bluuebunny/crossref_metadata_2025 huggingface dataset:
T-Bet Expression and Failure of GATA-3 Cross-Regulation Lead to Default Production of IFN-γ by γδ T Cells
Authors: Yin Zhinan , Chen ChangHung , Szabo Susanne J. , Glimcher Laurie H. , Ray Anuradha , Craft Joe
Published: April 2014

γδ T cells predominantly produce IFN-γ upon activation. To determine the basis for default production of IFN-γ by γδ T cells, we analyzed the transcription factors T-box expressed in T cells (T-bet) and GATA-3. T-bet, absent in naive γδ cells, was induced upon TCR signaling, with IFN-γ production. T-bet also regulated IL-4 synthesis, as γδ cells isolated from T-bet-deficient mice displayed enhanced IL-4 levels with reduced IFN-γ production. Notably, T-bet expression after TCR signaling in γδ cells was not down-regulated by IL-4, in conjunction with a higher ratio of T-bet:GATA-3 expression than that found in CD4+ T cells. Indeed, overexpression of GATA-3 failed to inhibit IFN-γ secretion in γδ cells to the degree seen in CD4+ T cells. These results indicate that T-bet enhances IFN-γ secretion and suppresses IL-4 secretion in γδ cells, and that GATA-3 fails to counterbalance T-bet-mediated IFN-γ production, accounting for the default synthesis of IFN-γ by these T lymphocytes.
Inhibitory Effects of Apoptotic Cell Ingestion upon Endotoxin-Driven Myeloid Dendritic Cell Maturation
Authors: Stuart Lynda M. , Lucas Mark , Simpson Cathy , Lamb Jonathan , Savill John , Lacy-Hulbert Adam
Published: April 2014

Dendritic cells (DCs) are the sentinels of the immune system, able to interact with both naive and memory T cells. The recent observation that DCs can ingest cells dying by apoptosis has raised the possibility that DCs may, in fact, present self-derived Ags, initiating both autoimmunity and tumor-specific responses, especially if associated with appropriate danger signals. Although the process of ingestion of apoptotic cells has not been shown to induce DC maturation, the exact fate of these phagocytosing DCs remains unclear. In this paper we demonstrate that DCs that ingest apoptotic cells are able to produce TNF-α but have a diminished ability to produce IL-12 in response to external stimuli, a property that corresponds to a failure to up-regulate CD86. By single-cell analysis we demonstrate that these inhibitory effects are restricted to those DCs that have engulfed apoptotic cells, with bystander DCs remaining unaffected. These changes were independent of the production of anti-inflammatory cytokines TGF-β1 and IL-10 and corresponded with a diminished capacity to stimulate naive T cells. Thus, the ingestion of apoptotic cells is not an immunologically null event but is capable of modulating DC maturation. These results have important implications for our understanding of the role of clearance of dying cells by DCs not only in the normal resolution of inflammation but also in control of subsequent immune responses to apoptotic cell-derived Ags.
CD8+, αβ-TCR+, and γδ-TCR+ Cells in the Recipient Hematopoietic Environment Mediate Resistance to Engraftment of Allogeneic Donor Bone Marrow
Authors: Xu Hong , Exner Beate G. , Cramer Daniel E. , Tanner Michael K. , Mueller Yvonne M. , Ildstad Suzanne T.
Published: April 2014

Historically, conditioning for engraftment of hematopoietic stem cells has been nonspecific. In the present study, we characterized which cells in the recipient hematopoietic microenvironment prevent allogeneic marrow engraftment. Mice defective in production of αβ-TCR+, γδ-TCR+, αβ- plus γδ-TCR+, CD8+, or CD4+ cells were transplanted with MHC-disparate allogeneic bone marrow. Conditioning with 500 cGy total body irradiation (TBI) plus a single dose of cyclophosphamide (CyP) on day +2 establishes chimerism in normal recipients. When mice were conditioned with 300 cGy TBI plus a single dose of CyP on day +2, all engrafted, except wild-type controls and those defective in production of CD4+ T cells. Mice lacking both αβ- and γδ-TCR+ cells engrafted without conditioning, suggesting that both αβ- and γδ-TCR T cells in the host play critical and nonredundant roles in preventing engraftment of allogeneic bone marrow. CD8 knockout (KO) mice engrafted without TBI, but only if they received CyP on day +2 relative to the marrow infusion, showing that a CD8− cell was targeted by the CyP conditioning. The CD8+ cell effector function is mechanistically different from that for conventional T cells, and independent of CD4+ T helper cells because CD4 KO mice require substantially higher levels of conditioning than the other KO phenotypes. These results suggest that a number of cell populations with different mechanisms of action mediate resistance to engraftment of allogeneic marrow. Targeting of specific recipient cellular populations may permit conditioning approaches to allow mixed chimerism with minimal morbidity and could potentially avoid the requirement for myelotoxic agents altogether.
Preferential Recognition of Self Antigens Despite Normal Thymic Deletion of CD4+CD25+ Regulatory T Cells
Authors: Romagnoli Paola , Hudrisier Denis , van Meerwijk Joost P. M.
Published: April 2014

T cell tolerance to self Ags is in part established in the thymus by induction of apoptosis or anergy of potentially autoreactive thymocytes. Some autospecific T cells nevertheless migrate to peripheral lymphoid organs but are kept under control by the recently identified CD4+CD25+ regulatory T cell subset. Because these cells inhibit autoimmunity more efficiently than useful non-self Ag-specific immune responses, they are probably autospecific, posing important questions as to how they develop in the thymus. In this study we show that significantly more peripheral CD4+CD25+ regulatory T cells recognize self than non-self Ags. However, we also show for a large panel of endogenous superantigens as well as for self peptide/MHC complexes that autospecific CD4+CD25+ thymocyte precursors are normally deleted during ontogeny. Combined, our data firmly establish that the repertoire of regulatory T cells is specifically enriched in autospecific cells despite the fact that their precursors are normally susceptible to thymic deletion.
Early Growth Response Transcription Factors Are Required for Development of CD4−CD8− Thymocytes to the CD4+CD8+ Stage
Authors: Carleton Michael , Haks Mariëlle C. , Smeele Sigrid A. A. , Jones Allan , Belkowski Stanley M. , Berger Marc A. , Linsley Peter , Kruisbeek Ada M. , Wiest David L.
Published: April 2014

