bluuebunny/crossref_metadata_2025 huggingface dataset:
TCR Vaccines against a Murine T Cell Lymphoma: A Primary Role for Antibodies of the IgG2c Class in Tumor Protection
Authors: Lambert Stacie L. , Okada Craig Y. , Levy Ronald
Published: April 2014

Tumor-associated proteins can act as effective immunotherapeutic targets. Immunization with tumor TCR protein conjugated to the immunogenic protein keyhole limpet hemocyanin (KLH) protects mice from tumor challenge with the murine T cell lymphoma C6VL. The immune mechanisms responsible for this tumor protection are of interest for designing more effective vaccine strategies. Previous studies using depletion experiments had suggested a CD8-mediated component of protection induced by TCR-KLH vaccines. In this study we used CD8α knockout, μMT, and FcγR knockout mice to investigate the relative roles of CD8+ T cells and Ab in protective immunity induced by TCR-KLH immunization. We found that CD8+ T cells are not required for tumor protection, although they may contribute to protection. Vaccine-induced Abs are sufficient to mediate protection against this murine T cell lymphoma through an FcR-dependent mechanism. This was confirmed with Ab transfers, which protect challenged mice. Additionally, recombinase-activating gene 1−/− splenocytes can mediate Ab-dependent cellular cytotoxicity against this tumor in the presence of bound anti-TCR Abs. IFN-γ knockout mice demonstrated a requirement for IFN-γ, probably via generation of IgG2c Abs, in vaccine-induced tumor protection. IFN-γ knockout mice were not protected by immunization and had a severe impairment in IgG2c Ab production in response to immunization. Although mock-depleted anti-TCR Abs could transfer tumor protection, IgG2c-deficient anti-TCR Abs were unable to transfer tumor protection to wild-type mice. These results suggest that TCR-KLH vaccine-induced tumor protection in the C6VL system is primarily attributable to the induction of IgG2c Abs and humoral immunity.
A Switch in Costimulation from CD28 to 4-1BB during Primary versus Secondary CD8 T Cell Response to Influenza In Vivo
Authors: Bertram Edward M. , Dawicki Wojciech , Sedgmen Bradley , Bramson Jonathan L. , Lynch David H. , Watts Tania H.
Published: April 2014

4-1BBL−/− mice exhibit normal primary CD8 T cell responses to influenza virus, but show decreased CD8 T cell numbers late in the primary response as well as decreased secondary responses. In contrast, CD28−/− mice are defective in initial CD8 T cell expansion. Using agonistic anti-4-1BB Ab to replace the CD28 or 4-1BB signal, we examined the timing of the required signals for CD28 vs 4-1BB costimulation. A single dose of agonistic anti-4-1BB Ab added only during priming restores the secondary CD8 T cell response in CD28−/− mice. Once the T cell numbers in the primary response reach a minimum threshold, a full secondary response is achieved even in the absence of CD28. In contrast, anti-4-1BB added during priming fails to correct the defective secondary response in 4-1BBL−/− mice, whereas addition of anti-4-1BB during challenge fully restores this response. Thus, there is a switch in costimulatory requirement from CD28 to 4-1BB during primary vs recall responses. Adoptive transfer studies show that T cells primed in 4-1BBL−/− or wild-type mice are equally capable of re-expansion when rechallenged in wild-type mice. These studies rule out a model in which signals delivered through 4-1BB during priming program the T cells to give a full recall response and suggest that 4-1BB-4-1BBL interactions take place at later stages in the immune response. The results indicate that anti-4-1BB or 4-1BBL therapy will be most effective during the boost phase of a prime-boost vaccination strategy.
Antigen-Specific Inhibition of CD8+ T Cell Response by Immature Myeloid Cells in Cancer Is Mediated by Reactive Oxygen Species
Authors: Kusmartsev Sergei , Nefedova Yulia , Yoder Daniel , Gabrilovich Dmitry I.
Published: April 2014

Tumor growth is associated with the accumulation of immature myeloid cells (ImC), which in mice are characterized by the expression of Gr-1 and CD11b markers. These cells suppress Ag-specific CD8+ T cells via direct cell-cell contact. However, the mechanism of immunosuppressive activity of tumor-derived ImC remains unclear. In this study we analyzed the function of ImC isolated from tumor-free control and tumor-bearing mice. Only ImC isolated from tumor-bearing mice, not those from their control counterparts, were able to inhibit the Ag-specific response of CD8+ T cells. ImC obtained from tumor-bearing mice had significantly higher levels of reactive oxygen species (ROS) than ImC isolated from tumor-free animals. Accumulation of H2O2, but not superoxide or NO, was a major contributor to this increased pool of ROS. It appears that arginase activity played an important role in H2O2 accumulation in these cells. Inhibition of ROS in ImC completely abrogated the inhibitory effect of these cells on T cells, indicating that ImC generated in tumor-bearing hosts suppress the CD8+ T cell response via production of ROS. Interaction of ImC with Ag-specific T cells in the presence of specific Ags resulted in a significant increase in ROS production compared with control Ags. That increase was independent of IFN-γ production by T cells, but was mediated by integrins CD11b, CD18, and CD29. Blocking of these integrins with specific Abs abrogated ROS production and ImC-mediated suppression of CD8+ T cell responses. This study demonstrates a new mechanism of Ag-specific T cell inhibition mediated by ROS produced by ImCs in cancer.
Cutting Edge: Distinct TCR- and CD28-Derived Signals Regulate CD95L, Bcl-xL, and the Survival of Primary T Cells
Authors: Kerstan Andreas , Hünig Thomas
Published: April 2014

