bluuebunny/crossref_metadata_2025 huggingface dataset:
CD25+ CD4+ T Cells Regulate the Expansion of Peripheral CD4 T Cells Through the Production of IL-10
Authors: Annacker Oliver , Pimenta-Araujo Ricardo , Burlen-Defranoux Odile , Barbosa Theolis C. , Cumano Ana , Bandeira Antonio
Published: April 2014

The mechanisms by which the immune system achieves constant T cell numbers throughout life, thereby controlling autoaggressive cell expansions, are to date not completely understood. Here, we show that the CD25+ subpopulation of naturally activated (CD45RBlow) CD4 T cells, but not CD25− CD45RBlow CD4 T cells, inhibits the accumulation of cotransferred CD45RBhigh CD4 T cells in lymphocyte-deficient mice. However, both CD25+ and CD25− CD45RBlow CD4 T cell subpopulations contain regulatory cells, since they can prevent naive CD4 T cell-induced wasting disease. In the absence of a correlation between disease and the number of recovered CD4+ cells, we conclude that expansion control and disease prevention are largely independent processes. CD25+ CD45RBlow CD4 T cells from IL-10-deficient mice do not protect from disease. They accumulate to a higher cell number and cannot prevent the expansion of CD45RBhigh CD4 T cells upon transfer compared with their wild-type counterparts. Although CD25+ CD45RBlow CD4 T cells are capable of expanding when transferred in vivo, they reach a homeostatic equilibrium at lower cell numbers than CD25− CD45RBlow or CD45RBhigh CD4 T cells. We conclude that CD25+ CD45RBlow CD4 T cells from nonmanipulated mice control the number of peripheral CD4 T cells through a mechanism involving the production of IL-10 by regulatory T cells.
Cell Surface-Expressed Moesin-Like Receptor Regulates T Cell Interactions with Tissue Components and Binds an Adhesion-Modulating IL-2 Peptide Generated by Elastase
Authors: Ariel Amiram , Hershkoviz Rami , Altbaum-Weiss Idit , Ganor Sharon , Lider Ofer
Published: April 2014

The adhesion of leukocytes to the extracellular matrix (ECM) depends on their responses to variations in the chemotactic signals in their milieu, as well as on the functioning of cytoskeletal and context-specific receptors. Ezrin, radixin, and moesin constitute a family of proteins that link the plasma membrane to the actin cytoskeleton. The surface expression of moesin on T cells and its role in cell adhesion has not been fully elucidated. Recently, we found that IL-2 peptides generated by elastase modified the adhesion of activated T cells to ECM ligands. Here, we further examined the adhesion regulatory effects of EFLNRWIT, one of the IL-2 peptides, as well as the existence and putative function of its receptor on T cells. We found that when presented to T cells in the absence of another activator, the EFLNRWIT peptide induced cell adhesion to vessel wall and ECM components. Binding of a radiolabeled peptide to T cells, precipitation with the immobilized peptide, and amino acid sequencing of the precipitated protein revealed that EFLNRWIT exerts its function via a cell surface-expressed moesin-like moiety, whose constitutive expression on T cells was increased after activation. This notion was further supported by our findings that: 1) anti-moesin mAb inhibited the binding of T cells to the immobilized EFLNRWIT peptide, 2) immobilized recombinant moesin bound the IL-2 peptide, and 3) soluble moesin inhibited the EFLNRWIT-induced T cell adhesion to fibronectin. Interestingly, moesin appears to be generally involved in T cell responses to adhesion-regulating signals. Thus, the IL-2 peptide EFLNRWIT appears to exert its modulating capacities via an adhesion-regulating moesin-like receptor.
Stromal Cell-Derived Factor 1α-Induced Chemotaxis in T Cells Is Mediated by Nitric Oxide Signaling Pathways
Authors: Cherla Rama P. , Ganju Ramesh K.
Published: April 2014

Stromal cell-derived factor 1α (SDF1α) and its cognate chemokine receptor CXCR4 act as potent chemoattractants and regulate trafficking and homing of hematopoietic progenitor cells and lymphocytes. However, the molecular mechanisms regulating SDF1α-driven cell migration are not well defined. In this study, we have explored the roles of the second messenger NO and the transcription factor NF-κB in SDF1α-induced T cell migration. SDF1α treatment of Jurkat T cells increased the activity of NO synthase, which catalyzes the generation of NO. We observed that pretreatment of Jurkat cells or activated PBLs with several NO donors significantly enhanced the SDF1α-induced migration, whereas various inhibitors of NO synthase markedly abrogated the chemotactic response in a concentration-dependent manner. Furthermore, we observed that inhibitors of the transcription factor NF-κB, which is linked to NO signaling pathways, also significantly blocked the SDF1α-induced chemotactic response. However, these compounds did not have a significant effect on SDF1α-induced mitogen-activated protein kinase activity. In addition, the MAP/Erk kinase kinase inhibitor PD98059 did not abrogate SDF1α-induced chemotaxis. AKT, which has been shown to mediate NO production, was also phosphorylated upon SDF1α stimulation. These studies suggest that NO-related signaling pathways may mediate SDF1α-induced chemotaxis, but not mitogen-activated protein kinase activation.
Src Homology 2 Domain-Containing Protein-Tyrosine Phosphatases, SHP-1 and SHP-2, Are Required for Platelet Endothelial Cell Adhesion Molecule-1/CD31-Mediated Inhibitory Signaling
Authors: Henshall Tanya L. , Jones Karen L. , Wilkinson Ray , Jackson Denise E.
Published: April 2014

