bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of Ly-49D+ NK Cells with H-2Dd Target Cells Leads to Dap-12 Phosphorylation and IFN-γ Secretion
Authors: Mason Llewellyn H. , Willette-Brown Jamie , Mason Anna T. , McVicar Daniel , Ortaldo John R.
Published: April 2014

Murine Ly-49D augments NK cell function upon recognition of target cells expressing H-2Dd. Ly-49D activation is mediated by the immunoreceptor tyrosine-based activation motif-containing signaling moiety Dap-12. In this report we demonstrate that Ly-49D receptor ligation can lead to the rapid and potent secretion of IFN-γ. Cytokine secretion can be induced from Ly-49D+ NK cells after receptor ligation with Ab or after interaction with target cells expressing their H-2Dd ligand. Consistent with the dominant inhibitory function of Ly-49G, NK cells coexpressing Ly-49D and Ly-49G show a profound reduction in IFN-γ secretion after interaction with targets expressing their common ligand, H-2Dd. Importantly, we are able to demonstrate for the first time that effector/target cell interactions using Ly-49D+ NK cells and H-2Dd targets result in the rapid phosphorylation of Dap-12. However, Dap-12 is not phosphorylated when Ly-49D+ NK cells coexpress the inhibitory receptor, Ly-49G. These studies are novel in describing Ly-49 activation vs inhibition, where two Ly-49 receptors recognize the same class I ligand, with the dominant inhibitory receptor down-regulating phosphorylation of Dap-12, cytokine secretion, and cytotoxicity in NK cells.
Analysis of the Requirements for the Induction of CD4+ T Cell Alloantigen Hyporesponsiveness by Ex Vivo Anti-CD40 Ligand Antibody
Authors: Taylor Patricia A. , Panoskaltsis-Mortari Angela , Noelle Randolph J. , Blazar Bruce R.
Published: April 2014

A major goal of the transplant field is to selectively tolerize only those donor T cells recognizing host alloantigen and mediating graft-vs-host disease (GVHD). Recently, we described an ex vivo approach in which the blockade of the CD40 ligand (CD40L):CD40 costimulatory pathway in bulk MLR cultures induces donor CD4+ T cells to become specifically tolerant to MHC class II-disparate alloantigenic-bearing stimulators, resulting in a profound reduction in GVHD generation in vivo. In studies presented in this work, we investigated the ex vivo requirements for tolerance induction. We found that CD4+ T cells become profoundly more hyporesponsive to alloantigen restimulation with prolonged culture duration such that 7 to 10 but not 4 days is needed to achieve maximum alloantigen hyporesponsiveness as assessed in secondary MLR cultures and GVHD generation. By day 7, both primed and tolerized cells had substantially increased blastogenesis and CD25 expression. Primed but not tolerized cells substantially down-regulated L-selectin expression, indicating that the tolerized cells do not become fully Ag experienced. Both Th1 and Th2 cytokine production is severely impaired by CD40L:CD40 blockade. Analysis of culture supernatants and results from IL-4 and IL-10 knockout mice indicated that GVHD prevention was not mediated by a skewing toward a Th2 phenotype. The addition of IL-4 to the cultures as a survival factor precluded the induction of tolerance in the anti-CD40L-cultured cells. These data provide further impetus for the ex vivo use of anti-CD40L mAb to block GVHD generation.
Characterization of the Roles of TNF Receptor-Associated Factor 6 in CD40-Mediated B Lymphocyte Effector Functions
Authors: Jalukar Sangita V. , Hostager Bruce S. , Bishop Gail A.
Published: April 2014

Signaling through CD40 in B cells leads to B cell proliferation, Ig and IL-6 secretion, isotype switching, and up-regulation of surface molecules. TNF receptor-associated factor (TRAF) proteins associate with the cytoplasmic tail of CD40 and act as adapter molecules. Of the six TRAFs identified to date, TRAFs 2, 3, 5, and 6 are reported to associate directly with the cytoplasmic tail of CD40, but previous studies have principally examined transient overexpression of TRAF6 in cells that do not normally express CD40. Thus, we examined the role of TRAF6 in CD40-mediated B lymphocyte effector functions using two approaches. We produced and stably expressed in mouse B cell lines a human CD40 molecule with two cytoplasmic domain point mutations (hCD40EEAA); this mutant fails to bind TRAF6, while showing normal association with TRAFs 2 and 3. We also inducibly expressed in B cells a transfected “dominant-negative” TRAF6 molecule which contains only the C-terminal TRAF-binding domain of TRAF6. Using both molecules, we found that TRAF6 association with CD40 is important for CD40-induced IL-6 and Ig secretion, and that TRAF6 mediates its effects on CD40-stimulated Ig secretion principally through its effects on IL-6 production by the B cell. TRAF6 association with CD40 was also found to be important for B7-1 up-regulation, but not for up-regulation of other surface molecules. Interestingly, however, although we could show TRAF6-dependent CD40-mediated activation of NF-κB in 293 kidney epithelial cells, no such effect was seen in B cells, suggesting that TRAF6 has cell-type-specific functions.
Effects of Src Homology Domain 2 (SH2)-Containing Inositol Phosphatase (SHIP), SH2-Containing Phosphotyrosine Phosphatase (SHP)-1, and SHP-2 SH2 Decoy Proteins on FcγRIIB1-Effector Interactions and Inhibitory Functions
Authors: Nakamura Kazuhiro , Brauweiler Anne , Cambier John C.
Published: April 2014

