bluuebunny/crossref_metadata_2025 huggingface dataset:
α-Actinin Immunization Elicits Anti-Chromatin Autoimmunity in Nonautoimmune Mice
Authors: Deocharan Bisram , Zhou Zhijie , Antar Kochnaf , Siconolfi-Baez Linda , Angeletti Ruth Hogue , Hardin John , Putterman Chaim
Published: April 2014

Anti-dsDNA Abs are characteristic of lupus and can be found deposited in the kidneys of lupus mice. Previously, we have shown that pathogenic anti-dsDNA Abs as well as Ig eluted from the kidneys of nephritic lupus mice cross-react with α-actinin. Moreover, cross-reactivity with α-actinin characterizes nephritogenic anti-dsDNA Abs in humans with lupus as well. To determine whether Abs generated against α-actinin in vivo cross-react with nuclear Ags, we s.c. immunized 10-wk-old female BALB/c mice (and several other nonautoimmune mice strains) with α-actinin in adjuvant. Immunized but not control mice displayed high titers of anti-nuclear Abs and IgG anti-chromatin autoantibodies, hypergammaglobulinemia, renal Ig deposition, and proteinuria. The specificity of the anti-chromatin response was determined by Western blotting of purified chromatin with serum from α-actinin immunized mice. By proteomic analysis, a 25-kDa doublet band was conclusively identified as high mobility group box (HMGB) proteins 1 and 3, and a 70-kDa band was identified as heat shock protein 70 (hsp70), both of which are known antigenic targets in murine lupus. Binding to purified HMGB1 and hsp70 by immunized mice sera was confirmed by ELISA and Western blot. Immunized mice sera binding to both 25- and 70-kDa bands were significantly inhibited by α-actinin and chromatin. Importantly, a panel of nephritogenic mAbs had significantly higher affinity for α-actinin, chromatin, HMGB, and hsp70 as compared with nonpathogenic Abs, suggesting a common motif in these Ags that is targeted by pathogenic autoantibodies.
Lupus Susceptibility Genes May Breach Tolerance to DNA by Impairing Receptor Editing of Nuclear Antigen-Reactive B Cells
Authors: Liu Yang , Li Liunan , Kumar Kirthi Raman , Xie Chun , Lightfoot Skip , Zhou Xin J. , Kearney John F. , Weigert Martin , Mohan Chandra
Published: April 2014

An NZM2410-derived lupus susceptibility locus on murine chromosome 4, Sle2z, has previously been noted to engender generalized B cell hyperactivity. To study how Sle2z impacts B cell tolerance, two Ig H chain site-directed transgenes, 3H9 and 56R, with specificity for DNA were backcrossed onto the C57BL/6 background with or without Sle2z. Interestingly, the presence of the NZM2410 “z” allele of Sle2 on the C57BL/6 background profoundly breached B cell tolerance to DNA, apparently by thwarting receptor editing. Whereas mAbs isolated from the spleens of B6.56R control mice demonstrated significant usage of the endogenous (i.e., nontargeted) H chain locus and evidence of vigorous L chain editing; Abs isolated from B6.Sle2z.56R spleens were largely composed of the transgenic H chain paired with a spectrum of L chains, predominantly recombined to Jk1 or Jk2. In addition, Sle2z-bearing B cells adopted divergent phenotypes depending on their Ag specificity. Whereas Sle2z-bearing anti-DNA transgenic B cells were skewed toward marginal zone B cells and preplasmablasts, B cells from the same mice that did not express the transgene were skewed toward the B1a phenotype. This work illustrates that genetic loci that confer lupus susceptibility may influence B cell differentiation depending on their Ag specificity and potentially contribute to antinuclear autoantibody formation by infringing upon B cell receptor editing. Taken together with a recent report on Sle1z, these studies suggest that dysregulated receptor-editing of nuclear Ag-reactive B cells may be a major mechanism through which antinuclear Abs arise in lupus.
B Cell Depletion Delays Collagen-Induced Arthritis in Mice: Arthritis Induction Requires Synergy between Humoral and Cell-Mediated Immunity
Authors: Yanaba Koichi , Hamaguchi Yasuhito , Venturi Guglielmo M. , Steeber Douglas A. , St. Clair E. William , Tedder Thomas F.
Published: April 2014

Rheumatoid arthritis is a systemic autoimmune disease. B cells are likely to play a critical role in arthritis pathogenesis, although it is unclear whether they are necessary for disease induction, autoantibody production, or disease progression. To assess the role of B cells in inflammatory arthritis, B cells were depleted using mouse anti-mouse CD20 mAbs in a mouse model of collagen-induced arthritis. CD20 mAbs effectively depleted mature B cells from adult DBA-1 mice. When B cells were depleted using CD20 mAbs before collagen immunization, there was a delay in disease onset and autoantibody production, with significantly diminished severity of arthritis both clinically and histologically. B cell depletion further delayed disease onset if initiated before, as well as after, collagen immunization. However, in both cases, the eventual reappearance of peripheral B cells triggered autoantibody production and the subsequent development of arthritis in collagen-sensitized mice. By contrast, B cell depletion after collagen immunizations did not have a significant effect on arthritis progression or severity. Thus, disease symptoms were only induced when peripheral B cells and their autoantibody products were present in collagen-immunized mice, documenting a critical role for B cells during the elicitation phase of collagen-induced arthritis. These studies suggest that B cell depletion strategies will be most effective when initiated early in the development of inflammatory arthritis, with sustained B cell depletion required to inhibit the production of isotype-switched pathogenic Abs and the evolution of joint inflammation and destruction.
Activating Fcγ Receptors Participate in the Development of Autoimmune Diabetes in NOD Mice
Authors: Inoue Yoshihiro , Kaifu Tomonori , Sugahara-Tobinai Akiko , Nakamura Akira , Miyazaki Jun-Ichi , Takai Toshiyuki
Published: April 2014