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a newly assigned member of the Ig immunoreceptor tyrosine-based inhibitory motif superfamily, and its functional role is suggested to be an inhibitory receptor that modulates immunoreceptor tyrosine-based activation motif-dependent signaling cascades. To test whether PECAM-1 is capable of delivering inhibitory signals in B cells and the functional requirement of protein-tyrosine phosphatases (PTPs) for this inhibitory signaling, we generated chimeric FcγRIIB1-PECAM-1 receptors containing the extracellular and transmembrane portions of murine FcγRIIB1 and the cytoplasmic domain of human PECAM-1. These chimeric receptors were stably expressed in chicken DT40 B cells either as wild-type or mutant cells deficient in SHP-1−/−, SHP-2−/−, SHIP−/−, or SHP-1/2−/− and then assessed for their ability to inhibit B cell Ag receptor (BCR) signaling. Coligation of wild-type FcγRIIB1-PECAM-1 with BCR resulted in inhibition of intracellular calcium release, suggesting that the cytoplasmic domain of PECAM-1 is capable of delivering an inhibitory signal that blocks BCR-mediated activation. This PECAM-1-mediated inhibitory signaling correlated with tyrosine phosphorylation of the FcγRIIB1-PECAM-1 chimera, recruitment of SHP-1 and SHP-2 PTPs by the phosphorylated chimera, and attenuation of calcium mobilization responses. Mutational analysis of the two tyrosine residues, 663 and 686, constituting the immunoreceptor tyrosine-based inhibitory motifs in PECAM-1 revealed that both tyrosine residues play a crucial role in the inhibitory signal. Functional analysis of various PTP-deficient DT40 B cell lines stably expressing wild-type chimeric FcγRIIB1-PECAM-1 receptor indicated that cytoplasmic Src homology 2-domain-containing phosphatases, SHP-1 and SHP-2, were both necessary and sufficient to deliver inhibitory negative regulation upon coligation of BCR complex with inhibitory receptor.
Oral Tolerance Revisited: Prior Oral Tolerization Abrogates Cholera Toxin-Induced Mucosal IgA Responses
Authors: Kato Hirotomo , Fujihashi Kohtaro , Kato Rie , Yuki Yoshikazu , McGhee Jerry R.
Published: April 2014

Oral delivery of a large dose or prolonged feeding of protein Ags induce systemic unresponsiveness most often characterized as reduced IgG and IgE Ab- and Ag-specific CD4+ T cell responses. It remains controversial whether oral tolerance extends to diminished mucosal IgA responses in the gastrointestinal tract. To address this issue, mice were given a high oral dose of OVA or PBS and then orally immunized with OVA and cholera toxin as mucosal adjuvant, and both systemic and mucosal immune responses were assessed. OVA-specific serum IgG and IgA and mucosal IgA Ab levels were markedly reduced in mice given OVA orally compared with mice fed PBS. Furthermore, when OVA-specific Ab-forming cells (AFCs) in both systemic and mucosa-associated tissues were examined, IgG AFCs in the spleen and IgA AFCs in the gastrointestinal tract lamina propria of mice given OVA orally were dramatically decreased. Furthermore, marked reductions in OVA-specific CD4+ T cell proliferative and cytokine responses in spleen and Peyer’s patches were seen in mice given oral OVA but were unaffected in PBS-fed mice. We conclude that high oral doses of protein induce both mucosal and systemic unresponsiveness and that use of mucosal adjuvants that induce both parenteral and mucosal immunity may be a better way to assess oral tolerance.
Antigen Receptor Proximal Signaling in Splenic B-2 Cell Subsets
Authors: Li Xiaoli , Martin Flavius , Oliver Alyce M. , Kearney John F. , Carter Robert H.
Published: April 2014

Splenic marginal zone (MZ) and follicular mantle (FO) B cells differ in their responses to stimuli in vitro and in vivo. We have previously shown that MZ cells exhibit greater calcium responses after ligation of membrane IgM (mIgM). We have now investigated the molecular mechanism underlying the difference in calcium responses following ligation of mIgM and studied the response to total B cell receptor ligation in these two subsets. We compared key cellular proteins involved in calcium signaling in MZ and FO cells. Tyrosine phosphorylation and activity of phospholipase C-γ2 and Syk protein tyrosine kinase were significantly higher in MZ cells than in FO cells after mIgM engagement, providing a likely explanation for our previous findings. Tyrosine phosphorylation of CD22 and expression of Src homology 2-containing inositol phosphatase and Src homology 2-containing protein tyrosine phosphatase-1 were also higher in the MZ cells. Expression and tyrosine phosphorylation of Btk, BLNK, Vav, or phosphatidylinositol 3-kinase were equivalent. In contrast, stimulation with anti-κ induced equivalent increases in calcium and activation of Syk in the two subsets. These signals were also equivalent in cells from IgM transgenic, JH knockout mice, which have equivalent levels of IgM in both subsets. With total spleen B cells, Btk was maximally phosphorylated at a lower concentration of anti-κ than Syk. Thus, calcium signaling in the subsets of mature B cells reflects the amount of Ig ligated more than the isotype or the subset and this correlates with the relative tyrosine phosphorylation of Syk.
Ligation of 4-1BB (CDw137) Regulates Graft-Versus-Host Disease, Graft-Versus-Leukemia, and Graft Rejection in Allogeneic Bone Marrow Transplant Recipients
Authors: Blazar Bruce R. , Kwon Byoung S. , Panoskaltsis-Mortari Angela , Kwak Kyu B. , Peschon Jacques J. , Taylor Patricia A.
Published: April 2014

