bluuebunny/crossref_metadata_2025 huggingface dataset:
Delayed Contraction of the CD8+ T Cell Response toward Lymphocytic Choriomeningitis Virus Infection in Mice Lacking Serglycin
Authors: Grujic Mirjana , Christensen Jan P. , Sørensen Maria R. , Abrink Magnus , Pejler Gunnar , Thomsen Allan R.
Published: April 2014

We previously reported that the lack of serglycin proteoglycan affects secretory granule morphology and granzyme B (GrB) storage in in vitro generated CTLs. In this study, the role of serglycin during viral infection was studied by infecting wild-type (wt) mice and serglycin-deficient (SG−/−) mice with lymphocytic choriomeningitis virus (LCMV). Wt and SG−/− mice cleared 103 PFU of highly invasive LCMV with the same kinetics, and the CD8+ T lymphocytes from wt and SG−/− animals did not differ in GrB, perforin, IFN-γ, or TNF-α content. However, when a less invasive LCMV strain was used, SG−/− GrB+ CD8+ T cells contained ∼30% less GrB than wt GrB+ CD8+ T cells. Interestingly, the contraction of the antiviral CD8+ T cell response to highly invasive LCMV was markedly delayed in SG−/− mice, and a delayed contraction of the virus-specific CD8+ T cell response was also seen after infection with vesicular stomatitis virus. BrdU labeling of cells in vivo revealed that the delayed contraction was associated with sustained proliferation of Ag-specific CD8+ T cells in SG−/− mice. Moreover, wt LCMV-specific CD8+ T cells from TCR318 transgenic mice expanded much more extensively in virus-infected SG−/− mice than in matched wt mice, indicating that the delayed contraction represents a T cell extrinsic phenomenon. In summary, the present report points to a novel, previously unrecognized role for serglycin proteoglycan in regulating the kinetics of antiviral CD8+ T cell responses.
CD4+CD25− T Cells Transduced to Express MHC Class I-Restricted Epitope-Specific TCR Synthesize Th1 Cytokines and Exhibit MHC Class I-Restricted Cytolytic Effector Function in a Human Melanoma Model
Authors: Chhabra Arvind , Yang Lili , Wang Pin , Comin-Anduix Begoña , Das Raja , Chakraborty Nitya G. , Ray Swagatam , Mehrotra Shikhar , Yang Haiguang , Hardee Cinnamon L. , Hollis Roger , Dorsky David I. , Koya Richard , Kohn Donald B. , Ribas Antoni , Economou James S. , Baltimore David , Mukherji Bijay
Published: April 2014

Cytolytic T cell-centric active specific and adoptive immunotherapeutic approaches might benefit from the simultaneous engagement of CD4+ T cells. Considering the difficulties in simultaneously engaging CD4+ and CD8+ T cells in tumor immunotherapy, especially in an Ag-specific manner, redirecting CD4+ T cells to MHC class I-restricted epitopes through engineered expression of MHC class I-restricted epitope-specific TCRs in CD4+ T cells has emerged as a strategic consideration. Such TCR-engineered CD4+ T cells have been shown to be capable of synthesizing cytokines as well as lysing target cells. We have conducted a critical examination of functional characteristics of CD4+ T cells engineered to express the α- and β-chains of a high functional avidity TCR specific for the melanoma epitope, MART-127–35, as a prototypic human tumor Ag system. We found that unpolarized CD4+CD25− T cells engineered to express the MART-127–35 TCR selectively synthesize Th1 cytokines and exhibit a potent Ag-specific lytic granule exocytosis-mediated cytolytic effector function of comparable efficacy to that of CD8+ CTL. Such TCR engineered CD4+ T cells, therefore, might be useful in clinical immunotherapy.
IL-15 Serves as a Costimulator in Determining the Activity of Autoreactive CD8 T Cells in an Experimental Mouse Model of Graft-versus-Host-Like Disease
Authors: Miyagawa Fumi , Tagaya Yutaka , Kim Brian S. , Patel Hiral J. , Ishida Kazuto , Ohteki Toshiaki , Waldmann Thomas A. , Katz Stephen I.
Published: April 2014

To elucidate the mechanisms controlling peripheral tolerance, we established two transgenic (Tg) mouse strains expressing different levels of membrane-bound OVA (mOVA) as a skin-associated self-Ag. When we transferred autoreactive TCR-Tg CD8 T cells (OT-I cells), keratin 14 (K14)-mOVAhigh Tg mice developed autoreactive skin disease (graft-vs-host disease (GVHD)-like skin lesions) while K14-mOVAlow Tg mice did not. OT-I cells in K14-mOVAhigh Tg mice were fully activated with full development of effector function. In contrast, OT-I cells in K14-mOVAlow Tg mice proliferated but did not gain effector function. Exogenous IL-15 altered the functional status of OT-I cells and concomitantly induced disease in K14-mOVAlow Tg mice. Conversely, neutralization of endogenous IL-15 activity in K14-mOVAhigh Tg mice attenuated GVHD-like skin lesions induced by OT-I cell transfer. Futhermore, K14-mOVAhigh Tg mice on IL-15 knockout or IL-15Rα knockout backgrounds did not develop skin lesions after adoptive transfer of OT-I cells. These results identify IL-15 as an indispensable costimulator that can determine the functional fate of autoreactive CD8 T cells and whether immunity or tolerance ensues, and they suggest that inhibition of IL-15 function may be efficacious in blocking expression of autoimmunity where a breach in peripheral tolerance is suspected.
SOCS1 Regulates CCR7 Expression and Migration of CD4+ T Cells into Peripheral Tissues
Authors: Yu Cheng-Rong , Mahdi Rashid M. , Liu Xuebin , Zhang Allen , Naka Tetsuji , Kishimoto Tadamitsu , Egwuagu Charles E.
Published: April 2014