Coaggregation of FcγRIIB1 with B cell Ag receptors (BCR) leads to inhibition of BCR-mediated signaling via recruitment of Src homology domain 2 (SH2)-containing phosphatases. In vitro peptide binding experiments using phosphotyrosine-containing sequences derived from the immunoreceptor tyrosine-based inhibitory motif (ITIM) known to mediate FcγRIIB1 effects suggest that the receptor uses SH2-containing inositol phophatase (SHIP) and SH2-containing phophotyrosine phosphatase (SHP)-1, as well as SHP-2 as effectors. In contrast, coimmunoprecipitation studies of receptor-effector associations suggest that the predominant FcγRIIB1 effector protein is SHIP. However, biologically significant interactions may be lost in such studies if reactants’ dissociation rates (Kd) are high. Thus, it is unclear to what extent these assays reflect the relative recruitment of SHIP, SHP-1, and SHP-2 to the receptor in vivo. As an alternative approach to this question, we have studied the effects of ectopically expressed SHIP, SHP-1, or SHP-2 SH2-containing decoy proteins on FcγRIIB1 signaling. Results demonstrate the SHIP is the predominant intracellular ligand for the phosphorylated FcγRIIB1 ITIM, although the SHP-2 decoy exhibits some ability to bind FcγRIIB1 and block Fc receptor function. The SHIP SH2, while not affecting FcγRIIB1 tyrosyl phosphorylation, blocks receptor-mediated recruitment of SHIP, SHIP phosphorylation, recruitment of p52 Shc, phosphatidylinositol 3,4,5-trisphosphate hydrolysis, inhibition of mitogen-activated protein kinase activation, and, albeit more modestly, FcγRIIB1 inhibition of Ca2+ mobilization. Taken together, results implicate ITIM interactions with SHIP as a major mechanism of FcγRIIB1-mediated inhibitory signaling.
Murine CCR9, a Chemokine Receptor for Thymus-Expressed Chemokine That Is Up-Regulated Following Pre-TCR Signaling
Authors: Norment Anne M. , Bogatzki Lisa Y. , Gantner Ben N. , Bevan Michael J.
Published: April 2014

Chemokines are likely to play an important role in regulating the trafficking of developing T cells within the thymus. By using anti-CD3ε treatment of recombinase-activating gene 2 (Rag2−/−) mice to mimic pre-TCR signaling and drive thymocyte development to the double positive stage, we have identified murine GPR-9-6 as a chemokine receptor whose expression is strongly induced following pre-TCR signaling. GPR-9-6 mRNA is present at high levels in the thymus, and by RT-PCR analysis its expression is induced as normal thymocytes undergo the double negative to double positive transition. Furthermore we show that TECK (thymus-expressed chemokine), a chemokine produced by thymic medullary dendritic cells, is a functional ligand for GPR-9-6. TECK specifically induces a calcium flux and chemotaxis of GPR-9-6-transfected cells. In addition, TECK stimulates the migration of normal double positive thymocytes, as well as Rag2−/− thymocytes following anti-CD3ε treatment. Hence, GPR-9-6 has been designated as CC chemokine receptor 9 (CCR9). Our results suggest that TECK delivers signals through CCR9 important for the navigation of developing thymocytes.
In Vivo CD4+ T Cell Tolerance Induction Versus Priming Is Independent of the Rate and Number of Cell Divisions
Authors: Adler Adam J. , Huang Ching-Tai , Yochum Gregory S. , Marsh David W. , Pardoll Drew M.
Published: April 2014

In vitro studies have suggested that tolerance induction (i.e., anergy) is associated with an inability of T cells to proliferate vigorously upon Ag recognition. In vivo, the relationship between T cell proliferation and tolerance induction is less clear. To clarify this issue, we have been studying a model system in which naive CD4+ T cells specific for the model Ag hemagluttinin (HA) are adoptively transferred into different transgenic founder lines of mice expressing HA as a peripheral self-Ag. When transferred into two lines whose HA expression differs by at least 1000-fold, HA-specific T cells undergo multiple rounds of cell division before reaching a nonresponsive (i.e., tolerant) state. While the proliferative response is more rapid in mice expressing higher levels of HA, the T cells become tolerant regardless of the level of peripheral HA expression. When the T cells encounter HA expressed as a viral Ag, they proliferate at a similar rate and undergo the same number of divisions as with self-HA, but they do not become tolerant. These results indicate that a tolerizing stimulus can induce similar T cell mitotic rates as a priming stimulus. Therefore, CD4+ T cell tolerance induction in vivo is not the result of an insufficient proliferative response elicited upon TCR engagement.
The p55 TNF-α Receptor Plays a Critical Role in T Cell Alloreactivity
Authors: Hill Geoffrey R. , Teshima Takanori , Rebel Vivienne I. , Krijanovski Oleg I. , Cooke Kenneth R. , Brinson Yani S. , Ferrara James L. M.
Published: April 2014

TNF-α is known to be an important mediator of tissue damage during allograft rejection and graft-vs-host disease (GVHD), but its role in supporting T cell responses to allogeneic Ags is unclear. We have studied this question by comparing normal mice with those lacking the p55 (p55 TNFR−/−) or p75 (p75 TNFR−/−) TNF-α receptors as donors in well-defined bone marrow transplant (BMT) models. Recipients of p55 TNFR−/− cells had significantly reduced mortality and morbidity from GVHD compared with the other two sources of T cells. In vitro, T cells lacking the p55 (but not the p75) TNF-α receptor exhibited decreased proliferation and production of Th1 cytokines in MLC. This defect was only partially restored by exogenous IL-2 and affected both CD4+ and CD8+ populations. CD8+ p55 TNFR−/− proliferation was impaired independently of IL-2 whereas CTL effector function was impaired in an IL-2-dependent fashion. Inhibition of TNF-α with TNFR:Fc in primary MLC also impaired the proliferation and Th1 differentiation of wild-type T cells. BMT mixing experiments demonstrated that the reduced ability of p55 TNFR−/− donor cells to induce GVHD was due to the absence of the p55 TNFR on T cells rather than bone marrow cells. These data highlight the importance of TNF-α in alloreactive T cell responses and suggest that inhibition of the T cell p55 TNF-α receptor may provide an additional useful therapeutic maneuver to inhibit alloreactive T cell responses following bone marrow and solid organ transplantation.
Blockade of CTLA-4 Signals Inhibits Th2-Mediated Murine Chronic Graft-Versus-Host Disease by an Enhanced Expansion of Regulatory CD8+ T Cells
Authors: Sakurai Jinkyo , Ohata Junko , Saito Kiyoshi , Miyajima Hiroaki , Hirano Takao , Kohsaka Takao , Enomoto Shoji , Okumura Ko , Azuma Miyuki
Published: April 2014