TCR-driven clonal expansion of T cells is limited by activation-induced cell death through CD95/CD95L interactions. This cell-contact dependent mechanism is attenuated by costimulation through CD28. Here, we show that primary rat lymph node T cells activated by “superagonistic” CD28-specific mAb, which do not require TCR-engagement for full T cell activation, do not up-regulate CD95L. CD28 superagonist activated T cells are highly resistant to artificial CD95 cross-linking, and display a marked up-regulation of the survival factor Bcl-xL. Consistently, NF-κB factors, known to promote Bcl-xL transcription, are strongly activated by superagonistic CD28 mAb stimulation. In contrast, a weaker induction of NFAT, which positively regulates the CD95L gene, in CD28 activated cells as compared with TCR- or TCR/CD28-stimulated cells was observed. Thus, by recruiting the mitogenic activity of CD28 in the absence of TCR engagement, the anti-apoptotic signals provided by costimulation are revealed without interfering proapoptotic effects induced by TCR stimulation.
Mature Human Thymocytes Migrate on Laminin-5 with Activation of Metalloproteinase-14 and Cleavage of CD44
Authors: Vivinus-Nebot Mylène , Rousselle Patricia , Breittmayer Jean-Philippe , Cenciarini Claire , Berrih-Aknin Sonia , Spong Suzanne , Nokelainen Pasi , Cottrez Françoise , Marinkovich M. Peter , Bernard Alain
Published: April 2014

We have previously shown that laminin-5 is expressed in the human thymic medulla, in which mature thymocytes are located. We now report that laminin-5 promotes migration of mature medullary thymocytes, whereas it has no effect on cortical immature thymocytes. Migration was inhibited by blocking mAbs directed against laminin-5 integrin receptors and by inhibitors of metalloproteinases. Interactions of thymocytes with laminin-5 induced a strong up-regulation of active metalloproteinase-14. However, we found that thymocytes did not cleave the laminin-5 γ2 chain, suggesting that they do not use the same pathway as epithelial cells to migrate on laminin-5. Interactions of thymocytes with laminin-5 also induced the release of a soluble fragment of CD44 cell surface molecule. Moreover, CD44-rich supernatants induced thymocyte migration in contrast with supernatants depleted in CD44 by immunoadsorption. CD44 cleavage was recently reported to be due to metalloproteinase-14 activation and led to increased migration in cancer cells. Thus, in this study, we show that laminin-5 promotes human mature thymocyte migration in vitro via a multimolecular mechanism involving laminin-5 integrin receptors, metalloproteinase-14 and CD44. These data suggest that, in vivo, laminin-5 may function in the migration of mature thymocytes within the medulla and be part of the thymic emigration process.
CCL17 and IL-10 as Effectors That Enable Alternatively Activated Macrophages to Inhibit the Generation of Classically Activated Macrophages
Authors: Katakura Tatsushi , Miyazaki Masaru , Kobayashi Makiko , Herndon David N. , Suzuki Fujio
Published: April 2014

Classically activated macrophages (CAMφ) have been described as a major effector cell on the host’s innate immunities. However, CAMφ are not generated in immunocompromised hosts whose alternatively activated macrophages (AAMφ) predominate. In this study, the mechanism by which AAMφ suppress the ability of resident macrophages (RMφ) to generate CAMφ was investigated. AAMφ were isolated from peritoneal exudates of mice 2 days after third-degree thermal injuries affecting 15% total body surface area. CAMφ were generated from RMφ (peritoneal Mφ from normal mice) through stimulation with CpG DNA, a typical CAMφ inducer. RMφ did not polarize to CAMφ when they were cultured with AAMφ in a dual-chamber Transwell even when supplemented with CpG DNA. In addition, RMφ stimulated with CpG DNA did not convert to CAMφ when they were cultured with the culture fluids of AAMφ (AAMφ Culture-Sup). AAMφ Culture-Sup contained IL-6, IL-10, CCL17, PGE2, and TGF-β. Among these, CCL17 and IL-10 inhibited CAMφ generation. The ability of AAMφ Culture-Sup to inhibit CAMφ generation was eliminated when the Culture-Sup was treated with a mixture of mAbs directed against CCL17 and IL-10. These results indicate that CCL17 and IL-10 released from AAMφ inhibit CAMφ generation from RMφ stimulated with CpG DNA.
Lck Is Required for Activation-Induced T Cell Death after TCR Ligation with Partial Agonists
Authors: Yu Xue-Zhong , Levin Steven D. , Madrenas Joaquin , Anasetti Claudio
Published: April 2014