4-1BB is expressed on activated CD4+ and CD8+ T cells; its ligand, 4-1BB ligand is expressed on APCs. Despite expression on both T cell subpopulations, 4-1BB has been reported to predominantly affect CD8+ T cell responses. By quantifying graft-vs-host disease alloresponses in vivo, we demonstrate that both CD4+ and CD8+ T cell-mediated alloresponses are regulated by 4-1BB/4-1BB ligand interactions to approximately the same extent. 4-1BB receptor-facilitated CD4+ T cell-mediated alloresponses were partly CD28 independent. In two distinct marrow graft rejection systems, host CD8+ and CD4+ T cells each separately contributed to host anti-donor T cell-mediated allograft rejection. α4-1BB mAb increased the graft-vs-leukemia effect of a suboptimal number of donor splenocytes given later post bone marrow transplantation by bolstering allogeneic responses resulting in leukemia elimination. In summary, 4-1BB ligation is a potent regulator of CD4+ and CD8+ T cell-mediated allogeneic responses in vivo. Modifying the ligation of 4-1BB represents a new approach to altering the graft-vs-host disease and graft-vs-leukemia effects of allogeneic T cells post bone marrow transplantation.
Immunobiology of Allograft Rejection in the Absence of IFN-γ: CD8+ Effector Cells Develop Independently of CD4+ Cells and CD40-CD40 Ligand Interactions
Authors: Bishop D. Keith , Wood Sherri Chan , Eichwald Ernst J. , Orosz Charles G.
Published: April 2014

Both wild-type (WT) and IFN-γ-deficient (IFN-γ−/−) C57BL/6 mice can rapidly reject BALB/c cardiac allografts. When depleted of CD8+ cells, both WT and IFN-γ−/− mice rejected their allografts, indicating that these mice share a common CD4-mediated, CD8-independent mechanism of rejection. However, when depleted of CD4+ cells, WT mice accepted their allografts, while IFN-γ−/− recipients rapidly rejected them. Hence, IFN-γ−/−, but not WT mice developed an unusual CD8-mediated, CD4-independent, mechanism of allograft rejection. Allograft rejection in IFN-γ−/− mice was associated with intragraft accumulation of IL-4-producing cells, polymorphonuclear leukocytes, and eosinophils. Furthermore, this form of rejection was resistant to treatment with anti-CD40 ligand (CD40L) mAb, which markedly prolonged graft survival in WT mice. T cell depletion studies verified that anti-CD40L treatment failed to prevent CD8-mediated allograft rejection in IFN-γ−/− mice. However, anti-CD40L treatment did prevent CD4-mediated rejection in IFN-γ−/− mice, although grafts were eventually rejected when CD8+ T cells repopulated the periphery. The IL-4 production and eosinophil influx into the graft that occurred during CD8-mediated rejection were apparently epiphenomenal, since treatment with anti-IL-4 mAb blocked intragraft accumulation of eosinophils, but did not interfere with allograft rejection. These studies demonstrate that a novel, CD8-mediated mechanism of allograft rejection, which is resistant to experimental immunosuppression, can develop when IFN-γ is limiting. An understanding of this mechanism is confounded by its association with Th2-like immune events, which contribute unique histopathologic features to the graft but are apparently unnecessary for the process of allograft rejection.
Functional Association of CD9 with the Fcγ Receptors in Macrophages
Authors: Kaji Keisuke , Takeshita Sunao , Miyake Kensuke , Takai Toshiyuki , Kudo Akira
Published: April 2014

CD9, a member of the tetraspan family of proteins, is highly expressed on macrophages. Although a clear function for the molecule has yet to be described, we have found that the anti-CD9 mAb activates mouse macrophages. The rat anti-CD9 mAb, KMC8.8, but not the F(ab′)2, induced tyrosine phosphorylation of proteins including syk and cbl and induced cell aggregation in the mouse macrophage cell line, J774, suggesting that co-cross-linking of CD9 and FcγR was required for the signal. Co-cross-linking of CD9-FcγR with KMC8.8 on macrophages from three different FcR-deficient mice, FcR γ-chain−/−, FcγRIIB−/−, and FcγRIII−/−, revealed that FcγRIII is specific and crucial for syk phosphorylation. Although both KMC8.8 and the anti-FcγRIIB/III mAb, 2.4G2, evoked similar phosphorylation patterns, only KMC8.8 induced cell aggregation. Additionally, KMC8.8 treatment led to reduce levels of TNF-α production and p42/44 extracellular signal-related kinase phosphorylation relative to 2.4G2 stimulation. Immunofluorescence staining showed that co-cross-linking of CD9-FcγR with KMC8.8 induced filopodium extension before cell aggregation, which was followed by simultaneous colocalization of CD9, FcγRIIB/III, Mac-1, ICAM-1, and F-actin at the cell-cell adhesion site. Moreover, KMC8.8 treatment of FcγR-deficient macrophages revealed that the colocalization of CD9, FcγRIII, Mac-1, and F-actin requires co-cross-linking of CD9-FcγRIII, whereas co-cross-linking of CD9-FcγRIIB induced the colocalization of only CD9 and FcγRIIB. Our results demonstrate that co-cross-linking of CD9 and FcγRs activates macrophages; therefore, CD9 may collaborate with FcRs functioning in infection and inflammation on macrophages.
Modes of Salmonid MHC Class I and II Evolution Differ from the Primate Paradigm
Authors: Shum Benny P. , Guethlein Lisbeth , Flodin Laura R. , Adkison Mark A. , Hedrick Ronald P. , Nehring R. Barry , Stet René J. M. , Secombes Christopher , Parham Peter
Published: April 2014

Rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) represent two salmonid genera separated for 15–20 million years. cDNA sequences were determined for the classical MHC class I heavy chain gene UBA and the MHC class II β-chain gene DAB from 15 rainbow and 10 brown trout. Both genes are highly polymorphic in both species and diploid in expression. The MHC class I alleles comprise several highly divergent lineages that are represented in both species and predate genera separation. The class II alleles are less divergent, highly species specific, and probably arose after genera separation. The striking difference in salmonid MHC class I and class II evolution contrasts with the situation in primates, where lineages of class II alleles have been sustained over longer periods of time relative to class I lineages. The difference may arise because salmonid MHC class I and II genes are not linked, whereas in mammals they are closely linked. A prevalent mechanism for evolving new MHC class I alleles in salmonids is recombination in intron II that shuffles α1 and α2 domains into different combinations.
Novel Control Motif Cluster in the IgH δ-γ3 Interval Exhibits B Cell-Specific Enhancer Function in Early Development
Authors: Mundt Cornelia A. , Nicholson Ian C. , Zou Xiangang , Popov Andrei V. , Ayling Christine , Brüggemann Marianne
Published: April 2014

