bluuebunny/crossref_metadata_2025 huggingface dataset:
The Plant Lectin Wheat Germ Agglutinin Inhibits the Binding of Pemphigus Foliaceus Autoantibodies to Desmoglein 1 in a Majority of Patients and Prevents Pathomechanisms of Pemphigus Foliaceus In Vitro and In Vivo
Authors: Ortiz-Urda Susana , Elbe-Bürger Adelheid , Smolle Josef , Marquart Yvonne , Chudnovsky Yakov , Ridky Todd W. , Bernstein Pamela , Wolff Klaus , Rappersberger Klemens
Published: April 2014

Pemphigus foliaceus (PF) is a life-threatening autoimmune blistering skin disease caused by pathogenic IgG autoantibodies against desmoglein 1 (dg1), a desmosomal cadherin-type adhesion glycoprotein. Using lectins and glycosidases, we have shown that dg1 displays an N-glycosylation pattern of the complex triantennary type. We have found that lectins and glycosidases interfere with N-bound sugar residues on the amino-terminal ectodomain of dg1 and completely abolish, in vitro, the antigenicity of dg1 in most of the patients’ sera. Moreover, in an ex vivo model using punch biopsies from normal human skin, we demonstrate that preincubation of the epidermis in wheat germ agglutinin (WGA) prevents PF autoantibody binding, acantholysis, and subcorneal blistering. In addition, we show that topical treatment with WGA inhibits PF autoantibody binding to keratinocytes in both newborn BALB/c mice and in organotypic human epidermis grafted onto the back of SCID mice. The epidermis of these pretreated animals displays a regular morphology, whereas control animals develop the immunopathologic phenotype of PF. These findings suggest that WGA may interfere with autoantibody binding to dg1, preventing experimental PF without affecting the adhesive function of dg1. Our observations may provide a new approach to the therapy of PF.
Activity and Safety of CTLA-4 Blockade Combined with Vaccines in Cynomolgus Macaques
Authors: Keler Tibor , Halk Ed , Vitale Laura , O’Neill Tom , Blanset Diann , Lee Steven , Srinivasan Mohan , Graziano Robert F. , Davis Thomas , Lonberg Nils , Korman Alan
Published: April 2014

The immune modulatory molecule CTLA-4 (CD152), through interactions with the B7 costimulatory molecules, has been shown to be a negative regulator of T cell activation in various murine model systems. Abs that block CTLA-4 function can enhance immune responses that mediate potent antitumor activity. However, CTLA-4 blockade can also exacerbate autoimmune disease. The safety and activity of anti-CTLA-4 Abs in primates has not been addressed. To that end, we generated human Abs against CTLA-4 using transgenic mice expressing human Ig genes. A high affinity Ab (10D1) that blocked the binding of CTLA-4 to the B7-1 and B7-2 ligands and had cross-reactivity with macaque CTLA-4 was chosen for further development. Administration of 10D1 to cynomolgus macaques significantly enhanced Ab responses to hepatitis surface Ag and a human melanoma cell vaccine. Anti-self Ab responses as measured by immunoassays using lysate from melanocyte-rich tissues were elicited in those animals receiving the melanoma cell vaccine and anti-CTLA-4 Ab. Remarkably, chronic administration of 10D1 did not result in measurable polyclonal T cell activation, significant alteration of the lymphocyte subsets, or induce clinically observable autoimmunity. Repeated dosing of the 10D1 did not elicit monkey anti-human Ab responses in the monkeys. These observations support the development of CTLA-4 blockade for human immunotherapy.
Chromatin Specificity of Anti-Double-Stranded DNA Antibodies and a Role for Arg Residues in the Third Complementarity-Determining Region of the Heavy Chain
Authors: Guth Amanda M. , Zhang Xianghua , Smith Diana , Detanico Thiago , Wysocki Lawrence J.
Published: April 2014

A spontaneous, autoreactive autoantibody called SN5–18 (IgG2b, κ) binds to a complex of H2A/H2B/dsDNA in chromatin, but erroneously appears to bind dsDNA when the Ab is used in a form that is not highly purified. Because of this finding, we evaluated the antigenic specificity of a prototypic anti-dsDNA Ab, 3H9/Vκ4, now used widely in transgenic studies of tolerance and autoimmunity. We found that the purified mAb 3H9/Vκ4 binds chromatin and specifically a complex of H2A/H2B/dsDNA, but not dsDNA in solid phase or in solution. When used in the form of culture supernatant or as a standard protein G preparation, mAb 3H9/Vκ4 appears to bind dsDNA, apparently due to nuclear proteins in the preparation that assemble on target DNA. Because of the reported role of VHCDR3 Arg residues in dsDNA binding and the near identity of the SN5–18 sequence to other dsDNA-specific Ab, we tested the contributions of two VHCDR3 Arg residues in SN5–18 to chromatin specificity. We found that both these Arg residues at positions 104 and 106 were required for detectable chromatin binding. These results indicate a role for VHCDR3 Arg residues in chromatin specificity of lupus-derived autoantibodies. Further, they provide an explanation for a possible discrepancy in the form of tolerance observed in different anti-DNA Ig transgene models.
Differential Regulation of Peripheral CD4+ T Cell Tolerance Induced by Deletion and TCR Revision
Authors: Ali Mohamed , Weinreich Michael , Balcaitis Stephanie , Cooper Cristine J. , Fink Pamela J.
Published: April 2014