CTLA-4 (CD152) is thought to be a negative regulator of T cell activation. Little is known about the function of CTLA-4 in Th2-type immune responses. We have investigated the effect of initial treatment with anti-CTLA-4 mAb on murine chronic graft-vs-host disease. Transfer of parental BALB/c splenocytes into C57BL/6 × BALB/c F1 mice induced serum IgE production, IL-4 expression by donor CD4+ T cells, and host allo-Ag-specific IgG1 production at 6–9 wk after transfer. Treatment with anti-CTLA-4 mAb for the initial 2 wk significantly reduced IgE and IgG1 production and IL-4 expression. Analysis of the splenic phenotype revealed the enhancement of donor T cell expansion, especially within the CD8 subset, and the elimination of host cells early after anti-CTLA-4 mAb treatment. This treatment did not affect early IFN-γ expression by CD4+ and CD8+ T cells and anti-host cytolytic activity. Thus, blockade of CTLA-4 greatly enhanced CD8+ T cell expansion, and this may result in the regulation of consequent Th2-mediated humoral immune responses. These findings suggest a new approach for regulating IgE-mediated allergic immune responses by blockade of CTLA-4 during a critical period of Ag sensitization.
Pathologic Role and Temporal Appearance of Newly Emerging Autoepitopes in Relapsing Experimental Autoimmune Encephalomyelitis
Authors: Vanderlugt Carol L. , Neville Katherine L. , Nikcevich Kelly M. , Eagar Todd N. , Bluestone Jeffrey A. , Miller Stephen D.
Published: April 2014

Relapsing experimental autoimmune encephalomyelitis (R-EAE) is a CD4+ T cell-mediated demyelinating disease model for multiple sclerosis. Myelin destruction during the initial relapsing phase of R-EAE in SJL mice initiated by immunization with the proteolipid protein (PLP) epitope PLP139–151 is associated with activation of T cells specific for the endogenous, non-cross-reactive PLP178–191 epitope (intramolecular epitope spreading), while relapses in R-EAE induced with the myelin basic protein (MBP) epitope MBP84–104 are associated with PLP139–151-specific responses (intermolecular epitope spreading). Here, we demonstrate that T cells specific for endogenous myelin epitopes play the major pathologic role in mediating clinical relapses. T cells specific for relapse-associated epitopes can serially transfer disease to naive recipients and are demonstrable in the CNS of mice with chronic R-EAE. More importantly, induction of myelin-specific tolerance to relapse-associated epitopes, by i.v. injection of ethylene carbodiimide-fixed peptide-pulsed APCs, either before disease initiation or during remission from acute disease effectively blocks the expression of the initial disease relapse. Further, blockade of B7-1-mediated costimulation with anti-B7-1 F(ab) during disease remission from acute PLP139–151-induced disease prevents clinical relapses by inhibiting activation of PLP178–191-specific T cells. The protective effects of anti-B7-1 F(ab) treatment are long-lasting and highly effective even when administered following the initial relapsing episode wherein spreading to a MBP epitope (MBP84–104) is inhibited. Collectively, these data indicate that epitope spreading is B7-1 dependent, plays a major pathologic role in disease progression, and follows a hierarchical order associated with the relative encephalitogenic dominance of the myelin epitopes (PLP139–151 > PLP178–191 > MBP84–104).
Vanadate Induces Calcium Signaling, Ca2+ Release-Activated Ca2+ Channel Activation, and Gene Expression in T Lymphocytes and RBL-2H3 Mast Cells Via Thiol Oxidation
Authors: Ehring George R. , Kerschbaum Hubert H. , Fanger Christopher M. , Eder Claudia , Rauer Heiko , Cahalan Michael D.
Published: April 2014

Using ratiometric Ca2+ imaging and patch-clamp measurement of Ca2+ channel activity, we investigated Ca2+ signaling induced by vanadium compounds in Jurkat T lymphocytes and rat basophilic leukemia cells. In the presence of external Ca2+, vanadium compounds produced sustained or oscillatory Ca2+ elevations; in nominally Ca2+-free medium, a transient Ca2+ rise was generated. Vanadate-induced Ca2+ signaling was blocked by heparin, a competitive inhibitor of the 1,4,5-inositol trisphosphate (IP3) receptor, suggesting that Ca2+ influx is secondary to depletion of IP3-sensitive Ca2+ stores. In Jurkat T cells, vanadate also activated the Ca2+-dependent transcription factor, NF-AT. Intracellular dialysis with vanadate activated Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels with kinetics comparable to those of dialysis with IP3. Neither phosphatase inhibitors nor nonhydrolyzable nucleotide analogues modified CRAC channel activation. The action of vanadate, but not IP3, was prevented by the thiol-reducing agent DTT. In addition, the activation of CRAC channels by vanadate was mimicked by the thiol-oxidizing agent chloramine T. These results suggest that vanadate enhances Ca2+ signaling via thiol oxidation of a proximal element in the signal transduction cascade.
Resting B Lymphocytes as APC for Naive T Lymphocytes: Dependence on CD40 Ligand/CD40
Authors: Evans Dean E. , Munks Michael W. , Purkerson Jeffrey M. , Parker David C.
Published: April 2014