TCR engagement can induce either T cell proliferation and differentiation or activation-induced T cell death (AICD) through apoptosis. The intracellular signaling pathways that dictate such a disparate fate after TCR engagement have only been partially elucidated. Non-FcR-binding anti-CD3 mAbs induce a partial agonist TCR signaling pattern and cause AICD on Ag-activated, cycling T cells. In this study, we examined TCR signaling during the induction of AICD by anti-CD3 fos, a non-FcR-binding anti-CD3 mAb. This mAb activates Fyn, Lck, and extracellular signal-regulated kinase, and induces phosphorylation of Src-like adapter protein, despite the inability to cause calcium mobilization or TCR polarization. Anti-CD3 fos also fails to effectively activate ζ-associated protein of 70 kDa or NF-κB. Using Ag-specific T cells deficient for Fyn or Lck, we provide compelling evidence that activation of Lck is required for the induction of AICD. Our data indicate that a selective and distinct TCR signaling pattern is required for AICD by TCR partial agonist ligands.
Importance of IL-10 for CTLA-4-Mediated Inhibition of Tumor-Eradicating Immunity
Authors: Jovasevic Vladimir M. , Gorelik Leonid , Bluestone Jeffrey A. , Mokyr Margalit B.
Published: April 2014

In this study, we show that engagement of CTLA-4 on tumor-infiltrating lymphocytes from low-dose melphalan (l-phenylalanine mustard (l-PAM))-treated MOPC-315 tumor bearers led to IL-10 secretion. In addition, the inhibitory activity of CTLA-4 ligation for IFN-γ secretion following stimulation with anti-CD3 plus anti-CD28 mAb depended on IL-10 production. Consistent with the importance of IL-10 for CTLA-4-mediated inhibition, administration of neutralizing anti-IL-10 mAb to low-dose l-PAM-treated MOPC-315 tumor bearers (administration of blocking anti-CTLA-4 mAb) resulted in enhanced tumor-infiltrating lymphocyte-mediated anti-MOPC-315 cytotoxicity and led to complete tumor eradication in a higher percentage of mice than that observed with low-dose l-PAM alone. The percentage of MOPC-315 tumor-bearing mice cured following administration of neutralizing anti-IL-10 mAb to low-dose l-PAM-treated MOPC-315 tumor bearers was comparable to that observed following administration of blocking anti-CTLA-4 mAb. Moreover, IL-10 neutralization together with CTLA-4 blockade did not provide added therapeutic benefits to low-dose l-PAM-treated MOPC-315 tumor bearers. Taken together, these results indicate that CTLA-4 blockade improves the therapeutic outcome of low-dose l-PAM for MOPC-315 tumor bearers by inhibiting IL-10 secretion as a consequence of blocking CTLA-4 ligation.
The Abundant NK Cells in Human Secondary Lymphoid Tissues Require Activation to Express Killer Cell Ig-Like Receptors and Become Cytolytic
Authors: Ferlazzo Guido , Thomas Dolca , Lin Shao-Lee , Goodman Kiera , Morandi Barbara , Muller William A. , Moretta Alessandro , Münz Christian
Published: April 2014

Natural killer cells are important cytolytic cells in innate immunity. We have characterized human NK cells of spleen, lymph nodes, and tonsils. More than 95% of peripheral blood and 85% of spleen NK cells are CD56dimCD16+ and express perforin, the natural cytotoxicity receptors (NCRs) NKp30 and NKp46, as well as in part killer cell Ig-like receptors (KIRs). In contrast, NK cells in lymph nodes have mainly a CD56brightCD16− phenotype and lack perforin. In addition, they lack KIRs and all NCR expression, except low levels of NKp46. The NK cells of tonsils also lack perforin, KIRs, NKp30, and CD16, but partially express NKp44 and NKp46. Upon IL-2 stimulation, however, lymph node and tonsilar NK cells up-regulate NCRs, express perforin, and acquire cytolytic activity for NK-sensitive target cells. In addition, they express CD16 and KIRs upon IL-2 activation, and therefore display a phenotype similar to peripheral blood NK cells. We hypothesize that IL-2 can mobilize the NK cells of secondary lymphoid tissues to mediate natural killing during immune responses. Because lymph nodes harbor 40% and peripheral blood only 2% of all lymphocytes in humans, this newly characterized perforin− NK cell compartment in lymph nodes and related tissues probably outnumbers perforin+ NK cells. These results also suggest secondary lymphoid organs as a possible site of NK cell differentiation and self-tolerance acquisition.
CpG-DNA Aided Cross-Priming by Cross-Presenting B Cells
Authors: Heit Antje , Huster Katharina M. , Schmitz Frank , Schiemann Matthias , Busch Dirk H. , Wagner Hermann
Published: April 2014

