bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutational Analysis of the Superantigen Staphylococcal Exfoliative Toxin A (ETA)
Authors: Rago James V. , Vath Gregory M. , Bohach Gregory A. , Ohlendorf Douglas H. , Schlievert Patrick M.
Published: April 2014

Exfoliative toxin A (ETA) is known to be a causative agent of staphylococcal scalded skin syndrome (SSSS). Although relatively little is known about exactly how the exfoliative toxins (ETs) cause SSSS, much has been discovered recently that may help elucidate the mechanism(s) by which ETA exhibits activities such as lymphocyte mitogenicity and epidermolytic activity. Here, we have shown that highly purified ETA does have T lymphocyte mitogenic activity in that wild-type ETA induced T cell proliferation whereas several single amino acid mutants lacked significant activity. Neither wild-type ETA nor any single amino acid mutants were proteolytic for a casein substrate, yet esterase activity was detected in wild-type ETA and several mutants, but eliminated in other mutants. A mutation in aa 164 (Asp to Ala) showed a 9-fold increase in esterase activity as well. Finally, we correlated esterase activity with epidermolytic activity. All mutants that lost esterase activity also lost epidermolytic activity. Conversely, mutants that retained esterase activity also retained exfoliative activity, implicating serine protease or serine protease-like activity in the causation of SSSS. Moreover, the mutants that displayed markedly reduced T cell superantigenic activity retained their epidermolytic activity (although some of these mutants required higher doses of toxin to cause disease), which suggests an ancillary role for this activity in SSSS causation.
Successful TCR-Based Immunotherapy for Autoimmune Myocarditis with DNA Vaccines After Rapid Identification of Pathogenic TCR
Authors: Matsumoto Yoh , Jee Youngheun , Sugisaki Mayumi
Published: April 2014

The identification of TCRs of autoimmune disease-inducing T cells within a short period of time is a key factor for designing TCR-based immunotherapy during the course of the disease. In this study, we show that experimental autoimmune carditis-associated TCRs, Vβ8.2 and Vβ10, were determined by complementarity-determining region 3 (CDR3)-spectratyping analysis and subsequent sequencing of the CDR3 region of spectratype-derived TCR clones. Immunotherapy targeting both Vβ8.2 and Vβ10 TCRs using mAbs and DNA vaccines significantly reduced the histological severity of experimental autoimmune carditis and completely suppressed the inflammation in some animals. Since depletion or suppression of one of two types of effector cells does not improve the severity of the disease significantly, combined TCR-based immunotherapy should be considered as a primary therapy for T cell-mediated autoimmune diseases. TCR-based immunotherapy after rapid identification of autoimmune disease-associated TCRs by CDR3 spectratyping can be applicable, not only to animal, but also to human autoimmune diseases whose pathomechanism is poorly understood.
Cutting Edge: Identification of a Novel Chemokine Receptor That Binds Dendritic Cell- and T Cell-Active Chemokines Including ELC, SLC, and TECK
Authors: Gosling Jennifa , Dairaghi Daniel J. , Wang Yu , Hanley Michael , Talbot Dale , Miao Zhenhua , Schall Thomas J.
Published: April 2014

Searching for new receptors of dendritic cell- and T cell-active chemokines, we used a combination of techniques to interrogate orphan chemokine receptors. We report here on human CCX CKR, previously represented only by noncontiguous expressed sequence tags homologous to bovine PPR1, a putative gustatory receptor. We employed a two-tiered process of ligand assignment, where immobilized chemokines constructed on stalks (stalkokines) were used as bait for adhesion of cells expressing CCX CKR. These cells adhered to stalkokines representing ELC, a chemokine previously thought to bind only CCR7. Adhesion was abolished in the presence of soluble ELC, SLC (CCR7 ligands), and TECK (a CCR9 ligand). Complete ligand profiles were further determined by radiolabeled ligand binding and competition with >80 chemokines. ELC, SLC, and TECK comprised high affinity ligands (IC50 <15 nM); lower affinity ligands include BLC and vMIP-II (IC50 <150 nM). With its high affinity for CC chemokines and homology to CC receptors, we provisionally designate this new receptor CCR10.
Cutting Edge: Ectopic Expression of the IL-12 Receptor-β2 in Developing and Committed Th2 Cells Does Not Affect the Production of IL-4 or Induce the Production of IFN-γ
Authors: Heath Victoria L. , Showe Louise , Crain Chad , Barrat Franck J. , Trinchieri Giorgio , O’Garra Anne
Published: April 2014

The IL-12 receptor-β2 (IL-12Rβ2) chain is expressed on Th1 cells and lost upon differentiation to the Th2 phenotype. This has been suggested as the basis for commitment of Th1 cells, because early differentiated Th2 cells do not reverse their phenotype and do not produce IFN-γ on restimulation in the presence of IL-12. In this study, we ectopically expressed the IL-12 receptor-β2 (IL-12Rβ2) bicistronically with enhanced green fluorescent protein by retroviral infection in developing and committed Th2 cells. Restimulation of Th2 cells expressing this ectopic IL-12Rβ2 in the presence of IL-12 led to levels of IL-4 production similar to those in control Th2 cells. The expression of IL-12Rβ2 in Th2 cells did not lead to significant levels of IFN-γ production, although IL-12-mediated STAT signaling and proliferation were restored. Thus, although the IL-12Rβ2 and IL-12-dependent STAT4 activation are required for Th1 responses, activation of this pathway is not sufficient to restore a Th1 phenotype in developing or committed Th2 cells.
Cutting Edge: WIP, a Binding Partner for Wiskott-Aldrich Syndrome Protein, Cooperates with Vav in the Regulation of T Cell Activation
Authors: Savoy Doris N. , Billadeau Daniel D. , Leibson Paul J.
Published: April 2014

Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP), specifically binds to a region of WASp that is frequently mutated in Wiskott-Aldrich syndrome. Due to the similar phenotypes of WASp- and Vav-deficient T cells, and the putative importance of the WIP/WASp complex in mediating normal signals from the TCR, we investigated the role of WIP in regulating NF-AT/AP-1-mediated gene transcription. We show that WIP has the ability to enhance Vav-mediated activation of NF-AT/AP-1 gene transcription. In addition, we provide evidence that the interaction of WIP with WASp is necessary, but not sufficient for the ability of WIP to regulate NF-AT/AP-1 activity. Finally, we have identified a region in WIP required for its regulation of NF-AT/AP-1 activity. Our data suggests that the WIP-WASp interaction is important for NF-AT/AP-1-mediated gene transcription, and that defects seen in the activation of T cells from WAS patients may be due to the inability of these cells to form a functional WIP/WASp-signaling complex.
TRAIL (Apo2 Ligand) and TWEAK (Apo3 Ligand) Mediate CD4+ T Cell Killing of Antigen-Presenting Macrophages
Authors: Kaplan Mariana J. , Ray Donna , Mo Ru-Ran , Yung Raymond L. , Richardson Bruce C.
Published: April 2014

The human marrow produces ∼1010 monocytes daily, and this production must be balanced by a similar rate of destruction. Monocytes/macrophages can undergo apoptosis after activating CD4+ T cells, suggesting one mechanism that may contribute to macrophage homeostasis. Previous reports indicate that Fas-Fas ligand interactions are the principle molecules mediating this response. However, D10, an Iak-restricted cloned Th2 line, will similarly induce apoptosis in Ag-presenting macrophages, and D10 cells lack Fas ligand. To confirm that D10 cells kill macrophages through Fas-independent pathways, D10 cells were shown to kill MRL lpr/lpr (Iak) macrophages in an Ag-dependent fashion, indicating additional mechanisms. Recent reports demonstrate that TNF-related apoptosis-inducing ligand (TRAIL), interacting with Apo2, and TNF-like weak inducer of apoptosis (TWEAK), interacting with Apo3, will induce apoptosis in some cells. Using Abs to TRAIL and an Apo3-IgG Fc fusion protein, we demonstrated that D10 cells express both TRAIL and TWEAK. The Apo3 fusion protein, but not human IgG, inhibited D10-induced macrophage apoptosis, as did anti-TRAIL. Further studies demonstrated that AE7, a cloned Th1 line, and splenic T cells express TWEAK, TRAIL, and Fas ligand, and inhibiting these molecules also inhibited macrophage killing. These results indicate that D10 cells induce macrophage apoptosis through TRAIL- and TWEAK-dependent pathways. Because normal T cells also express these molecules, these results support the concept that T cells have multiple pathways by which to induce macrophage apoptosis. These pathways may be important in immune processes such as macrophage homeostasis as well as in down-regulation of immune responses and elimination of macrophages infected with intracellular organisms.
Identification of a Precursor to Phosphatidyl Choline-Specific B-1 Cells Suggesting That B-1 Cells Differentiate from Splenic Conventional B Cells In Vivo: Cyclosporin A Blocks Differentiation to B-1
Authors: Arnold Larry W. , McCray Suzanne K. , Tatu Calin , Clarke Stephen H.
Published: April 2014

The origin of B-1 cells is controversial. The initial paradigm posited that B-1 and B-2 cells derive from separate lineages. More recently it has been argued that B-1 cells derive from conventional B cells as a result of T-independent Ag activation. To understand B-1 cell differentiation, we have generated Ig transgenic (Tg) mice using the H and L chain genes (VH12 and Vκ4) of anti-phosphatidyl choline (anti-PtC) B cells. In normal mice anti-PtC B cells segregate to B-1. Segregation is intact in VH12 (6-1) and VH12/Vκ4 (double) Tg mice that develop large numbers of PtC-specific B cells. However, if B-1 cell differentiation is blocked, anti-PtC B cells in these Tg mice are B-2-like in phenotype, suggesting the existence of an Ag-driven differentiative pathway from B-2 to B-1. In this study, we show that double Tg mice have a population of anti-PtC B cells that have the phenotypic characteristics of both B-2 and B-1 cells and that have the potential to differentiate to B-1 (B-1a and B-1b). Cyclosporin A blocks this differentiation and induces a more B-2-like phenotype in these cells. These findings indicate that these cells are intermediate between B-2 and B-1, further evidence of a B-2 to B-1 differentiative pathway.
Inhibition of Autoimmune Diabetes by Fas Ligand: The Paradox Is Solved
Authors: Kim Sunshin , Kim Kyoung-Ah , Hwang Dae-Youn , Lee Tae H. , Kayagaki Nobuhiko , Yagita Hideo , Lee Myung-Shik
Published: April 2014

Previous reports that diabetogenic lymphocytes did not induce diabetes in nonobese diabetic (NOD)-lpr mice suggested the critical role of Fas-Fas ligand (FasL) interaction in pancreatic β cell apoptosis. However, recent works demonstrated that FasL is not an effector molecule in islet β cell death. We addressed why diabetes cannot be transferred to NOD-lpr mice despite the nonessential role of Fas in β cell apoptosis. Lymphocytes from NOD-lpr mice were constitutively expressing FasL. A decrease in the number of FasL+ lymphocytes by neonatal thymectomy facilitated the development of insulitis. Cotransfer of FasL+ lymphocytes from NOD-lpr mice completely abrogated diabetes after adoptive transfer of lymphocytes from diabetic NOD mice. The inhibition of diabetes by cotransferred lymphocytes was reversed by anti-FasL Ab, indicating that FasL on abnormal lymphocytes from NOD-lpr mice was responsible for the inhibition of diabetes transfer. Pretreatment of lymphocytes with soluble FasL (sFasL) also inhibited diabetes transfer. sFasL treatment decreased the number of CD4+CD45RBlow cells and increased the number of propidium iodide-stained cells among CD4+CD45RBlow cells, suggesting that sFasL induces apoptosis on CD4+CD45RBlow “memory” cells. These results resolve the paradox between previous findings and suggest a new role for FasL in the treatment of autoimmune disorders. Our data also suggest that sFasL is involved in the deletion of potentially hazardous peripheral “memory” cells, contrary to previous reports that Fas on unmanipulated peripheral lymphocytes is nonfunctional.
Induction of T Cell Anergy in the Absence of CTLA-4/B7 Interaction
Authors: Frauwirth Kenneth A. , Alegre Maria-Luisa , Thompson Craig B.
Published: April 2014