Although resting B cells as APC are tolerogenic for naive T cells in vivo, we show here that they can provide all the costimulatory signals necessary for naive T cell proliferation in vivo and in vitro. In the absence of an activating signal through the B cell Ag receptor, T cell proliferation after Ag recognition on resting B cells depends on CD40 expression on the B cells, implying that naive T cells use the membrane-bound cytokine, CD40 ligand (CD154), to induce the costimulatory signals that they need. Induction of B7-1 (CD80) and increased or sustained expression of CD44H, ICAM-1 (CD54), and B7-2 (CD86) are dependent on the interaction of CD40 ligand with CD40. Transient expression (12 h) of B7-2 is T cell- and peptide Ag-dependent, but CD40-independent. Only sustained (≥24 h) expression of B7-2 and perhaps increased expression of ICAM-1 could be shown to be functionally important in this system. T cells cultured with CD40-deficient B cells and peptide remain about as responsive as fresh naive cells upon secondary culture with whole splenic APC. Therefore, B cells, and perhaps other APC, may be tolerogenic not because they fail to provide sufficient costimulation for T cell proliferation, but because they are deficient in some later functions necessary for a productive T cell response.
Vaccination with Mouse Mammary Adenocarcinoma Cells Coexpressing B7-1 (CD80) and B7-2 (CD86) Discloses the Dominant Effect of B7-1 in the Induction of Antitumor Immunity
Authors: Martín-Fontecha Alfonso , Moro Monica , Crosti Maria Cristina , Veglia Fabrizio , Casorati Giulia , Dellabona Paolo
Published: April 2014

Nonreplicating TS/A mammary adenocarcinoma cells expressing B7-2 (CD86) (TS/A-2) are more immunogenic than those expressing B7-1 (CD80) (TS/A-1), indicating that B7-1 and B7-2 display nonredundant costimulatory effects in inducing antitumor responses. Whereas transfection of B7-2 cDNA into TS/A-1 cells does not improve their immunogenicity, transfection of B7-1 cDNA into TS/A-2 cells (TS/A-2/1) decreases their immunogenicity in a manner that is directly related to the surface levels of B7-1. Ab blocking of B7-1 on TS/A-2/1 cells before their injection in vivo restores the higher immunogenicity characteristic of single B7-2 transfectants, indicating therefore that B7-1 actively modulates the B7-2-dependent costimulation. The expression of B7-1 also modifies quantitatively the balance of endogenous IFN-γ and IL-4 induced in vivo by TS/A-2 vaccines. In fact, we find that vaccination with TS/A-2/1 cells results in the production of more IFN-γ and less IL-4 than TS/A-2 vaccines, a pattern comparable to that induced by TS/A-1 cells. Thus, in the TS/A model of antitumor response, B7-1 modulates B7-2-dependent costimulatory effects in a dominant, noncompetitive way.
Hierarchical Costimulator Thresholds for Distinct Immune Responses: Application of a Novel Two-Step Fc Fusion Protein Transfer Method
Authors: Chen Aoshuang , Zheng Guoxing , Tykocinski Mark L.
Published: April 2014

Activation of T cells is dependent upon coordinate engagement of Ag and costimulator receptors on their surfaces. In the case of the Ag receptors (TCRs), activation thresholds have been defined, with the number of TCRs that must be triggered to stimulate cytokine secretion by individual activated T cells differing for the various cytokines. In the present study, we have determined whether comparable activation thresholds exist for the costimulator receptors on T cells. To facilitate this type of quantitative costimulator analysis, we developed a novel two-step protein transfer approach that permits delivery of graded amounts of proteins to APC surfaces. By adding a human B7-1 · Fcγ1 (Fc domain of human IgG1) fusion protein to cells precoated with palmitated protein A, fine titration of the B7-1 extracellular domain was achieved. The B7-1 · Fcγ1 reincorporated into cell membranes by this method retained costimulator function, as measured by an in vitro proliferation assay. The degree of proliferation was dependent on the surface density of B7-1 · Fcγ1. Significantly, the threshold B7-1 · Fcγ1 density required for cytokine production differed between IFN-γ and IL-2 and mirrored the hierarchy (IFN-γ < IL-2) described previously for the TCR activation threshold. Hence, this study invokes a novel protein transfer strategy to establish that the levels of surface costimulator on APCs can dictate both the magnitude and the quality of evoked T cell responses. The notion of costimulator receptor activation thresholds emerges.
uPA/uPAR System Is Active in Immature Dendritic Cells Derived from CD14+CD34+ Precursors and Is Down-Regulated upon Maturation
Authors: Ferrero Elisabetta , Vettoretto Katuscia , Bondanza Attilio , Villa Antonello , Resnati Massimo , Poggi Alessandro , Zocchi Maria Raffaella
Published: April 2014

We recently described a subset of peripheral CD14+CD34+ cells able to migrate across endothelial cell monolayers and differentiate into immunostimulatory dendritic cells (DC). In this paper we show that immature DC derived from CD14+CD34+ precursors are also capable of reverse transendothelial migration and extracellular matrix (ECM) invasion using the urokinase plasminogen activator receptor (uPAR). We found that these cells respond to macrophage-inflammatory protein (MIP)-1α, enhancing their ability to invade ECM and supporting the idea that immature DC are selectively recruited at the site of inflammation to expand the pool of APCs. Interestingly, MIP-1α was also capable of preventing the decreased matrix invasion observed by blocking uPAR, suggesting that the uPA/uPAR system and MIP-1α cooperate in driving immature DC migration through the subendothelial matrix. Upon exposure to maturating stimuli, such as TNF-α, CD14+CD34+-derived DC enhance their APC function and decrease the capacity of invading ECM; these changes are accompanied by altered expression and function of uPAR. Moreover, mature DC shift their sensitivity from MIP-1α to MIP-3β, enhancing their transendothelial migration capability in response to the latter chemokine. Our data support the hypothesis that bloodborne DC can move through ECM toward the site of pathogen entry where they differentiate into fully mature APCs with their motility and function regulated by microenvironmental stimuli, including MIP-1α, MIP-3β, and TNF-α.
Effect of Molecular Size on the Ability of Zwitterionic Polysaccharides to Stimulate Cellular Immunity
Authors: Kalka-Moll Wiltrud M. , Tzianabos Arthur O. , Wang Ying , Carey Vincent J. , Finberg Robert W. , Onderdonk Andrew B. , Kasper Dennis L.
Published: April 2014