Suppressors of cytokine signaling (SOCS) proteins control many aspects of lymphocyte function through regulation of STAT pathways. SOCS1-deficient mice develop severe skin and eye diseases that result from massive infiltration of inflammatory cells into these tissues. In this study, we have used SOCS1-, STAT1-, or STAT6-deficient mice, as well as, T cells with stable overexpression or deletion of SOCS1, to examine whether SOCS1 is involved in regulating lymphocyte trafficking to peripheral tissues. We show that SOCS1-deficient mice have increased numbers of T cells with characteristics of effector memory cells and expression of CCR7, a protein that promotes retention of T cells in lymphoid tissues, is markedly reduced in these cells. The decrease in CCR7 expression correlates with hyperactivation of STAT6, suggesting that aberrant recruitment of T cells into SOCS1-deficient mouse skin or eye results from abrogation of negative feedback regulation of STAT6 activation and CCR7 expression. Consistent with in vivo regulation of CCR7 expression and lymphocyte migration by SOCS1, forced overexpression of SOCS1 in T cells up-regulates CCR7 expression and enhances chemotaxis toward CCL19 or CCL21. CCR6 and CXCR3 are also up-regulated on SOCS1-deficient T cells and in situ analysis of the cornea or retina further reveal that these cells may mediate the chronic skin and eye inflammation through recruitment of Th1 and Th17 cells into these tissues. Collectively, these results suggest that SOCS1 regulates steady-state levels of chemokine receptors through its inhibitory effects on STAT pathways and this may underscore its role in regulating recruitment and retention of effector cells into nonlymphoid tissues.
The Potency of Erythropoietin-Mimic Antibodies Correlates Inversely with Affinity
Authors: Lacy Susan E. , DeVries Peter J. , Xie Nancy , Fung Emma , Lesniewski Richard R. , Reilly Edward B.
Published: April 2014

Preclinical animal studies have shown that Ab12.6, an agonistic human Ab targeting the erythropoietin receptor (EPOR), exhibits several potential dosing and safety features that make it an attractive clinical candidate for the treatment of anemia. Ab12.6 was derived by yeast display affinity maturation of parental Ab12, a strategy initially intended to improve off-rate and affinity for EPOR, thereby enhancing erythropoietic activity. Analysis of full-length IgGs derived from yeast clones identified sequences within Ab12 CDRH2 that independently influenced both affinity and potency. The Ab12.6 derivative displayed improved in vitro potency and in vivo efficacy, although its binding affinity to the EPOR was lower than that of the parent Ab12. Additional Ab12 derivatives also exhibited an inverse correlation between affinity and potency. These results suggest that for this class of agonistic Abs, faster off-rates may permit continuous receptor stimulation and more efficient erythropoiesis.
IL-17A Inhibits the Expansion of IL-17A-Producing T Cells in Mice through “Short-Loop” Inhibition via IL-17 Receptor
Authors: Smith Emily , Stark Matthew A. , Zarbock Alexander , Burcin Tracy L. , Bruce Anthony C. , Vaswani Devin , Foley Patricia , Ley Klaus
Published: April 2014

IL-23 and IL-17A regulate granulopoiesis through G-CSF, the main granulopoietic cytokine. IL-23 is secreted by activated macrophages and dendritic cells and promotes the expansion of three subsets of IL-17A-expressing neutrophil-regulatory T (Tn) cells; CD4−CD8−αβlow, CD4+CD8−αβ+ (Th17), and γδ+ T cells. In this study, we investigate the effects of IL-17A on circulating neutrophil levels using IL-17R-deficient (Il17ra−/−) mice and Il17ra−/−Itgb2−/− mice that lack both IL-17R and all four β2 integrins. IL-17R deficiency conferred a reduction in neutrophil numbers and G-CSF levels, as did Ab blockade against IL-17A in wild-type mice. Bone marrow transplantation revealed that IL-17R expression on nonhemopoietic cells had the greatest effects on regulating blood neutrophil counts. Although circulating neutrophil numbers were reduced, IL-17A expression, secretion, and the number of IL-17A-producing Tn cells were elevated in Il17ra−/− and Il17ra−/−Itgb2−/− mice, suggesting a negative feedback effect through IL-17R. The negative regulation of IL-17A-producing T cells and IL-17A and IL-17F gene expression through the interactions of IL-17A or IL-17F with IL-17R was confirmed in splenocyte cultures in vitro. We conclude that IL-17A regulates blood neutrophil counts by inducing G-CSF production mainly in nonhemopoietic cells. IL-17A controls the expansion of IL-17A-producing Tn cell populations through IL-17R.
Parenchymal Cell TNF Receptors Contribute to Inflammatory Cell Recruitment and Respiratory Failure in Pneumocystis carinii-Induced Pneumonia
Authors: Pryhuber Gloria S. , Huyck Heidie L. , Bhagwat Samir , O'Reilly Michael A. , Finkelstein Jacob N. , Gigliotti Francis , Wright Terry W.
Published: April 2014