In Vβ5 transgenic mice, mature Vβ5+CD4+ T cells are tolerized upon recognition of a self Ag, encoded by a defective endogenous retrovirus, whose expression is confined to the lymphoid periphery. Cells are driven by the tolerogen to enter one of two tolerance pathways, deletion or TCR revision. CD4+ T cells entering the former pathway are rendered anergic and then eliminated. In contrast, TCR revision drives gene rearrangement at the endogenous TCR β locus and results in the appearance of Vβ5−, endogenous Vβ+, CD4+ T cells that are both self-tolerant and functional. An analysis of the molecules that influence each of these pathways was conducted to understand better the nature of the interactions that control tolerance induction in the lymphoid periphery. These studies reveal that deletion is efficient in reconstituted radiation chimeras and is B cell, CD28, inducible costimulatory molecule, Fas, CD4, and CD8 independent. In contrast, TCR revision is radiosensitive, B cell, CD28, and inducible costimulatory molecule dependent, Fas and CD4 influenced, and CD8 independent. Our data demonstrate the differential regulation of these two divergent tolerance pathways, despite the fact that they are both driven by the same tolerogen and restricted to mature CD4+ T cells.
Cutting Edge: CD4+ T Cell Help Can Be Essential for Primary CD8+ T Cell Responses In Vivo
Authors: Wang Jyh-Chiang E. , Livingstone Alexandra M.
Published: April 2014

Recent studies have shown that CD4+ T cell help is required for the generation of memory CD8+ T cells that can proliferate and differentiate into effector cells on Ag restimulation. The importance of help for primary CD8+ T cell responses remains controversial. It has been suggested that help is not required for the initial proliferation and differentiation of CD8+ T cells in vivo and that classical models of helper-dependent responses describe impaired secondary responses to Ag in vitro. We have measured primary CD8+ T cell responses to peptide-pulsed dendritic cells in mice by cytokine ELISPOT and tetramer staining. No responses were detected in the absence of help, either when normal dendritic cells were injected into MHC II-deficient mice or when MHC II-deficient dendritic cells were injected into normal mice. Thus, the primary in vivo CD8+ T cell response depends absolutely on help from CD4+ T cells in our experimental system.
Homing to Nonlymphoid Tissues Is Not Necessary for Effector Th1 Cell Differentiation
Authors: Bajénoff Marc , Guerder Sylvie
Published: April 2014

The differentiation of naive T cells into effector Th1 cells is a complex process that may proceed in two steps, commitment and development. Initial TCR engagement and IFN-γ signaling instruct the T cells to commit to the Th1 lineage, while subsequent IL-12 and potentially TCR signaling induces final differentiation into irreversible, Th1 effector cells. In agreement with a multistep process of Th1 cell differentiation, effector Th1 cell generation requires repeated TCR and cytokine signaling, thus raising the possibility that commitment and differentiation processes may occur in two distinct anatomical sites, the lymphoid organ and the site of infection, respectively. We tested this possibility using a model of skin sensitization that permits a direct analysis of Ag-specific T cells both within lymphoid organs and at the site of sensitization. We show in this study that Ag presentation in the skin does not induce further differentiation of skin-infiltrating T cells that are highly divided and fully differentiated effector cells. Thus, effector Th1 cell differentiation is completed within lymphoid organs. In addition, we examined the heterogeneity of CD4 T cell responses in vivo through the analysis of the expression, by activated T cells, of different selectins, including P-selectin ligand and CD62L known to define separable effector populations. We delineated, in lymph nodes, at least five distinct subpopulations of activated CD4 T cells with different phenotypes and recirculation properties. Collectively, these results show that the lymphoid environment orchestrates T cell activation to generate a repertoire of effector T cells with a diversity of effector functions.
Evidence for a Ligand-Mediated Positive Selection Signal in Differentiation to a Mature B Cell
Authors: Wang Hongsheng , Clarke Stephen H.
Published: April 2014

Positive selection is required for B cell differentiation, as indicated by the requirement for expression of the pre-B cell receptor (pre-BCR) and the BCR at the pre-B and immature B cell stages, respectively. Positive selection mediated by a tonic signal from these receptors is sufficient to drive B cell differentiation beyond the pre-B and immature B cell stages, but it is unclear whether additional positive selection signals are required for differentiation to a mature B-2 cell. We have identified a population of Ig transgenic B cells that differentiatively arrest at a transitional B cell stage in the spleen. They exhibit no evidence of Ag encounter or negative selection and can differentiate to mature B-2 cells in vivo upon weak BCR stimulation or adoptive transfer to irradiated hosts. These data are consistent with a requirement for a ligand-mediated BCR signal for differentiation to a mature B-2 cell.
Proinflammatory Actions of Thromboxane Receptors to Enhance Cellular Immune Responses
Authors: Thomas Dennis W. , Rocha Paulo N. , Nataraj Chandra , Robinson Lisa A. , Spurney Robert F. , Koller Beverly H. , Coffman Thomas M.
Published: April 2014

Metabolism of arachidonic acid by the cyclo-oxygenase (COX) pathway generates a family of prostanoid mediators. Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting COX, thereby reducing prostanoid synthesis. The efficacy of these agents in reducing inflammation suggests a dominant proinflammatory role for the COX pathway. However, the actions of COX metabolites are complex, and certain prostanoids, such as PGE2, in some circumstances actually inhibit immune and inflammatory responses. In these studies, we examine the hypothesis that anti-inflammatory actions of NSAIDs may be due, in part, to inhibition of thromboxane A2 synthesis. To study the immunoregulatory actions of thromboxane A2, we used mice with a targeted disruption of the gene encoding the thromboxane-prostanoid (TP) receptor. Both mitogen-induced responses and cellular responses to alloantigen were substantially reduced in TP−/− spleen cells. Similar attenuation was observed with pharmacological inhibition of TP signaling in wild-type splenocytes, suggesting that reduced responsiveness was not due to subtle developmental abnormalities in the TP-deficient mice. The absence of TP receptors reduced immune-mediated tissue injury following cardiac transplant rejection, an in vivo model of intense inflammation. Taken together, these findings show that thromboxane augments cellular immune responses and inflammatory tissue injury. Specific inhibition of the TP receptor may provide a more precise approach to limit inflammation without some of the untoward effects associated with NSAIDs.
Effective Genetic Vaccination with a Widely Shared Endogenous Retroviral Tumor Antigen Requires CD40 Stimulation during Tumor Rejection Phase
Authors: Bronte Vincenzo , Cingarlini Sara , Apolloni Elisa , Serafini Paolo , Marigo Ilaria , De Santo Carmela , Macino Beatrice , Marin Oriano , Zanovello Paola
Published: April 2014