The large-molecular-sized zwitterionic capsular polysaccharide of the anaerobe Bacteroides fragilis NCTC 9343, designated polysaccharide (PS) A, stimulates T cell proliferation in vitro and induces T cell-dependent protection against abscess formation in vivo. In the present study, we utilized a modification of a recently developed ozonolytic method for depolymerizing polysaccharides to examine the influence of the molecular size of PS A on cell-mediated immunity. Ozonolysis successfully depolymerized PS A into structurally intact fragments. PS A with average molecular sizes of 129.0 (native), 77.8, 46.9, and 17.1 kDa stimulated CD4+-cell proliferation in vitro to the same degree, whereas the 5.0-kDa fragment was much less stimulatory than the control 129.0-kDa PS A. Rats treated with 129.0-kDa, 46.9-kDa, and 17.1-kDa PS A molecules, but not those treated with the 5.0-kDa molecule, were protected against intraabdominal abscesses induced by challenge with viable B. fragilis. These results demonstrate that a zwitterionic polysaccharide as small as 22 repeating units (88 monosaccharides) elicits a T cell-dependent immune response. These findings clearly distinguish zwitterionic T cell-dependent polysaccharides from T cell-independent polysaccharides and give evidence of the existence of a novel mechanism for a polysaccharide-induced immune response.
Soluble Antigen and CD40 Triggering Are Sufficient to Induce Primary and Memory Cytotoxic T Cells
Authors: Lefrançois Leo , Altman John D. , Williams Kristina , Olson Sara
Published: April 2014

The signals directing induction of tolerance rather than immunity are largely unknown. The CD8 T cell response to soluble Ags generally results in deletional tolerance following transient, costimulation-dependent activation. We demonstrated that CD40 signaling reversed the outcome of this response. Adoptive transfer of OVA-specific CD8 T cells followed by soluble OVA immunization resulted in induction of lytic activity and optimal clonal expansion only when CD40 was triggered via an agonistic mAb. Activation of CD8 T cells by CD40 signaling was indirect, because CD40 expression by host cells was required. CD40 signaling along with soluble Ag immunization also induced expansion of secondary lymphoid and intestinal mucosal endogenous OVA-specific CD8 T cells as detected by MHC tetramer reactivity. When CD40 activation was included, long-lived secondary lymphoid and mucosal memory CD8 cells were generated from adoptively transferred and endogenous CD8 T cells. Mucosal and peripheral CD8 memory cells exhibited constitutive Ag-specific lytic activity, with mucosal memory cells being 10-fold more lytic than splenic or lymph node memory cells. These results demonstrated that CD40 signaling during a response to a poorly immunogenic soluble Ag was necessary and sufficient for CTL and memory T cell induction.
Defective Expression of the Monocyte Chemotactic Protein-1 Receptor CCR2 in Macrophages Associated with Human Ovarian Carcinoma
Authors: Sica Antonio , Saccani Alessandra , Bottazzi Barbara , Bernasconi Sergio , Allavena Paola , Gaetano Brancatelli , Fei Francesca , LaRosa Graig , Scotton Chris , Balkwill Frances , Mantovani Alberto
Published: April 2014

Monocyte chemotactic protein-1 (MCP-1, CCL2) is an important determinant of macrophage infiltration in tumors, ovarian carcinoma in particular. MCP-1 binds the chemokine receptor CCR2. Recent results indicate that proinflammatory and anti-inflammatory signals regulate chemokine receptor expression in monocytes. The present study was designed to investigate the expression of CCR2 in tumor-associated macrophages (TAM) from ovarian cancer patients. TAM isolated from ascitic or solid ovarian carcinoma displayed defective CCR2 mRNA (Northern blot and PCR) and surface expression and did not migrate in response to MCP-1. The defect was selective for CCR2 in that CCR1 and CCR5 were expressed normally in TAM. CCR2 gene expression and chemotactic response to MCP-1 were decreased to a lesser extent in blood monocytes from cancer patients. CCR2 mRNA levels and the chemotactic response to MCP-1 were drastically reduced in fresh monocytes cultured in the presence of tumor ascites from cancer patients. Ab against TNF-α restored the CCR2 mRNA level in monocytes cultured in the presence of ascitic fluid. The finding of defective CCR2 expression in TAM, largely dependent on local TNF production, is consistent with previous in vitro data on down-regulation of chemokine receptors by proinflammatory molecules. Receptor inhibition may serve as a mechanism to arrest and retain recruited macrophages and to prevent chemokine scavenging by mononuclear phagocytes at sites of inflammation and tumor growth. In the presence of advanced tumors or chronic inflammation, systemic down-regulation of receptor expression by proinflammatory molecules leaking in the systemic circulation may account for defective chemotaxis and a defective capacity to mount inflammatory responses associated with advanced neoplasia.
Comparative Contribution of CD1 on the Development of CD4+ and CD8+ T Cell Compartments
Authors: Wang Bin , Chun Taehoon , Wang Chyung-Ru
Published: April 2014