The opportunistic organism Pneumocystis carinii (Pc) produces a life-threatening pneumonia (PcP) in patients with low CD4+ T cell counts. Animal models of HIV-AIDS-related PcP indicate that development of severe disease is dependent on the presence of CD8+ T cells and the TNF receptors (TNFR) TNFRsf1a and TNFRsf1b. To distinguish roles of parenchymal and hematopoietic cell TNF signaling in PcP-related lung injury, murine bone marrow transplant chimeras of wild-type, C57BL6/J, and TNFRsf1a/1b double-null origin were generated, CD4+ T cell depleted, and inoculated with Pc. As expected, C57 → C57 chimeras (donor marrow → recipient) developed significant disease as assessed by weight loss, impaired pulmonary function (lung resistance and dynamic lung compliance), and inflammatory cell infiltration. In contrast, TNFRsf1a/1b−/− → TNFRsf1a/1b−/− mice were relatively mildly affected despite carrying the greatest organism burden. Mice solely lacking parenchymal TNFRs (C57 → TNFRsf1a/1b−/−) had milder disease than did C57 → C57 mice. Both groups of mice with TNFR-deficient parenchymal cells had low bronchoalveolar lavage fluid total cell counts and fewer lavageable CD8+ T cells than did C57 → C57 mice, suggesting that parenchymal TNFR signaling contributes to PcP-related immunopathology through the recruitment of damaging immune cells. Interestingly, mice with wild-type parenchymal cells but TNFRsf1a/1b−/− hematopoietic cells (TNFRsf1a/1b−/− → C57) displayed exacerbated disease characterized by increased MCP-1 and KC production in the lung and increased macrophage and lymphocyte numbers in the lavage, indicating a dysregulated immune response. This study supports a key role of parenchymal cell TNFRs in lung injury induced by Pc and a potential protective effect of receptors on radiosensitive, bone marrow-derived cells.
Malaria-Specific and Nonspecific Activation of CD8+ T Cells during Blood Stage of Plasmodium berghei Infection
Authors: Miyakoda Mana , Kimura Daisuke , Yuda Masao , Chinzei Yasuo , Shibata Yoshisada , Honma Kiri , Yui Katsuyuki
Published: April 2014

Cerebral malaria is one of the severe complications of Plasmodium falciparum infection. Studies using a rodent model of Plasmodium berghei ANKA infection established that CD8+ T cells are involved in the pathogenesis of cerebral malaria. However, it is unclear whether and how Plasmodium-specific CD8+ T cells can be activated during the erythrocyte stage of malaria infection. We generated recombinant Plasmodium berghei ANKA expressing OVA (OVA-PbA) to investigate the parasite-specific T cell responses during malaria infection. Using this model system, we demonstrate two types of CD8+ T cell activations during the infection with malaria parasite. Ag (OVA)-specific CD8+ T cells were activated by TAP-dependent cross-presentation during infection with OVA-PbA leading to their expression of an activation phenotype and granzyme B and the development to functional CTL. These highly activated CD8+ T cells were preferentially sequestered in the brain, although it was unclear whether these cells were involved in the pathogenesis of cerebral malaria. Activation of OVA-specific CD8+ T cells in RAG2 knockout TCR-transgenic mice during infection with OVA-PbA did not have a protective role but rather was pathogenic to the host as shown by their higher parasitemia and earlier death when compared with RAG2 knockout mice. The OVA-specific CD8+ T cells, however, were also activated during infection with wild-type parasites in an Ag-nonspecific manner, although the levels of activation were much lower. This nonspecific activation occurred in a TAP-independent manner, appeared to require NK cells, and was not by itself pathogenic to the host.
Systemic Release of High Mobility Group Box 1 Protein during Severe Murine Influenza
Authors: Alleva Lisa M. , Budd Alison C. , Clark Ian A.
Published: April 2014

Hypercytokinemia is gaining recognition as the mechanism of fatality from influenza. No work to date has addressed the role of high mobility group box 1 protein (HMGB1) in influenza, the parallel being that in other severe proinflammatory cytokine syndromes (e.g., sepsis and malaria) levels of circulating HMGB1 are elevated and may correlate with death. Using a commercially available ELISA for HMGB1, we found that HMGB1 was not increased in the plasma of influenza virus-infected mice (A/Japan/305/57) on day 7 post infection, about the time of peak mortality, and peak levels of HMGB1 in the plasma did not occur until relatively late in infection, on day 9 post infection. In keeping with the late peak of HMGB1 being unassociated with mortality, administration of ethyl pyruvate, which inhibits active secretion but not passive release of HMGB1, to influenza virus-infected mice, did not affect their survival. Further work is required to determine whether influenza virus infection induces passive release of HMGB1, and whether HMGB1 neutralization with a specific Ab would improve survival.
Exosomes from Bronchoalveolar Fluid of Tolerized Mice Prevent Allergic Reaction
Authors: Prado Noela , Marazuela Eva G. , Segura Elodie , Fernández-García Héctor , Villalba Mayte , Théry Clotilde , Rodríguez Rosalía , Batanero Eva
Published: April 2014

Exosomes are nanovesicles originating from multivesicular bodies that are secreted by a variety of cell types. The dual capability of exosomes to promote immunity or to induce tolerance has prompted their clinical use as vehicles for vaccination against different human diseases. In the present study, the effect of allergen-specific exosomes from tolerized mice on the development of allergen-induced allergic response was determined using a mouse model. Mice were tolerized by respiratory exposure to the olive pollen allergen Ole e 1. Exosome-like vesicles were isolated from bronchoalveolar lavage fluid of the animals by the well-established filtration and ultracentrifugation procedure, characterized by electron microscopy, Western blot, and FACS analyses, and assessed in a prophylactic protocol. To this end, BALB/c mice were intranasally treated with tolerogenic exosomes or naive exosomes as control, 1 wk before sensitization/challenge to Ole e 1. Blood, lungs, and spleen were collected and analyzed for immune responses. Intranasal administration of tolerogenic exosomes inhibited the development of IgE response, Th2 cytokine production, and airway inflammation—cardinal features of allergy— and maintained specific long-term protection in vivo. This protective effect was associated with a concomitant increase in the expression of the regulatory cytokine TGF-β. These observations demonstrate that exosomes can induce tolerance and protection against allergic sensitization in mice. Thus, exosome-based vaccines could represent an alternative to conventional therapy for allergic diseases in humans.
Novel System Evaluating In Vivo Pathogenicity of Desmoglein 3-Reactive T Cell Clones Using Murine Pemphigus Vulgaris
Authors: Takahashi Hayato , Amagai Masayuki , Nishikawa Takeji , Fujii Yoshiko , Kawakami Yutaka , Kuwana Masataka
Published: April 2014