Covalent linkage of immunostimulatory CpG-DNA to OVA (CpG-OVA complex) results in CpG-DNA-aided cross-presentation of OVA by dendritic cells (DCs). In this study, we analyzed the thesis that CpG-OVA complexes may be cross-presented by B cells to route internalized Ag into the class I MHC presentation pathway. First, we describe that conjugation of CpG-DNA to OVA enhances up to 40-fold internalization of OVA by B cells, which in turn generate the CD8 T cell epitope SIINFEKL complexed to MHC class I, albeit less efficiently than DCs. Furthermore, upon internalization, CpG-DNA conjugated to OVA stimulates B cells to up-regulate costimulatory molecules and cytokines including IL-12. Adoptive transfer of CpG-OVA complex-loaded wild-type B cells cross-primes naive CD8 T cells both in wild-type mice and in MyD88-deficient mice. Overall, these findings disclose attributes of B cells, including cross-presentation of exogenous Ag and cross-priming of naive CD8 T cells that hitherto have been considered as hallmarks restricted to DCs.
Tumor-Infiltrating Lymphocyte Secretion of IL-6 Antagonizes Tumor-Derived TGF-β1 and Restores the Lymphokine-Activated Killing Activity
Authors: Hsiao Ya-Wen , Liao Kuang-Wen , Hung Shao-Wen , Chu Rea-Min
Published: April 2014

IL-6 is a multifunctional cytokine that regulates cell growth, differentiation, and cell survival. Many tumor cells produce TGF-β1, which allows them to evade CTL-mediated immune responses. IL-6 antagonizes TGF-β1 inhibition of CD3 cell activation. However, whether IL-6 restores NK activity, which also is suppressed by TGF-β1, is not known. We used canine transmissible venereal tumor (CTVT), which produces TGF-β1, as a model to determine whether IL-6 restores lymphokine-activated killer (LAK) activity. During the progression phase, CTVT cells stop expressing MHC molecules. During the regression phase, the number of surface MHC molecules increases dramatically on about one-third of tumor cells. Tumor cells that stop expressing MHC should be targeted by NK cells. In this study, we found that TGF-β1 secreted by CTVT cells suppressed LAK cytotoxicity. Interestingly, tumor-infiltrating lymphocytes (TIL) isolated from regressing CTVT secrete high concentrations of IL-6 and antagonize the anti-LAK activity of tumor cell TGF-β1. TIL also produce IL-6 during progression phase, but the concentration is too low to block the anti-LAK activity of TGF-β1. There is probably a threshold concentration of IL-6 needed to reverse TGF-β1-inhibited LAK activity. In addition, in the absence of TGF-β1, IL-6 derived from TIL does not promote the activity of LAK. This new mechanism, in which TIL manufacture high concentrations of IL-6 to block tumor TGF-β1 anti-LAK activity, has potential applications in cancer immunotherapy and tumor prognosis.
Phosphatidylinositol 3-Kinase Is a Determinant of Responsiveness to B Cell Antigen Receptor-Mediated Epstein-Barr Virus Activation
Authors: Iwakiri Dai , Takada Kenzo
Published: April 2014

B cell Ag receptor (BCR) cross-linking with anti-Ig Abs efficiently induces activation of latently infected EBV in some B cell lines, but not in others. The present study was aimed at defining the molecular mechanisms that determine the response to BCR-mediated EBV activation. Comparison of Burkitt’s lymphoma-derived Akata, Mutu-I, and Daudi cells, which are representative responders and nonresponders to BCR-mediated EBV activation, respectively, indicated that three signaling pathways, phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK), were activated in anti-Ig-treated Akata and Mutu-I cells. However, in anti-Ig-treated Daudi cells PI3K was not activated, ERK was faintly activated, and p38 MAPK was constitutively phosphorylated irrespective of anti-Ig treatment. Restoration of PI3K activity with insulin-like growth factor 1 restored ERK and p38 MAPK pathways, and was accompanied by EBV activation in anti-Ig-treated Daudi cells. In contrast, a specific inhibitor for PI3K, wortmannin, inhibited EBV activation by anti-Ig Abs in Akata and Mutu-I cells. Transfection assays in EBV-negative Daudi cells revealed that PI3K activated a promoter for BZLF1, which is a switch of EBV activation from a latent infection, in the absence of other EBV products suggesting that the BZLF promoter was a target of BCR signaling, and that PI3K was important for BCR-mediated BZLF1 activation. These results indicate that the absence of PI3K impedes the progression of signals through the BCR and becomes a determinant of unresponsiveness to BCR-mediated EBV activation.
E-Selectin, Thymus- and Activation-Regulated Chemokine/CCL17, and Intercellular Adhesion Molecule-1 Are Constitutively Coexpressed in Dermal Microvessels: A Foundation for a Cutaneous Immunosurveillance System
Authors: Chong Benjamin F. , Murphy Jo-Ellen , Kupper Thomas S. , Fuhlbrigge Robert C.
Published: April 2014