CD1 molecules are MHC class I-like glycoproteins whose expression is essential for the development of a unique subset of T cells, the NK T cells. To evaluate to what extent CD1 contributes to the development of CD4+ and CD8+ T cells, we generated CD1oIIo and CD1oTAPo mice and compared the generation of T cells in these double-mutant mice and IIo or TAPo mice. FACS analysis showed that the number of CD4+ T cells in CD1oIIo mice was reduced significantly compared with the corresponding population in IIo mice. Both CD4+ NK1.1+ and the CD4+ NK1.1− population were reduced in CD1oIIo mice, suggesting that CD1 can select not only CD4+ NK1.1+ T cells but also some NK1.1− CD4+ T cells. Functional analysis showed that the residual CD4+ cells in CD1oIIo can secrete large amounts of IFN-γ and a significant amount of IL-4 during primary stimulation with anti-CD3, suggesting that this population may be enriched for NK T cells restricted by other class I molecules. In contrast to the CD4+ population, no significant differences in the CD8+ T cell compartment can be detected between TAPo and CD1oTAPo mice in all lymphoid tissues tested, including intestinal intraepithelial lymphocytes. Our data suggest that, unlike other MHC class I molecules, CD1 does not contribute in a major way to the development of CD8+ T cells.
α4β1 Integrin/VCAM-1 Interaction Activates αLβ2 Integrin-Mediated Adhesion to ICAM-1 in Human T Cells
Authors: Chan Jason R. , Hyduk Sharon J. , Cybulsky Myron I.
Published: April 2014

Modulation of integrin affinity and/or avidity provides a regulatory mechanism by which leukocyte adhesion to endothelium is strengthened or weakened at different stages of emigration. In this study, we demonstrate that binding of high-affinity α4β1 integrins to VCAM-1 strengthens αLβ2 integrin-mediated adhesion. The strength of adhesion of Jurkat cells, a human leukemia T cell line, or MnCl2-treated peripheral blood T cells to immobilized chimeric human VCAM-1/Fc, ICAM-1/Fc, or both was quantified using parallel plate flow chamber leukocyte detachment assays in which shear stress was increased incrementally (0.5–30 dynes/cm2). The strength of adhesion to VCAM-1 plus ICAM-1, or to a 40-kDa fragment of fibronectin containing the CS-1 exon plus ICAM-1, was greater than the sum of adhesion to each molecule alone. Treatment of Jurkat or blood T cells with soluble cross-linked VCAM-1/Fc or HP2/1, a mAb to α4, significantly increased adhesion to ICAM-1. These treatments induced clustering of αLβ2 integrins, but not the high-affinity β2 integrin epitope recognized by mAb 24. Up-regulated adhesion to ICAM-1 was abolished by cytochalasin D, an inhibitor of cytoskeletal rearrangement. Taken together, our data suggest that the binding of VCAM-1 or fibronectin to α4β1 integrins initiates a signaling pathway that increases β2 integrin avidity but not affinity. A role for the cytoskeleton is implicated in this process.
The Multidrug Resistance Protein 1: A Functionally Important Activation Marker for Murine Th1 Cells
Authors: Prechtl Stefan , Roellinghoff Martin , Scheper Rik , Cole Susan P. C. , Deeley Roger G. , Lohoff Michael
Published: April 2014

Previously, we described the expression of an energy-dependent pump in resting murine Th2 (but not resting Th1) cells which extruded the fluorescent dye Fluo-3. After stimulation with Ag and APCs, Th1 cells also expressed this pump. Furthermore, expression of the murine multidrug resistance protein 1 (mrp1) correlated with the presence of the pump. In this study, we report that Fluo-3 is indeed transported by murine mrp1 or its human ortholog MRP1, as revealed by transfection of HEK 293 cells with mrp1 or MRP1 cDNA. Like antigenic activation, IL-2 dose-dependently enhanced the Fluo-3-extruding activity in murine Th1 cells. Although TNF-α and IL-12 by themselves only weakly enhanced Fluo-3 extrusion, each of them did so in strong synergism with IL-2. An Ab directed against mrp1 was used to quantify the expression of mrp1 protein in T cells at the single-cell level. Like the Fluo-3 pump, mrp1 protein expression was enhanced by IL-2. Immunohistochemical studies using confocal laser microscopy indicated that mrp1 is localized mainly at the plasma membrane. In addition, protein expression of mrp1 was induced in Vβ8+CD4+ T cells 12 h after in vivo application of Staphylococcal enterotoxin B. Finally, mrp1 was functionally relevant during the activation process of Th1 cells, because T cell activation could be suppressed by exposure of cells to the mrp1 inhibitor MK571. Thus, we present mrp1 as a novel, functionally important activation marker for Th1 cells and short-term in vivo activated CD4+ T cells, whereas its expression seems to be constitutive in Th2 cells.
Autocrine Production of IL-10 Mediates Defective IL-12 Production and NF-κB Activation in Tumor-Associated Macrophages
Authors: Sica Antonio , Saccani Alessandra , Bottazzi Barbara , Polentarutti Nadia , Vecchi Annunciata , Damme Jo Van , Mantovani Alberto
Published: April 2014

IL-12 is a central cytokine in the activation of inflammation and immunity and in the generation of Th1-type responses. Tumor-associated macrophages (TAM) from mouse and human tumors showed defective production of IL-12. Defective IL-12 production was associated with lack of p50/p65 NF-κB activation. TAM produced increased amounts of the immunosuppressive cytokine IL-10. Abs against IL-10 restored the defective capacity of TAM to produce IL-12. Our data suggest that during tumor growth an IL-10-dependent pathway of diversion of macrophage function can be activated into the tumor microenvironment and results in the promotion of the IL-10+ IL-12− phenotype of TAM. Blocking IL-10, as well as other immunosuppressive cytokines present in the tumor microenvironment, such as TGF-β, may complement therapeutic strategies aimed at activating type I antitumor immune responses.
Antiviral B Cell Memory in the Absence of Mature Follicular Dendritic Cell Networks and Classical Germinal Centers in TNFR1−/− Mice
Authors: Karrer U. , López-Macías C. , Oxenius A. , Odermatt B. , Bachmann M. F. , Kalinke U. , Bluethmann H. , Hengartner H. , Zinkernagel R. M.
Published: April 2014