Progression of immature CD4−CD8− thymocytes beyond the β-selection checkpoint to the CD4+CD8+ stage requires activation of the pre-TCR complex; however, few of the DNA-binding proteins that serve as molecular effectors of those pre-TCR signals have been identified. We demonstrate in this study that members of the early growth response (Egr) family of transcription factors are critical effectors of the signals that promote this developmental transition. Specifically, the induction of three Egr family members (Egr1, 2, and 3) correlates with pre-TCR activation and development of CD4−CD8− thymocytes beyond the β-selection checkpoint. Enforced expression of each of these Egr factors is able to bypass the block in thymocyte development associated with defective pre-TCR function. However, Egr family members may play somewhat distinct roles in promoting thymocyte development, because there are differences in the genes modulated by enforced expression of particular Egr factors. Finally, interfering with Egr function using dominant-negative proteins disrupts thymocyte development from the CD4−CD8− to the CD4+CD8+ stage. Taken together, these data demonstrate that the Egr proteins play an essential role in executing the differentiation program initiated by pre-TCR signaling.
Notch1 Perturbation of Hemopoiesis Involves Non-Cell- Autonomous Modifications
Authors: Kawamata Shin , Du Changchun , Li Kaijun , Lavau Catherine
Published: April 2014

To study the effects of Notch on hemopoiesis we used a bone marrow transduction/transplantation model and compared the transduced and nontransduced populations in reconstituted mice. While cells expressing a constitutively active form of murine Notch1 (Notch1IC) completely lacked B cells, a profound suppression of the B lineage was also seen in the nontransduced compartment. Experiments performed with retroviral supernatants of varying titers showed that the perturbations of B cell development among the nontransduced population correlated with the percentage of Notch1IC-transduced cells inoculated into the mice. The myeloid lineage of the Notch1IC-transplanted mice was altered as well, and this also affected the nontransduced population that had features of excessive maturation. To explore the basis of these non-cell-autonomous modifications we prepared conditioned medium from ex vivo cultures of Notch1IC-transplanted mice bone marrow and showed that it inhibited B cell maturation and promoted myeloid differentiation in a dose-dependent manner. Finally, we found that the T cell leukemia/lymphomas that occur in Notch1IC-transplanted mice were accompanied by abnormal maturation of nontransduced T cells in the bone marrow. These findings indicate that modifications of neighboring cells through non-cell-autonomous modifications take part in multiple facets of the activity of Notch on hemopoiesis.
Mutagenesis Within Human FcεRIα Differentially Affects Human and Murine IgE Binding
Authors: Mackay Graham A. , Hulett Mark D. , Cook Justin P. D. , Trist Halina M. , Henry Alistair J. , McDonnell James M. , Beavil Andrew J. , Beavil Rebecca L. , Sutton Brian J. , Hogarth P. Mark , Gould Hannah J.
Published: April 2014

Soluble fragments of the α-chain of FcεRI, the high-affinity receptor for IgE, compete with membrane-bound receptors for IgE and may thus provide a means to combat allergic responses. Mutagenesis within FcεRIα is used in this study, in conjunction with the crystal structure of the FcεRIα/IgE complex, to define the relative importance of specific residues within human FcεRIα for IgE binding. We have also compared the effects of these mutants on binding to both human and mouse IgE, with a view to evaluating the mouse as an appropriate model for the analysis of future agents designed to mimic the human FcεRIα and attenuate allergic disease. Three residues within the C-C′ region of the FcεRI α2 domain and two residues within the α2 proximal loops of the α1 domain were selected for mutagenesis and tested in binding assays with human and mouse IgE. All three α2 mutations (K117D, W130A, and Y131A) reduced the affinity of human IgE binding to different extents, but K117D had a far more pronounced effect on mouse IgE binding, and although Y131A had little effect, W130A modestly enhanced binding to mouse IgE. The mutations in α1 (R15A and F17A) diminished binding to both human and mouse IgE, with these effects most likely caused by disruption of the α1/α2 interface. Our results demonstrate that the effects of mutations in human FcεRIα on mouse IgE binding, and hence the inhibitory properties of human receptor-based peptides assayed in rodent models of allergy, may not necessarily reflect their activity in a human IgE-based system.
Innate Direct Anticancer Effector Function of Human Immature Dendritic Cells. I. Involvement of an Apoptosis-Inducing Pathway
Authors: Janjic Bratislav M. , Lu Ganwei , Pimenov Alexei , Whiteside Theresa L. , Storkus Walter J. , Vujanovic Nikola L.
Published: April 2014

Dendritic cells (DCs) mediate cross-priming of tumor-specific T cells by acquiring tumor Ags from dead cancer cells. The process of cross-priming would be most economical and efficient if DCs also induce death of cancer cells. In this study, we demonstrate that normal human in vitro generated immature DCs consistently and efficiently induce apoptosis in cancer cell lines, freshly isolated noncultured cancer cells, and normal proliferating endothelial cells, but not in most normal cells. In addition, in vivo generated noncultured peripheral blood immature DCs mediate similar tumoricidal activity as their in vitro counterpart, indicating that this DC activity might be biologically relevant. In contrast to immature DCs, freshly isolated monocytes (myeloid DC precursors) and in vitro generated mature DCs are not cytotoxic or are less cytotoxic, respectively, suggesting that DC-mediated killing of cancer cells is developmentally regulated. Comparable cytotoxic activity is mediated by untreated DCs, paraformaldehyde-fixed DCs, and soluble products of DCs, and is destructible by proteases, indicating that both cell membrane-bound and secreted proteins mediate this DC function. Overall, our data demonstrate that human immature DCs are capable of inducing apoptosis in cancer cells and thus to both directly mediate anticancer activity and initiate processing of cellular tumor Ags.
Deficiency in the Anti-Apoptotic Protein A1-a Results in a Diminished Acute Inflammatory Response
Authors: Orlofsky Amos , Weiss Louis M. , Kawachi Nicole , Prystowsky Michael B.
Published: April 2014