The success of the cutaneous immune system reflects its ability to rapidly and efficiently recruit leukocytes to areas of trauma and infection. Skin-homing memory T cells expressing cutaneous lymphocyte-associated Ag tether on the walls of postcapillary venules in inflamed skin via interaction with endothelial E-selectin and roll in response to the shear stress imparted by flowing blood. Rolling cells sample the vascular surface for chemoattractant compounds (e.g., thymus- and activation-regulated chemokine/CCL17 interacting with CCR4 on the leukocyte surface) and, if successfully stimulated, progress to firm arrest and transmigration mediated by LFA-1 and vascular ICAM-1. Although it is established that this sequence of events draws T cells into inflamed skin, the mechanisms directing trafficking of T cells to noninflamed skin are less well characterized. We hypothesized that basal expression and colocalization of E-selectin, chemokine (e.g., CCL17), and ICAM-1 in dermal vessels could serve to recruit T cells to noninflamed human skin. Immunohistochemical staining for E-selectin and CD31 demonstrated E-selectin expression in a restricted subset of dermal vessels in noninflamed human skin from three different sites. Confocal multicolor immunofluorescence imaging revealed a nonuniform distribution of E-selectin in dermal vessels as well as colocalization of E-selectin with CCL17 and ICAM-1. Coexpression of these molecules on blood vessels in noninflamed skin provides the basis for a model of cutaneous immunosurveillance system active in the absence of pathologic inflammation.
Escherichia coliExpressing Recombinant Antigen and Listeriolysin O Stimulate Class I-Restricted CD8+ T Cells following Uptake by Human APC
Authors: Hu Paul Q. , Tuma-Warrino Renee J. , Bryan Marianne A. , Mitchell Kathleen G. , Higgins Darren E. , Watkins Simon C. , Salter Russell D.
Published: April 2014

Vaccination against cancer or intracellular pathogens requires stimulation of class I-restricted CD8+ T cells. It is therefore important to develop Ag delivery vectors that will promote cross-presentation by APCs and stimulate appropriate inflammatory responses. Toward this goal, we tested the potential of Escherichia coli as an Ag delivery vector in in vitro human culture. Bacteria expressing enhanced green fluorescent protein were internalized efficiently by dendritic cells, as shown by flow cytometry and fluorescence microscopy. Phenotypic changes in DC were observed, including up-regulation of costimulatory molecules and IL-12p40 production. We tested whether bacteria expressing recombinant Ags could stimulate human T cells using the influenza matrix protein as a model Ag. Specific responses against an immunodominant epitope were seen using IFN-γ ELISPOT assays when the matrix protein was coexpressed with listeriolysin O, but not when expressed alone. THP-1 macrophages were also capable of stimulating T cells after uptake of bacteria, but showed slower kinetics and lower overall levels of T cell stimulation than dendritic cells. Increased phagocytosis of bacteria induced by differentiation of THP-1 increased their ability to stimulate T cells, as did opsonization. Presentation was blocked by proteasome inhibitors, but not by lysosomal protease inhibitors leupeptin and E64. These results demonstrate that recombinant E. coli can be engineered to direct Ags to the cytosol of human phagocytic APCs, and suggest possible vaccine strategies for generating CD8+ T cell responses against pathogens or tumors.
Regulation of c-Jun N-Terminal Kinase by MEKK-2 and Mitogen-Activated Protein Kinase Kinase Kinases in Rheumatoid Arthritis
Authors: Hammaker Deepa R. , Boyle David L. , Chabaud-Riou Martine , Firestein Gary S.
Published: April 2014

The mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) is a critical regulator of collagenase-1 production in rheumatoid arthritis (RA). The MAPKs are regulated by upstream kinases, including MAPK kinases (MAPKKs) and MAPK kinase kinases (MAP3Ks). The present study was designed to evaluate the expression and regulation of the JNK pathway by MAP3K in arthritis. RT-PCR studies of MAP3K gene expression in RA and osteoarthritis synovial tissue demonstrated mitogen-activated protein kinase/ERK kinase kinase (MEKK) 1, MEKK2, apoptosis-signal regulating kinase-1, TGF-β activated kinase 1 (TAK1) gene expression while only trace amounts of MEKK3, MEKK4, and MLK3 mRNA were detected. Western blot analysis demonstrated immunoreactive MEKK2, TAK1, and trace amounts of MEKK3 but not MEKK1 or apoptosis-signal regulating kinase-1. Analysis of MAP3K mRNA in cultured fibroblast-like synoviocytes (FLS) showed that all of the MAP3Ks examined were expressed. Western blot analysis of FLS demonstrated that MEKK1, MEKK2, and TAK1 were readily detectable and were subsequently the focus of functional studies. In vitro kinase assays using MEKK2 immunoprecipitates demonstrated that IL-1 increased MEKK2-mediated phosphorylation of the key MAPKKs that activate JNK (MAPK kinase (MKK)4 and MKK7). Furthermore, MEKK2 immunoprecipitates activated c-Jun in an IL-1 dependent manner and this activity was inhibited by the selective JNK inhibitor SP600125. Of interest, MEKK1 immunoprecipitates from IL-1-stimulated FLS appeared to activate c-Jun through the JNK pathway and TAK1 activation of c-Jun was dependent on JNK, ERK, and p38. These data indicate that MEKK2 is a potent activator of the JNK pathway in FLS and that signal complexes including MEKK2, MKK4, MKK7, and/or JNK are potential therapeutic targets in RA.
The Role of TNF-Related Activation-Induced Cytokine–Receptor Activating NF-κB Interaction in Acute Allograft Rejection and CD40L-Independent Chronic Allograft Rejection
Authors: Guillonneau Carole , Louvet Cédric , Renaudin Karine , Heslan Jean-Marie , Heslan Michèle , Tesson Laurent , Vignes Caroline , Guillot Cécile , Choi Yongwon , Turka Lawrence A. , Cuturi Maria-Cristina , Anegon Ignacio , Josien Régis
Published: April 2014