TNFR1−/− mice have been shown to lack networks of mature follicular dendritic cells (FDCs) and they do not form germinal centers. With nonreplicating Ags, IgG titers were inefficiently induced and not maintained. In this study, the neutralizing Ab response and the establishment of B cell memory in TNFR1−/− mice after infection with vesicular stomatitis virus (VSV) were analyzed histologically and functionally. Immunization with VSV-derived protein Ags without adjuvant induced only IgM but no IgG Abs in TNFR1−/− mice, whereas VSV glycoprotein emulsified in CFA or IFA induced IgM and IgG responses that were short-lived and of moderate titer. However, infection with live VSV induced excellent neutralizing IgM and IgG responses in TNFR1−/− mice, and adoptively transferable B cell memory was generated and persisted for more than 300 days. In contrast, IgG levels and Ab-forming cells in the bone marrow declined within 300 days by 90–95% compared with controls. These findings suggest that 1) increased Ag dose and time of Ag availability can substitute for FDC-stored Ab-complexed Ag in the induction of efficient IgG responses in TNFR1−/− mice devoid of classical germinal centers; 2) the induction and maintenance of adoptively transferable B cell memory can occur in the absence of Ag bound to mature FDCs; and 3) the long-term maintenance of elevated IgG titers is largely dependent on FDC-associated persisting Ag. However, about 5–10% of the Ab production remained in the absence of detectable persisting Ag in TNFR1−/− mice, probably either due to immature FDCs being partially functional and/or due to long-lived plasma cells.
IFN-γ Is Necessary But Not Sufficient for Anti-CD40 Antibody-Mediated Inhibition of the Th2 Response to Schistosoma mansoni Eggs
Authors: Martin Diana L. , King Christopher L. , Pearlman Eric , Strine Ellen , Heinzel Frederick P.
Published: April 2014

The injection of Schistosoma mansoni eggs into the footpads of mice results in a localized Th2 cytokine response and tissue eosinophilia. We examined whether treatment with CD40-activating Abs would block the development of Th2 cytokine responses and eosinophilic tissue pathology in this model. Seven days after C57BL/6 mice were injected with eggs and the FGK45 anti-CD40 Ab, Ag-specific synthesis of IL-4, IL-5, and IL-13 in lymph node culture was reduced (>10-fold) relative to control mice treated with eggs and rat IgG. In contrast, IFN-γ and IL-12 were increased in both culture supernatants and in the serum. Similar changes in lymph node cytokine mRNA were observed in vivo, and tissue eosinophilia was reduced nearly 20-fold. Th2 cytokine responses in anti-CD40-treated IFN-γ−/− and IL-12 p40−/− C57BL/6 mice were unaffected, although anti-CD40 induced high levels of systemic and local IFN-γ production in both wild-type and IL-12 p40−/− mice. We conclude that CD40-activating treatments strongly reverse the immune phenotype generated in response to a classic, Th2-biasing stimulus and stimulate IFN-γ through a novel IL-12-independent pathway. This model for Th1-deviating immune therapy may have relevance to the treatment of Th2-dependent diseases in general.
Modulation of Renal Disease in MRL/lpr Mice Genetically Deficient in the Alternative Complement Pathway Factor B
Authors: Watanabe Hiroshi , Garnier Gérard , Circolo Antonella , Wetsel Rick A. , Ruiz Phil , Holers V. Michael , Boackle Susan A. , Colten Harvey R. , Gilkeson Gary S.
Published: April 2014

In systemic lupus erythematosus, the renal deposition of complement-containing immune complexes initiates an inflammatory cascade resulting in glomerulonephritis. Activation of the classical complement pathway with deposition of C3 is pathogenic in lupus nephritis. Although the alternative complement pathway is activated in lupus nephritis, its role in disease pathogenesis is unknown. To determine the role of the alternative pathway in lupus nephritis, complement factor B-deficient mice were backcrossed to MRL/lpr mice. MRL/lpr mice develop a spontaneous lupus-like disease characterized by immune complex glomerulonephritis. We derived complement factor B wild-type (B+/+), homozygous knockout (B−/−), and heterozygous (B+/−) MRL/lpr mice. Compared with B+/− or B+/+ mice, MRL/lpr B−/− mice developed significantly less proteinuria, less glomerular IgG deposition, and decreased renal scores as well as lower IgG3 cryoglobulin production and vasculitis. Serum C3 levels were normal in the B−/− mice compared with significantly decreased levels in the other two groups. These results suggest that: 1) factor B plays an important role in the pathogenesis of glomerulonephritis and vasculitis in MRL/lpr mice; and 2) activation of the alternative pathway, either by the amplification loop or by IgA immune complexes, has a prominent effect on serum C3 levels in this lupus model.
Comparison of Mouse and Rabbit Eiκ Enhancers Indicates That Different Elements Within the Enhancer May Mediate Activation of Transcription and Recombination
Authors: Coquilleau Isabelle , Cavelier Patricia , Rougeon François , Goodhardt Michele
Published: April 2014

The intronic Ig κ-light chain enhancer (Eiκ) has been implicated in regulation of transcription and Vκ-Jκ recombination at the κ locus. To identify sequences within the Eiκ enhancer which are involved in control of recombination, we have made use of the finding that the Eiκ element from the rabbit b9 κ locus is capable of inducing rearrangement, but not transcription of κ genes in mouse lymphoid cells. We have therefore compared the binding of murine nuclear proteins to the mouse and rabbit Eiκ elements. DNase I footprinting and gel mobility shift assays indicate that only the κB, κE1, and κE2 sites of the rabbit enhancer are able to interact with murine trans-acting factors. Moreover, although the rabbit κB site binds murine NF-κB p50/p50 and p50/p65 complexes with high affinity, this site is not capable of mediating transcriptional activation of transient transfection reporter constructs in mouse B lineage cells. These results therefore suggest that, in contrast to the maintenance of κ enhancer transcription which requires all of the Eiκ sites, only the κB, κE1, and κE2 sites may be necessary for the recombinational activity of the enhancer. Furthermore, NF-κB-mediated effects on transcription and recombination appear to involve separate downstream activation pathways.
Human Cytomegalovirus Gene Products US3 and US6 Down-Regulate Trophoblast Class I MHC Molecules
Authors: Jun Youngsoo , Kim Eunok , Jin Mirim , Sung Ha Chin , Han Hoon , Geraghty Daniel E. , Ahn Kwangseog
Published: April 2014