Type 1 diabetes mellitus (T1D) in humans is an organ-specific autoimmune disease in which pancreatic islet β cells are ruptured by autoreactive T cells. NOD mice, the most commonly used animal model of T1D, show early infiltration of leukocytes in the islets (insulitis), resulting in islet destruction and diabetes later. NOD mice produce various islet β cell-specific autoantibodies, although it remains a subject of debate regarding whether these autoantibodies contribute to the development of T1D. FcγRs are multipotent molecules that play important roles in Ab-mediated regulatory as well as effector functions in autoimmune diseases. To investigate the possible role of FcγRs in NOD mice, we generated several FcγR-less NOD lines, namely FcR common γ-chain (FcRγ)-deficient (NOD.γ−/−), FcγRIII-deficient (NOD.III−/−), FcγRIIB-deficient (NOD.IIB−/−), and both FcRγ and FcγRIIB-deficient NOD (NOD.null) mice. In this study, we show significant protection from diabetes in NOD.γ−/−, NOD.III−/−, and NOD.null, but not in NOD.IIB−/− mice even with grossly comparable production of autoantibodies among them. Insulitis in NOD.γ−/− mice was also alleviated. Adoptive transfer of bone marrow-derived dendritic cells or NK cells from NOD mice rendered NOD.γ−/− animals more susceptible to diabetes, suggesting a possible scenario in which activating FcγRs on dendritic cells enhance autoantigen presentation leading to the activation of autoreactive T cells, and FcγRIII on NK cells trigger Ab-dependent effector functions and inflammation. These findings highlight the critical roles of activating FcγRs in the development of T1D, and indicate that FcγRs are novel targets for therapies for T1D.
A Novel Alloantigen-Specific CD8+PD1+ Regulatory T Cell Induced by ICOS-B7h Blockade In Vivo
Authors: Izawa Atsushi , Yamaura Kazuhiro , Albin Monica J. , Jurewicz Mollie , Tanaka Katsunori , Clarkson Michael R. , Ueno Takuya , Habicht Antje , Freeman Gordon J. , Yagita Hideo , Abdi Reza , Pearson Todd , Greiner Dale L. , Sayegh Mohamed H. , Najafian Nader
Published: April 2014

Delayed ICOS-B7h signal blockade promotes significant prolongation of cardiac allograft survival in wild-type but not in CD8-deficient C57BL/6 recipients of fully MHC-mismatched BALB/c heart allografts, suggesting the possible generation of CD8+ regulatory T cells in vivo. We now show that the administration of a blocking anti-ICOS mAb results in the generation of regulatory CD8+ T cells. These cells can transfer protection and prolong the survival of donor-specific BALB/c, but not third party C3H, heart grafts in CD8-deficient C57BL/6 recipients. This is unique to ICOS-B7h blockade, because B7 blockade by CTLA4-Ig prolongs graft survival in CD8-deficient mice and does not result in the generation of regulatory CD8+ T cells. Those cells localize to the graft, produce both IFN-γ and IL-4 after allostimulation in vitro, prohibit the expansion of alloreactive CD4+ T cells, and appear to mediate a Th2 switch of recipient CD4+ T cells after adoptive transfer in vivo. Finally, these cells are not confined to the CD28-negative population but express programmed death 1, a molecule required for their regulatory function in vivo. CD8+PD1+ T cells suppress alloreactive CD4+ T cells but do not inhibit the functions by alloreactive CD8+ T cells in vitro. These results describe a novel allospecific regulatory CD8+PD1+ T cell induced by ICOS-B7h blockade in vivo.
Transcriptional Analysis of Clonal Deletion In Vivo
Authors: Baldwin Troy A. , Hogquist Kristin A.
Published: April 2014

Engagement of the TCR on CD4+CD8+ thymocytes initiates either a program of survival and differentiation (positive selection) or death (clonal deletion), which is dictated in large part by the affinity of the TCR for self-peptide-MHC complexes. Although much is known about the factors involved in positive selection, little is understood about the molecular mechanism leading to clonal deletion. To gain further insight into this process, we used a highly physiological TCR transgenic mouse model to compare gene expression changes under conditions of nonselection, positive selection, and negative selection. We identified 388 genes that were differentially regulated in negative selection compared with either nonselection or positive selection. These regulated genes fall into many functional categories including cell surface and intracellular signal transduction, survival and apoptosis, transcription and translation, and adhesion and migration. Additionally, we have compared our transcriptional profile to profiles of negative selection in other model systems in an effort to identify those genes with a higher probability of being functionally relevant. These included three up-regulated genes, bim, nur77, and ian1, and one down-regulated gene, lip1. Collectively, these data provide a framework for understanding the molecular basis of clonal deletion.
Epidermal Langerhans Cells Are Dispensable for Humoral and Cell-Mediated Immunity Elicited by Gene Gun Immunization
Authors: Stoecklinger Angelika , Grieshuber Ines , Scheiblhofer Sandra , Weiss Richard , Ritter Uwe , Kissenpfennig Adrien , Malissen Bernard , Romani Nikolaus , Koch Franz , Ferreira Fatima , Thalhamer Josef , Hammerl Peter
Published: April 2014

Gene gun immunization, i.e., bombardment of skin with DNA-coated particles, is an efficient method for the administration of DNA vaccines. Direct transfection of APC or cross-presentation of exogenous Ag acquired from transfected nonimmune cells enables MHC-I-restricted activation of CD8+ T cells. Additionally, MHC-II-restricted presentation of exogenous Ag activates CD4+ Th cells. Being the principal APC in the epidermis, Langerhans cells (LC) seem ideal candidates to accomplish these functions. However, the dependence on LC of gene gun-induced immune reactions has not yet been demonstrated directly. This was primarily hampered by difficulties to discriminate the contributions of LC from those of other dermal dendritic cells. To address this problem, we have used Langerin-diphtheria toxin receptor knockin mice that allow for selective inducible ablation of LC. LC deficiency, even over the entire duration of experiments, did not affect any of the gene gun-induced immune functions examined, including proliferation of CD4+ and CD8+ T cells, IFN-γ secretion by spleen cells, Ab production, CTL activity, and development of protective antitumor immunity. Together, our data show that gene gun immunization is capable of inducing humoral and cell-mediated immune reactions independently of LC.
Blockade of IDO Inhibits Nasal Tolerance Induction
Authors: van der Marel Arnold P. J. , Samsom Janneke N. , Greuter Mascha , van Berkel Lisette A. , O’Toole Tom , Kraal Georg , Mebius Reina E.
Published: April 2014

