , Barbosa Rita , Peixoto António , Brown Marion H. , Parnes Jane R. , Bismuth Georges , Moreira Alexandra , Rocha Benedita , Carmo Alexandre M.
Published: April 2014

The great majority of mammalian genes yield multiple transcripts arising from differential mRNA processing, but in very few instances have alternative forms been assigned distinct functional properties. We have cloned and characterized a new isoform of the accessory molecule CD6 that lacks the CD166 binding domain and is expressed in rat and human primary cells. The novel isoform, CD6Δd3, results from exon 5 skipping and consequently lacks the third scavenger receptor cysteine-rich (SRCR) domain of CD6. Differential expression of the SRCR domain 3 resulted in a remarkable functional difference: whereas full-length CD6 targeted to the immunological synapse, CD6Δd3 was unable to localize at the T cell:APC interface during Ag presentation. Analysis of expression of CD6 variants showed that, while being more frequent in coexpression with full-length CD6, the CD6Δd3 isoform constituted the sole species in a small percentage of T cells. In the rat thymus, CD6Δd3 is less represented in double-positive thymocytes but is detectable in nearly 50% of single-positive CD4 or CD8 thymocytes, suggesting that CD6 switching between full-length and Δd3 isoforms may be involved in thymic selection. Strikingly, CD6Δd3 is markedly up-regulated upon activation of T lymphocytes, partially substituting full-length CD6, as evaluated by RT-PCR analysis at the single-cell level, by immunoblotting, and by flow cytometry using Abs recognizing SRCR domains 1 and 3 of human CD6. This elegant mechanism controlling the expression of the CD166 binding domain may help regulate signaling delivered by CD6, through different types of extracellular engagement.