Immunologic tolerance in T lymphocytes is maintained through both thymic and peripheral contributions. One peripheral tolerance mechanism is the induction of T cell anergy, a form of nonresponsiveness resulting from incomplete T cell activation, such as stimulation through the TCR in the absence of costimulation. Recent reports have suggested that engagement of the inhibitory receptor CTLA-4 by its B7 ligand is critical for the initiation of anergy. We tested the importance of CTLA-4 in anergy induction in primary T cells with an in vitro anergy system. Using both CTLA-4/B7-blocking agents and CTLA-4-deficient T cells, we found that T cell anergy can be established in the absence of CTLA-4 expression and/or function. Even in the absence of CTLA-4 signal transduction, T cells activated solely through TCR ligation lose the ability to proliferate as a result of autocrine IL-2 production upon subsequent receptor engagement. Thus, CTLA-4 signaling is not required for the development of T cell anergy.
Selective Suppression of IL-12 Production by Chemoattractants
Authors: Braun Michael C. , Lahey Edward , Kelsall Brian L.
Published: April 2014

We investigated the ability of chemoattractants to affect IL-12 production by human monocytes and dendritic cells. We found that pretreatment of monocytes with macrophage chemoattractant proteins (MCP-1 to -4), or C5a, but not stromal-derived factor-1, macrophage inflammatory protein-1α, RANTES, or eotaxin, inhibited IL-12 p70 production in response to stimulation with Staphylococcus aureus, Cowan strain 1 (SAC), and IFN-γ. The production of TNF-α and IL-10, however, was minimally affected by any of the chemoattractants. The degree of inhibition of IL-12 p70 production by MCP-1 to -4 was donor dependent and was affected by the autocrine inhibitory effects of IL-10. In contrast, C5a profoundly suppressed IL-12 production in an IL-10-independent fashion. Neither TGF-β1 nor PGE2 was important for the suppression of IL-12 by any of the chemoattractants tested. The accumulation of mRNA for both IL-12 p35 and p40 genes was inhibited by chemokine pretreatment. Interestingly, MCP-1 to -4 and C5a did not suppress IL-12 production by monocyte-derived dendritic cells (DC) stimulated with CD40 ligand and IFN-γ or by SAC and IFN-γ, suggesting that these factors may act at the site of inflammation to suppress IL-12 and IFN-γ production rather than in the lymph node to affect T cell priming. Despite the inability of C5a to inhibit IL-12 production by DCs, the receptor for C5a (CD88) was expressed by these cells, and recombinant C5a induced a Ca2+ flux. Taken together, these results define a range of chemoattractant molecules with the ability to suppress IL-12 production by human monocytes and have broad implications for the regulation of immune responses in vivo.
Role of Mitogen-Activated Protein Kinase Cascades in Mediating Lipopolysaccharide-Stimulated Induction of Cyclooxygenase-2 and IL-1β in RAW264 Macrophages
Authors: Caivano Matilde , Cohen Philip
Published: April 2014

LPS stimulation of RAW264 macrophages triggered the activation of mitogen- and stress-activated protein kinases-1 and -2 (MSK1, MSK2) and their putative substrates, the transcription factors cyclic AMP response element-binding protein (CREB) and activating transcription factor-1 (ATF1). The activation of MSK1/MSK2 was prevented by preincubating the cells with a combination of two drugs that suppress activation of the classical mitogen-activated protein kinase cascade and stress-activated protein kinase/p38, respectively, but inhibition was only partial in the presence of either inhibitor. The LPS-stimulated activation of CREB and ATF1, the transcription of the cyclooxygenase-2 (COX-2) and IL-1β genes (the promoters of which contain a cyclic AMP response element), and the induction of the COX-2 protein were prevented by the same drug combination, as well as by Ro 318220 or H89, potent inhibitors of MSK1/MSK2. Two other transcription factors, C/EBPβ and NF-κB, have been implicated in the transcription of the COX-2 gene. However, PD 98059 and/or SB 203580 did not prevent the LPS-induced increase in the level of the transcription factor C/EBPβ, and none of the four inhibitors used in this study prevented the activation of NF-κB. Our results demonstrate that two different mitogen-activated protein kinase cascades are rate limiting for the LPS-induced activation of CREB/ATF1 and the transcription of the COX-2 and IL-1β genes. They also suggest that MSK1 and MSK2 may play a role in these processes and hence are potential targets for the development of novel antiinflammatory drugs.
Intratumoral Coinjection of Two Adenoviruses, One Encoding the Chemokine IFN-γ-Inducible Protein-10 and Another Encoding IL-12, Results in Marked Antitumoral Synergy
Authors: Narvaiza Iñigo , Mazzolini Guillermo , Barajas Miguel , Duarte Marina , Zaratiegui Mikel , Qian Cheng , Melero Ignacio , Prieto Jesús
Published: April 2014