Autoreactive T cells are thought to be involved in the pathogenesis of autoimmune diseases, but evidence for their direct pathogenicity is almost lacking. Herein we established a unique system for evaluating the in vivo pathogenicity of desmoglein 3 (Dsg3)-reactive T cells at a clonal level in a mouse model for pemphigus vulgaris (PV), an autoimmune blistering disease induced by anti-Dsg3 autoantibodies. Dsg3-reactive CD4+ T cell lines generated in vitro were adoptively transferred into Rag-2−/− mice with primed B cells derived from Dsg3-immunized Dsg3−/− mice. Seven of 20 T cell lines induced IgG anti-Dsg3 Ab production and acantholytic blister, a typical disease phenotype, in recipient mice. Comparison of the characteristics between pathogenic and nonpathogenic Dsg3-reactive T cell lines led to the identification of IL-4 and IL-10 as potential factors associated with pathogenicity. Further in vitro analysis showed that IL-4, but not IL-10, promoted IgG anti-Dsg3 Ab production by primed B cells. Additionally, adenoviral expression of soluble IL-4Rα in vivo suppressed IgG anti-Dsg3 Ab production and the PV phenotype, indicating a pathogenic role of IL-4. This strategy is useful for evaluating the effector function of autoreactive T cells involved in the pathogenesis of various autoimmune diseases.
Cell Surface Expression of the Bovine Leukemia Virus-Binding Receptor on B and T Lymphocytes Is Induced by Receptor Engagement
Authors: Lavanya Madakasira , Kinet Sandrina , Montel-Hagen Amélie , Mongellaz Cédric , Battini Jean-Luc , Sitbon Marc , Taylor Naomi
Published: April 2014

Bovine leukemia virus (BLV), one of the most common infectious viruses of cattle, is endemic in many herds. Approximately 30–40% of adult cows in the United States are infected by this oncogenic C-type retrovirus and 1–5% of animals will eventually develop a malignant lymphoma. BLV, like the human and simian T cell leukemia viruses, is a deltaretrovirus but, in contrast with the latter, the BLV receptor remains unidentified. In this study, we demonstrate that the amino-terminal 182 residues of the BLV envelope glycoprotein surface unit encompasses the receptor-binding domain. A bona fide interaction of this receptor-binding domain with the BLV receptor was demonstrated by specific interference with BLV, but not human T cell leukemia virus, envelope glycoprotein-mediated binding. We generated a rabbit Ig Fc-tagged BLV receptor-binding domain construct and ascertained that the ligand binds the BLV receptor on target cells from multiple species. Using this tool, we determined that the BLV-binding receptor is expressed on differentiating pro/pre-B cells in mouse bone marrow. However, the receptor was not detected on mature/quiescent B cells but was induced upon B cell activation. Activation of human B and T lymphocytes also induced surface BLV-binding receptor expression and required de novo protein synthesis. Receptor levels were down-regulated as activated lymphocytes returned to quiescence. In the human thymus, BLV-binding receptor expression was specifically detected on thymocytes responding to the IL-7 cytokine. Thus, expression of the BLV-binding receptor is a marker of enhanced metabolic activity in B cells, T cells, and thymocytes.
Regulation of Thymic NKT Cell Development by the B7-CD28 Costimulatory Pathway
Authors: Williams Joy A. , Lumsden Joanne M. , Yu Xiang , Feigenbaum Lionel , Zhang Jingjing , Steinberg Seth M. , Hodes Richard J.
Published: April 2014

Invariant NKT (iNKT) cells are a population of TCRαβ-expressing cells that are unique in several respects. In contrast to conventional T cells, iNKT cells are selected in the thymus for recognition of CD1, rather than conventional MHC class I or II, and are selected by CD1-expressing double-positive thymocytes, rather than by the thymic stromal cells responsible for positive selection of conventional T cells. We have probed further the requirements for thymic iNKT cell development and find that these cells are highly sensitive to B7-CD28 costimulatory interactions, as evidenced by the substantially decreased numbers of thymic iNKT cells in CD28 and in B7 knockout mice. In contrast to the requirement for CD1, B7-CD28 signaling does not affect early iNKT cell lineage commitment, but exerts its influence on the subsequent intrathymic expansion and differentiation of iNKT cells. CD28 wild-type/CD28-deficient mixed bone marrow chimeras provided evidence of both cell-autonomous and non-cell-autonomous roles for CD28 during iNKT cell development. Paradoxically, transgenic mice in which thymic expression of B7 is elevated have essentially no measurable thymic iNKT cells. Taken together, these results demonstrate that the unique pathway involved in iNKT cell development is marked by a critical role of B7-CD28 interactions and that disruption or augmentation of this costimulatory interaction has substantial effects on iNKT cell development in the thymus.
Cell Type-Specific Regulation of ITAM-Mediated NF-κB Activation by the Adaptors, CARMA1 and CARD9
Authors: Hara Hiromitsu , Ishihara Chitose , Takeuchi Arata , Xue Liquan , Morris Stephan W. , Penninger Josef M. , Yoshida Hiroki , Saito Takashi
Published: April 2014