The amino acid tryptophan is essential for the proliferation and survival of cells. Modulation of tryptophan metabolism has been described as an important regulatory mechanism for the control of immune responses. The enzyme IDO degrades the indole moiety of tryptophan, not only depleting tryptophan but also producing immunomodulatory metabolites called kynurenines, which have apoptosis-inducing capabilities. In this study, we show that IDO is more highly expressed in nonplasmacytoid dendritic cells of the nose draining lymph nodes (LNs), which form a unique environment to induce tolerance to inhaled Ags, when compared with other peripheral LNs. Upon blockade of IDO during intranasal OVA administration, Ag-specific immune tolerance was abrogated. Analysis of Ag-specific T cells in the LNs revealed that inhibition of IDO resulted in enhanced survival at 48 h after antigenic stimulation, although this result was not mediated through alterations in apoptosis or cell proliferation. Furthermore, no differences were found in CD4+ T cells expressing FoxP3. Our data suggest that the level of IDO expression in dendritic cells, present in nose draining LNs, allows for the generation of a sufficient number of regulatory T cells to control and balance effector T cells in such a way that immune tolerance is induced, whereas upon IDO blockade, effector T cells will outnumber regulatory T cells, leading to immunity.
Foxp3+CD25+ T Regulatory Cells Stimulate IFN-γ-Independent CD152-Mediated Activation of Tryptophan Catabolism That Provides Dendritic Cells with Immune Regulatory Activity in Mice Unresponsive to Staphylococcal Enterotoxin B
Authors: Feunou Pascal , Vanwetswinkel Sophie , Gaudray Florence , Goldman Michel , Matthys Patrick , Braun Michel Y.
Published: April 2014

Mice made unresponsive by repeated injection of staphylococcal enterotoxin B (SEB) contained SEB-specific CD25+CD4+TCRBV8+ T cells that were able to transfer their state of unresponsiveness to primary-stimulated T cells. About one-half of these cells stably up-regulated the expression of CD152. We undertook the present study to determine whether CD152high cells seen in this system were T regulatory cells responsible for suppression or whether they represented SEB-activated CD4+ T effector cells. Our results show that, among SEB-specific TCRBV8+ T cells isolated from unresponsive mice, all CD152highCD25+CD4+ T cells expressed Foxp3, the NF required for differentiation and function of natural T regulatory cells. Moreover, suppression by CD25+CD4+TCRBV8+ T cells was fully inhibited by anti-CD152 Abs. Following stimulation by soluble CD152-Ig, dendritic cells (DC) isolated from unresponsive mice strongly increased the expression and the function of indoleamine-2,3-dioxygenase (IDO), the enzyme responsible for the catabolism of tryptophan. This capacity to activate IDO was independent of IFN-γ production by DC because CD152-Ig stimulation of DC isolated from SEB-treated IFN-γ-deficient animals activated IDO expression and function. Finally, adding 1-methyl-tryptophan, an inhibitor of tryptophan catabolism, increased substantially the capacity of DC from unresponsive animals to stimulate primary T cell response toward SEB. Thus, we conclude that IFN-γ-independent CD152-mediated activation of tryptophan catabolism by Foxp3+CD25+ T regulatory cells provides DC with immune regulatory activity in mice unresponsive to SEB.
Tristetraprolin, a Negative Regulator of mRNA Stability, Is Increased in Old B Cells and Is Involved in the Degradation of E47 mRNA
Authors: Frasca Daniela , Landin Ana Marie , Alvarez Juan P. , Blackshear Perry J. , Riley Richard L. , Blomberg Bonnie B.
Published: April 2014

We have previously shown that the E2A-encoded transcription factor E47, which regulates class switch in splenic B cells, is down-regulated in old B cells, due to increased E47 mRNA decay. At least part of the decreased stability of E47 mRNA seen in aged B cells is mediated by proteins. We have herein looked at the specific proteins responsible for the degradation of the E47 mRNA and found that tristetraprolin (TTP), a physiological regulator of mRNA expression and stability, is involved in the degradation of the E47 mRNA. Although many studies have characterized TTP expression and function in macrophages, monocytes, mast cells, and T cells, little is known about the expression and function of TTP in primary B cells. We show herein that TTP mRNA and protein expression are induced by LPS in B cells from young and old mice, the levels of TTP in old B cells always being higher than those in young B cells. Although TTP mRNA is degraded at a significantly higher rate in old B cells, TTP mRNA expression is higher in old than in young, likely due to its increased transcription. Like in macrophages, TTP protein expression and function in B cells are dependent upon p38 MAPK. We found that there is less phospho-TTP (inactive form), as well as phospho-p38, in old than in young splenic-activated B cells. This is the first report showing that TTP is involved in the degradation of the E47 mRNA and is up-regulated in old B cells.
Cutting Edge: OX40 Inhibits TGF-β- and Antigen-Driven Conversion of Naive CD4 T Cells into CD25+Foxp3+ T cells
Authors: So Takanori , Croft Michael
Published: April 2014