We have constructed a recombinant defective adenovirus that expresses functional murine IFN-γ-inducible protein-10 (IP-10) chemokine (AdCMVIP-10). Injection of AdCMVIP-10 into s.c. tumor nodules derived from the CT26 murine colorectal adenocarcinoma cell line displayed some antitumor activity but it was not curative in most cases. Previous studies have shown that injection of similar s.c. CT26 tumor nodules with adenovirus-encoding IL-12 (AdCMVIL-12) induces tumor regression in nearly 70% of cases in association with generation of antitumor CTL activity. AdCMVIP-10 synergizes with the antitumor effect of suboptimal doses of AdCMVIL-12, reaching 100% of tumor eradication not only against injected, but also against distant noninjected tumor nodules. Colocalization of both adenoviruses at the same tumor nodule was required for the local and distant therapeutic effects. Importantly, intratumoral gene transfer with IL-12 and IP-10 generated a powerful tumor-specific CTL response in a synergistic fashion, while both CD4 and CD8 T cells appeared in the infiltrate of regressing tumors. Moreover, the antitumor activity of IP-10 plus IL-12 combined gene therapy was greatly diminished by simultaneous in vivo depletion of CD4+ and CD8+ T cells but was largely unaffected by single depletion of each T cell subset. An important role for NK cells was also suggested by asialo GM1 depletion experiments. From a clinical point of view, the effects of IP-10 permit one to lower the required gene transfer level of IL-12, thus preventing dose-dependent IL-12-mediated toxicity while improving the therapeutic efficacy of the elicited antitumor response.
Salmonella-Type Heptaacylated Lipid A Is Inactive and Acts as an Antagonist of Lipopolysaccharide Action on Human Line Cells
Authors: Tanamoto Ken-ichi , Azumi Satoko
Published: April 2014

The stimulation of both THP-1 and U937 human-derived cells by Salmonella lipid A preparations from various strains, as assessed by TNF-α induction and NF-κB activation, was found to be very low (almost inactive) compared with Escherichia coli lipid A, but all of the lipid As exerted strong activity on mouse cells and on Limulus gelation activity. Experiments using chemically synthesized E. coli-type hexaacylated lipid A (506) and Salmonella-type heptaacylated lipid A (516) yielded clearer results. Both lipid A preparations strongly induced TNF-α release and activated NF-κB in mouse peritoneal macrophages and mouse macrophage-like cell line J774-1 and induced Limulus gelation activity, although the activity of the latter was slightly weaker than that of the former. However, 516 was completely inactive on both THP-1 and U937 cells in terms of both induction of TNF-α and NF-κB activation, whereas 506 displayed strong activity on both cells, the same as natural E. coli LPS. In contrast to the action of the lipid A preparations, all the Salmonella LPSs also exhibited full activity on human cells. However, the polysaccharide portion of the LPS neither exhibited TNF-α induction activity on the cells when administered alone or together with lipid A nor inhibited the activity of the LPS. These results suggest that the mechanism of activation by LPS or the recognition of lipid A structure by human and mouse cells may differ. In addition, both 516 and lipid A from Salmonella were found to antagonize the 506 and E. coli LPS action that induced TNF-α release and NF-κB activation in THP-1 cells.
The Role of High-Mobility Group I(Y) Proteins in Expression of IL-2 and T Cell Proliferation
Authors: Himes S. Roy , Reeves Raymond , Attema Joanne , Nissen Mark , Li Ying , Shannon M. Frances
Published: April 2014

The high-mobility group I(Y) (HMGI(Y)) family of proteins plays an important architectural role in chromatin and have been implicated in the control of inducible gene expression. We have previously shown that expression of HMGI antisense RNA in Jurkat T cells inhibits the activity of the IL-2 promoter. Here we have investigated the role of HMGI(Y) in controlling IL-2 promoter-reporter constructs as well as the endogenous IL-2 gene in both Jurkat T cells and human PBL. We found that the IL-2 promoter has numerous binding sites for HMGI(Y), which overlap or are adjacent to the known transcription factor binding sites. HMGI(Y) modulates binding to the IL-2 promoter of at least three transcription factor families, AP-1, NF-AT and NF-κB. By using a mutant HMGI that cannot bind to DNA but can still interact with the transcription factors, we found that DNA binding by HMGI was not essential for the promotion of transcription factor binding. However, the non-DNA binding mutant acts as a dominant negative protein in transfection assays, suggesting that the formation of functional HMGI(Y)-containing complexes requires DNA binding as well as protein:protein interactions. The alteration of HMGI(Y) levels affects IL-2 promoter activity not only in Jurkat T cells but also in PBL. Importantly, we also show here that expression of the endogenous IL-2 gene as well as proliferation of PBL are affected by changes in HMGI(Y) levels. These results demonstrate a major role for HMGI(Y) in IL-2 expression and hence T cell proliferation.
Tumor Rejection and Immune Memory Elicited by Locally Released LEC Chemokine Are Associated with an Impressive Recruitment of APCs, Lymphocytes, and Granulocytes
Authors: Giovarelli Mirella , Cappello Paola , Forni Guido , Salcedo Theodora , Moore Paul A. , LeFleur David W. , Nardelli Bernadetta , Carlo Emma Di , Lollini Pier-Luigi , Ruben Steve , Ullrich Stephen , Garotta Gianni , Musiani Piero
Published: April 2014