A1 is an anti-apoptotic member of the Bcl-2 family that is up-regulated in inflammatory myeloid cells. In the present study, we investigated the role of A1 in the maintenance of acute inflammation in mice. Mice possess three genes encoding highly related isoforms of A1. A1-a isoform mRNA was minimally expressed in resident peritoneal macrophages, but was present at a 300-fold higher level in inflammatory macrophages elicited by i.p. infection with Toxoplasma gondii. In comparison, A1-b and A1-d levels were 3- and 10-fold higher, respectively. Peritoneal leukocytosis was decreased in infected A1-a-deficient mice compared with wild-type, and this reduction was associated with a small but reproducible enhancement of survival. These effects could not be explained by an alteration in peritoneal parasite load, nor by increased apoptosis of infected inflammatory cells, which were protected from cell death by an A1-a-independent mechanism. Increased apoptosis in inflammatory neutrophils was observed sporadically in A1-a-deficient mice. Regulation of apoptosis by A1-a may be an important proinflammatory event in acute host responses.
Cell-Mediated Immunity Induced by RecombinantMycobacterium bovisBacille Calmette-Guérin Strains Against an Intracellular Bacterial Pathogen: Importance of Antigen Secretion or Membrane-Targeted Antigen Display as Lipoprotein for Vaccine Efficacy
Authors: Grode Leander , Kursar Mischo , Fensterle Joachim , Kaufmann Stefan H. E. , Hess Jürgen
Published: April 2014

Live recombinant vaccines expressing defined pathogen-derived Ags represent powerful candidates for future vaccination strategies. In this study, we report on the differential induction of protective cell-mediated immunity elicited by different recombinant Mycobacterium bovis Bacille Calmette-Guérin (BCG) strains displaying p60 Ag of Listeria monocytogenes in secreted, cytosolic, or membrane-attached form for T cell recognition. Anti-listerial protection evoked by the membrane-linked p60 lipoprotein of rBCG Mp60 and that of the p60 derivative secreted by rBCG Sp60-40 were nearly equal, whereas cytosolic p60 displayed by rBCG Np60 failed to protect mice from listeriosis. In vivo depletion of CD4 or CD8 T cell subpopulations in rBCG Mp60-vaccinated mice before listerial challenge revealed interactions of both T cell subsets in anti-listerial protection. In rBCG Sp60-40-vaccinated animals, CD4 T cells predominantly contributed to anti-listerial control as shown by the failure of anti-CD8 mAb treatment to impair the outcome of listeriosis in rBCG Sp60-40-vaccinated mice after L. monocytogenes challenge. Hence, differential Ag display by rBCG influences cell-mediated immunity, which in turn may impact vaccine efficacy due to the different requirements of CD4 or CD8 T cells for pathogen elimination.
Oral DNA Vaccination In Utero Induces Mucosal Immunity and Immune Memory in the Neonate
Authors: Gerdts Volker , Snider Marlene , Brownlie Robert , Babiuk Lorne A. , Griebel Philip J.
Published: April 2014

Infectious diseases are responsible for a significant number of deaths during the first weeks of life. Some of the salient pathogens include HSV, HIV, hepatitis B virus, group B streptococcus, Haemophilus sp., and Chlamydia sp. The vertical transmission of many of these pathogens significantly increases the risk of neonatal infection. We recently reported that oral DNA immunization in utero induced high serum Ab titers and cell-mediated immunity in fetal lambs. In this study, we demonstrate immune memory and mucosal immunity in newborn lambs following oral DNA immunization of the fetus. A single oral exposure in utero to plasmid DNA encoding a truncated form of glycoprotein D of bovine herpesvirus-1 induced detectable immune responses in 80% (12 of 15) of newborn lambs. There was no evidence for the induction of immune tolerance in nonresponding lambs. Responding lambs displayed both systemic and mucosal immune responses and reduced virus shedding following intranasal challenge. Furthermore, strong anamnestic responses were evident for at least 3 mo after birth. The efficacy of in utero oral DNA immunization was further demonstrated with the hepatitis B surface Ag, and protective serum Ab titers occurred in 75% of immunized lambs. Thus, the present investigation confirms that oral DNA immunization in utero can induce both mucosal and systemic immune responses in the neonate and that this immunity has the potential to prevent vertical disease transmission.
Streptococcus pneumoniaeEvades Complement Attack and Opsonophagocytosis by Expressing thepspCLocus-Encoded Hic Protein That Binds to Short Consensus Repeats 8–11 of Factor H
Authors: Jarva Hanna , Janulczyk Robert , Hellwage Jens , Zipfel Peter F. , Björck Lars , Meri Seppo
Published: April 2014