Activating NK cell receptors transduce signals through ITAM-containing adaptors, including FcRγ and DAP12. Although the caspase recruitment domain (CARD)9-Bcl10 complex is essential for FcRγ/DAP12-mediated NF-κB activation in myeloid cells, its involvement in NK cell receptor signaling is unknown. Herein we show that the deficiency of CARMA1 or Bcl10, but not CARD9, resulted in severe impairment of cytokine/chemokine production mediated by activating NK cell receptors due to a selective defect in NF-κB activation, whereas cytotoxicity mediated by the same receptors did not require CARMA1-Bcl10-mediated signaling. IκB kinase (IKK) activation by direct protein kinase C (PKC) stimulation with PMA plus ionomycin (P/I) was abrogated in CARMA1-deficient NK cells, similar to T and B lymphocytes, whereas CARD9-deficient dendritic cells (DCs) exhibited normal P/I-induced IKK activation. Surprisingly, CARMA1 deficiency also abrogated P/I-induced IKK activation in DCs, indicating that CARMA1 is essential for PKC-mediated NF-κB activation in all cell types, although the PKC-CARMA1 axis is not used downstream of myeloid ITAM receptors. Consistently, PKC inhibition abrogated ITAM receptor-mediated activation only in NK cells but not in DCs, suggesting PKC-CARMA1-independent, CARD9-dependent ITAM receptor signaling in myeloid cells. Conversely, the overexpression of CARD9 in CARMA1-deficient cells failed to restore the PKC-mediated NF-κB activation. Thus, NF-κB activation signaling through ITAM receptors is regulated by a cell type-specific mechanism depending on the usage of adaptors CARMA1 and CARD9, which determines the PKC dependence of the signaling.
Induction of Tolerance to Cardiac Allografts Using Donor Splenocytes Engineered to Display on Their Surface an Exogenous Fas Ligand Protein
Authors: Yolcu Esma S. , Gu Xiao , Lacelle Chantale , Zhao Hong , Bandura-Morgan Laura , Askenasy Nadir , Shirwan Haval
Published: April 2014

The critical role played by Fas ligand (FasL) in immune homeostasis renders this molecule an attractive target for immunomodulation to achieve tolerance to auto- and transplantation Ags. Immunomodulation with genetically modified cells expressing FasL was shown to induce tolerance to alloantigens. However, genetic modification of primary cells in a rapid, efficient, and clinically applicable manner proved challenging. Therefore, we tested the efficacy of donor splenocytes rapidly and efficiently engineered to display on their surface a chimeric form of FasL protein (SA-FasL) for tolerance induction to cardiac allografts. The i.p. injection of ACI rats with Wistar-Furth rat splenocytes displaying SA-FasL on their surface resulted in tolerance to donor, but not F344 third-party cardiac allografts. Tolerance was associated with apoptosis of donor reactive T effector cells and induction/expansion of CD4+CD25+FoxP3+ T regulatory (Treg) cells. Treg cells played a critical role in the observed tolerance as adoptive transfer of sorted Treg cells from long-term graft recipients into naive unmanipulated ACI rats resulted in indefinite survival of secondary Wistar-Furth grafts. Immunomodulation with allogeneic cells rapidly and efficiently engineered to display on their surface SA-FasL protein provides an effective and clinically applicable means of cell-based therapy with potential application to regenerative medicine, transplantation, and autoimmunity.
SCART Scavenger Receptors Identify a Novel Subset of Adult γδ T Cells
Authors: Kisielow Jan , Kopf Manfred , Karjalainen Klaus
Published: April 2014

Although there has been great progress in the characterization of αβ T cell differentiation, selection, and function, γδ T cells have remained poorly understood. One of the main reasons for this is the lack of γδ T cell-specific surface markers other than the TCR chains themselves. In this study we describe two novel surface receptors, SCART1 and SCART2. SCARTs are related to CD5, CD6, and CD163 scavenger receptors but, unlike them, are found primarily on developing and mature γδ T cells. Characterization of SCART2 positive immature and peripheral γδ T cells suggests that they undergo lineage specification in the thymus and belong to a new IL-17-producing subset with distinct homing capabilities.
Identification of Initiator B Cells, a Novel Subset of Activation-Induced Deaminase-Dependent B-1-Like Cells That Mediate Initiation of Contact Sensitivity
Authors: Kerfoot Steven M. , Szczepanik Marian , Tung James W. , Askenase Philip W.
Published: April 2014

Contact sensitivity (CS) is related to delayed-type hypersensitivity and is a well-characterized prototype of T cell-mediated inflammation. However, the inflammatory response associated with CS is additionally dependent on Ag-specific IgM produced by a subpopulation of B cells in response to sensitization. Upon re-exposure to hapten, this IgM mediates rapid vascular activation and subsequent recruitment of proinflammatory T cells to the local site. Interference with this pathway prevents the full development of the classic delayed inflammatory response and is therefore termed the “CS initiation” pathway. In this study, we show that CS initiation is defective in mice deficient in activation-induced deaminase, an enzyme central to the process of somatic hypermutation. Using adoptive transfer experiments, we demonstrate that the defect is specific to a B-1-like population of B cells and that transfer of WT cells reconstitutes CS initiation mechanisms in deficient recipients. We went on to identify a novel subpopulation of Ag-binding B cells in the spleens of sensitized mice that possess initiation activity (CD19+CD5+Thy-1intIgMhighIgDhigh) that we name “initiator B cells.” Analysis of BCR H chain genes isolated from these cells revealed evidence of activation-induced deaminase-mediated somatic hypermutation. The sensitivity of CS initiation to very low amounts of sensitizing hapten suggests that the responsible B cells have increased IgM receptor gene mutations enabling selection to generate Abs with sufficient affinity to mediate the response.
An Endogenous Prostaglandin Enhances Environmental Phthalate-Induced Apoptosis in Bone Marrow B Cells: Activation of Distinct but Overlapping Pathways
Authors: Bissonnette Stephanie L. , Teague Jessica E. , Sherr David H. , Schlezinger Jennifer J.
Published: April 2014