Endogenous retrovirus (ERV) products are recognized by T lymphocytes in mice and humans. As these Ags are preferentially expressed by neoplastic tissues, they might represent an ideal target for active immunization by genetic vaccination. However, i.m. inoculation of plasmid DNA encoding mouse gp70 or p15E, two products of the env gene of an endogenous murine leukemia virus, elicited a weak Ag-specific T lymphocyte response and resulted in partial protection from challenge with mouse tumors possessing these Ags. Depletion experiments showed that CD8+, but not CD4+, T lymphocytes were crucial for the antitumor activity of the vaccines. Systemic administration of agonistic anti-CD40 mAb increased the therapeutic potential of genetic vaccination, but only when given during the tumor rejection phase and not at the time of immunization. This effect correlated with a dramatic increase in the number of ERV-specific CD8+ T lymphocytes. Adjuvant activity of CD40 agonists thus seems to be relevant to enhance the CD8+ T cell-dependent response in tumor-bearing hosts, suggesting that sustaining tumor-specific T lymphocyte survival in subjects undergoing vaccination might be a key event in the successful vaccination with weak tumor Ags.
Newly Activated T Cells Promote Maturation of Bystander Dendritic Cells but Not IL-12 Production
Authors: Spörri Roman , Reis e Sousa Caetano
Published: April 2014

The activation of dendritic cells (DC) leads to increased costimulatory activity (termed DC maturation) and, in some instances, production of immunomodulatory cytokines such as IL-12. Both innate and T cell-derived signals can promote DC activation but it is unclear to what extent the two classes of stimuli are interchangeable or regulate distinct aspects of DC function. In this study, we show that signals from newly activated CD4+ T cells cannot initiate IL-12 synthesis although they can amplify secretion of bioactive IL-12 p70 by DC exposed to an appropriate innate stimulus. This occurs exclusively in cis and does not influence IL-12 synthesis by bystander DC that do not present Ag. In marked contrast, signals from newly activated CD4+ T cells can induce an increase in DC costimulatory activity in the absence of any innate priming. This occurs both in cis and in trans, affecting all DC in the microenvironment, including those that do not bear specific Ag. Consistent with the latter, we show that newly activated CD4+ T cells in vivo can deliver “help” in trans, effectively lowering the number of MHC/peptide complexes required for proliferation of third-party naive CD4+ T cells recognizing Ag on bystander DC. These results demonstrate that DC maturation and cytokine production are regulated distinctly by innate stimuli vs signals from CD4+ T cells and reveal a process of trans activation of DC without secretion of polarizing cytokines that takes place during T cell priming and may be involved in amplifying immune responses.
Phosphodiesterase 7A-Deficient Mice Have Functional T Cells
Authors: Yang Guchen , McIntyre Kim W. , Townsend Robert M. , Shen Henry H. , Pitts William J. , Dodd John H. , Nadler Steven G. , McKinnon Murray , Watson Andrew J.
Published: April 2014

Phosphodiesterases (PDEs) are enzymes which hydrolyze the cyclic nucleotide second messengers, cAMP and cGMP. In leukocytes, PDEs are responsible for depletion of cAMP which broadly suppresses cell functions and cellular responses to many activation stimuli. PDE7A has been proposed to be essential for T lymphocyte activation based on its induction during cell activation and the suppression of proliferation and IL-2 production observed following inhibition of PDE7A expression using a PDE7A antisense oligonucleotide. These observations have led to the suggestion that selective PDE7 inhibitors could be useful in the treatment of T cell-mediated autoimmune diseases. In the present report, we have used targeted gene disruption to examine the role PDE7A plays in T cell activation. In our studies, PDE7A knockout mice (PDE7A−/−) showed no deficiencies in T cell proliferation or Th1- and Th2-cytokine production driven by CD3 and CD28 costimulation. Unexpectedly, the Ab response to the T cell-dependent Ag, keyhole limpet hemocyanin, in the PDE7A−/− mice was found to be significantly elevated. The results from our studies strongly support the notion that PDE7A is not essential for T cell activation.
Cyclic Nucleotides Promote Monocyte Differentiation Toward a DC-SIGN+ (CD209) Intermediate Cell and Impair Differentiation into Dendritic Cells
Authors: Giordano Daniela , Magaletti Dario M. , Clark Edward A. , Beavo Joseph A.
Published: April 2014