Naive CD4 T cells can develop into regulatory T cells by acquiring the transcription factor Foxp3. Combined signals from the TCR, CD28, IL-2R, and TGF-βR promote Foxp3 expression in activated naive CD25− CD4 T cells. Here we show that OX40 (CD134) signaling inhibits TGF-β-driven Foxp3 mRNA and suppresses the conversion of naive Ag-specific transgenic CD4 T cells into CD25+Foxp3+ T cells. These data identify OX40 as a negative regulator of Foxp3 and suggest that OX40 can concomitantly promote effector T cell generation while antagonizing the differentiation of adaptive Foxp3+ regulatory T cells.
Controls for Lung Dendritic Cell Maturation and Migration during Respiratory Viral Infection
Authors: Grayson Mitchell H. , Ramos Madeleine S. , Rohlfing Michelle M. , Kitchens Robert , Wang Howard D. , Gould Aaron , Agapov Eugene , Holtzman Michael J.
Published: April 2014

Dendritic cells are ideally suited to orchestrate the innate and adaptive immune responses to infection, but we know little about how these cells respond to infection with common respiratory viruses. Paramyxoviral infections are the most frequent cause of serious respiratory illness in childhood and are associated with an increased risk of asthma. We therefore used a high-fidelity mouse model of paramyxoviral respiratory infection triggered by Sendai virus to examine the response of conventional and plasmacytoid dendritic cells (cDCs and pDCs, respectively) in the lung. We found that pDCs are scarce at baseline but become the predominant population of lung dendritic cells during infection. This recruitment allows for a source of IFN-α locally at the site of infection. In contrast, cDCs rapidly differentiate into myeloid cDCs and begin to migrate from the lung to draining lymph nodes within 2 h after viral inoculation. These events cause the number of lung cDCs to decrease rapidly and remain decreased at the site of viral infection. Maturation and migration of lung cDCs depends on Ccl5 and Ccr5 signals because these events are significantly impaired in Ccl5−/− and Ccr5−/− mice. cDCs failure to migrate to draining lymph nodes in Ccl5−/− or Ccr5−/− mice is associated with impaired up-regulation of CCR7 that would normally direct this process. Our results indicate that pDCs and cDCs respond distinctly to respiratory paramyxoviral infection with patterns of movement that should serve to coordinate the innate and adaptive immune responses, respectively.
Steady State Dendritic Cells Present Parenchymal Self-Antigen and Contribute to, but Are Not Essential for, Tolerization of Naive and Th1 Effector CD4 Cells
Authors: Hagymasi Adam T. , Slaiby Aaron M. , Mihalyo Marianne A. , Qui Harry Z. , Zammit David J. , Lefrancois Leo , Adler Adam J.
Published: April 2014

Bone marrow-derived APC are critical for both priming effector/memory T cell responses to pathogens and inducing peripheral tolerance in self-reactive T cells. In particular, dendritic cells (DC) can acquire peripheral self-Ags under steady state conditions and are thought to present them to cognate T cells in a default tolerogenic manner, whereas exposure to pathogen-associated inflammatory mediators during the acquisition of pathogen-derived Ags appears to reprogram DCs to prime effector and memory T cell function. Recent studies have confirmed the critical role of DCs in priming CD8 cell effector responses to certain pathogens, although the necessity of steady state DCs in programming T cell tolerance to peripheral self-Ags has not been directly tested. In the current study, the role of steady state DCs in programming self-reactive CD4 cell peripheral tolerance was assessed by combining the CD11c-diphtheria toxin receptor transgenic system, in which DC can be depleted via treatment with diphtheria toxin, with a TCR-transgenic adoptive transfer system in which either naive or Th1 effector CD4 cells are induced to undergo tolerization after exposure to cognate parenchymally derived self-Ag. Although steady state DCs present parenchymal self-Ag and contribute to the tolerization of cognate naive and Th1 effector CD4 cells, they are not essential, indicating the involvement of a non-DC tolerogenic APC population(s). Tolerogenic APCs, however, do not require the cooperation of CD4+CD25+ regulatory T cells. Similarly, DC were required for maximal priming of naive CD4 cells to vaccinia viral-Ag, but priming could still occur in the absence of DC.
Listeriolysin O Expressed in a Bacterial Vaccine Suppresses CD4+CD25high Regulatory T Cell Function In Vivo
Authors: Nitcheu-Tefit Josianne , Dai Ming-Shen , Critchley-Thorne Rebecca J. , Ramirez-Jimenez Francisco , Xu Man , Conchon Sophie , Ferry Nicolas , Stauss Hans J. , Vassaux Georges
Published: April 2014

CD4+CD25high regulatory T cells (Treg) protect the host from autoimmune diseases but are also obstacles against cancer therapies. An ideal cancer vaccine would stimulate specific cytotoxic responses and reduce/suppress Treg function. In this study, we showed that Escherichia coli expressing listeriolysin O and OVA (E. coli LLO/OVA) demonstrated remarkable levels of protection against OVA-expressing tumor cells. By contrast, E. coli expressing OVA only (E. coli OVA) showed poor protection. High-avidity OVA-specific CTL were induced in E. coli LLO/OVA-vaccinated mice, and CD8+ depletion—but not NK cell depletion, abolished the antitumor activity of the E. coli LLO/OVA vaccine. Phenotypic analysis of T cells following vaccination with either vaccine revealed preferential generation of CD44highCD62Llow CD8+ effector memory T cells over CD44highCD62Lhigh central memory T cells. Unexpectedly, CD4+ depletion turned E. coli OVA into a vaccine as effective as E. coli LLO/OVA suggesting that a subset of CD4+ cells suppressed the CD8+ T cell-mediated antitumor response. Further depletion experiments demonstrated that these suppressive cells consisted of CD4+CD25high regulatory T cells. We therefore assessed these vaccines for Treg function and found that although CD4+CD25high expansion and Foxp3 expression within this population was similar in all groups of mice, Treg cells from E. coli LLO/OVA-vaccinated animals were unable to suppress conventional T cells proliferation. These findings provide the first evidence that LLO expression affects Treg cell function and may have important implications for enhancing antitumor vaccination strategies in humans.
Regulation of MHC Class II Expression and Antigen Processing in Murine and Human Mesenchymal Stromal Cells by IFN-γ, TGF-β, and Cell Density
Authors: Romieu-Mourez Raphaëlle , François Moïra , Boivin Marie-Noëlle , Stagg John , Galipeau Jacques
Published: April 2014