The majority of the human Ig heavy chain (IgH) constant (C) region locus has been cloned and mapped. An exception is the region between Cδ and Cγ3, which is unstable and may be a recombination hot spot. We isolated a pBAC clone (pHuIgH3′δ-γ3) that established a 52-kb distance between Cδ and Cγ3. Sequence analysis identified a high number of repeat elements, explaining the instability of the region, and an unusually large accumulation of transcription factor-binding motifs, for both lymphocyte-specific and ubiquitous transcription activators (IKAROS, E47, Oct-1, USF, Myc/Max), and for factors that may repress transcription (ΔEF1, Gfi-1, E4BP4, C/EBPβ). Functional analysis in reporter gene assays revealed the importance of the Cδ-Cγ3 interval in lymphocyte differentiation and identified independent regions capable of either enhancement or silencing of reporter gene expression and interaction with the IgH intron enhancer Eμ. In transgenic mice, carrying a construct that links the β-globin reporter to the novel δ-γ3 intron enhancer (Eδ-γ3), transgene transcription is exclusively found in bone marrow B cells from the early stage when IgH rearrangement is initiated up to the successful completion of H and L locus recombination, resulting in Ab expression. These findings suggest that the Cδ-Cγ3 interval exerts regulatory control on Ig gene activation and expression during early lymphoid development.
Gorillas with Spondyloarthropathies Express an MHC Class I Molecule with Only Limited Sequence Similarity to HLA-B27 that Binds Peptides with Arginine at P2
Authors: Urvater Julie A. , Hickman Heather , Dzuris John L. , Prilliman Kiley , Allen Todd M. , Schwartz Kevin J. , Lorentzen David , Shufflebotham Clare , Collins Edward J. , Neiffer Donald L. , Raphael Bonnie , Hildebrand William , Sette Alessandro , Watkins David I.
Published: April 2014

The human MHC class I gene, HLA-B27, is a strong risk factor for susceptibility to a group of disorders termed spondyloarthropathies (SpAs). HLA-B27-transgenic rodents develop SpAs, implicating HLA-B27 in the etiology of these disorders. Several nonhuman primates, including gorillas, develop signs of SpAs indistinguishable from clinical signs of humans with SpAs. To determine whether SpAs in gorillas have a similar HLA-B27-related etiology, we analyzed the MHC class I molecules expressed in four affected gorillas. Gogo-B01, isolated from three of the animals, has only limited similarity to HLA-B27 at the end of the α1 domain. It differs by several residues in the B pocket, including differences at positions 45 and 67. However, the molecular model of Gogo-B*0101 is consistent with a requirement for positively charged residues at the second amino acid of peptides bound by the MHC class I molecule. Indeed, the peptide binding motif and sequence of individual ligands eluted from Gogo-B*0101 demonstrate that, like HLA-B27, this gorilla MHC class I molecule binds peptides with arginine at the second amino acid position of peptides bound by the MHC class I molecule. Furthermore, live cell binding assays show that Gogo-B*0101 can bind HLA-B27 ligands. Therefore, although most gorillas that develop SpAs express an MHC class I molecule with striking differences to HLA-B27, this molecule binds peptides similar to those bound by HLA-B27.
Structural and Functional Consequences of Altering a Peptide MHC Anchor Residue
Authors: Kersh Gilbert J. , Miley Michael J. , Nelson Christopher A. , Grakoui Arash , Horvath Stephen , Donermeyer David L. , Kappler John , Allen Paul M. , Fremont Daved H.
Published: April 2014

To better understand TCR discrimination of multiple ligands, we have analyzed the crystal structures of two Hb peptide/I-Ek complexes that differ by only a single amino acid substitution at the P6 anchor position within the peptide (E73D). Detailed comparison of multiple independently determined structures at 1.9 Å resolution reveals that removal of a single buried methylene group can alter a critical portion of the TCR recognition surface. Significant variance was observed in the peptide P5-P8 main chain as well as a rotamer difference at LeuP8, ∼10 Å distal from the substitution. No significant variations were observed in the conformation of the two MHC class II molecules. The ligand alteration results in two peptide/MHC complexes that generate bulk T cell responses that are distinct and essentially nonoverlapping. For the Hb-specific T cell 3.L2, substitution reduces the potency of the ligand 1000-fold. Soluble 3.L2 TCR binds the two peptide/MHC complexes with similar affinity, although with faster kinetics. These results highlight the role of subtle variations in MHC Ag presentation on T cell activation and signaling.
Soluble CD16 Inhibits CR3 (CD11b/CD18)-Mediated Infection of Monocytes/Macrophages by Opsonized Primary R5 HIV-1
Authors: Bouhlal Hicham , Galon Jérôme , Kazatchkine Michel D. , Fridman Wolf-Herman , Sautès-Fridman Catherine , Haeffner Cavaillon Nicole
Published: April 2014