Recruitment of monocytes into tissues and their differentiation into macrophages or dendritic cells (DCs) depend on the microenvironment of the inflammatory site. Although many factors affecting this process have been identified, the intracellular signaling pathways implicated are poorly understood. We found that cyclic nucleotides regulate certain steps of monocyte differentiation into DCs. Increased levels of the cyclic nucleotides, cAMP or cGMP, inhibit differentiation of CD14+/CD1alow monocytes into CD14−/CD1ahigh DCs. However, DC-specific ICAM-3-grabbing nonintegrin (CD209) up-regulation was not affected by cyclic nucleotides, indicating that DC development was not blocked at the monocyte stage. Interestingly, Ag-presenting function was increased by cyclic nucleotides, as measured by the higher expression of MHC class II, CD86, and an increased ability to stimulate CD4+ T cell proliferation in allogeneic MLRs. Although cyclic nucleotides do not completely block DC differentiation, they do block the ability of DCs to be induced to mature by LPS. Treatment during DC differentiation with either cAMP or cGMP analogues hampered LPS-induced expression of CD83, DC-LAMP, and CCR7 and the ability of DCs to migrate toward CCL19/macrophage-inflammatory protein 3β. Interestingly, the induction of a CD16+ subpopulation of cells was also observed. Thus, signals causing an increase in either cAMP or cGMP levels during monocyte recruitment to inflammatory sites may restrain the activation of acquired immunity by blocking DC development and migration to lymph nodes. At the same time, these signals promote development of an active intermediate cell type having properties between those of macrophages and DCs, which might contribute to the innate immune response in the periphery.
Genetic Complementation in Female (BXSB × NZW)F2 Mice
Authors: Kono Dwight H. , Park Miyo S. , Theofilopoulos Argyrios N.
Published: April 2014

F1 hybrids among New Zealand Black (NZB), New Zealand White (NZW), and BXSB lupus-prone strains develop accelerated autoimmunity in both sexes regardless of the specific combination. To identify BXSB susceptibility loci in the absence of the Y chromosome accelerator of autoimmunity (Yaa) and to study the genetics of this complementation, genome-wide quantitative trait locus (QTL) mapping was performed on female (BXSB × NZW)F2 mice. Six QTL were identified on chromosomes 1, 4, 5, 6, 7, and 17. Survival mapped to chromosomes 5 and 17, anti-chromatin Ab to chromosomes 4 and 17, glomerulonephritis to chromosomes 6 and 17, and splenomegaly to chromosomes 1, 7, and 17. QTL on chromosomes 4 and 6 were new and designated as Lxw1 and -2, respectively. Two non-MHC QTL (chromosomes 1 and 4) were inherited from the BXSB and the rest were NZW-derived, including two similar to previously defined loci. Only two of 11 previously defined non-MHC BXSB QTL using male (Yaa+) crosses were implicated, suggesting that some male-defined BXSB QTL may require coexpression of the Yaa. Findings from this and other studies indicate that BXSB and NZB backgrounds contribute completely different sets of genes to complement NZW mice. Identification of susceptibility genes and complementing genes in several lupus-prone strain combinations will be important for defining the epistatic effects and background influences on the heterogeneous genetic factors responsible for lupus induction.
Differential Effects of IL-1α and IL-1β on Tumorigenicity Patterns and Invasiveness
Authors: Song Xiaoping , Voronov Elena , Dvorkin Tatyana , Fima Eyal , Cagnano Emanuela , Benharroch Daniel , Shendler Yaakov , Bjorkdahl Olle , Segal Shraga , Dinarello Charles A. , Apte Ron N.
Published: April 2014

In this study, we show that distinct compartmentalization patterns of the IL-1 molecules (IL-1α and IL-1β), in the milieu of tumor cells that produce them, differentially affect the malignant process. Active forms of IL-1, namely precursor IL-1α (pIL-1α), mature IL-1β (mIL-1β), and mIL-1β fused to a signal sequence (ssIL-1β), were transfected into an established fibrosarcoma cell line, and tumorigenicity and antitumor immunity were assessed. Cell lines transfected with pIL-1α, which expresses IL-1α on the membrane, fail to develop local tumors and activate antitumor effector mechanisms, such as CTLs, NK cells, and high levels of IFN-γ production. Cells transfected with secretable IL-1β (mIL-1β and ssIL-1β) were more aggressive than wild-type and mock-transfected tumor cells; ssIL-1β transfectants even exhibited metastatic tumors in the lungs of mice after i.v. inoculation (experimental metastasis). In IL-1β tumors, increased vascularity patterns were observed. No detectable antitumor effector mechanisms were observed in spleens of mice injected with IL-1β transfectants, mock-transfected or wild-type fibrosarcoma cells. Moreover, in spleens of mice injected with IL-1β transfectants, suppression of polyclonal mitogenic responses (proliferation, IFN-γ and IL-2 production) to Con A was observed, suggesting the development of general anergy. Histologically, infiltrating mononuclear cells penetrating the tumor were seen at pIL-1α tumor sites, whereas in mIL-1β and ssIL-1β tumor sites such infiltrating cells do not penetrate inside the tumor. This is, to our knowledge, the first report on differential, nonredundant, in vivo effects of IL-1α and IL-1β in malignant processes; IL-1α reduces tumorigenicity by inducing antitumor immunity, whereas IL-1β promotes invasiveness, including tumor angiogenesis, and also induces immune suppression in the host.
The Interaction of γδ T Cells with Activated Macrophages Is a Property of the Vγ1 Subset
Authors: Dalton Jane E. , Pearson Jayne , Scott Phillip , Carding Simon R.
Published: April 2014