Phthalate esters are ubiquitous environmental contaminants that are produced for a variety of common industrial and commercial purposes. We have shown that mono-(2-ethylhexyl) phthalate (MEHP), the toxic metabolite of di-(2-ethylhexyl) phthalate, induces bone marrow B cell apoptosis that is enhanced in the presence of the endogenous prostaglandin 15-deoxy-Δ(12, 14)-PGJ2 (15d-PGJ2). Here, studies were performed to determine whether 15d-PGJ2-mediated enhancement of MEHP-induced apoptosis represents activation of an overlapping or complementary apoptosis pathway. MEHP and 15d-PGJ2 induced significant apoptosis within 8 and 5 h, respectively, in a pro/pre-B cell line and acted cooperatively to induce apoptosis in primary pro-B cells. Apoptosis induced with each chemical was accompanied by activation of a combination of initiator caspases (caspases-2, -8, and -9) and executed by caspase-3. Apoptosis induced with MEHP and 15d-PGJ2 was reduced in APAF1 null primary pro-B cells and accompanied by alteration of mitochondrial membranes, albeit with different kinetics, indicating an intrinsically activated apoptosis pathway. Significant Bax translocation to the mitochondria supports its role in initiating release of cytochrome c. Both chemicals induced Bid cleavage, a result consistent with a truncated Bid-mediated release of cytochrome c in an apoptosis amplification feedback loop; however, significantly more Bid was cleaved following 15d-PGJ2 treatment, potentially differentiating the two pathways. Indeed, Bid cleavage and cytochrome c release following 15d-PGJ2 but not MEHP treatment was profoundly inhibited by Z-VAD-FMK, suggesting that 15d-PGJ2 activates apoptosis via two pathways, Bax mobilization and protease-dependent Bid cleavage. Thus, endogenous 15d-PGJ2-mediated enhancement of environmental chemical-induced apoptosis represents activation of an overlapping but distinct signaling pathway.
TCR Antagonism by Peptide Requires High TCR Expression
Authors: Jones Daniel S. , Reichardt Peter , Ford Mandy L. , Edwards Lindsay J. , Evavold Brian D.
Published: April 2014

Current models of T cell activation focus on the kinetics of TCR-ligand interactions as the central parameter governing T cell responsiveness. However, these kinetic parameters do not adequately predict all T cell behavior, particularly the response to antagonist ligands. Recent studies have demonstrated that TCR number is a critical parameter influencing the responses of CD4+ T cells to weak agonist ligands, and receptor density represents an important means of regulating tissue responsiveness in other receptor ligand systems. To systematically address the impact of TCR expression on CD8+ T cell responses, mAbs to the TCR α-chain and T cells expressing two TCR species were used as two different methods to manipulate the number of available TCRs on P14 and OT-I transgenic T cells. Both methods of TCR reduction demonstrated that the efficacy of antagonist peptides was significantly reduced on T cells bearing low numbers of available receptors. In addition, the ability of weak agonists to induce proliferation was critically dependent on the availability of high numbers of TCRs. Therefore, in this report we show that TCR density is a major determinant of CD8+ T cell reactivity to weak agonist and antagonist ligands but not agonist ligands.
Activated CD8 T Cells Redistribute to Antigen-Free Lymph Nodes and Exhibit Effector and Memory Characteristics
Authors: Brinkman C. Colin , Sheasley-O'Neill Stacey L. , Ferguson Andrew R. , Engelhard Victor H.
Published: April 2014

Exogenous dendritic cells display restricted trafficking when injected in vivo and stimulate CD8 T cell responses that are localized to a small number of lymphoid compartments. By examining these responses in the presence and absence of FTY720, a drug that causes sequestration of T cells in lymph nodes, we demonstrate that a significant fraction of divided CD8 T cells redistribute into Ag-free lymph nodes within 3 days of activation. Despite variation in the level of expression of CD62L, redistribution of these cells is CD62L-dependent. Redistributed CD8 T cells exhibit characteristics of differentiated effectors. However, when re-isolated from Ag-free lymph nodes 3 days after activation and transferred into naive mice, they persist for at least 3 wk and expand upon Ag challenge. Thus, CD8 T cells that redistribute to Ag-free lymph nodes 3 days after immunization contain memory precursors. We suggest that this redistribution process represents an important mechanism for establishment of lymph node resident central memory, and that redistribution to Ag-free nodes is an additional characteristic to be added to those that distinguish memory precursors from terminal effectors.
Pemphigus Vulgaris IgG Directly Inhibit Desmoglein 3-Mediated Transinteraction
Authors: Heupel Wolfgang-Moritz , Zillikens Detlef , Drenckhahn Detlev , Waschke Jens
Published: April 2014

The autoimmune blistering skin disease pemphigus is caused by autoantibodies against keratinocyte surface Ags. In pemphigus vulgaris (PV), autoantibodies are primarily directed against desmosomal cadherins desmoglein (Dsg) 3 and Dsg 1, whereas pemphigus foliaceus (PF) patients only have Abs against Dsg 1. At present, it is unclear whether Dsg autoantibodies contribute to pemphigus pathogenesis by direct inhibition of Dsg transinteraction. Using atomic force microscopy, we provide evidence that PV-IgG directly interfere with homophilic Dsg 3 but, similar to PF-IgG, not with homophilic Dsg 1 transinteraction, indicating that the molecular mechanisms in PV and PF pathogenesis substantially differ. PV-IgG (containing Dsg 3 or Dsg 1 and Dsg 3 autoantibodies) as well as PV-IgG Fab reduced binding activity of Dsg 3 by ∼60%, comparable to Ca2+ depletion. Similarly, the mouse monoclonal PV Ab AK 23 targeting the N-terminal Dsg 3 domain and AK 23 Fab reduced Dsg 3 transinteraction. In contrast, neither PV-IgG nor PF-IgG blocked Dsg 1 transinteraction. In HaCaT monolayers, however, both PV- and PF-IgG caused keratinocyte dissociation as well as loss of Dsg 1 and Dsg 3 transinteraction as revealed by laser tweezer assay. These data demonstrate that PV-IgG and PF-IgG reduce Dsg transinteraction by cell-dependent mechanisms and suggest that in addition, Abs to Dsg 3 contribute to PV by direct inhibition of Dsg transinteraction.
Expression of Macrophage Migration Inhibitory Factor by Neuroblastoma Leads to the Inhibition of Antitumor T Cell Reactivity In Vivo
Authors: Zhou Qiang , Yan Xiaocai , Gershan Jill , Orentas Rimas J. , Johnson Bryon D.
Published: April 2014