Streptococcus pneumoniae is an important cause of upper and lower respiratory tract infections, meningitis, peritonitis, bacterial arthritis, and sepsis. Here we have studied a novel immune evasion mechanism of serotype 3 pneumococci, which are particularly resistant to phagocytosis. On their surfaces the bacteria express the factor H-binding inhibitor of complement (Hic), a protein of the pneumococcal surface protein C family. Using radioligand binding, microtiter plate assays, surface plasmon resonance analysis, and recombinant constructs of factor H, we located the binding site of Hic to short consensus repeats (SCRs) 8–11 in the middle part of factor H. This represents a novel microbial interaction region on factor H. The only other ligand known so far for SCRs 8–11 of factor H is C-reactive protein (CRP), an acute phase protein that binds to the pneumococcal C-polysaccharide. The binding sites of Hic and CRP within the SCR8–11 region were different, however, because CRP did not inhibit the binding of Hic and required calcium for binding. Binding of factor H to Hic-expressing pneumococci promoted factor I-mediated cleavage of C3b and restricted phagocytosis of pneumococci. Thus, virulent pneumococci avoid complement attack and opsonophagocytosis by recruiting functionally active factor H with the Hic surface protein. Hic binds to a previously unrecognized microbial interaction site in the middle part of factor H.
IL-4 Up-Regulates the Expression of Tissue Inhibitor of Metalloproteinase-2 in Dermal Fibroblasts Via the p38 Mitogen-Activated Protein Kinase-Dependent Pathway
Authors: Ihn Hironobu , Yamane Kenichi , Asano Yoshihide , Kubo Masahide , Tamaki Kunihiko
Published: April 2014

Tissue inhibitor of metalloproteinase-2 (TIMP-2) is a potent inhibitor of activated matrix metalloproteinases such as gelatinase and collagenase, and thus helps to control extracellular matrix metabolism and deposition by connective tissue cells. We examined the responsiveness of the expression of TIMP-2 to various cytokines in dermal fibroblasts and studied the regulatory and signaling mechanisms of the response. TIMP-2 protein and mRNA expression was induced by IL-4 in a dose- and time-dependent manner, but not by TGF-β, oncostatin M, or IL-6. IL-4 induction of TIMP-2 expression was dependent upon transcription. The p38 mitogen-activated protein kinase (MAPK) inhibitors SB202190 and SB203580 suppressed IL-4-induced TIMP-2 expression, suggesting the involvement of p38 MAP kinase in the signaling of IL-4 leading to TIMP-2 expression. Immunoblotting analysis using a specific Ab against phosphorylated p38 MAP kinase (Thr180/Tyr182) showed that IL-4 induced phosphorylation of p38 MAP kinase in human dermal fibroblasts. Furthermore, the p38 MAP kinase assay showed that IL-4 induces p38 MAPK activation in human dermal fibroblasts. The expression of the dominant-negative mutant p38 MAPK represses the IL-4-induced TIMP-2 expression in human dermal fibroblasts. Thus, IL-4 can potentially alter the dermal matrix metabolism by regulating TIMP-2.
Functional IL-10 Deficiency in the Lung of Cystic Fibrosis (cftr−/−) and IL-10 Knockout Mice Causes Increased Expression and Function of B7 Costimulatory Molecules on Alveolar Macrophages
Authors: Soltys Jindrich , Bonfield Tracey , Chmiel James , Berger Melvin
Published: April 2014

Alveolar macrophages are poor APCs that only minimally express B7 costimulatory molecules. Because our previous data suggest that bronchial epithelial cells constitutively secrete IL-10, and IL-10 inhibits B7 expression in vitro, we hypothesized that this IL-10 is responsible for suppressing B7 expression on macrophages that enter the airways. Furthermore, because we have shown that cystic fibrosis (CF) lungs are deficient in IL-10, we hypothesized that bronchoalveolar macrophages (BALMs) from cystic fibrosis transmembrane conductance regulator (CFTR)−/− as well as IL-10−/− mice might express increased B7. Immunofluorescence for B7 was positive on BALMs from CF patients and CFTR−/− and IL-10−/− mice, but was negative on controls. FACS showed that 63.9% of BALMs from IL-10−/− mice were B7-1 positive, as were 67.4% of BALMs from CFTR−/− mice, whereas <7% of BALMs from wild-type controls were positive. Using BALMs to costimulate splenic T cells with anti-CD3 as a mitogen showed 9202 ± 2107 cpm [3H]thymidine incorporation for BALMs from IL-10−/− mice and 4082 ± 1036 cpm for BALMs from CFTR−/− mice, but <200 cpm with BALMs from either type of +/+ mouse. Treatment of CFTR−/− mice with recombinant mouse IL-10 reduced the B7 expression and costimulatory activity of the BALMs. These data suggest that the IL-10 secreted in the healthy lung may be responsible for the absence of B7 and poor costimulatory activity of BALMs and that reductions of pulmonary IL-10 in CF may enhance B7 expression and local immune responses.
Molecular Mechanisms Involved in Lymphocyte Recruitment in Inflamed Brain Microvessels: Critical Roles for P-Selectin Glycoprotein Ligand-1 and Heterotrimeric Gi-Linked Receptors
Authors: Piccio Laura , Rossi Barbara , Scarpini Elio , Laudanna Carlo , Giagulli Cinzia , Issekutz Andrew C. , Vestweber Dietmar , Butcher Eugene C. , Constantin Gabriela
Published: April 2014

Lymphocyte recruitment into the brain is a critical event in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis. We developed a novel intravital microscopy model to directly analyze through the skull the interactions between lymphocytes and the endothelium in cerebral venules of mice. No adhesive interactions were observed between lymphocytes and the nonactivated endothelium in the cerebral microcirculation. When brain venules were activated by pretreating mice with TNF-α or LPS, proteolipid protein 139–151 autoreactive T lymphocytes rolled and arrested; notably, only a few peripheral lymph node cells rolled and firmly adhered. Abs anti-P-selectin glycoprotein ligand-1 and anti-E- and P-selectin blocked tethering and rolling of autoreactive lymphocytes, suggesting that P-selectin glycoprotein ligand-1/endothelial selectins are critical in the recruitment of lymphocytes in inflamed brain venules. E- and P-selectin were expressed on cerebral vessels upon in vivo activation and had a patchy distribution during the preclinical phase of active and passive experimental autoimmune encephalomyelitis. LFA-1/ICAM-1 and α4 integrins/VCAM-1 supported rolling, but were not relevant to rolling velocity. Firm arrest was mainly mediated by LFA-1 and ICAM-1. Pretreatment of autoreactive lymphocytes with pertussis toxin blocked integrin-dependent arrest, implicating a requirement for Gi protein-dependent signaling in vessels from nonlymphoid districts. In conclusion, our data unveils the molecular mechanisms controlling the recruitment of autoreactive lymphocytes in inflamed cerebral vessels and suggest new insights into the pathogenesis of autoimmune inflammatory diseases of the CNS.
Long-Term Survival of Corneal Allografts Is Dependent on Intact CD1d-Reactive NKT Cells
Authors: Sonoda Koh-Hei , Taniguchi Masaru , Stein-Streilein Joan
Published: April 2014