Immunoregulation is an emerging paradigm of γδ T cell function. The mechanisms by which γδ T cells mediate this function, however, are not clear. Studies have identified a direct role for γδ T cells in resolving the host immune response to infection, by eliminating populations of activated macrophages. The aim of this study was to identify macrophage-reactive γδ T cells and establish the requirements/outcomes of macrophage-γδ T cell interactions during the immune response to the intracellular bacterium, Listeria monocytogenes (Lm). Using a macrophage-T cell coculture system in which peritoneal macrophages from naive or Lm-infected TCRδ−/− mice were incubated with splenocytes from naive and Lm-infected αβ/γδ T cell-deficient and wild-type mice, the ability to bind macrophages was shown to be restricted to γδ T cells and the GV5S1 (Vγ1) subset of γδ T cells. Macrophage adherence resulted in a 4- to 10-fold enrichment of Vγ1+ T cells. Enrichment of Vγ1 T cells was dependent upon the activation status of macrophages, but independent of the activation status of γδ T cells. Vγ1 T cells were cytotoxic for activated macrophages with both the binding to and killing of macrophages being TCR dependent because anti-TCRγδ Abs inhibited both Vγ1 binding and killing activities. These studies establish the identity of macrophage cytotoxic γδ T cells, the conditions under which this interaction occurs, and the outcome of this interaction. These findings are concordant with the involvement of Vγ1 T cells in macrophage homeostasis during the resolution of pathogen-mediated immune responses.
The Male Minor Transplantation Antigen Preferentially Activates Recipient CD4+ T Cells through the Indirect Presentation Pathway In Vivo
Authors: Chen Yifa , Demir Yilmaz , Valujskikh Anna , Heeger Peter S.
Published: April 2014

To evaluate the priming and trafficking of male Ag-reactive CD4+ T cells in vivo, we developed an adoptive transfer model, using Marilyn (Mar) TCR transgenic T cells that are specific for the H-Y minor transplantation Ag plus I-Ab. By manipulating donor and recipient strain combinations, we permitted the Mar CD4+ T cells to respond to the H-Y Ag after processing and presentation by recipient APCs (indirect pathway), or to the male Ag as expressed on donor APCs (direct pathway). Mar CD4+ T cells responding through the indirect pathway specifically proliferated and expressed activation markers between days 2 and 4 posttransplant, migrated to the graft 2–3 days before cessation of graft heartbeat, and were detected in close proximity to transplant-infiltrating recipient APCs. Intriguingly, adoptively transferred Mar T cells did not respond to male heart or skin grafts placed onto syngeneic MHC class II-deficient female recipients, demonstrating that activation of Mar T cell preferentially occurs through cognate interactions with processed male Ag expressed on recipient APCs. The data highlight the potency of indirect processing and presentation pathways in vivo and underscore the importance of indirectly primed CD4+ T cells as relevant participants in both the priming and effector phases of acute graft rejection.
Nasal Vaccination with Myelin Oligodendrocyte Glycoprotein Reduces Stroke Size by Inducing IL-10-Producing CD4+ T Cells
Authors: Frenkel Dan , Huang Zhihong , Maron Ruth , Koldzic Djordje N. , Hancock Wayne W. , Moskowitz Michael A. , Weiner Howard L.
Published: April 2014

Inflammation plays an important role in ischemic stroke and in humans IL-10 may have a beneficial effect in stroke. We mucosally administered myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide to C57BL/6 mice before middle cerebral artery occlusion (MCAO) to induce an anti-inflammatory T cell response directed at CNS myelin. Nasal and oral administration of MOG35–55 peptide decreased ischemic infarct size at 24 and 72 h after MCAO surgery. Nasal MOG35–55 peptide was most efficacious and reduced infarct size by 70% at 24 h and by 50% at 72 h (p ≤ 0.0001 vs control) and also improved behavior score. Immunohistochemistry demonstrated increased IL-10 and reduced IFN-γ in the area surrounding the ischemic infarct following nasal treatment. Nasal MOG did not reduce infarct size in IL-10-deficient mice. Adoptive transfer of CD4+ T cells to untreated mice from nasally tolerized mice before MCAO surgery decreased stroke size (p < 0.001 vs control), whereas, CD4+ T cells from nasally tolerized IL-10-deficient mice had no effect. Our results demonstrate that IL-10-secreting CD4+ T cells induced by nasal MOG reduce injury following stroke. In addition, we observed a dramatic reduction of CD11b+ cells in nasal MOG-treated animals. CD11b+ cells may contribute to secondary infarct expansion by enhancing NO synthesis that may be reduced by elevated IL-10 levels. Modulation of cerebral inflammation by nasal vaccination with myelin Ags that increase IL-10 in the brain may improve outcome after stroke and enhance mechanisms of recovery.
Reduced Switching inSCIDB Cells Is Associated with Altered Somatic Mutation of Recombined S Regions
Authors: Cook Adam J. L. , Oganesian Liana , Harumal Pearly , Basten Antony , Brink Robert , Jolly Christopher J.
Published: April 2014

Deoxyribonucleic acid double-stranded breaks act as intermediates in Ig V(D)J recombination and probably perform a similar function in class switch recombination between IgH C genes. In SCID mice, V(D)J recombination is blocked because the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) protein is defective. We show in this study that switching to all isotypes examined was detectable when the SCID mutation was introduced into anti-hen egg lysozyme transgenic B cells capable of undergoing class switch recombination, but switching was significantly reduced in comparison with control B cells of the same specificity lacking the RAG1 gene. Thus, DNA-PKcs is involved in switching to all isotypes, but plays a lesser role in the switching process than it does in V(D)J-coding joint formation. The higher level of switching observed by us in SCID B cells compared with that observed by others in DNA-PKcsnull cells raises the possibility that kinase-deficient DNA-PKcs can function in switching. Point mutation of G:C base pairs with cytidines on the sense strand was greatly reduced in recombined switch regions from SCID cells compared with control RAG1−/− B cells. The preferential loss of sense strand cytidine mutations from hybrid S regions in SCID cells suggests the possibility that nicks might form in S regions of activated B cells on the template strand independently of activation-induced cytidine deaminase and are converted to double-strand breaks when activation-induced cytidine deaminase deaminates the non-template strand.
Identification of a Critical Ig-Like Domain in IL-18 Receptor α and Characterization of a Functional IL-18 Receptor Complex
Authors: Azam Tania , Novick Daniela , Bufler Philip , Yoon Do-Young , Rubinstein Menachem , Dinarello Charles A. , Kim Soo Hyun
Published: April 2014