Mesenchymal stromal cells (MSC) possess immunosuppressive properties, yet when treated with IFN-γ they acquire APC functions. To gain insight into MSC immune plasticity, we explored signaling pathways induced by IFN-γ required for MHC class II (MHC II)-dependent Ag presentation. IFN-γ-induced MHC II expression in mouse MSC was enhanced by high cell density or serum deprivation and suppressed by TGF-β. This process was regulated by the activity of the type IV CIITA promoter independently of STAT1 activation and the induction of the IFN regulatory factor 1-dependent B7H1/PD-L1 encoding gene. The absence of direct correlation with the cell cycle suggested that cellular connectivity modulates IFN-γ responsiveness for MHC II expression in mouse MSC. TGF-β signaling in mouse MSC involved ALK5 and ALK1 TGF-βRI, leading to the phosphorylation of Smad2/Smad3 and Smad1/Smad5/Smad8. An opposite effect was observed in human MSC where IFN-γ-induced MHC II expression occurred at the highest levels in low-density cultures; however, TGF-β reduced IFN-γ-induced MHC II expression and its signaling was similar as in mouse MSC. This suggests that the IFN-γ-induced APC features of MSC can be modulated by TGF-β, serum factors, and cell density in vitro, although not in the same way in mouse and human MSC, via their convergent effects on CIITA expression.
Ca2+ Signals in CD4+ T Cells during Early Contacts with Antigen-Bearing Dendritic Cells in Lymph Node
Authors: Wei Sindy H. , Safrina Olga , Yu Ying , Garrod Kym R. , Cahalan Michael D. , Parker Ian
Published: April 2014

T cell activation by APC requires cytosolic Ca2+ ([Ca2+]i) elevation. Using two-photon microscopy, we visualized Ca2+ signaling and motility of murine CD4+ T cells within lymph node (LN) explants under control, inflammatory, and immunizing conditions. Without Ag under basal noninflammatory conditions, T cells showed infrequent Ca2+ spikes associated with sustained slowing. Inflammation reduced velocities and Ca2+ spiking in the absence of specific Ag. During early Ag encounter, most T cells engaged Ag-presenting dendritic cells in clusters, and showed increased Ca2+ spike frequency and elevated basal [Ca2+]i. These Ca2+ signals persisted for hours, irrespective of whether T cells were in contact with visualized dendritic cells. We propose that sustained increases in basal [Ca2+]i and spiking frequency constitute a Ca2+ signaling modality that, integrated over hours, distinguishes immunogenic from basal state in the native lymphoid environment.
IFN-γ and Fas Ligand Are Required for Graft-versus-Tumor Activity against Renal Cell Carcinoma in the Absence of Lethal Graft-versus-Host Disease
Authors: Ramirez-Montagut Teresa , Chow Andrew , Kochman Adam A. , Smith Odette M. , Suh David , Sindhi Hamad , Lu Sydney , Borsotti Chiara , Grubin Jeremy , Patel Neel , Terwey Theis H. , Kim Theo D. , Heller Glenn , Murphy George F. , Liu Chen , Alpdogan Onder , van den Brink Marcel R. M.
Published: April 2014

To determine the mechanisms of graft-versus-tumor (GVT) activity in the absence of graft-versus-host disease (GVHD) against a solid tumor, we established two allogeneic bone marrow transplantation models with a murine renal cell carcinoma (RENCA). The addition of 0.3 × 106 donor CD8+ T cells to the allograft increased the survival of tumor-bearing mice without causing GVHD. The analysis of CD8+ T cells deficient in cytotoxic molecules demonstrated that anti-RENCA activity is dependent on IFN-γ and Fas ligand (FasL), but does not require soluble or membrane-bound TNF-α, perforin, or TRAIL. Recipients of IFN-γ−/− CD8+ T cells are unable to reject RENCA compared with recipients of wild-type CD8+ T cells and, importantly, neither group develops severe GVHD. IFN-γ−/− CD8+ T cells derived from transplanted mice are less able to kill RENCA cells in vitro, while pretreatment of RENCA cells with IFN-γ enhances class I and FasL expression and rescues the lytic capacity of IFN-γ−/− CD8+ T cells. These results demonstrate that the addition of low numbers of selected donor CD8+ T cells to the allograft can mediate GVT activity without lethal GVHD against murine renal cell carcinoma, and this GVT activity is dependent on IFN-γ and FasL.
OX40 Gene Expression Is Up-Regulated by Chromatin Remodeling in Its Promoter Region Containing Sp1/Sp3, YY1, and NF-κB Binding Sites
Authors: Tone Yukiko , Kojima Yoshitsugu , Furuuchi Keiji , Brady Maya , Yashiro-Ohtani Yumi , Tykocinski Mark L. , Tone Masahide
Published: April 2014