BALB/c mice that tolerate the allogeneic grafts develop allogeneic-specific anterior chamber-associated immune deviation. Because CD1d-reactive NKT cells are essential for anterior chamber-associated immune deviation, we postulated that the survival of C57BL/6 (B6) cornea graft in BALB/c mice was also dependent on CD1d-reactive NKT cells. The B6 corneal graft rejection rate in BALB/c vs Jα281 knockout (KO) mice, which lack NKT cells, was measured. While there were no difference in the early phase of rejection, the survival rates at 12 wk after grafting for BALB/c and Jα281 KO mice were 50 and 0%, respectively. Because anti-CD1d mAb abrogated the corneal graft survival in the wild-type mice we concluded that CD1d-reactive NKT cells were essential for graft survival. Moreover, allospecific T regulatory (Tr) cells correlated with acceptance of B6 grafts in BALB/c mice, and the adoptive transfer of these allospecific Tr cells to Jα281 KO mice allowed a 50% survival rate of B6 cornea grafts. In conclusion, CD1d-reactive NKT cells are required for induction of allospecific Tr cells and are essential for survival of corneal allografts. Mechanisms that contribute to cornea graft acceptance may lead to new therapies for improvement in graft survival in high-risk corneas and other transplanted tissues and grafts.
Cross-Linking Surface Ig Delays CD40 Ligand- and IL-4-Induced B Cell Ig Class Switching and Reveals Evidence for Independent Regulation of B Cell Proliferation and Differentiation
Authors: Rush James S. , Hasbold Jhagvaral , Hodgkin Philip D.
Published: April 2014

T cells stimulate B cells to divide and differentiate by providing activating signals in the form of inducible membrane-bound molecules and secreted cytokines. Provision of these signals in vitro reproduces many of the consequences of T-B collaboration in the absence of any form of Ag stimulation. Although clearly not obligatory, Ag signals appear to play an important regulatory role in numerous aspects of the B cell response. To examine directly the effect of an Ag signal, naive B cells were stimulated in the presence of rCD40 ligand, with or without IL-4 in the presence or absence of different anti-Ig mAbs. Anti-Ig mAbs exerted variable effects on the B cell division rate, from enhancement to no effect to inhibition. In contrast, all anti-Ig mAbs tested inhibited division-linked isotype switching to IgG1 and IgE. Thus, B cell Ag receptor ligands could modify the rates of B cell expansion and class switching independently. The ability of anti-Ig reagents to modify class switching suggests the B cell Ag receptor may play an important role in the selection of Ig isotypes during T cell-dependent humoral immune responses to Ags of different physical structure.
IL-4 and T Cells Are Required for the Generation of IgG1 Isotype Antibodies Against Cardiolipin
Authors: Fischer Karsten , Collins Helen , Taniguchi Masaru , Kaufmann Stefan H. E. , Schaible Ulrich E.
Published: April 2014

Infection with Mycobacterium tuberculosis induces Abs against a vast array of mycobacterial lipids and glycolipids. One of the most prominent lipid Ags recognized is cardiolipin (CL). The kinetics of the generation of anti-CL Abs during infection reveals that IgM titers to CL increase over time. Interestingly, at day 30 postinfection CL-specific IgG1 appears, an isotype usually dependent on T cell help. Using an immunization schedule with CL/anti-CL Ab complexes, which induces antiphospholipid syndrome in mice, we show that the generation of IgG1 to CL requires IL-4 and that optimal production is T cell dependent. IgG1 production to CL was impaired in nude (nu/nu) mice devoid in conventional T cells, but was not affected in mice deficient for either αβ TCR+, γδ TCR+, CD4+, CD8+, or NK1.1+ T cells. We conclude that IgG1 production to CL depends on T cell help and IL-4, which can be provided by different T cell populations. This is the first report that IL-4 is indispensable for the induction of IgG1 Abs to lipid Ags.
Impaired Precursor B Cell Differentiation in Bruton’s Tyrosine Kinase-Deficient Mice
Authors: Middendorp Sabine , Dingjan Gemma M. , Hendriks Rudolf W.
Published: April 2014

Bruton’s tyrosine kinase (Btk) is a cytoplasmic signaling molecule that is crucial for precursor (pre-B) cell differentiation in humans. In this study, we show that during the transition of large cycling to small resting pre-B cells in the mouse, Btk-deficient cells failed to efficiently modulate the expression of CD43, surrogate L chain, CD2, and CD25. In an analysis of the kinetics of pre-B cell differentiation in vivo, Btk-deficient cells manifested a specific developmental delay within the small pre-B cell compartment of ∼3 h, when compared with wild-type cells. Likewise, in in vitro bone marrow cultures, Btk-deficient large cycling pre-B cells showed increased IL-7 mediated expansion and reduced developmental progression into noncycling CD2+CD25+ surrogate L chain-negative small pre-B cells and subsequently into Ig-positive B cells. Furthermore, the absence of Btk resulted in increased proliferative responses to IL-7 in recombination-activating gene-1-deficient pro-B cells. These findings identify a novel role for Btk in the regulation of the differentiation stage-specific modulation of IL-7 responsiveness in pro-B and pre-B cells. Moreover, our results show that Btk is critical for an efficient transit through the small pre-B cell compartment, thereby regulating cell surface phenotype changes during the developmental progression of cytoplasmic μ H chain expressing pre-B cells into immature IgM+ B cells.
Characterization of the Fas Ligand/Fas-Dependent Apoptosis of Antiretroviral, Class I MHC Tetramer-Defined, CD8+ CTL by In Vivo Retrovirus-Infected Cells
Authors: Rich Robert F. , Green William R.
Published: April 2014