We analyzed the role of TNF-related activation-induced cytokine (TRANCE), a member of the TNF family expressed on activated T cells that shares functional properties with CD40L, and its receptor-activating NF-κB (RANK) which is mostly expressed on mature dendritic cells, during allogenic responses in vivo using a rodent heart allograft model. TRANCE mRNA was strongly up-regulated in acutely rejected allografts on days 4 and 5 posttransplantation whereas RANK was detected as early as day 1 but did not show further up-regulation during the first week. Immunofluoresence analyses of heart allografts showed that 80 and 100% of TRANCE and RANK-expressing cells were T cells and APCs, respectively. We show for the first time that short-term TRANCE blockade using a mouse RANKIg fusion molecule can significantly prolong heart allograft survival in both rat and mouse models. Similarly, rat heart allografts transduced with a RANKIg encoding recombinant adenovirus exhibited a significant prolongation of survival (14.3 vs 7.6 days, p < 0.0001). However, TRANCE blockade using RANKIg did not appear to inhibit allogeneic T and B cell priming humoral responses against RANKIg. Interestingly, TRANCE blockade induced strong up-regulation of CD40 ligand (CD40L) mRNA in allografts. Combined CD40L and TRANCE blockade resulted in significantly decreased chronic allograft rejection lesions as well as allogeneic humoral responses compared with CD40L blockade alone. We conclude that TRANCE-RANK interactions play an important role during acute allograft rejection and that CD40L-independent allogeneic immune responses can be, at least in part, dependent on the TRANCE pathway of costimulation.
Hypoxia Selectively Inhibits Monocyte Chemoattractant Protein-1 Production by Macrophages
Authors: Bosco Maria Carla , Puppo Maura , Pastorino Sandra , Mi Zenghui , Melillo Giovanni , Massazza Stefano , Rapisarda Annamaria , Varesio Luigi
Published: April 2014

Hypoxia, a local decrease in oxygen tension occurring in inflammatory and tumor lesions, modulates gene expression in macrophages. Because macrophages are important chemokine producers, we investigated the regulatory effects of hypoxia on macrophage-derived chemokines. We demonstrated that hypoxia inhibits the production of the macrophage and T lymphocyte chemotactic and activating factor, monocyte chemoattractant protein-1 (MCP-1). Exposure of mouse macrophages to low oxygen tension resulted in the down-regulation of constitutive MCP-1 mRNA expression and protein secretion. Hypoxia inhibitory effects were selective for MCP-1 because the chemokines macrophage inflammatory protein-1β (MIP-1β), RANTES, IFN-γ-inducible protein-10, and MIP-2 were not affected, and MIP-1α was induced. Hypoxia also inhibited, in a time-dependent fashion, MCP-1 up-regulation by IFN-γ and LPS. Moreover, the inhibitory action of hypoxia was exerted on human monocytic cells. MCP-1 down-regulation was associated with inhibition of gene transcription and mRNA destabilization, suggesting a dual molecular mechanism of control. Finally, we found that the triptophan catabolite picolinic acid and the iron chelator desferrioxamine, which mimic hypoxia in the induction of gene expression, differentially regulated the expression of MCP-1. This study characterizes a novel property of hypoxia as a selective inhibitor of MCP-1 production induced by different stimuli in macrophages and demonstrates that down-regulation of gene expression by hypoxia can be controlled at both transcriptional and posttranscriptional levels. Inhibition of MCP-1 may represent a negative regulatory mechanism to control macrophage-mediated leukocyte recruitment in pathological tissues.
HLA Class II Polymorphisms Determine Responses to Bacterial Superantigens
Authors: Llewelyn Martin , Sriskandan Shiranee , Peakman Mark , Ambrozak David R. , Douek Daniel C. , Kwok William W. , Cohen Jonathan , Altmann Daniel M.
Published: April 2014