We demonstrate that soluble CD16 (sCD16; soluble FcγRIII), a natural ligand of CR3, inhibits the infection of monocytes by primary R5 HIV-1 strain opsonized with serum of seronegative individuals. Inhibition of monocyte infection by sCD16 was similar to that observed with anti-CR3 mAbs, indicating that opsonized HIV may use a CR3-dependent pathway for entry in monocytic cells. Cultured human monocytes express both CR3 (CD11b/CD18) and CCR5 receptors. RANTES, the natural ligand of CCR5, inhibited infection of monocytes with unopsonized HIV particles and partially that of monocytes infected with HIV particles opsonized with complement-derived fragments. Although HIV-infected monocytes from homozygous CCR5 Δ 32/Δ 32 (CCR5−/−) individuals produce low levels of p24, cells infected with opsonized particles produced higher levels of p24 than cells infected with unopsonized particles. Our results thus suggest that CR3 may represent an alternative coreceptor to CCR5 of opsonized primary R5 virus entry into monocytes/macrophages. We also observed that the concentration of sCD16 is greatly decreased in sera of HIV-infected patients with low lymphocyte CD4+ counts. Taken together, our findings suggest that sCD16, present in plasma, may play an important role in controlling HIV-1 spread.
Contribution of Th1 and Th2 Cells to Protection and Pathology in Experimental Models of Granulomatous Lung Disease
Authors: Wangoo Arun , Sparer Tim , Brown Ivor N. , Snewin Valerie A. , Janssen Riny , Thole Jelle , Cook H. Terence , Shaw Rory J. , Young Douglas B.
Published: April 2014

Mice that had received adoptive transfer of DO11.10 TCR transgenic T cells polarized toward a Th1 or a Th2 phenotype were challenged with Ag-coated beads or with recombinant Mycobacterium tuberculosis expressing the OVA determinant. The resulting bead-induced pulmonary granulomas reflected the phenotype of the adoptively transferred T cells, with the Th2 cells promoting a fibrotic reaction. Mice receiving Th1 cells mounted an epitope-specific protective response to challenge with recombinant M. tuberculosis. Th2 recipients were characterized by enhanced weight loss and lung fibrosis during acute high-dose infection. The combination of TCR transgenic T cells and epitope-tagged mycobacteria provides a novel experimental model for investigation of the pathogenesis of tuberculosis.
Prophylactic Tumor Vaccination: Comparison of Effector Mechanisms Initiated by Protein Versus DNA Vaccination
Authors: Zöller Margot , Christ Oliver
Published: April 2014

Clinical success in tumor vaccination frequently does not reach expectation. Since vaccination protocols are quite variable, we used the murine renal cell carcinoma line RENCA transfected with the lacZ gene (RENCA-β-gal) to compare the efficacy of two different vaccination strategies or their combination and to elaborate on the underlying mechanisms. BALB/c mice were vaccinated either with naked lacZ DNA or with attenuated Salmonella typhimurium transformed with lacZ DNA or with dendritic cells (DC) loaded with the β-galactosidase protein or mice were vaccinated with both DNA and protein. Although all regimens led to a prolongation of survival time, oral vaccination with transfected S. typhimurium followed by i.v. transfer of protein-loaded DC provided the optimal schedule. In this setting, >50% of mice remained tumor free after challenge with 10 times the lethal tumor dose of RENCA-β-gal. As explored in transfer experiments, the superior efficacy of combining DNA and protein vaccination is due to the facts that 1) optimal protection depends on both activated CD4+ and CD8+ cells and 2) CD8+ CTL are most strongly activated by vaccination with transformed Salmonella, whereas vaccination with protein-loaded DC is superior for the activation of Th. The latter induced sustained activation of CTL and recruitment of nonadaptive defense mechanisms. The data demonstrate the strength of DNA vaccination, particularly by the oral route, and provide evidence that a combined treatment with protein-loaded DC can significantly increase the therapeutic efficacy.
Maturation Versus Death of Developing Double-Positive Thymocytes Reflects Competing Effects on Bcl-2 Expression and Can Be Regulated by the Intensity of CD28 Costimulation
Authors: McKean David J. , Huntoon Catherine J. , Bell Michael P. , Tai Xuguang , Sharrow Susan , Hedin Karen E. , Conley Abigail , Singer Alfred
Published: April 2014

Immature double-positive (DP) thymocytes mature into CD4+CD8− cells in response to coengagement of TCR with any of a variety of cell surface “coinducer” receptors, including CD2. In contrast, DP thymocytes are signaled to undergo apoptosis by coengagement of TCR with CD28 costimulatory receptors, but the molecular basis for DP thymocyte apoptosis by TCR plus CD28 coengagement is not known. In the present study, we report that TCR plus CD28 coengagement does not invariably induce DP thymocyte apoptosis but, depending on the intensity of CD28 costimulation, can induce DP thymocyte maturation. We demonstrate that distinct but interacting signal transduction pathways mediate DP thymocyte maturation signals and DP thymocyte apoptotic signals. Specifically, DP maturation signals are transduced by the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and up-regulate expression of the antiapoptotic protein Bcl-2. In contrast, the apoptotic response stimulated by CD28 costimulatory signals is mediated by ERK/MAPK-independent pathways. Importantly, when TCR-activated thymocytes are simultaneously coengaged by both CD28 and CD2 receptors, CD28 signals can inhibit ERK/MAPK-dependent Bcl-2 protein up-regulation. Thus, there is cross-talk between the signal transduction pathways that transduce apoptotic and maturation responses, enabling CD28-initiated signal transduction pathways to both stimulate DP thymocyte apoptosis and also negatively regulate maturation responses initiated by TCR plus CD2 coengagement.
Integrin αE(CD103)β7 Mediates Adhesion to Intestinal Microvascular Endothelial Cell Lines Via an E-Cadherin-Independent Interaction
Authors: Strauch Ulrike G. , Mueller Ruth C. , Li Xiao Y. , Cernadas Manuela , Higgins Jonathan M. G. , Binion David G. , Parker Christina M.
Published: April 2014