C57BL/6 (B6; H-2b) mice mount strong AKR/Gross murine leukemia virus (MuLV)-specific CD8+ CTL responses to the immunodominant Kb-restricted epitope, KSPWFTTL, of endogenous AKR/Gross MuLV. In sharp contrast, spontaneous virus-expressing AKR.H-2b congenic mice are low/nonresponders for the generation of AKR/Gross MuLV-specific CTL. Furthermore, when viable AKR.H-2b spleen cells are cocultured with primed responder B6 antiviral precursor CTL, the AKR.H-2b cells function as “veto” cells that actively mediate the inhibition of antiviral CTL generation. AKR.H-2b veto cell inhibition is virus specific, MHC restricted, contact dependent, and mediated through veto cell Fas ligand/responder T cell Fas interactions. In this study, following specific priming and secondary in vitro restimulation, antiretroviral CD8+ CTL were identified by a labeled Kb/KSPWFTTL tetramer and flow cytometry, enabling direct visualization of AKR.H-2b veto cell-mediated depletion of these CTL. A 65–93% reduction in the number of B6 Kb/KSPWFTTL tetramer+ CTL correlated with a similar reduction in antiviral CTL cytotoxicity. Addition on sequential days to the antiviral CTL restimulation cultures of either 1) AKR.H-2b veto cells or 2) a blocking Fas-Ig fusion protein (to cultures also containing AKR.H-2b veto cells) to block inhibition demonstrated that AKR.H-2b veto cells begin to inhibit B6 precursor CTL/CTL expansion during days 2 and 3 of the 6-day culture. Shortly thereafter, a high percentage of B6 tetramer+ CTL cocultured with AKR.H-2b veto cells was annexin V positive and Fashigh, indicating apoptosis as the mechanism of veto cell inhibition. Experiments using the irreversible inhibitor emetine demonstrated that AKR.H-2b cells had to be metabolically active and capable of protein synthesis to function as veto cells. Of the tetramer-positive CTL that survived veto cell-mediated apoptosis, there was no marked skewing from the preferential usage of Vβ4, 8.1/8.2, and 11 TCR normally observed. These findings provide further insight into the complexity of host/virus interactions and suggest a fail-safe escape mechanism by virus-infected cells for epitopes residing in critical areas of viral proteins that cannot accommodate variations of amino acid sequence.
Complement Receptor Type 1 (CD35) Mediates Inhibitory Signals in Human B Lymphocytes
Authors: Józsi Mihály , Prechl József , Bajtay Zsuzsa , Erdei Anna
Published: April 2014

The complement system—particularly component C3—has been demonstrated to be a key link between innate and adaptive immunity. The trimolecular complex of complement receptor type 2 (CR2), CD19, and CD81 is known to promote B cell activation when coligated with the B cell Ag receptor. In the present study, we aimed to elucidate the role of human complement receptor type 1 (CR1), the other C3-receptor on B cells. As ligand, aggregated C3 and aggregated C3(H2O), i.e., multimeric “C3b-like C3”, are used, which bind to CR1, but not to CR2. In experiments studying the functional consequences of CR1-clustering, the multimeric ligand is shown to inhibit the proliferation of tonsil B cells activated with a suboptimal dose of anti-IgM F(ab′)2. Importantly, this inhibitory activity also occurs in the presence of the costimulatory cytokines IL-2 and IL-15. The anti-IgM-induced transient increase in the concentration of intracellular free Ca2+ and phosphorylation of several cytoplasmic proteins are strongly reduced in the presence of the CR1 ligand. Data presented indicate that CR1 has a negative regulatory role in the B cell Ag receptor mediated activation of human B lymphocytes.
Two New Isotype-Specific Switching Activities Detected for Ig Class Switching
Authors: Ma Limei , Wortis Henry H. , Kenter Amy L.
Published: April 2014

Ig class switch recombination (CSR) occurs by an intrachromosomal deletional process between switch (S) regions in B cells. To facilitate the study of CSR, we derived a new B cell line, 1.B4.B6, which is uniquely capable of μ→γ3, μ→ε, and μ→α, but not μ→γ1 CSR at its endogenous loci. The 1.B4.B6 cell line was used in combination with plasmid-based isotype-specific S substrates in transient transfection assays to test for the presence of trans-acting switching activities. The 1.B4.B6 cell line supports μ→γ3, but not μ→γ1 recombination, on S substrates. In contrast, normal splenic B cells activated with LPS and IL-4 are capable of plasmid-based μ→γ1 CSR and demonstrate that this S plasmid is active. Activation-induced deaminase (AID) was used as a marker to identify existing B cell lines as possible candidates for supporting CSR. The M12 and A20 cell lines were identified as AID positive and, following activation with CD40L and other activators, were found to differentially support μ→ε and μ→α plasmid-based CSR. These studies provide evidence for two new switching activities for μ→γ1 and μ→ε CSR, which are distinct from μ→γ3 and μ→α switching activities previously described. AID is expressed in all the B cell lines capable of CSR, but cannot account for the isotype specificity defined by the S plasmid assay. These results are consistent with a model in which isotype-specific switching factors are either isotype-specific recombinases or DNA binding proteins with sequence specificity for S DNA.
Phosphorylation and O-Linked Glycosylation of Elf-1 Leads to Its Translocation to the Nucleus and Binding to the Promoter of the TCR ζ-Chain
Authors: Juang Yuang-Taung , Solomou Elena E. , Rellahan Barbara , Tsokos George C.
Published: April 2014