The excessive immunological response triggered by microbial superantigens has been implicated in the etiology of a wide range of human diseases but has been most clearly defined for the staphylococcal and streptococcal toxic shock syndromes. Because MHC class II presentation of superantigens to T cells is not MHC-restricted, the possibility that HLA polymorphisms could influence superantigenicity, and thus clinical susceptibility to the toxicity of individual superantigens, has received little attention. In this study, we demonstrate that binding of streptococcal and staphylococcal superantigens to HLA class II is influenced by allelic differences in class II. For the superantigen streptococcal pyrogenic exotoxin A, class II binding is dependent on DQ α-chain polymorphisms such that HLA-DQA1*01 α-chains show greater binding than DQA1*03/05 α-chains. The functional implications of differential binding on T cell activation were investigated in various experimental systems using human T cells and murine Vβ8.2 transgenic cells as responders. These studies showed quantitative and qualitative differences resulting from differential HLA-DQ binding. We observed changes in T cell proliferation and cytokine production, and in the Vβ specific changes in T cell repertoire that have hitherto been regarded as a defining feature of an individual superantigen. Our observations reveal a mechanism for the different outcomes seen following infection by toxigenic bacteria.
Cysteinyl Leukotrienes Regulate Dendritic Cell Functions in a Murine Model of Asthma
Authors: Machida Ikuko , Matsuse Hiroto , Kondo Yuki , Kawano Tetsuya , Saeki Sachiko , Tomari Shinya , Obase Yasushi , Fukushima Chizu , Kohno Shigeru
Published: April 2014

Dendritic cells (DCs) act as APCs in the airway and play a critical role in allergy. Cysteinyl leukotrienes (cysLTs) synthesized from arachidonic acid are primary mediators of immediate asthmatic reaction. The aim of this study was to investigate the effects of cysLTs on Dermatophagoides farinae (Der f)-pulsed mouse myeloid DCs in inducing allergic airway inflammation in vitro and in vivo. Control DC (medium-pulsed), Der f-pulsed DC, cysLT-pulsed DC, Der f- and cysLT-pulsed DC, and Der f-pulsed and cysLT receptor antagonist (LTRA)-treated DC were prepared from murine bone marrow, and the production of cytokines ws compared. Subsequently, these DCs were intranasally instilled into another group of naive mice, followed by intranasal Der f challenge to induce allergic airway inflammation in vivo. Der f-pulsed DC produced significantly higher amounts of IL-10 and IL-12 compared with control DC. Der f- and cysLT-pulsed DC further increased IL-10 production compared with Der f-pulsed DC. In contrast, treatment of Der f-pulsed DC with LTRA increased IL-12 and decreased IL-10. Intranasal instillation of Der f-pulsed DC resulted in airway eosinophilia associated with a significant rise in IL-5 levels in the airway compared with control DC. Pulmonary eosinophilia and excess IL-5 were further enhanced in Der f- and cysLT-pulsed DC-harboring mice. In contrast, Der f-pulsed and LTRA-treated DC significantly inhibited airway eosinophilia, reduced IL-5, and increased IFN-γ in the airway. Our results suggest that cysLTs play an important role in the development of allergic airway inflammation by regulating the immunomodulatory functions of DCs.
TNF-α-Induced Apoptosis of Macrophages Following Inhibition of NF-κB: A Central Role for Disruption of Mitochondria
Authors: Liu Hongtao , Ma Yingyu , Pagliari Lisa J. , Perlman Harris , Yu Chenfei , Lin Anning , Pope Richard M.
Published: April 2014

Previously, we established that suppressing the constitutive activation of NF-κB in in vitro matured human macrophages resulted in apoptosis initiated by a decrease of the Bcl-2 family member, A1, and the loss of mitochondrial transmembrane potential (Δψm). This study was performed to characterize the mechanism of TNF-α-induced apoptosis in macrophages following the inhibition of NF-κB. The addition of TNF-α markedly enhanced the loss of Δψm and the induction of apoptotic cell death. Although caspase 8 was activated and contributed to DNA fragmentation, it was not necessary for the TNF-α-induced loss of Δψm. The inhibition of NF-κB alone resulted in the release of cytochrome c from the mitochondria, while both cytochrome c and second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI were released following the addition of TNF-α. Furthermore, c-Jun N-terminal kinase activation, which was sustained following treatment with TNF-α when NF-κB was inhibited, contributed to DNA fragmentation. These observations demonstrate that cytochrome c and second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI may be differentially released from the mitochondria, and that the sustained activation of c-Jun N-terminal kinase modulated the DNA fragmentation independent of the loss of Δψm.
Ets-1 Regulates TNF-α-Induced Matrix Metalloproteinase-9 and Tenascin Expression in Primary Bronchial Fibroblasts
Authors: Nakamura Yutaka , Esnault Stéphane , Maeda Takashi , Kelly Elizabeth A. B. , Malter James S. , Jarjour Nizar N.
Published: April 2014