Integrins are important for T cell interactions with endothelial cells. Because the integrin αEβ7 is expressed on some circulating gut-homing T cells and as T cell numbers are reduced in the intestinal lamina propria of αE-deficient mice, we evaluated whether αEβ7 mediates binding to intestinal endothelial cells. We found that anti-αEβ7 mAbs partially blocked the binding of cultured intraepithelial T cells to human intestinal microvascular endothelial cells (HIMEC). Furthermore, αEβ7-transfected K562 cells bound more efficiently than vector-transfected K562 cells to HIMEC. Finally, HIMEC bound directly to an αEβ7-Fc fusion protein. These interactions were partially blocked by anti-αEβ7 mAbs, and endothelial cell binding to the αEβ7-Fc was dependent upon the metal ion-dependent adhesion site within the αE A domain. Of note, the HIMEC lacked expression of E-cadherin, the only known αEβ7 counterreceptor as assessed by functional studies, flow cytometry, and RT-PCR. Thus, HIMEC/αEβ7 binding was independent of E-cadherin. In addition, this interaction appeared to be tissue selective, as HIMEC bound to the αEβ7-Fc, whereas microvascular endothelial cells from the skin did not. Finally, there was evidence for an αEβ7 ligand on intestinal endothelial cells in vivo, as αEβ7 expression enhanced lymphocyte binding around vessels in the lamina propria in tissue sections. Thus, we have defined a novel interaction for αEβ7 at a nonepithelial location. These studies suggest a role for αEβ7 in interactions with the intestinal endothelium that may have implications for intestinal T cell homing or functional responses.
HLA-DP Allele-Specific T Cell Responses to Beryllium Account for DP-Associated Susceptibility to Chronic Beryllium Disease
Authors: Lombardi Giovanna , Germain Conrad , Uren Julia , Fiorillo Maria Teresa , du Bois Ronald M. , Jones-Williams William , Saltini Cesare , Sorrentino Rosa , Lechler Robert
Published: April 2014

Occupational exposure to small molecules, such as metals, is frequently associated with hypersensitivity reactions. Chronic beryllium (Be) disease (CBD) is a multisystem granulomatous disease that primarily affects the lung, and occurs in ∼3% of individuals exposed to this element. Immunogenetic studies have demonstrated a strong association between CBD and possession of alleles of HLA-DP containing glutamic acid (Glu) at position 69 in the HLA-DPβ-chain. T cell clones were raised from three patients with CBD in whom exposure occurred 10 and 30 years previously. Of 25 Be-specific clones that were obtained, all were restricted by HLA-DP alleles with Glu at DPβ69. Furthermore, the proliferative responses of the clones were absolutely dependent upon DPβ Glu69 in that a single amino acid substitution at this position abolished the response. As befits a disease whose pathogenesis involves a delayed type hypersensitivity response, the large majority of Be-specific clones secreted IFN-γ (Th1) and little or no IL-4 (Th2) cytokines. This study provides insights into the molecular basis of DP2-associated susceptibility to CBD.
CD40/CD40 Ligand Interactions Are Critical for Elicitation of Autoimmune-Mediated Fibrosis in the Lung
Authors: Zhang-Hoover Jie , Sutton Annika , Stein-Streilein Joan
Published: April 2014

Pulmonary interstitial fibrosis (PIF), associated with persistent inflammation and increased collagen deposition in the interstitium, is often considered an autoimmune disease. Hapten immune PIF (HIPIF), a model for PIF, is elicited in the lung by a single intratracheal (i.t.) challenge in mice sensitized with hapten (2,4,6-trinitrobenzene sulfonic acid, TNBS). In this study, we characterized the role of CD40/CD40 ligand (CD40L) interactions in the elicitation of secondary cell-mediated immune responses that lead to development of fibrosis in the lung using an adoptive transfer model of HIPIF. The expression of CD40 was detected on bronchoalveolar lavage (BAL) cells 1–3 days after i.t. challenge with hapten in the HIPIF lung, but not lungs from the control mice. The CD40bright BAL cells morphologically resembled infiltrating monocytes. Furthermore, blocking CD40/CD40L interactions with blocking Ab decreased BAL production of Th1-mediators (IL-12 and TNF-α). Moreover, either blocking CD40/CD40L interactions with the Ab or using IL-12 knockout recipient mice prevented the increased collagen deposition (accumulation of hydroxyproline) in the lungs during HIPIF induction. We conclude that second signals (CD40/CD40L interactions) are required for elicitation of secondary immune responses that lead to PIF in vivo. The results support the notion that CD40/CD40L interactions are involved in the pathogenesis of an ongoing autoimmune disease.
Gene Expression of Osteoclast Differentiation Factor Is Induced by Lipopolysaccharide in Mouse Osteoblasts Via Toll-Like Receptors
Authors: Kikuchi Takeshi , Matsuguchi Tetsuya , Tsuboi Naotake , Mitani Akio , Tanaka Shigehisa , Matsuoka Masanori , Yamamoto Genta , Hishikawa Toshimitsu , Noguchi Toshihide , Yoshikai Yasunobu
Published: April 2014

Osteoclast differentiation factor (ODF), a recently identified cytokine of the TNF family, is expressed as a membrane-associated protein in osteoblasts and stromal cells. ODF stimulates the differentiation of osteoclast precursors into osteoclasts in the presence of M-CSF. Here we investigated the effects of LPS on the gene expression of ODF in mouse osteoblasts and an osteoblast cell line and found that LPS increased the ODF mRNA level. A specific inhibitor of extracellular signal-regulated kinase or protein kinase C inhibited this up-regulation, indicating that extracellular signal-regulated kinase and protein kinase C activation was involved. A protein synthesis inhibitor, cycloheximide, rather enhanced the LPS-mediated increase of ODF mRNA, and both a neutralizing Ab of TNF-α and a specific inhibitor of PGE synthesis failed to block the ODF mRNA increase by native LPS. Thus, LPS directly induced ODF mRNA. Mouse osteoblasts and an osteoblast cell line constitutively expressed Toll-like receptor (TLR) 2 and 4, which are known as putative LPS receptors. ODF mRNA increases in response to synthetic lipid A were defective in primary osteoblasts from C3H/HeJ mice that contain a nonfunctional mutation in the TLR4 gene, suggesting that TLR4 plays an essential role in the process. Altogether, our results indicate that ODF gene expression is directly increased in osteoblasts by LPS treatment via TLR, and this pathway may play an important role in the pathogenesis of LPS-mediated bone disorders, such as periodontitis.
IC14, an Anti-CD14 Antibody, Inhibits Endotoxin-Mediated Symptoms and Inflammatory Responses in Humans
Authors: Verbon Annelies , Dekkers Pascale E. P. , ten Hove Tessa , Hack C. Erik , Pribble John P. , Turner Terence , Souza Sonia , Axtelle Tim , Hoek Frans J. , van Deventer Sander J. H. , van der Poll Tom
Published: April 2014