Steady state mRNA levels in various human tissues reveal that the proinflammatory cytokine IL-18 is constitutively and ubiquitously expressed. However, limited IL-18R α-chain (IL-18Rα) expression in tissues may restrict ligand-acting sites and contribute to a specific response for IL-18. To study the IL-18R complex, [125I]IL-18 was studied for binding to the cell surface receptors of IL-18-responsive NK and macrophagic KG-1 cells. After cross-linking, [125I]IL-18 formed three IL-18R complexes with sizes of approximately 93, 160, and 220 kDa. In KG-1 cells, Scatchard analysis revealed the presence of 135 binding sites/cell, with an apparent dissociation constant (Kd) of 250 pM; in NK cells, there were 350 binding sites per cell with an apparent Kd of 146 pM. Each domain of extracellular IL-18Rα was cloned and individually expressed in Escherichia coli. An mAb specifically recognized the membrane-proximal third domain; this mAb blocked IL-18-induced IFN-γ production in NK cells. Furthermore, deletion of the membrane-proximal third domain of IL-18Rα prevented the formation of IL-18R ternary complex with IL-18R β-chain. The present studies demonstrate that the biologically active IL-18R complex requires the membrane-proximal third Ig-like domain in IL-18Rα for the formation of IL-18R ternary complex as well as for signal transduction involved in IL-18-induced IFN-γ in NK cells.
Critical Role for the Oct-2/OCA-B Partnership in Ig-Secreting Cells
Authors: Salas Mabel , Eckhardt Laurel A.
Published: April 2014

B and T lymphocytes arise from a common precursor in the bone marrow, but ultimately acquire very different functions. The difference in function is largely attributable to the expression of tissue-specific transcription factors that activate discrete sets of genes. In previous studies we and others have shown that the specialized genes expressed by Ig-secreting cells cease transcription when these cells are fused to a T lymphoma. The extinguished genes include those encoding Ig, J chain, and the transcription factors Oct-2, PU.1, and the coactivator OCA-B. Remarkably, if we sustain Oct-2 expression during cell fusion, all the other tissue-specific genes of the Ig-secreting cell simultaneously escape silencing. This suggests that Oct-2 plays a central role in maintaining the gene expression program of these cells. In the present studies we have investigated the roles of the transcription factor PU.1 and the coactivator OCA-B within the hierarchy of regulatory factors that sustain Ig-secreting cell function. Our results show that OCA-B and Oct-2 are regulatory partners in this process and that PU.1 plays a subordinate role at this cell stage.
The B12/23 Restriction Is Critically Dependent on Recombination Signal Nonamer and Spacer Sequences
Authors: Hughes Maureen M. , Tillman Robert E. , Wehrly Tara D. , White J. Michael , Sleckman Barry P.
Published: April 2014

Ag receptor variable region gene assembly is initiated through the formation of a synaptic complex which minimally includes the recombination-activating gene (RAG) 1/2 proteins and a pair of recombination signals (RSs) flanking the recombining gene segments. RSs are composed of conserved heptamer and nonamer sequences flanking relatively nonconserved spacers of 12 or 23 bp. RSs regulate variable region gene assembly within the context of the 12/23 rule which mandates that recombination only occurs between RSs of dissimilar spacer length. RSs can exert additional constraints on variable region gene assembly beyond imposing spacer length requirements. At a minimum this restriction, termed B12/23, is imposed on the Vβ to DJβ rearrangement step by the 5′ Dβ RS and is enforced at or before the DNA cleavage step of the V(D)J recombination reaction. In this study, the components of the 5′ Dβ RS required for enforcing the B12/23 rule are assessed on chromosomal substrates in vivo in the context of normal murine thymocyte development and on extrachromosomal substrates induced to undergo recombination in nonlymphoid cell lines. These analyses reveal that the integrity of the nonamer sequence as well as the highly conserved spacer nucleotides of the 5′ Dβ1 RS are critical for enforcing the B12/23 restriction. These findings have important implications for understanding the B12/23 restriction and the manner in which RS synaptic complexes are assembled in vivo.
Transcriptional Regulation of ILT Family Receptors
Authors: Nakajima Hideo , Asai Azusa , Okada Aki , Ping Lin , Hamajima Fumiyasu , Sata Tetsutaro , Isobe Kenichi
Published: April 2014

Ig-like transcripts (ILT/leukocyte Ig-like receptor/monocyte/macrophage Ig-like receptor or CD85) are encoded on human chromosome 19q13.4, designated the human leukocyte receptor complex, and are predominantly expressed on myeloid lineage cells. We investigated the transcriptional regulation of ILT1, ILT2, and ILT4 genes to elucidate control mechanisms operating on the specific expression of ILT receptors. Inhibitory ILT2 and ILT4 both have a similar genomic structure, in which the ∼160-bp 5′-flanking regions function as core promoters with critically important PU.1 binding sites. However, an Sp1 family-binding GC-box is more influential in trans-activation of ILT2 than ILT4. Additionally, ILT4 transcription is tightly regulated by chromatin modifications accompanied by histone acetylation, which strictly controls expression within myeloid lineage cells. Activating ILT1 carries a core promoter corresponding to the intronic region of ILT2 and ILT4, where PU.1 and Runx1 binding sites are essential, but a downstream heat shock element also augments promoter activity. Thus, each ILT is regulated by a distinct transcriptional mechanism, although PU.1 acts as a common trans-acting factor. We also found that human CMV infection strongly trans-activates inhibitory ILT2 and ILT4 genes through the expression of immediate-early proteins.
Transcriptional Regulation of Src Homology 2 Domain-Containing Leukocyte Phosphoprotein of 76 kDa: Dissection of Key Promoter Elements
Authors: Zhong Xiao-Ping , Maltzman Jonathan S. , Hainey Ehmonie A. , Koretzky Gary A.
Published: April 2014

SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa) is an adaptor molecule expressed in all hemopoietic cell lineages except mature B cells and is known to play critical roles in the function of T cells, mast cells, and platelets and in vascular differentiation. Although great progress has been achieved in our understanding of SLP-76 function, little is known about the mechanisms regulating its expression. In this study we report the initial characterization of essential elements that control SLP-76 transcription. We identify several DNase I-hypersensitive sites in the SLP-76 locus, with a prominent site located in its promoter region. This site exists in T cells and monocytic cells, but not in B cells or fibroblasts. Using transient transfection assays, we identify a 507-bp fragment containing the 5′-untranslated region of the first exon and the immediate upstream sequence that confers transcriptional activation in T cells and monocytic cells, but not in B cells. Analysis of the 5′ ends of SLP-76 transcripts reveals differential regulation of SLP-76 transcription initiation between T cells and monocytic cells. Mutational and gel-shift analyses further indicate a critical role within this region for a binding site for Ets family transcription factors. The present study provides the first data to address the mechanisms controlling SLP-76 transcription by providing evidence for several key cis-regulatory elements in the promoter region.
The Protein Made from a Common Allele of KIR3DL1 (3DL1*004) Is Poorly Expressed at Cell Surfaces due to Substitution at Positions 86 in Ig Domain 0 and 182 in Ig Domain 1
Authors: Pando Marcelo J. , Gardiner Clair M. , Gleimer Michael , McQueen Karina L. , Parham Peter
Published: April 2014

KIR3DL1 is an inhibitory HLA-B receptor of human NK and T cells that exhibits genetic and phenotypic polymorphism. KIR3DL1*004, a common allotype, cannot be detected on the surface of PBLs using the KIR3DL1-specific Ab DX9. The nature of this phenotype was investigated through comparison of 3DL1*004 with 3DL1*002, an allele giving high DX9 binding to cell surfaces. Analysis of Jurkat T cell transfectants with 3DL1*004 cDNA showed that 3DL1*004 is poorly expressed at the cell surface, but detectable intracellularly. Analysis of recombinant mutants made between 3DL1*004 and 3DL1*002 showed that polymorphism in Ig domains 0 and 1 (D0 and D1) causes the intracellular retention of 3DL1*004. Reciprocal point mutations were introduced into 3DL1*004 and 3DL1*002 at positions 44 and 86 of the D0 domain, where 3DL1*004 has unique residues, and at position 182 of the D1 domain, where 3DL1*004 resembles 3DL1*005, an allotype giving low DX9-binding phenotype. Leucine 86 in 3DL1*004 is the principal cause of its intracellular retention, with a secondary and additive contribution from serine 182. By contrast, glycine 44, which is naturally present in 3DL1*004, slightly increased cell surface expression when introduced into 3DL1*002. In 3DL1*004, the presence of leucine at position 86 corrupts the WSXPS motif implicated in proper folding of the KIR D0 Ig-like domain. This study demonstrates how a difference between KIR3DL1 allotypes in the D0 domain profoundly affects cell surface expression and function.
Homotypic Secretory Vesicle Fusion Induced by the Protein Tyrosine Phosphatase MEG2 Depends on Polyphosphoinositides in T Cells
Authors: Huynh Huong , Wang Xiaodong , Li Weizhong , Bottini Nunzio , Williams Scott , Nika Konstantina , Ishihara Hisamitsu , Godzik Adam , Mustelin Tomas
Published: April 2014

Sec14p homology domains are found in a large number of proteins from plants, yeast, invertebrates, and higher eukaryotes. We report that the N-terminal Sec14p homology domain of the human protein tyrosine phosphatase PTP-MEG2 binds phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) in vitro and colocalizes with this lipid on secretory vesicle membranes in intact cells. Point mutations that prevented PtdIns(3,4,5)P3 binding abrogated the capacity of PTP-MEG2 to induce homotypic secretory vesicle fusion in cells. Inhibition of cellular PtdIns(3,4,5)P3 synthesis also rapidly reversed the effect of PTP-MEG2 on secretory vesicles. Finally, we show that several different phosphoinositide kinases colocalize with PTP-MEG2, thus allowing for local synthesis of PtdIns(3,4,5)P3 in secretory vesicle membranes. We suggest that PTP-MEG2 through its Sec14p homology domain couples inositide phosphorylation to tyrosine dephosphorylation and the regulation of intracellular traffic of the secretory pathway in T cells.
Basal Chromatin Modification at the IL-4 Gene in Helper T Cells
Authors: Grogan Jane L. , Wang Zhi-En , Stanley Sarah , Harmon Brian , Loots Gaby G. , Rubin Edward M. , Locksley Richard M.
Published: April 2014