Neuroblastomas and many other solid tumors produce high amounts of macrophage migration inhibitory factor (MIF), which appears to play a role in tumor progression. We found that MIF expression in neuroblastoma inhibits T cell proliferation in vitro, raising the possibility that MIF promotes tumorigenesis, in part, by suppressing antitumor immunity. To examine whether tumor-derived MIF leads to suppression of T cell immunity in vivo, we generated MIF-deficient neuroblastoma cell lines using short hairpin small interfering RNAs (siRNA). The MIF knockdown (MIFKD) AGN2a neuroblastoma cells were more effectively rejected in immune-competent mice than control siRNA-transduced or wild-type AGN2a. However, the increased rejection of MIFKD AGN2a was not observed in T cell-depleted mice. MIFKD tumors had increased infiltration of CD8+ and CD4+ T cells, as well as increased numbers of macrophages, dendritic cells, and B cells. Immunization with MIFKD AGN2a cells significantly increased protection against tumor challenge as compared with immunization with wild-type AGN2a, and the increased protection correlated with elevated frequencies of tumor-reactive CD8+ T cells in the lymphoid tissue of treated animals. Increased numbers of infiltrating tumor-reactive CD8+ T cells were also observed at the site of tumor vaccination. In vitro, treatment of AGN2a-derived culture supernatants with neutralizing MIF-specific Ab failed to reverse T cell suppressive activity, suggesting that MIF is not directly responsible for the immune suppression in vivo. This supports a model whereby MIF expression in neuroblastoma initiates a pathway that leads to the suppression of T cell immunity in vivo.
Bilirubin Possesses Powerful Immunomodulatory Activity and Suppresses Experimental Autoimmune Encephalomyelitis
Authors: Liu Yingru , Li Ping , Lu Jie , Xiong Wei , Oger Joel , Tetzlaff Wolfram , Cynader Max
Published: April 2014

Bilirubin, an abundant bile pigment in mammalian serum, was once considered a toxic waste product and has more recently been recognized as a potent antioxidant of physiological importance. However, its potential biological functions in other fields are not well understood. Herein we show that bilirubin is also a powerful immunomodulatory agent. Bilirubin significantly inhibited Ag-specific and polyclonal T cell responses, while other similar antioxidants completely lacked this effect. Bilirubin suppressed CD4+ T cell responses at multiple steps. High levels of bilirubin could induce apoptosis in reactive CD4+ T cells. Bilirubin at nonapoptotic concentrations suppressed CD4+ T cell reactivity through a wide range of actions, including inhibition of costimulator activities, suppression of immune transcription factor activation, and down-regulation of inducible MHC class II expression. Further studies suggest that bilirubin actions were direct, rather than via induction of immune deviation or regulatory T cells. In vivo, treatment with bilirubin effectively suppressed experimental autoimmune encephalomyelitis in SJL/J mice. In contrast, depletion of endogenous bilirubin dramatically exacerbated this disease. In summary, our results identify bilirubin as an important immunomodulator that may protect mammals against autoimmune diseases, thereby indicating its potential in the treatment of multiple sclerosis and other immune disorders.
Stromal Cells Confer Lymph Node-Specific Properties by Shaping a Unique Microenvironment Influencing Local Immune Responses
Authors: Ahrendt Manuela , Hammerschmidt Swantje Iris , Pabst Oliver , Pabst Reinhard , Bode Ulrike
Published: April 2014

Lymph nodes (LN) consist not only of highly motile immune cells coming from the draining area or from the systemic circulation, but also of resident stromal cells building the backbone of the LN. These two cell types form a unique microenvironment which is important for initiating an optimal immune response. The present study asked how the unique microenvironment of the mesenteric lymph node (mLN) is influenced by highly motile cells and/or by the stromal cells. A transplantation model in rats and mice was established. After resecting the mLN, fragments of peripheral lymph node (pLN) or mLN were inserted into the mesentery. The pLN and mLN have LN-specific properties, resulting in differences of, for example, the CD103+ dendritic cell subset, the adhesion molecule mucosal addressin cell adhesion molecule 1, the chemokine receptor CCR9, the cytokine IL-4, and the enzyme retinal dehydrogenase 2. This new model clearly showed that during regeneration stromal cells survived and immune cells were replaced. Surviving high endothelial venules retained their site-specific expression (mucosal addressin cell adhesion molecule 1). In addition, the low expression of retinal dehydrogenase 2 and CCR9 persisted in the transplanted pLN, suggesting that stromal cells influence the lymph node-specific properties. To examine the functional relevance of this different expression pattern in transplanted animals, an immune response against orally applied cholera toxin was initiated. The data showed that the IgA response against cholera toxin is significantly diminished in animals transplanted with pLN. This model documents that stromal cells of the LN are active players in shaping a unique microenvironment and influencing immune responses in the drained area.
ICOS Ligation Recruits the p50α PI3K Regulatory Subunit to the Immunological Synapse
Authors: Fos Camille , Salles Audrey , Lang Valérie , Carrette Florent , Audebert Stéphane , Pastor Sonia , Ghiotto Marguerite , Olive Daniel , Bismuth Georges , Nunès Jacques A.
Published: April 2014