OX40 is a member of the TNFR superfamily (CD134; TNFRSF4) that is expressed on activated T cells and regulates T cell-mediated immune responses. In this study, we have examined the regulation of OX40 gene expression in T cells. Low-level OX40 mRNA expression was detected in both resting T cells and the nonactivated EL4 T cell line, and was up-regulated in both types of T cells upon activation with anti-CD3 Ab. We have shown in this study that basal OX40 promoter activity is regulated by constitutively expressed Sp1/Sp3 and YY1 transcription factors. NF-κB (p50 and p65) also binds to the OX40 promoter region, but the level of direct enhancement of the OX40 promoter activity by this transcription factor is not sufficient to account for the observed up-regulation of OX40 mRNA expression associated with activation. We have detected by chromatin immunoprecipitation that histone H4 molecules in the OX40 promoter region are highly acetylated by activation and NF-κB binds to the OX40 promoter in vivo. These findings suggest that OX40 gene expression is regulated by chromatin remodeling, and that NF-κB might be involved in initiation of chromatin remodeling in the OX40 promoter region in activated T cells. CD4+CD25+ regulatory T (Treg) cells also express OX40 at high levels, and signaling through this receptor can neutralize suppressive activity of this Treg cell. In CD4+CD25+ Treg cells, histone H4 molecules in the OX40 promoter region are also highly acetylated, even in the absence of in vitro activation.
A Natural Hypomorphic Variant of the Apoptosis Regulator Gimap4/IAN1
Authors: Carter Christine , Dion Carine , Schnell Silke , Coadwell W. John , Graham Margaret , Hepburn Lucy , Morgan Geoffrey , Hutchings Amanda , Pascall John C. , Jacobs Heinz , Miller J. Ross , Butcher Geoffrey W.
Published: April 2014

The Gimap/IAN family of GTPases has been implicated in the regulation of cell survival, particularly in lymphomyeloid cells. Prosurvival and prodeath properties have been described for different family members. We generated novel serological reagents to study the expression in rats of the prodeath family member Gimap4 (IAN1), which is sharply up-regulated at or soon after the stage of T cell-positive selection in the thymus. During these investigations we were surprised to discover a severe deficiency of Gimap4 expression in the inbred Brown Norway (BN) rat. Genetic analysis linked this trait to the Gimap gene cluster on rat chromosome 4, the probable cause being an AT dinucleotide insertion in the BN Gimap4 allele (AT(+)). This allele encodes a truncated form of Gimap4 that is missing 21 carboxyl-terminal residues relative to wild type. The low protein expression associated with this allele appears to have a posttranscriptional cause, because mRNA expression was apparently normal. Spontaneous and induced apoptosis of BN and wild-type T cells was analyzed in vitro and compared with the recently described mouse Gimap4 knockout. This revealed a “delayed” apoptosis phenotype similar to but less marked than that of the knockout. The Gimap4 AT(+) allele found in BN was shown to be rare in inbred rat strains. Nevertheless, when wild rat DNA samples were studied the AT(+) allele was found at a high overall frequency (∼30%). This suggests an adaptive significance for this hypomorphic allele.
Early Innate Immune Responses to Sin Nombre Hantavirus Occur Independently of IFN Regulatory Factor 3, Characterized Pattern Recognition Receptors, and Viral Entry
Authors: Prescott Joseph B. , Hall Pamela R. , Bondu-Hawkins Virginie S. , Ye Chunyan , Hjelle Brian
Published: April 2014

Sin Nombre virus (SNV) is a highly pathogenic New World virus and etiologic agent of hantavirus cardiopulmonary syndrome. We have previously shown that replication-defective virus particles are able to induce a strong IFN-stimulated gene (ISG) response in human primary cells. RNA viruses often stimulate the innate immune response by interactions between viral nucleic acids, acting as a pathogen-associated molecular pattern, and cellular pattern-recognition receptors (PRRs). Ligand binding to PRRs activates transcription factors which regulate the expression of antiviral genes, and in all systems examined thus far, IFN regulatory factor 3 (IRF3) has been described as an essential intermediate for induction of ISG expression. However, we now describe a model in which IRF3 is dispensable for the induction of ISG transcription in response to viral particles. IRF3-independent ISG transcription in human hepatoma cell lines is initiated early after exposure to SNV virus particles in an entry- and replication-independent fashion. Furthermore, using gene knockdown, we discovered that this activation is independent of the best-characterized RNA- and protein-sensing PRRs including the cytoplasmic caspase recruitment domain-containing RNA helicases and the TLRs. SNV particles engage a heretofore unrecognized PRR, likely located at the cell surface, and engage a novel IRF3-independent pathway that activates the innate immune response.
Inhibition of Phagosome Maturation by Mycobacteria Does Not Interfere with Presentation of Mycobacterial Antigens by MHC Molecules
Authors: Majlessi Laleh , Combaluzier Benoit , Albrecht Imke , Garcia Jessica E. , Nouze Clémence , Pieters Jean , Leclerc Claude
Published: April 2014

Pathogenic mycobacteria escape host innate immune responses by surviving within phagosomes of host macrophages and blocking their delivery to lysosomes. Avoiding lysosomal delivery may also be involved in the capacity of living mycobacteria to modulate MHC class I- or II-dependent T cell responses, which may contribute to their pathogenicity in vivo. In this study, we show that the presentation of mycobacterial Ags is independent of the site of intracellular residence inside professional APCs. Infection of mouse macrophages or dendritic cells in vitro with mycobacterial mutants that are unable to escape lysosomal transfer resulted in an identical efficiency of Ag presentation compared with wild-type mycobacteria. Moreover, in vivo, such mutants induced CD4+ Th1 or CD8+ CTL responses in mice against various mycobacterial Ags that were comparable to those induced by their wild-type counterparts. These results suggest that the limiting factor for the generation of an adaptive immune response against mycobacteria is not the degree of lysosomal delivery. These findings are important in the rational design of improved vaccines to combat mycobacterial diseases.
Cutting Edge: The ST2 Ligand IL-33 Potently Activates and Drives Maturation of Human Mast Cells
Authors: Allakhverdi Zoulfia , Smith Dirk E. , Comeau Michael R. , Delespesse Guy
Published: April 2014