The epidemiological correlation between human CMV (HCMV) infection and spontaneous fetal loss has been suggested, but the underlying mechanism is not well understood. Fetal cytotrophoblasts, which are in direct contact with the maternal immune system in the uterus during pregnancy, do not express HLA-A and HLA-B, but express the nonclassical class I HLA-G and HLA-C. It has been shown that both HLA-G and HLA-C are capable of inhibiting NK-mediated cell lysis. In our present study, using human trophoblast cell lines as well as other cell lines stably transfected with the human class I genes, we have demonstrated that HCMV US3 and US6 down-regulate the cell-surface expression of both HLA-G and HLA-C by two different mechanisms. HCMV US3 physically associates with both trophoblast class I MHC species, retaining them in the endoplasmic reticulum. In contrast, HCMV US6 inhibits peptide transport by TAP and thus specifically the intracellular trafficking of class I molecules. Therefore, these findings suggest for the first time a possible molecular mechanism underlying HCMV-related spontaneous pregnancy loss.
High-Level Rearrangement and Transcription of Yeast Artificial Chromosome-Based Mouse Igκ Transgenes Containing Distal Regions of the Contig
Authors: Li Shuyu , Hammer Robert E. , George-Raizen Julia B. , Meyers Katherine C. , Garrard William T.
Published: April 2014

The mouse Igκ L chain gene locus has been extensively studied, but to date high-level expression of germline transgenes has not been achieved. Reasoning that each end of the locus may contain regulatory elements because these regions are not deleted upon Vκ-Jκ joining, we used yeast artificial chromosome-based techniques to fuse distal regions of the contig to create transgene miniloci. The largest minilocus (290 kb) possessed all members of the upstream Vκ2 gene family including their entire 5′ and 3′ flanking sequences, along with one member of a downstream Vκ21 gene family. In addition, again using yeast artificial chromosome-based technology, we created Igκ miniloci that contained differing lengths of sequences 5′ of the most distal Vκ2 gene family member. In transgenic mice, Igκ miniloci exhibited position-independent and copy number-dependent germline transcription. Igκ miniloci were rearranged in tissue and developmental stage-specific manners. The levels of rearrangement and transcription of the distal and proximal Vκ gene families were similar to their endogenous counterparts and appeared to be responsive to allelic exclusion, but were differentially sensitive to numerous position effects. The minilocus that contained the longest 5′ region exhibited significantly greater recombination of the upstream Vκ2 genes but not the downstream Vκ21 gene, providing evidence for a local recombination stimulating element. These results provide evidence that our miniloci contain nearly all regulatory elements required for bona fide Igκ gene expression, making them useful substrates for functional analyses of cis-acting sequences in the future.
Glucocorticoids Inhibit Calcium- and Calcineurin-Dependent Activation of the Human IL-4 Promoter
Authors: Chen Rongbing , Burke Thomas F. , Cumberland John E. , Brummet Mary , Beck Lisa A. , Casolaro Vincenzo , Georas Steve N.
Published: April 2014

The mechanism by which glucocorticoids (GC) inhibit IL-4 gene expression is currently unknown. In T lymphocytes, IL-4 gene expression is regulated at the level of transcription by increases in intracellular calcium concentration and by the calcium-activated phosphatase calcineurin. In this paper we report that dexamethasone (Dex) inhibits calcium ionophore-induced activation of the human IL-4 promoter in transiently transfected Jurkat T cells. Inhibition of the promoter by Dex is dependent on expression of the GC receptor (GR), because it does not occur in GR-deficient cells. Dex also represses activation of the promoter induced by cotransfecting cells with a constitutively active mutant of calcineurin. Using a series of deletion constructs, we show that the proximal 95 bp of the IL-4 promoter contain a Dex-sensitive regulatory element. This region contains the P1 sequence, a proximal binding site for NF-AT. A calcium-induced but Dex-inhibited nuclear complex containing NF-AT binds to the P1 element in EMSA. Using immunoprecipitation under nondenaturing conditions, we found that the GRα isoform coprecipitates with NF-ATc in nuclear extracts of calcium ionophore- and Dex-treated cells. Taken together, our results show that GC inhibit IL-4 gene expression by interfering with NF-AT-dependent transactivation of the proximal human IL-4 promoter.
Human CD8β, But Not Mouse CD8β, Can Be Expressed in the Absence of CD8α as a ββ Homodimer
Authors: Devine Lesley , Kieffer Lynda J. , Aitken Victoria , Kavathas Paula B.
Published: April 2014

The T cell coreceptor CD8 exists on mature T cells as disulfide-linked homodimers of CD8α polypeptide chains and heterodimers of CD8α- and CD8β-chains. The function of the CD8α-chain for binding to MHC class I and associating with the tyrosine kinase p56lck was demonstrated with CD8αα homodimers. CD8αβ functions as a better coreceptor, but the actual function of CD8β is less clear. Addressing this issue has been hampered by the apparent inability of CD8β to be expressed without CD8α. This study demonstrates that human, but not mouse, CD8β can be expressed on the cell surface without CD8α in both transfected COS-7 cells and murine lymphocytes. By creating chimeric proteins, we show that the murine Ig domain of CD8β is responsible for the lack of expression of murine CD8ββ dimers. In contrast to CD8αα, CD8ββ is unable to bind MHC class I in a cell-cell adhesion assay. Detection of this form of CD8 should facilitate studies on the function of the CD8 β-chain and indicates that caution should be used when interpreting studies on CD8 function using chimeric protein with the murine CD8ββ Ig domain. In addition, we demonstrate that the Ig domains of CD8α are also involved in controlling the ability of CD8 to be expressed. Mutation of B- and F-strand cysteine residues in CD8α reduced the ability of the protein to fold properly and, therefore, to be expressed.