The human β chemokine known as LEC (also called NCC-4, HCC-4, or LMC) displays chemotactic activity for monocytes and dendritic cells. The possibility that its local presence increases tumor immunogenicity is addressed in this paper. TSA parental cells (TSA-pc) are poorly immunogenic adenocarcinoma cells that grow progressively, kill both nu/nu and syngeneic BALB/c mice, and give rise to lung metastases. TSA cells engineered to release LEC (TSA-LEC) are still able to grow in nu/nu mice, but are promptly rejected and display a marginal metastatic phenotype in BALB/c mice. Rejection is associated with a marked T lymphocyte and granulocyte infiltration, along with extensive macrophage and dendritic cell recruitment. NK cells and CD4+ T lymphocytes are uninfluential in TSA-LEC cell rejection, whereas both CD8+ lymphocytes and polymorphonuclear leukocytes play a major role. An antitumor immune memory is established very quickly after rejection, since 6 days later 75% of BALB/c mice were already resistant to a TSA-pc challenge. Spleen cells from rejecting mice display specific cytotoxic activity against TSA-pc and secrete IFN-γ and IL-2 when restimulated by TSA-pc. The ability of LEC to markedly improve recognition of poorly immunogenic cells by promoting APC-T cell cross-talk suggests that it could be an effective component of antitumor vaccines.
Tumor Immunity in Perforin-Deficient Mice: A Role for CD95 (Fas/APO-1)
Authors: Rosen Dalia , Li Jie-Hui , Keidar Sergey , Markon Ilya , Orda Ruben , Berke Gideon
Published: April 2014

CTL and NK cells use two distinct cytocidal pathways: 1) perforin and granzyme based and 2) CD95L/CD95 mediated. The former requires perforin expression by the effectors (CTL or NK), whereas the latter requires CD95 (Fas/APO-1) expression by the target. We have investigated how these two factors contribute to tumor immune surveillance by studying the immunity of perforin-deficient mice against the progressor C57BL/6 Lewis lung carcinoma 3LL, which expresses no CD95 when cultured in vitro. Unexpectedly, the results indicated that the perforin-independent CD95L/CD95 pathway of CTL/NK plays a role in acting against D122 and Kb39.5 (39.5) high and low metastatic sublines, respectively, derived from the 3LL tumor. Although no membrane-bound CD95 was detected on cultured D122 and 39.5 cells, surface CD95 expression on both D122 and 39.5 was considerably up-regulated when the tumors were grown in vivo. A similarly enhanced expression of CD95 was observed with three additional tumors; LF−, BW, and P815, injected into syngeneic and allogeneic mice. The finding of up-regulated CD95 expression on tumor cells placed in vivo suggests that a CD95-based mechanism plays a role in tumor immunity at early stages of tumor growth. Consequently, the progressive down-regulation of CD95 expression during tumor progression may indeed be an escape mechanism as previously reported. Together, these results suggest a role for CD95-dependent, perforin-independent immunity against certain tumors.
CD4+ T Cell Priming Accelerates the Clearance of Sendai Virus in Mice, but Has a Negative Effect on CD8+ T Cell Memory
Authors: Zhong Weimin , Marshall Dana , Coleclough Christopher , Woodland David L.
Published: April 2014

Current vaccines designed to promote humoral immunity to respiratory virus infections also induce potent CD4+ T cell memory. However, little is known about the impact of primed CD4+ T cells on the immune response to heterologous viruses that are serologically distinct, but that share CD4+ T cell epitopes. In addition, the protective capacity of primed CD4+ T cells has not been fully evaluated. In the present study, we addressed these two issues using a murine Sendai virus model. Mice were primed with an HN421–436 peptide that represents the dominant CD4+ T cell epitope on the hemagglutinin-neuraminidase (HN) of Sendai virus. This vaccination strategy induced strong CD4+ T cell memory to the peptide, but did not induce Abs specific for the Sendai virus virion. Subsequent Sendai virus infection of primed mice resulted in 1) a substantially accelerated virus-specific CD4+ T cell response in the pneumonic lung; 2) enhanced primary antiviral Ab-forming cell response in the mediastinal lymph nodes; and 3) accelerated viral clearance. Interestingly, the virus-specific CD8+ T cell response in the lung and the development of long-term memory CD8+ T cells in the spleen were significantly reduced. Taken together, our data demonstrate that primed CD4+ T cells, in the absence of pre-existing Ab, can have a significant effect on the subsequent immune responses to a respiratory virus infection.
The Tryptophan Catabolite Picolinic Acid Selectively Induces the Chemokines Macrophage Inflammatory Protein-1α and -1β in Macrophages
Authors: Bosco Maria Carla , Rapisarda Annamaria , Massazza Stefano , Melillo Giovanni , Young Howard , Varesio Luigi
Published: April 2014

We previously found that the tryptophan catabolite picolinic acid (PA) is a costimulus for the activation of macrophage effector functions. In this study, we have investigated the ability of PA to modulate the expression of chemokines in macrophages. We demonstrate that PA is a potent activator of the inflammatory chemokines MIP (macrophage inflammatory protein)-1α and MIP-1β (MIPs) mRNA expression in mouse macrophages in a dose- and time-dependent fashion and through a de novo protein synthesis-dependent process. The induction by PA occurred within 3 h of treatment and reached a peak in 12 h. The stimulatory effects of PA were selective for MIPs because other chemokines, including monocyte chemoattractant protein-1, RANTES, IFN-γ-inducible protein-10, MIP-2, and macrophage-derived chemokine, were not induced under the same experimental conditions and were not an epiphenomenon of macrophage activation because IFN-γ did not affect MIPs expression. Induction of both MIP-1α and MIP-1β by PA was associated with transcriptional activation and mRNA stabilization, suggesting a dual molecular mechanism of control. Iron chelation could be involved in MIPs induction by PA because iron sulfate inhibited the process and the iron-chelating agent, desferrioxamine, induced MIPs expression. We propose the existence of a new pathway leading to inflammation initiated by tryptophan catabolism that can communicate with the immune system through the production of PA, followed by secretion of chemokines by macrophages. These results establish the importance of PA as an activator of macrophage proinflammatory functions, providing the first evidence that this molecule can be biologically active without the need for a costimulatory agent.
Distinct T Cell/Renal Tubular Epithelial Cell Interactions Define Differential Chemokine Production: Implications for Tubulointerstitial Injury in Chronic Glomerulonephritides
Authors: Kuroiwa Takashi , Schlimgen Ryan , Illei Gabor G. , McInnes Iain B. , Boumpas Dimitrios T.
Published: April 2014