Chromatin immunoprecipitations in naive CD4, but not CD8, T cells, demonstrated association of the IL-4 promoter with acetylated histone. Histone modifications and rapid IL-4 transcription were absent in conserved noncoding sequence 1 (CNS-1)−/− cells lacking an 8-kb-distant enhancer in the IL-4/IL-13 intergenic region, but also in CD4−/− and Itk−/− cells, which have similar Th2 deficiencies. Histones associated with the IL-13 promoter were not similarly acetylated in naive T cells, but became acetylated in differentiated Th2 cells. Conversely, Th1 differentiation induced histone methylation at the type 2 cytokine locus. Like CD4−/− and Itk−/− mice, CNS-1−/− BALB/c mice were highly resistant to the Th2-inducing protozoan, Leishmania major. CNS-1 deficiency led to failure of IL-4 gene repositioning to heterochromatin after Th1 polarization, possibly related to the presence of reiterative Ikaros binding sites in the intergenic element. Hyperacetylation of nonexpressed genes may serve to mark lineage-specific loci for rapid expression and further modification.
CTL Are Inactivated by Herpes Simplex Virus-Infected Cells Expressing a Viral Protein Kinase
Authors: Sloan Derek D. , Zahariadis George , Posavad Christine M. , Pate Nichlos T. , Kussick Steven J. , Jerome Keith R.
Published: April 2014

Numerous cell-to-cell signals tightly regulate CTL function. Human fibroblasts infected with HSV type 1 or 2 can generate such a signal and inactivate human CTL. Inactivated CTL lose their ability to release cytotoxic granules and synthesize cytokines when triggered through the TCR. Inactivation requires cell-to-cell contact between CTL and HSV-infected cells. However, inactivated CTL are not infected with HSV. The inactivation of CTL is sustainable, as CTL function remains impaired when the CTL are removed from the HSV-infected cells. IL-2 treatment does not alter inactivation, and the inactivated phenotype is not transferable between CTL, distinguishing this phenotype from traditional anergy and T regulatory cell models. CTL inactivated by HSV-infected cells are not apoptotic, and the inactivated state can be overcome by phorbol ester stimulation, suggesting that inactivated CTL are viable and that the signaling block is specific to the TCR. HSV-infected cells require the expression of US3, a viral protein kinase, to transmit the inactivating signal. Elucidation of the molecular nature of this signaling pathway may allow targeted manipulation of CTL function.
Human Cytomegalovirus-Encoded US2 Differentially Affects Surface Expression of MHC Class I Locus Products and Targets Membrane-Bound, but Not Soluble HLA-G1 for Degradation
Authors: Barel Martine T. , Ressing Maaike , Pizzato Nathalie , van Leeuwen Daphne , Le Bouteiller Philippe , Lenfant Francoise , Wiertz Emmanuel J. H. J.
Published: April 2014

Human CMV (HCMV) can elude CTL as well as NK cells by modulating surface expression of MHC class I molecules. This strategy would be most efficient if the virus would selectively down-regulate viral Ag-presenting alleles, while at the same time preserving other alleles to act as inhibitors of NK cell activation. We focused on the HCMV unique short (US) region encoded protein US2, which binds to newly synthesized MHC class I H chains and supports their dislocation to the cytosol for subsequent degradation by proteasomes. We studied the effect of US2 on surface expression of individual class I locus products using flow cytometry. Our results were combined with crystal structure data of complexed US2/HLA-A2/β2-microglobulin and alignments of 948 HLA class I database sequences of the endoplasmic reticulum lumenal region inplicated in US2 binding. This study suggests that surface expression of all HLA-A and -G and most HLA-B alleles will be affected by US2. Several HLA-B alleles and all HLA-C and -E alleles are likely to be insensitive to US2-mediated degradation. We also found that the MHC class I endoplasmic reticulum-lumenal domain alone is not sufficient for degradation by US2, as illustrated by the stability of soluble HLA-G1 in the presence of US2. Furthermore, we showed that the membrane-bound HLA-G1 isoform, but also tailless HLA-A2, are targeted for degradation. This indicates that the cytoplasmic tail of the MHC class I H chain is not required for its dislocation to the cytosol by US2.
Differential Activation of Formyl Peptide Receptor-Like 1 by Peptide Ligands
Authors: Bae Yoe-Sik , Yi Hee Jeong , Lee Ha-Young , Jo Eun Jin , Kim Jung Im , Lee Taehoon G. , Ye Richard D. , Kwak Jong-Young , Ryu Sung Ho
Published: April 2014

Formyl peptide receptor-like 1 (FPRL1) plays a key role in the regulation of immune responses. The activation of FPRL1 induces a complicated pattern of cellular signaling, which results in the regulation of several immune responses, such as chemotactic migration and the production of reactive oxygen species (ROS). Because some of these cellular responses are not beneficial to the host, ligands that selectively modulate these cellular responses are useful. His-Phe-Tyr-Leu-Pro-Met (HFYLPM) is a synthetic peptide that binds to FPRL1. In this study, we generated various HFYLPM analogues and examined their effects on cellular responses via FPRL1 in FPRL1-expressing rat basophilic leukemia-2H3 cells or in primary human neutrophils. Among the HXYLPM analogues, His-Arg-Tyr-Leu-Pro-Met (HRYLPM) activated a broad spectrum of cellular signaling events, including an intracellular Ca2+ concentration increase, phosphoinositide 3-kinase, extracellular signal-regulated kinase, and Akt activation, however, His-Glu-Tyr-Leu-Pro-Met (HEYLPM) activated only intracellular Ca2+ concentration and Akt but did not increase Ca2+. In addition, HRYLPM was found to stimulate chemotaxis and ROS generation via phosphoinositide 3-kinase and an intracellular Ca2+ concentration increase, respectively, whereas HEYLPM stimulated chemotaxis but not ROS generation. With respect to the molecular mechanisms involved in the differential action of HRYLPM and HEYLPM, we found that HRYLPM but not HEYLPM competitively inhibited the binding of 125I-labeled Trp-Lys-Tyr-Met-Val-d-Met-NH2 (WKYMVm, a FPRL1 ligand) to FPRL1. This study demonstrates that the important chemoattractant receptor, FPRL1, may be differentially modulated by distinct peptide ligands. We also suggest that HRYLPM and HEYLPM may be used to selectively modulate FPRL1.