Elf-1, a member of the E 26-specific transcription factor family with a predicted molecular mass of 68 kDa, is involved in the transcriptional regulation of several hematopoietic cell genes. We demonstrate that Elf-1 exists primarily as a 98-kDa form in the nucleus and as an 80-kDa form in the cytoplasm. Phosphorylation and O-linked glycosylation contribute to the increased posttranslational molecular mass of Elf-1. The 98-kDa Elf-1 is released from the cytoplasm tethering retinoblastoma protein and moves to the nucleus, where it binds to the promoter of the TCR ζ-chain gene. Finally, the cytoplasmic 98-kDa form enters the proteasome pathway and undergoes degradation. In conclusion, different forms of Elf-1 are the products of posttranslational modifications that determine its subcellular localization, activity, and metabolic degradation.
Optimal T Cell Responses to Cryptococcus neoformans Mannoprotein Are Dependent on Recognition of Conjugated Carbohydrates by Mannose Receptors
Authors: Mansour Michael K. , Schlesinger Larry S. , Levitz Stuart M.
Published: April 2014

Cryptococcosis is a leading cause of death among individuals with compromised T cell function. Soluble Cryptococcus neoformans mannoproteins (MP) have emerged as promising vaccine candidates due to their capacity to elicit delayed-type hypersensitivity and Th type 1-like cytokines, both critical to the clearance of this pathogenic yeast. In this study, the mechanisms responsible for the potent immunostimulatory properties of MP were explored. Using Chinese hamster ovary cells expressing human macrophage mannose receptor (MMR), we determined that MP is a MMR ligand. Functionally, competitive blockade of multilectin mannose receptors (MR) on APCs diminished MP-dependent stimulation of primary T cells from immunized mice and the MP-reactive CD4+ T cell hybridoma, P1D6, by 72 and 99%, respectively. Removal of O-linked saccharides from MP by β-elimination inhibited MP-dependent stimulation of P1D6 and primary T cells by 89 and 90%, respectively. In addition, MP-dependent stimulation of P1D6 was abrogated after digestion with proteinase K, suggesting the protein core of MP contributed the antigenic moiety presented by APC. Stimulation of P1D6 by MP also was abolished using APC obtained from invariant chain-deficient mice, demonstrating Ag presentation was MHC class II restricted. Our data suggest that MP is a ligand for the MMR and that T cell stimulation is functionally inhibited either by competitive blockade of MR or by removal of carbohydrate residues critical for recognition. The demonstration that efficient T cell responses to MP require recognition of terminal mannose groups by MMR provides both a molecular basis for the immunogenicity of cryptococcal MP and support for vaccination strategies that target MR.
Toll-Like Receptor 4-MD-2 Complex Mediates the Signal Transduction Induced by Flavolipin, an Amino Acid-Containing Lipid Unique toFlavobacterium meningosepticum
Authors: Gomi Kazunori , Kawasaki Kiyoshi , Kawai Yohko , Shiozaki Masao , Nishijima Masahiro
Published: April 2014

Flavolipin, an amino acid-containing lipid isolated from Flavobacterium meningosepticum, induces many immune responses. It has been shown that flavolipin does not induce an immune response of macrophages derived from C3H/HeJ mice, which possess a point mutation in Toll-like receptor 4 (TLR4). To determine whether TLR4 or the molecular complex of TLR4 and TLR4 association molecule MD-2 mediates the flavolipin signal, flavolipin responsiveness was examined by measuring NF-κB activation in Ba/F3 cells and Ba/F3 transfectants expressing TLR4 or both TLR4 and MD-2. Flavolipin-induced NF-κB activation was detected in the cells expressing both TLR4 and MD-2, but not in the other cells. Expression of CD14 in the transfectant expressing both TLR4 and MD-2 increased the sensitivity to flavolipin. Furthermore, flavolipin stereoisomers were chemically synthesized, and their abilities to induce NF-κB activation were examined. (R)-Flavolipin, in which the configuration of the lipid moiety is R, induced NF-κB activation via the TLR4-MD-2 complex, but (S)-flavolipin did not. In this study, we demonstrated the involvement of TLR4-MD-2 and CD14 in flavolipin signaling and the importance of the (R)-configuration of the flavolipin lipid moiety for the induction of an immune response via TLR4-MD-2.
The Role of IFN in Respiratory Syncytial Virus Pathogenesis
Authors: Durbin Joan E. , Johnson Teresa R. , Durbin Russell K. , Mertz Sara E. , Morotti Rafaella A. , Peebles R. Stokes , Graham Barney S.
Published: April 2014

Formalin-inactivated respiratory syncytial virus (RSV) vaccine preparations have been shown to cause enhanced disease in naive hosts following natural infection. In this study we demonstrate a similar pattern of enhanced disease severity following primary RSV infection of IFN-nonresponsive STAT1−/− mice. STAT1−/− mice showed markedly increased illness compared with wild-type BALB/c animals following RSV inoculation despite similar lung virus titers and rates of virus clearance. Histologically, STAT1−/− animals had eosinophilic and neutrophilic pulmonary infiltrates not present in wild-type or IFN-γ−/−-infected mice. In cytokine analyses of infected lung tissue, IFN-γ was induced in both STAT1−/− and wild-type mice, with preferential IL-4, IL-5, and IL-13 induction only in the STAT1−/− animals. Eotaxin was detected in the lungs of both wild-type and STAT1−/− mice following infection, with a 1.7-fold increase over wild-type in the STAT1−/− mice. Using a peptide epitope newly identified in the RSV fusion protein, we were able to demonstrate that wild-type memory CD4+ T cells stimulated by this peptide produce primarily IFN-γ, while STAT1−/−CD4+ cells produce primarily IL-13. These findings suggest that STAT1 activation by both type I (αβ) and type II (γ) IFNs plays an important role in establishing a protective, Th1 Ag-specific immune response to RSV infection.
Role of Lipopolysaccharide-Binding Protein in Early Alcohol-Induced Liver Injury in Mice
Authors: Uesugi Takehiko , Froh Matthias , Arteel Gavin E. , Bradford Blair U. , Wheeler Michael D. , Gäbele Erwin , Isayama Fuyumi , Thurman Ronald G.
Published: April 2014