Chemokines can promote interstitial fibrosis that is, in turn, a strong predictor of renal failure in chronic glomerulonephritides (GN). Resident renal cells, including renal tubular epithelial cells (RTEC), represent a prominent source of chemokine expression. Evaluating those factors responsible for sustained chemokine production by RTEC during GN is therefore crucial. The contribution of interstitial T cells to such expression, and in particular the precise nature of their interactions with RTEC, are poorly understood. Activated T cell/RTEC coculture induced production of high levels of monocyte chemoattractant protein-1 (MCP-1), RANTES, and IFN-inducible protein-10 from RTEC. Using double-chamber cultures and activated T cell plasma membrane preparations we demonstrated that both cell contact and soluble factors contributed to RTEC chemokine production. Importantly, different chemokines exhibited distinct activation requirements. Thus, for RANTES cell contact was essential, but not sufficient. In contrast, either soluble factors or cell contact induced MCP-1 and IFN-inducible protein-10 production, although both pathways were required for a maximal response. Neutralization experiments identified critical roles in this process for proinflammatory cytokines such as TNF-α, IL-1β, and IFN-γ as well as membrane molecules such as LFA-1, CD40 ligand, and membrane bound TNF-α. Finally, chemotactic bioassays of T cell/RTEC coculture supernatants demonstrated 80% reduction of monocyte migration following MCP-1 neutralization, indicating a dominant role for this chemokine. In summary, activation of renal tubular cells by infiltrating T cells can amplify and perpetuate local inflammatory responses through chemokine production differentially mediated by soluble and cell contact-dependent factors. Recognition of this regulatory diversity has important implications in the choice of potential therapeutic targets in GN.
High Susceptibility for Cystic Fibrosis Human Airway Gland Cells to Produce IL-8 Through the IκB Kinase α Pathway in Response to Extracellular NaCl Content
Authors: Tabary Olivier , Escotte Sandie , Couetil Jean Paul , Hubert Dominique , Dusser Daniel , Puchelle Edith , Jacquot Jacky
Published: April 2014

Increasing evidence suggests that in airways from cystic fibrosis (CF) patients, inflammation may precede bacterial infection and be related to an endogenous dysregulation of proinflammatory cytokines in airway epithelial cells. Several investigators have reported that, in CF airway fluids, elevated NaCl concentrations may also contribute to the diseased state by inhibiting the bactericidal properties of airway fluid. Because many proinflammatory cytokines are transcriptionally regulated by the NF-κB, we investigated whether an elevated extracellular NaCl content in airway fluids significantly impaired the regulation of the NF-κB/IκBα complex and the chemokine IL-8 production in primary non-CF and CF human bronchial gland epithelial cells. Exposure of non-CF gland cells to hypotonic (85 mM) NaCl solution, compared with isotonic (115 mM) NaCl and hypertonic (170 mM) NaCl solutions, resulted in a significant decrease in IL-8 production that was paralleled by a strong inhibition of activated NF-κB associated with an increased cytosolic expression of IκBα and a decrease in the IκB kinase α protein level. In CF gland cells, we demonstrated that, compared with the high IL-8 in an hypertonic solution, the release of IL-8 was significantly reduced 2-fold in an isotonic solution and 5-fold in a hypotonic solution. Strikingly, exposure of CF bronchial gland cells to either hypotonic or isotonic milieu did not result in a marked inhibition of the activated NF-κB/IκBα system. This is the first demonstration that primary human CF bronchial gland cells exhibit abnormally high IL-8 production through constitutively activated NF-κB and high IκB kinase α level, whatever the hypo-, iso-, and hypertonic NaCl milieu.
In Vitro Priming with Adenovirus/gp100 Antigen-Transduced Dendritic Cells Reveals the Epitope Specificity of HLA-A*0201-Restricted CD8+ T Cells in Patients with Melanoma
Authors: Linette Gerald P. , Shankara Srinivas , Longerich Simonne , Yang Sixun , Doll Rhonda , Nicolette Charles , Preffer Frederic I. , Roberts Bruce L. , Haluska Frank G.
Published: April 2014

Replication-deficient recombinant adenovirus (Ad) encoding human gp100 or MART-1 melanoma Ag was used to transduce human dendritic cells (DC) ex vivo as a model system for cancer vaccine therapy. A second generation E1/E4 region deleted Ad which harbors the CMV immediate-early promoter/enhancer and a unique E4-ORF6/pIX chimeric gene was employed as the backbone vector. We demonstrate that human monocyte-derived DC are permissive to Ad infection at multiplicity of infection between 100 and 500 and occurs independent of the coxsackie Ad receptor. Fluorescent-labeled Ad was used to assess the kinetics and distribution of viral vector within DC. Ad-transduced DC show peak transgene expression at 24–48 h and expression remains detectable for at least 7 days. DC transduced with replication-deficient Ad do not exhibit any unusual phenotypic characteristics or cytopathic effects. DC transduced with Ad2/gp100v2 can elicit tumor-specific CTL in vitro from patients bearing gp100+ metastatic melanoma. Using a panel of gp100-derived synthetic peptides, we show that Ad2/gp100v2-transduced DC elicit Ag-specific CTL that recognize only the G209 and G280 epitopes, both of which display relatively short half-lives (∼7–8 h) on the surface of HLA-A*0201+ cells. Thus, patients with metastatic melanoma are not tolerant to gp100 Ag based on the detection of CD8+ T cells specific for multiple HLA-A*0201-restricted, gp100-derived epitopes.
Prediction of the Immunodominant Epitope of the Pyruvate Dehydrogenase Complex E2 in Primary Biliary Cirrhosis Using Phage Display
Authors: Rowley Merrill J. , Scealy Marita , Whisstock James C. , Jois Jennifer A. , Wijeyewickrema Lakshmi C. , Mackay Ian R.
Published: April 2014