ICOS ligation in concert with TCR stimulation results in strong PI3K activation in T lymphocytes. The ICOS cytoplasmic tail contains an YMFM motif that binds the p85α subunit of class IA PI3K, similar to the YMNM motif of CD28, suggesting a redundant function of the two receptors in PI3K signaling. However, ICOS costimulation shows greater PI3K activity than CD28 in T cells. We show in this report that ICOS expression in activated T cells triggers the participation of p50α, one of the regulatory subunits of class IA PI3Ks. Using different T-APC cell conjugate systems, we report that p50α accumulates at the immunological synapse in activated but not in resting T cells. Our results demonstrate that ICOS membrane expression is involved in this process and that p50α plasma membrane accumulation requires a functional YMFM Src homology 2 domain-binding motif in ICOS. We also show that ICOS triggering with its ligand, ICOSL, induces the recruitment of p50α at the synapse of T cell/APC conjugates. In association with the p110 catalytic subunit, p50α is known to carry a stronger lipid kinase activity compared with p85α. Accordingly, we observed that ICOS engagement results in a stronger activation of PI3K. Together, these findings provide evidence that p50α is likely a determining factor in ICOS-mediated PI3K activity in T cells. These results also suggest that a differential recruitment and activity of class IA PI3K subunits represents a novel mechanism in the control of PI3K signaling by costimulatory molecules.
HIV Envelope Binding by Macrophage-Expressed gp340 Promotes HIV-1 Infection
Authors: Cannon Georgetta , Yi Yanjie , Ni Houping , Stoddard Earl , Scales David A. , Van Ryk Donald I. , Chaiken Irwin , Malamud Daniel , Weissman Drew
Published: April 2014

The scavenger receptor cysteine-rich protein gp340 functions as part of the host innate immune defense system at mucosal surfaces. In the genital tract, its expression by cervical and vaginal epithelial cells promotes HIV trans-infection and may play a role in sexual transmission. Gp340 is an alternatively spliced product of the deleted in malignant brain tumors 1 (DMBT1) gene. In addition to its innate immune system activity, DMBT1 demonstrates instability in multiple types of cancer and plays a role in epithelial cell differentiation. We demonstrate that monocyte-derived macrophages express gp340 and that HIV-1 infection is decreased when envelope cannot bind it. Inhibition of infection occurred at the level of fusion of M-, T-, and dual-tropic envelopes. Additional HIV-1 envelope binding molecules, such as dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), mannose-binding lectin, and heparan sulfate, enhance the efficiency of infection of the cells that express them by increasing the local concentration of infectious virus. Our data suggest that gp340, which is expressed by macrophages in vivo, may function to enhance infection in much the same manner. Its expression on tissue macrophages and epithelial cells suggests important new opportunities for HIV-1 pathogenesis investigation and therapy.
Activation of Human Alveolar Macrophages via P2 Receptors: Coupling to Intracellular Ca2+ Increases and Cytokine Secretion
Authors: Myrtek Daniel , Müller Tobias , Geyer Verena , Derr Natalie , Ferrari Davide , Zissel Gernot , Dürk Thorsten , Sorichter Stephan , Luttmann Werner , Kuepper Michael , Norgauer Johannes , Di Virgilio Francesco , Virchow J. Christian , Idzko Marco
Published: April 2014

Alveolar macrophages play a crucial role in the pathogenesis of inflammatory airway diseases. By the generation and release of different inflammatory mediators they contribute to both recruitment of different leukocytes into the lung and to airway remodeling. A potent stimulus for the release of inflammatory cytokines is ATP, which mediates its cellular effects through the interaction with different membrane receptors, belonging to the P2X and P2Y families. The aim of this study was to characterize the biological properties of purinoceptors in human alveolar macrophages obtained from bronchoalveolar lavages in the context of inflammatory airway diseases. The present study is the first showing that human alveolar macrophages express mRNA for different P2 subtypes, namely P2X1, P2X4, P2X5, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y13, and P2Y14. We also showed that extracellular ATP induced Ca2+ transients and increased IL-1β secretion via P2X receptors. Furthermore, extracellular nucleotides inhibited production of IL-12p40 and TNF-α, whereas IL-6 secretion was up-regulated. In summary, our data further support the hypothesis that purinoceptors are involved in the pathogenesis of inflammatory lung diseases.
IL-6 Regulates Neutrophil Trafficking during Acute Inflammation via STAT3
Authors: Fielding Ceri A. , McLoughlin Rachel M. , McLeod Louise , Colmont Chantal S. , Najdovska Meri , Grail Dianne , Ernst Matthias , Jones Simon A. , Topley Nicholas , Jenkins Brendan J.
Published: April 2014

The successful resolution of inflammation is dependent upon the coordinated transition from the initial recruitment of neutrophils to a more sustained population of mononuclear cells. IL-6, which signals via the common receptor subunit gp130, represents a crucial checkpoint regulator of neutrophil trafficking during the inflammatory response by orchestrating chemokine production and leukocyte apoptosis. However, the relative contribution of specific IL-6-dependent signaling pathways to these processes remains unresolved. To define the receptor-mediated signaling events responsible for IL-6-driven neutrophil trafficking, we used a series of gp130 knockin mutant mice displaying altered IL-6-signaling capacities in an experimental model of acute peritoneal inflammation. Hyperactivation of STAT1 and STAT3 in gp130Y757F/Y757F mice led to a more rapid clearance of neutrophils, and this coincided with a pronounced down-modulation in production of the neutrophil-attracting chemokine CXCL1/KC. By contrast, the proportion of apoptotic neutrophils in the inflammatory infiltrate remained unaffected. In gp130Y757F/Y757F mice lacking IL-6, neutrophil trafficking and CXCL1/KC levels were normal, and this corresponded with a reduction in the level of STAT1/3 activity. Furthermore, monoallelic ablation of Stat3 in gp130Y757F/Y757F mice specifically reduced STAT3 activity and corrected both the rapid clearance of neutrophils and impaired CXCL1/KC production. Conversely, genetic deletion of Stat1 in gp130Y757F/Y757F mice failed to rescue the altered responses observed in gp130Y757F/Y757F mice. Collectively, these data genetically define that IL-6-driven signaling via STAT3, but not STAT1, limits the inflammatory recruitment of neutrophils, and therefore represents a critical event for the termination of the innate immune response.