IL-33, the natural ligand of the IL-1 receptor family member ST2L, is known to enhance experimental allergic-type inflammatory responses by costimulating the production of cytokines from activated Th2 lymphocytes. Although ST2L has long been known to be expressed by mast cells, its role in their biology has not been explored. In this study we report that IL-33 directly stimulates primary human mast cells (MCs) to produce several proinflammatory cytokines and chemokines and also exerts a permissive effect on the MCs response to thymic stromal lymphopoietin, a recently described potent MCs activator. IL-33 also acts both alone and in concert with thymic stromal lymphopoietin to accelerate the in vitro maturation of CD34+ MC precursors and induce the secretion of Th2 cytokines and Th2-attracting chemokines. Taken together, these results suggest that IL-33 may play an important role in mast cell-mediated inflammation and further emphasize the role of innate immunity in allergic diseases.
Generation of CD8 T Cell Memory Is Regulated by IL-12
Authors: Pearce Erika L. , Shen Hao
Published: April 2014

Various signals during infection influence CD8 T cell memory generation, but these factors have yet to be fully defined. IL-12 is a proinflammatory cytokine that has been shown to enhance IFN-γ-producing T cell responses and has been widely tested as a vaccine adjuvant. In this study, we show that IL-12-deficient mice generate a weaker primary CD8 T cell response and are more susceptible to Listeria monocytogenes infection, but have substantially more memory CD8 T cells and greater protective immunity against reinfection. Kinetic analyses show that in the absence of IL-12 there is a reduced contraction of Ag-specific CD8 T cells and a gradual increase in memory CD8 T cells as a result of increased homeostatic renewal. By signaling directly through its receptor on CD8 T cells, IL-12 influences their differentiation to favor the generation of fully activated effectors, but hinders the formation of CD8 T cell memory precursors and differentiation of long-term CD8 T cell memory. These results have implications for understanding memory T cell development and enhancing vaccine efficacy, and offer new insight into the role of IL-12 in coordinating the innate and adaptive immune response.
NK Cell Lysis of HIV-1-Infected Autologous CD4 Primary T Cells: Requirement for IFN-Mediated NK Activation by Plasmacytoid Dendritic Cells
Authors: Tomescu Costin , Chehimi Jihed , Maino Vernon C. , Montaner Luis J.
Published: April 2014

In vivo, several mechanisms have been postulated to protect HIV-1-infected cells from NK surveillance. In vitro, previous research indicates HIV-1-infected autologous CD4+ primary T cells are resistant to NK lysis. We hypothesized that NK lysis of HIV-1-infected target cells would be augmented by the presence of accessory cells and/or accessory cell factors. In this study, we show that stimulation of plasmacytoid dendritic cells (PDC) with the TLR9 agonist, CpG ODN 2216, triggered NK lysis of HIV-1-infected autologous CD4+ primary T cells. PDC-stimulated NK lysis was dependent upon MHC class I (MHC-I) down-regulation on infected cells, and primary HIV-1 isolates that exhibited enhanced MHC-I down-regulation were more susceptible to NK-mediated lysis. PDC-stimulated NK lysis of HIV-1-infected autologous CD4+ primary T cells was blocked by neutralizing Abs to type 1 IFN and was perforin/granzyme dependent. Overall, our data suggest that HIV-infected cells are not innately resistant to NK lysis, and that exogenous NK stimulation derived from PDC can trigger NK cytotoxicity against HIV-1-infected autologous CD4+ primary T cells.
Antigen, in the Presence of TGF-β, Induces Up-Regulation ofFoxP3gfp+ in CD4+ TCR Transgenic T Cells That Mediate Linked Suppression of CD8+ T Cell Responses
Authors: Kapp Judith A. , Honjo Kazuhito , Kapp Linda M. , Goldsmith Kelly , Bucy R. Pat
Published: April 2014

CD4+CD25+ regulatory T cells (Tregs) inhibit immune responses to a variety of Ags, but their specificity and mechanism of suppression are controversial. This controversy is largely because many studies focused on natural Tregs with undefined specificities and suppression has frequently been measured on polyclonal T cell responses. To address the issue of specificity further, we have bred Kd-specific, CD4+ TCR (TCR75) transgenic mice to Foxp3gfp knockin reporter mice to permit sorting of Tregs with a known specificity. Foxp3gfp.TCR75 mice did not express significant numbers of natural FoxP3+ Tregs expressing the TCR75 transgenes, but FoxP3 expression was induced by stimulating with Kd plus TGF-β. The resulting GFP+ TCR75 cells were anergic, whereas the GFP− TCR75 cells proliferated upon restimulation with Kd peptide. Yet both exhibited severely reduced expression of intracellular IFN-γ and TNF-α upon restimulation. GFP+, but not GFP−, TCR75 T cells suppressed responses by naive TCR75 T cells and by nontransgenic spleen cells stimulated with anti-CD3. GFP+ TCR75 cells also inhibited polyclonal C57BL/6 anti-Kd CTL responses if the APC expressed Kd and both MHC class I and class II, and responses by OT1 T cells to B6.Kd.OVA but not B6.Kd plus OVA expressing APC, demonstrating linked-suppression of CD8 responses. Thus, Tregs exhibit a greater degree of specificity in vitro than previously appreciated. The observation that Tregs and responder T cells must recognize the same APC provides a mechanistic explanation for the observation that Tregs must be in direct contact with effector T cells to suppress their responses.
Role of Direct Effects of IFN-γ on T Cells in the Regulation of CD8 T Cell Homeostasis
Authors: Tewari Kavita , Nakayama Yumi , Suresh M.
Published: April 2014

It is well recognized that IFN-γ plays a critical role in the control of CD8 T cell expansion and contraction during immune responses to several intracellular pathogens. However, our understanding of the mechanisms underlying the regulation of T cell fate by IFN-γ is sorely incomplete. Specifically, it is unclear whether regulation of CD8 T cell homeostasis occurs by a T cell intrinsic IFN-γ pathway. In this study, we have determined the role of the direct effects of IFN-γ on T cells in regulating the expansion, contraction, and memory phases of the polyclonal CD8 T cell response to an acute viral infection. Using two complementary approaches we demonstrate that the direct effects of IFN-γ suppress IL-7R expression on Ag-specific effector CD8 T cells, but clonal expansion or deletion of activated CD8 T cells in vivo can occur in the apparent absence of IFN-γR signaling in T cells. These findings have clarified fundamental features of control of T cell homeostasis by IFN-γ in the context of CD8 T cell memory and protective immunity.
Induction of FoxP3+CD4+25+ Regulatory T Cells Following Hemopoietic Stem Cell Transplantation: Role of Bone Marrow-Derived Facilitating Cells
Authors: Taylor Kendra N. , Shinde-Patil Vivek R. , Cohick Evan , Colson Yolonda L.
Published: April 2014