CD14 is a receptor for cell wall components of Gram-negative and Gram-positive bacteria that has been implicated in the initiation of the inflammatory response to sepsis. To determine the role of CD14 in LPS-induced effects in humans, 16 healthy subjects received an i.v. injection of LPS (4 ng/kg) preceded (−2 h) by i.v. IC14, a recombinant chimeric mAb against human CD14, at a dose of 1 mg/kg over 1 h, or placebo. In subjects receiving IC14, saturation of CD14 on circulating monocytes and granulocytes was >90% at the time of LPS injection. IC14 attenuated LPS-induced clinical symptoms and strongly inhibited LPS-induced proinflammatory cytokine release, while only delaying the release of the anti-inflammatory cytokines soluble TNF receptor type I and IL-1 receptor antagonist. IC14 also inhibited leukocyte activation, but more modestly reduced endothelial cell activation and the acute phase protein response. The capacity of circulating monocytes and granulocytes to phagocytose Escherichia coli was only marginally reduced after infusion of IC14. These data provide the first proof of principle that blockade of CD14 is associated with reduced LPS responsiveness in humans in vivo.
Antigen-Independent Suppression of the Allergic Immune Response to Bee Venom Phospholipase A2 by DNA Vaccination in CBA/J Mice
Authors: Jilek Samantha , Barbey Catherine , Spertini François , Corthésy Blaise
Published: April 2014

Phospholipase A2 (PLA2) is one of the major honey bee venom allergens for humans. To assess the long-term prevention of allergic reactions by DNA vaccination, a PLA2-CBA/J mouse model was employed using empty or PLA2 sequence-carrying DNA plasmids. Early skin application of either DNA construct before (prophylactic approach) or after (therapeutic approach) sensitization with PLA2/alum led to reduced PLA2-specific IgE and IgG1 titers at 7 mo, with concomitant rise in IgG2a and IgG3. Splenocytes recovered at 5–6 mo after the last DNA administration exhibited a sustained IFN-γ and IL-10 secretion and reduced IL-4 production. Recall challenge with PLA2 boosted IFN-γ and IL-10 secretion, suggesting the reactivation of quiescent memory Th1 lymphocytes. Mice from the prophylactic groups were fully protected against anaphylaxis, whereas 65% of the animals recovered in the therapeutic groups. Th1-polarized immune responses were also active in mice vaccinated with an empty plasmid 32 wk before sensitization with another Ag (OVA). This is the first demonstration that the Ag-coding sequence in DNA vaccine is not necessary to promote immune modulation in naive and sensitized animals for a prolonged period, and has relevance for the understanding of the innate and induced mechanisms underlying gene immunotherapy in long-term treatment of allergy.
Regulatory Activity of Autocrine IL-10 on Dendritic Cell Functions
Authors: Corinti Silvia , Albanesi Cristina , la Sala Andrea , Pastore Saveria , Girolomoni Giampiero
Published: April 2014

IL-10 is a critical cytokine that blocks the maturation of dendritic cells (DCs), but the relevance of autocrine IL-10 on DC functions has not been investigated. In this study, we found that immature monocyte-derived DCs released low but sizeable amounts of IL-10. After stimulation with bacteria, LPS, lipoteichoic acid, or soluble CD40 ligand, DCs secreted high levels of IL-10. Addition of an anti-IL-10-neutralizing Ab to immature DCs as well as to soluble CD40 ligand- or LPS-maturing DCs led to enhanced expression of surface CD83, CD80, CD86, and MHC molecules and markedly augmented release of TNF-α and IL-12, but diminished IL-10 mRNA expression. Moreover, DCs treated with anti-IL-10 Ab showed an increased capacity to activate allogeneic T cells and primed naive T cells to a more prominent Th1 polarization. DC maturation and IL-10 neutralization were associated with enhanced accumulation of the IL-10 receptor binding chain (IL-10R1) mRNA and intracellular IL-10R1 protein. In contrast, surface IL-10R1 and IL-10 binding activity diminished in mature DCs. These results indicate that autocrine IL-10 prevents spontaneous maturation of DCs in vitro, limits LPS- and CD40-mediated maturation, and increases IL-10 production by DCs. Moreover, IL-10R expression appears to be regulated by both transcriptional and posttranscriptional mechanisms. Endogenous IL-10 and IL-10R can be relevant targets for the manipulation of DC functions.
Identification of an IL-2 Binding Protein in the Saliva of the Lyme Disease Vector Tick, Ixodes scapularis
Authors: Gillespie R. Dean , Dolan Marc C. , Piesman Joseph , Titus Richard G.
Published: April 2014