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by autoantibodies reactive with the pyruvate dehydrogenase complex. A conformational epitope has been mapped to aa 91–227 within the inner lipoyl domain of the E2 subunit (pyruvate dehydrogenase complex E2 (PDC-E2)). We have used phage display to further localize this epitope. A random heptapeptide library was screened using IgG from two patients with PBC, with negative selection using pooled normal IgG. Phage that contained peptide inserts (phagotopes) selected using PBC sera differed from those selected using IgG from patients with RA or polychondritis. Two motifs occurred only among the PBC-selected phagotopes; these were MH (13 sequences, 16 phagotopes) and FV (FVEHTRW, FVEIYSP, FVLPWRI). The phagotopes selected were tested for reactivity with anti-PDC-E2 affinity purified from four patients with PBC. Phagotopes that contained 1 of 15 different peptide sequences were reactive with one or more of these four anti-PDC-E2 preparations, whereas phagotopes that contained 1of the remaining 28 sequences were negative. The peptides (FVLPWRI, MHLNTPP, MHLTQSP) encoded by three phagotopes that were strongly reactive with all four preparations of anti-PDC-E2 were synthesized. Each of the selected peptides, but not an irrelevant peptide, inhibited the reactivity by ELISA of PBC serum with recombinant PDC-E2 and reduced the inhibition of the enzyme activity of PDC by a PBC serum. The peptide sequences, along with the known NMR structure of the inner lipoyl domain of PDC-E2, allow the prediction of nonsequential residues 131HM132 and 178FEV180 that contribute to a conformational epitope.
Cutting Edge: Infection by the Agent of Human Granulocytic Ehrlichiosis Prevents the Respiratory Burst by Down-Regulatinggp91phox
Authors: Banerjee Rila , Anguita Juan , Roos Dirk , Fikrig Erol
Published: April 2014

The agent of human granulocytic ehrlichiosis (HGE) is an emerging tick-borne pathogen that resides in neutrophils and can be cultured in a promyelocytic (HL-60) cell line. In response to microbes, polymorphonuclear leukocytes normally activate the NADPH oxidase enzyme complex and generate superoxide anion (O2−). However, HL-60 cells infected with HGE bacteria did not produce O2− upon activation with PMA. RT-PCR demonstrated that HGE organisms inhibited mRNA expression of a single component of NADPH oxidase, gp91phox, and FACS analysis showed that plasma membrane-associated gp91phox protein was reduced on the infected cells. Infection with HGE organisms also decreased gp91phox mRNA levels in splenic neutrophils in a murine model of HGE, demonstrating this phenomenon in vivo. Therefore, HGE bacteria repress the respiratory burst by down-regulating gp91phox, the first direct inhibition of NADPH oxidase by a pathogen.
Cutting Edge: Increased Expression of Bcl-2 in Antigen-Specific Memory CD8+ T Cells
Authors: Grayson Jason M. , Zajac Allan J. , Altman John D. , Ahmed Rafi
Published: April 2014

Bcl-2 plays a critical role in regulating cell survival and apoptosis. We examined Bcl-2 expression in virus-specific CD8 T cells during the expansion, death, and memory phases of the T cell response following infection of mice with lymphocytic choriomeningitis virus (LCMV). Naive CD8 T cells expressed a basal level of Bcl-2 that was down-regulated in effector CD8 T cells just before the death phase. Bcl-2 levels remained low during the death phase but surviving memory CD8 T cells expressed higher levels of Bcl-2 than naive cells. These changes were shown to occur in LCMV TCR transgenic cells as well as virus-specific CD8 T cells in C57BL/6 and BALB/c mice identified by MHC class I tetramers. In all instances, memory CD8 T cells expressed higher levels of Bcl-2, suggesting that increased Bcl-2 expression plays a role in the long-term maintenance of memory CD8 T cells in vivo.
Cutting Edge: Ectopic Expression of the Chemokine TCA4/SLC Is Sufficient to Trigger Lymphoid Neogenesis
Authors: Fan Lian , Reilly Christina R. , Luo Yi , Dorf Martin E. , Lo David
Published: April 2014

To test whether accumulation of naive lymphocytes is sufficient to trigger lymphoid development, we generated mice with islet expression of the chemokine TCA4/SLC. This chemokine is specific for naive lymphocytes and mature dendritic cells (DC) which express the CCR7 receptor. Islets initially developed accumulations of T cells with DC, with scattered B cells at the perimeter. These infiltrates consolidated into organized lymphoid tissue, with high endothelial venules and stromal reticulum. Infiltrate lymphocytes showed a naive CD44low CD25− CD69− phenotype, though half were CD62L negative. When backcrossed to RAG-1 knockout, DC were not recruited. Interestingly, islet lymphoid tissue developed in backcrosses to Ikaros knockout mice despite the absence of normal peripheral nodes. Our results indicate that TCA4/SLC can induce the development and organization of lymphoid tissue through diffential recruitment of T and B lymphocytes and secondary effects on stromal cell development.
Persistence of Physiological Self Antigen Is Required for the Regulation of Self Tolerance
Authors: Garza Kristine M. , Agersborg Sally S. , Baker Elizabeth , Tung Kenneth S. K.
Published: April 2014