The establishment of donor cell lineages following allogeneic bone marrow transplantation is frequently associated with the development of graft-vs-host disease (GVHD). The identification of cell populations that are capable of supporting allogeneic stem cell (SC) engraftment and the induction of tolerance without inducing GVHD could expand the use of this therapy. CD8+TCR− facilitating cells (FC) have been shown to promote allogeneic SC engraftment with resulting transplantation tolerance across complete MHC barriers without inducing GVHD. Although donor reconstitution in SC plus FC recipients is associated with the induction of regulatory T cell-associated factors, it is not known whether an induction of regulatory T cells and subsequent tolerance is a direct effect of the FC. The current study demonstrates that 1) SC plus FC transplantation results in the induction of donor CD4+25+ regulatory T cells and that FC are present in the spleen of recipients before the induction of these cells, 2) activation of FC with CpG-oligodeoxynucleotide promotes CD4+25− T cell differentiation into CD4+25+ regulatory T cells in vitro, as demonstrated by cytokine and forkhead/winged helix transcription factor (FoxP3) gene and protein expression, and 3) direct contact between FC and CD4+25− T cells is required for FoxP3+CD4+25+ regulatory T cell induction and is dependent on CD86 expression on FC. This is the first report to demonstrate a mechanism for FC in the induction of regulatory T cells following allogeneic SC plus FC transplantation. The transplantation of donor FC may provide an alternative approach to permit clinical SC engraftment and induction of transplantation tolerance in the future.
Selective Expansion of Foxp3-Positive Regulatory T Cells and Immunosuppression by Suppressors of Cytokine Signaling 3-Deficient Dendritic Cells
Authors: Matsumura Yumiko , Kobayashi Takashi , Ichiyama Kenji , Yoshida Ryoko , Hashimoto Masayuki , Takimoto Tomohito , Tanaka Kentaro , Chinen Takatoshi , Shichita Takashi , Wyss-Coray Tony , Sato Katsuaki , Yoshimura Akihiko
Published: April 2014

Dendritic cells (DCs) induce immunity and immunological tolerance as APCs. It has been shown that DCs secreting IL-10 induce IL-10+ Tr1-type regulatory T (Treg) cells, whereas Foxp3-positive Treg cells are expanded from naive CD4+ T cells by coculturing with mature DCs. However, the regulatory mechanism of expansion of Foxp3+ Treg cells by DCs has not been clarified. In this study, we demonstrated that suppressors of cytokine signaling (SOCS)-3-deficient DCs have a strong potential as Foxp3+ T cell-inducing tolerogenic DCs. SOCS3−/− DCs expressed lower levels of class II MHC, CD40, CD86, and IL-12 than wild-type (WT)-DCs both in vitro and in vivo, and showed constitutive activation of STAT3. Foxp3− effector T cells were predominantly expanded by the priming with WT-DCs, whereas Foxp3+ Treg cells were selectively expanded by SOCS3−/− DCs. Adoptive transfer of SOCS3−/− DCs reduced the severity of experimental autoimmune encephalomyelitis. Foxp3+ T cell expansion was blocked by anti-TGF-β Ab, and SOCS3−/− DCs produced higher levels of TGF-β than WT-DCs, suggesting that TGF-β plays an essential role in the expansion of Foxp3+ Treg cells. These results indicate an important role of SOCS3 in determining on immunity or tolerance by DCs.
Acute Rejection of Allografted CTL-Susceptible Leukemia Cells from Perforin/Fas Ligand Double-Deficient Mice
Authors: Nomi Hayahito , Tashiro-Yamaji Junko , Yamamoto Yumiko , Miura-Takeda Sayako , Miyoshi-Higashino Masako , Takahashi Takeshi , Azuma Haruhito , Ueda Haruhiko , Katsuoka Yoji , Kubota Takahiro , Yoshida Ryotaro
Published: April 2014

The generation of knockout mice demonstrated that CD4+, but not CD8+, T cells were essential for the rejection of allografted skin or heart, presumably because these targets were CTL resistant. In the case of CTL-susceptible targets (e.g., P815 mastocytoma cells and EL-4 or RLmale1 T lymphoma cells), however, it is assumed that the CTL is the effector cell responsible for allograft rejection and that perforin and Fas ligand (FasL) pathways are the killing mechanisms. In the present study, we examined the role of these cytotoxic molecules in the rejection of i.p. allografted CTL-susceptible leukemia cells. Unexpectedly, the allografted leukemia cells were acutely rejected from gld (a mutation of FasL), perforin−/−, or double-deficient mice. The peritoneal exudate cells from gld or normal mice showed T cell-, TCRαβ-, and perforin-dependent cytotoxic activity against the allograft, whereas the exudate cells from perforin−/− mice exhibited almost full cytotoxic activity in the presence of Fas-Fc. Furthermore, the infiltrates from double-deficient mice showed a high cytotoxic activity against the allografted cells even in the presence of anti-TCRαβ Ab or in the absence of T cells. The cytotoxic cells appeared to be macrophages, because they were Mac-1+ mononuclear cells with a kidney- or horseshoe-shaped nucleus and because the cytotoxic activity was completely suppressed by the addition of NG-monomethyl-l-arginine, an inhibitor of inducible NO synthase. These results indicate that macrophages are ready and available to kill CTL-susceptible allografts when CTLs lack both perforin and FasL molecules.