A potent inhibitor of mitogen-stimulated T cell proliferation exists in the saliva of several species of hard ticks, including the Lyme disease vector tick, Ixodes scapularis. Our characterization of this phenomenon has led to the identification of a possible mechanism for the T cell inhibitory activity of I. scapularis saliva. The T cell inhibitor can overcome stimulation of mouse spleen cells with anti-CD3 mAb; however, a direct and avid interaction with T cells does not appear to be necessary. Tick saliva inhibits a mouse IL-2 capture ELISA, suggesting that a soluble IL-2 binding factor is present in the saliva. This hypothesis was verified by using a direct binding assay in which plate-immobilized tick saliva was shown to bind both mouse and human IL-2. Elimination of the IL-2 binding capacity of saliva in the in vitro assays by trypsin digestion demonstrated that the IL-2 binding factor is a protein. These experiments comprise the first demonstration of the existence of such a secreted IL-2 binding protein from any parasite or pathogen. This arthropod salivary IL-2 binding capacity provides a simple mechanism for the suppression of T cell proliferation as well as for the activity of other immune effector cells that are responsive to IL-2 stimulation. Relevance of the tick T cell inhibitory activity to the human immune system is demonstrated by the ability of tick saliva to inhibit proliferation of human T cells and CTLL-2 cells grown in the presence of human IL-2.
Biological Activity of Soluble CD100. II. Soluble CD100, Similarly to H-SemaIII, Inhibits Immune Cell Migration
Authors: Delaire Stéphanie , Billard Christian , Tordjman Rafaèle , Chédotal Alain , Elhabazi Abdellah , Bensussan Armand , Boumsell Laurence
Published: April 2014

CD100 is a human 150-kDa homodimer expressed at the surface of most hemopoietic cells, and its gene belongs to the Ig and semaphorin gene families. Semaphorin genes encode soluble and membrane-bound proteins, most of which have been shown to act as chemorepellents on growth cone guidance. CD100 is discrete, as it is a transmembrane leukocyte surface molecule that can also exist in a soluble form. While our previous studies using mAbs suggested that the transmembrane form of CD100 plays a role in lymphocyte activation, no function was shown for its soluble form. Here, we investigated the effect of soluble CD100 in a cell migration assay; both CD100 spontaneously shed from a stable transfectant and soluble recombinant CD100 inhibited spontaneous and chemokine-induced migration of human monocytes. Interestingly, only the dimeric form of CD100 exerted an effect. Moreover, soluble CD100 inhibited migration of cells from monocytic and B cell lineages. A similar inhibitory effect on migration was observed with H-SemaIII, but not H-SemaIV, semaphorins. In addition, both CD100 and H-SemaIII were recognized by two CD100 mAbs in an ELISA, and one of these mAb abolished the inhibitory effect of each of these semaphorins. We also provide evidence that CD100 and H-SemaIII act through the same receptor on immune cells, which is not neuropilin-1. Furthermore, we describe a function on immune cells for H-SemaIII, a semaphorin to date only studied in the nervous system.
IL-9 and IL-13 Production by Activated Mast Cells Is Strongly Enhanced in the Presence of Lipopolysaccharide: NF-κB Is Decisively Involved in the Expression of IL-9
Authors: Stassen Michael , Müller Christian , Arnold Martina , Hültner Lothar , Klein-Hessling Stefan , Neudörfl Christine , Reineke Tanja , Serfling Edgar , Schmitt Edgar
Published: April 2014

Mast cells, due to their ability to produce a large panel of mediators and cytokines, participate in a variety of processes in adaptive and innate immunity. Herein we report that in primary murine bone marrow-derived mast cells activated with ionomycin or IgE-Ag the bacterial endotoxin LPS strongly enhances the expression of IL-9 and IL-13, but not IL-4. This costimulatory effect of LPS is absent in activated mast cells derived from the LPS-hyporesponsive mouse strain BALB/c-LPSd, although in these cells the proinflammatory cytokine IL-1 can still substitute for LPS. The enhanced production of mast cell-derived IL-13 in the presence of IL-1 is a novel observation. Coactivation of mast cells with LPS leads to a synergistic activation of NF-κB, which is shown by an NF-κB-driven reporter gene construct. In the presence of an inhibitor of NF-κB activation, the production of IL-9 is strongly decreased, whereas the expression of IL-13 is hardly reduced, and that of IL-4 is not affected at all. NF-κB drives the expression of IL-9 via three NF-κB binding sites within the IL-9 promoter, which we characterize using gel shift analyses and reporter gene assays. In the light of recent reports that strongly support critical roles for IL-9 and IL-13 in allergic lung inflammation, our results emphasize the potential clinical importance of LPS as an enhancer of mast cell-derived IL-9 and IL-13 production in the course of inflammatory reactions and allergic diseases.
Regulation of Activator Protein-1 and NF-κB in CD8+ T Cells Exposed To Peripheral Self-Antigens
Authors: Guerder Sylvie , Rincòn Mercedes , Schmitt-Verhulst Anne-Marie
Published: April 2014

The transcriptional events that control T cell tolerance to peripheral self Ags are still unknown. In this study, we analyzed the regulation of AP-1- and NF-κB-mediated transcription during in vivo induction of tolerance to a self Ag expressed exclusively on hepatocytes. Naive CD8+Désiré (Des)+ T cells isolated from the Des TCR-transgenic mice that are specific for the H-2Kb class I Ag were transferred into Alb-Kb-transgenic mice that express the H-2Kb Ag on hepatocytes only. Tolerance develops in these mice. We found that the self-reactive CD8+Des+ T cells were transiently activated, then became unresponsive and were further deleted. In contrast to CD8+Des+ T cells activated in vivo with APCs, which express high AP-1 and high NF-κB transcriptional activity, the unresponsive CD8+Des+ T cells expressed no AP-1 and only weak NF-κB transcriptional activity. The differences in NF-κB transcriptional activity correlated with the generation of distinct NF-κB complexes. Indeed, in vivo primed T cells predominantly express p50/p50 and p65/p50 dimers, whereas these p50-containing complexes are barely detectable in tolerant T cells that express p65- and c-Rel-containing complexes. These observations suggest that fine regulation of NF-κB complex formation may determine T cell fate.