Cellular responses to endotoxins are enhanced markedly by LPS-binding protein (LBP). Furthermore, it has been demonstrated that endotoxins and proinflammatory cytokines such as TNF-α participate in early alcohol-induced liver injury. Therefore, in this study, a long-term intragastric ethanol feeding model was used to test the hypothesis that LBP is involved in alcoholic hepatitis by comparing LBP knockout and wild-type mice. Two-month-old female mice were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 wk. There was no difference in mean urine alcohol concentrations between the groups fed ethanol. Dietary alcohol significantly increased liver to body weight ratios and serum alanine aminotransferase levels in wild-type mice (189 ± 31 U/L) over high-fat controls (24 ± 7 U/L), effects which were blunted significantly in LBP knockout mice (60 ± 17 U/L). Although no significant pathological changes were observed in high-fat controls, 4 wk of dietary ethanol caused steatosis, mild inflammation, and focal necrosis in wild-type animals as expected (pathology score, 5.9 ± 0.5). These pathological changes were reduced significantly in LBP knockout mice fed ethanol (score, 2.6 ± 0.5). Endotoxin levels in the portal vein were increased significantly after 4 wk in both groups fed ethanol. Moreover, ethanol increased TNF-α mRNA expression in wild-type, but not in LBP knockout mice. These data are consistent with the hypothesis that LBP plays an important role in early alcohol-induced liver injury by enhancing LPS-induced signal transduction, most likely in Kupffer cells.
The Cutaneous Reverse Arthus Reaction Requires Intercellular Adhesion Molecule 1 and L-Selectin Expression
Authors: Kaburagi Yuko , Hasegawa Minoru , Nagaoka Tetsuya , Shimada Yuka , Hamaguchi Yasuhito , Komura Kazuhiro , Saito Eriko , Yanaba Koichi , Takehara Kazuhiko , Kadono Takafumi , Steeber Douglas A. , Tedder Thomas F. , Sato Shinichi
Published: April 2014

The deposition of immune complexes (IC) induces an acute inflammatory response with tissue injury. IC-induced inflammation is mediated by inflammatory cell infiltration, a process highly regulated by expression of multiple adhesion molecules. To assess the role of L-selectin and ICAM-1 in this pathogenetic process, the cutaneous reverse passive Arthus reaction was examined in mice lacking L-selectin (L-selectin−/−), ICAM-1 (ICAM-1−/−), or both (L-selectin/ICAM-1−/−). Edema and hemorrhage, which peaked 4 and 8 h after IC challenge, respectively, were significantly reduced in L-selectin−/−, ICAM-1−/−, and L-selectin/ICAM-1−/− mice compared with wild-type littermates. In general, edema and hemorrhage were more significantly inhibited in ICAM-1−/− mice than in L-selectin−/− mice, but were most significantly reduced in L-selectin/ICAM-1−/− mice compared with ICAM-1−/− or L-selectin−/− mice. Decreased edema and hemorrhage correlated with reduced neutrophil and mast cell infiltration in all adhesion molecule-deficient mice, but leukocyte infiltration was most affected in L-selectin/ICAM-1−/− mice. Reduced neutrophil and mast cell infiltration was also observed for all mutant mice in the peritoneal Arthus reaction. Furthermore, cutaneous TNF-α production was inhibited in each deficient mouse, which paralleled the reductions in cutaneous inflammation. These results indicate that ICAM-1 and L-selectin cooperatively contribute to the cutaneous Arthus reaction by regulating neutrophil and mast cell recruitment and suggest that ICAM-1 and L-selectin are therapeutic targets for human IC-mediated disease.
Lamina Propria CD4+ T Lymphocytes Synergize with Murine Intestinal Epithelial Cells to Enhance Proinflammatory Response Against an Intracellular Pathogen
Authors: Mennechet Franck J. D. , Kasper Lloyd H. , Rachinel Nicolas , Li Wen , Vandewalle Alain , Buzoni-Gatel Dominique
Published: April 2014

Acute and lethal ileitis can be elicited in certain strains of inbred mice after oral infection with the intracellular protozoan parasite Toxoplasma gondii. The development of this inflammatory process is dependent upon the induction of a robust Th1 response, including overproduction of IFN-γ, TNF-α, and NO, as has been reported in other experimental models of human inflammatory bowel disease. In this study we have investigated the role of CD4+ T cells from the lamina propria (LP) in the early inflammatory events after T. gondii infection using isolated and primary cultured intestinal cells from infected mice and immortalized mouse mICcl2 intestinal epithelial cells. Primed LP CD4+ T cells isolated from parasite-infected mice produce substantial quantities of both IFN-γ and TNF-α. IFN-γ- and TNF-α-producing LP CD4+ T cells synergize with infected mICcl2 and enhance the production of several inflammatory chemokines including macrophage-inflammatory protein-2, monocyte chemoattractant protein-1, monocyte chemoattractant protein-3, macrophage-inflammatory protein-1αβ, and IFN-γ-inducible protein-10. Furthermore, primed LP CD4+ T cells cocultured with infected mICcl2 inhibited replication of the parasite in the intestinal epithelial cells. Thus, LP CD4+ T cells can interact with parasite-infected intestinal epithelial cells and alter the expression of several proinflammatory products that have been associated with the development of intestinal inflammation. The interaction between these two components of the gut mucosal compartment (CD4+ T cells and enterocytes) may play a role in the immunopathogenesis of this pathogen-driven experimental inflammatory bowel disease model.