Endogenous Ag requirement for induction and maintenance of T cell tolerance has been extensively investigated in mice that express a transgenic Ag and/or its cognate transgenic TCR. In contrast, studies on tolerance for physiologically expressed self Ag and normal T cells are limited. Herein, we showed that the murine ovarian-specific ZP3 Ag is detectable from birth. Tolerance to ZP3 is detected in female relative to male mice. In comparison to males, 100-fold more ovarian peptide (pZP3) is required to elicit a comparable pathogenic response in females. Female tolerance to pZP3 was dependent on the presence of endogenous ovarian Ag, because neonatal ovariectomy converted the female response to that of males. Moreover, in female mice that were ovariectomized from the ages of 1–6 wk, the pZP3 responses were enhanced to the male level if ovaries were removed up to 7 days, but not 3 days, before adult challenge with pZP3. Thus, the physiologically expressed ZP3 Ag induces tolerance to pZP3, and the maintenance of tolerance is critically dependent on the continuous presence of the endogenous ovarian Ag. In contrast, exposure to endogenous ovarian Ag confined to the neonatal period is insufficient for the induction and maintenance of tolerance to ZP3.
Engagement of CD28 Modulates CXC Chemokine Receptor 4 Surface Expression in Both Resting and CD3-Stimulated CD4+ T Cells
Authors: Secchiero Paola , Zella Davide , Curreli Sabrina , Mirandola Prisco , Capitani Silvano , Gallo Robert C. , Zauli Giorgio
Published: April 2014

Optimal CD4+ T cell activation requires the cooperation of multiple signaling pathways coupled to the TCR-CD3 complex and to the CD28 costimulatory molecule. In this study, we have investigated the expression of surface CXC chemokine receptor 4 (CXCR4) in enriched populations of CD4+ T PBL, stimulated with anti-CD3 and anti-CD28 mAbs, immobilized on plastic. Anti-CD3 alone induced a progressive down-regulation of surface CXCR4, accompanied by a significant decline in the entry of the HXB2 T cell line-tropic (X4-tropic) HIV-1 clone in CD4+ T cells. Of note, this effect was strictly dependent on the presence in culture of CD14+ monocytes. On the other hand, anti-CD28 alone induced a small but reproducible increase in the expression of surface CXCR4 as well as in the entry of HXB2 HIV-1 clone in resting CD4+ T cells. When the two mAbs were used in combination, anti-CD28 potently synergized with anti-CD3 in inducing the expression of CD69 activation marker and stimulating the proliferation of CD4+ T cells. On the other hand, anti-CD28 counteracted the CXCR4 down-modulation induced by anti-CD3. The latter effect was particularly evident when anti-CD28 was associated to suboptimal concentrations of anti-CD3. Because CXCR4 is the major coreceptor for the highly cytopathic X4-tropic HIV-1 strains, which preferentially replicate in proliferating CD4+ T cells, the ability of anti-CD28 to up-regulate the surface expression of CXCR4 in both resting and activated CD4+ T cells provides one relevant mechanism for the progression of HIV-1 disease.
The CC Chemokine CKβ-11/MIP-3β/ELC/Exodus 3 Mediates Tumor Rejection of Murine Breast Cancer Cells Through NK Cells
Authors: Braun Stephen E. , Chen Keyue , Foster Richard G. , Kim Chang H. , Hromas Robert , Kaplan Mark H. , Broxmeyer Hal E. , Cornetta Kenneth
Published: April 2014

CKβ-11 chemoattracts T cells, B cells, dendritic cells, macrophage progenitors, and NK cells and facilitates dendritic cell and T cell interactions in secondary lymphoid tissues. We hypothesized that expression of CKβ-11 in tumor cells may generate antitumor immunity through these interactions. After transduction with the retroviral vector L(CKβ11)SN, the murine breast cancer cell line C3L5 (C3L5-CKβ11) showed expression of retroviral mRNA by Northern analysis and production of functional CKβ-11 by chemotaxis of human NK cells to C3L5-CKβ11 supernatant. Only 10% of mice injected with C3L5-CKβ11 developed tumors, compared with 100% of mice injected with a transduced control C3L5 line (C3L5-G1N). Importantly, the in vitro growth characteristics of the CKβ-11-transduced cell line were unaffected, suggesting the difference in growth in vivo was a result of chemokine production. Vaccination with C3L5-CKβ11 partially protected animals from parental C3L5 challenge. Immunodepletion with anti-asialo-GM1 or anti-CD4 during C3L5-CKβ11 vaccination significantly reduced CKβ-11 antitumor activity compared with control and anti-CD8-treated groups. Splenocytes from NK-depleted animals transferred the acquired immunity generated with C3L5-CKβ11 vaccination, while splenocytes from the CD4-depleted animals did not. These results indicate, for the first time, that expression of CKβ-11 in a breast cancer cell line mediates rejection of the transduced tumor through a mechanism involving NK and CD4+ cells. Furthermore, CKβ-11-transduced tumor cells generate long-term antitumor immunity that requires CD4+ cells. These studies demonstrate the potential role of CKβ-11 as an adjuvant in stimulating antitumor responses.
Caspase Enzyme Activity Is Not Essential for Apoptosis During Thymocyte Development
Authors: Doerfler Petra , Forbush Katherine A. , Perlmutter Roger M.
Published: April 2014

Caspases, a family of cysteine proteases, are critical mediators of apoptosis. To address the importance of caspases in thymocyte development, we have generated transgenic mice that express the baculovirus protein p35, a viral caspase inhibitor, specifically in the thymus. p35 expression inhibited Fas (CD95)-, CD3-, or peptide-induced caspase activity in vitro and conferred resistance to Fas-induced apoptosis. However, p35 did not block specific peptide-induced negative selection in OT1 and HY TCR transgenic mouse models. Even the potent pharmacological caspase inhibitor zVAD-FMK (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone) could not prevent peptide-induced deletion of OT1 thymocytes, although it improved basal thymocyte survival in vitro. Moreover, the developmental block observed in rag1−/− thymocytes, which lack pre-TCR signaling, was also not rescued by p35 expression. These results indicate that caspase-independent signal transduction pathways can mediate thymocyte death during normal T cell development.