Increased subepithelial deposition of extracellular matrix proteins is a key feature in bronchial asthma. Matrix metalloproteinase-9 (MMP-9) is a proteolytic enzyme that degrades the extracellular matrix. Tenascin is an extracellular matrix glycoprotein that is abundant in thickened asthmatic subbasement membrane. The expression of MMP-9 and tenascin reflects disease activity in asthma and airway remodeling. The molecular mechanisms regulating the expression of these proteins remain unknown. Both MMP-9 and tenascin promoters contain an Ets binding site, suggesting control by Ets-1. Thus, we hypothesized that Ets-1 expression is increased in asthma and that it contributed to enhanced MMP-9 and tenascin expression. To test this hypothesis, we determined the expression of Ets-1 in bronchial biopsies obtained from asthmatic subjects and determined the expression of Ets-1, MMP-9, and tenascin by bronchial fibroblasts activated ex vivo. We observed that nuclear extracts from TNF-α-activated fibroblasts showed increased Ets-binding activity. In addition, TNF-α-activated fibroblasts had increased expression of Ets-1 mRNA and protein, which preceded an increase in MMP-9 and tenascin mRNA. Furthermore, treatment of fibroblasts with Ets-1 antisense oligonucleotides down-regulated TNF-α-induced Ets-1, MMP-9, and, to a lesser extent, tenascin protein expression or activity. Taken together, these data demonstrate that TNF-α increases MMP-9 and tenascin expression in bronchial fibroblasts via the transcription factor Ets-1, and suggest a role for Ets-1 in airway remodeling in asthma.
Mycobacterium tuberculosis Recall Antigens Suppress HIV-1 Replication in Anergic Donor Cells via CD8+ T Cell Expansion and Increased IL-10 Levels
Authors: Ranjbar Shahin , Ly Nary , Thim Sok , Reynes Jean-Marc , Goldfeld Anne E.
Published: April 2014

Mycobacterium tuberculosis (MTb) is the leading cause of death in the setting of AIDS. MTb enhances the pathogenicity and accelerates the course of HIV disease and, furthermore, infection with HIV-1 increases the risk of reactivation or reinfection with MTb. In this study, we show that host-specific recall responses to one pathogen, MTb, has a direct effect upon the regulation of a second pathogen, HIV-1. Using cells from immunocompetent former tuberculosis (TB) patients who displayed either a persistently positive (responsive) or negative (anergic), delayed-type hypersensitivity (DTH) reaction to intradermal injection of purified protein derivative (PPD), we investigated the effect of recall Ags to MTb upon the replication of HIV-1 primary isolates in vitro. We show that HIV-1 replication of a T cell-tropic isolate was significantly impaired in MTb-stimulated PBMC from PPD-anergic donors. Furthermore, these donors displayed a significant increase in CD8+ T cells and IL-10 levels and lower levels of IL-2 and TNF-α relative to PPD-responsive donors in response to PPD stimulation. Strikingly, CD8+ T cell depletion and blocking of IL-10 significantly increased HIV-1 replication in these PPD-anergic donors, indicating that an immunosuppressive response to MTb recall Ags inhibits HIV-1 replication in PPD-anergic individuals. Therefore, immunotherapeutic approaches aimed at recapitulating Ag-specific MTb anergy in vivo could result in novel and effective approaches to inhibit HIV-1 disease progression in MTb/HIV-1 coinfection.
Molecular Analysis of TCR Clonotypes in LGL: A Clonal Model for Polyclonal Responses
Authors: O’Keefe Christine L. , Plasilova Magdalena , Wlodarski Marcin , Risitano Antonio M. , Rodriguez Alexander R. , Howe Evan , Young Neal S. , Hsi Eric , Maciejewski Jaroslaw P.
Published: April 2014

Large granular lymphocytic (LGL) leukemia is a clonal lymphoproliferative disorder of CTL associated with cytopenias resulting from an immune and cytokine attack on hemopoietic progenitor cells. Extreme clonality of CTL expansions seen in LGL leukemia makes it an ideal model to study the role of the T cell repertoire in other less-polarized immune-mediated disorders. Complementarity-determining region 3 (CDR3) of the TCR is a unique Ag-specific region that can serve as a molecular marker, or clonotype, of the disease-specific T cells. We studied the variable portion of the β-chain spectrum in a cohort of LGL leukemia patients. The CDR3 sequences were determined for the immunodominant clones and used to design clonotype-specific primers. By direct and semi-nested amplification, clonotype amplicons were found to be shared by multiple patients and controls. Analysis of the generated sequences demonstrated that the original clonotypes are rarely encountered in normal control samples; however, high levels of homology were found in both controls and patients. Clonotypes derived from individual LGL patients can be used as tumor markers for the malignant clone. More generally, clonotypic analysis and comparison of the variable portion of the β-chain CDR3-specific sequences from a large number of patients may lead to better subclassification of not only LGL but also other immune-mediated disorders.
Cutting Edge: Activation of Toll-Like Receptor 2 Induces a Th2 Immune Response and Promotes Experimental Asthma
Authors: Redecke Vanessa , Häcker Hans , Datta Sandip K. , Fermin Agnes , Pitha Paula M. , Broide David H. , Raz Eyal
Published: April 2014

Recognition of microbial components by APCs and their activation through Toll-like receptors (TLR) leads to the induction of adaptive immune responses. In this study, we show that activation of TLR2 by its synthetic ligand Pam3Cys, in contrast to activation of TLR9 by immunostimulatory DNA (ISS-ODN), induces a prominent Th2-biased immune response. Activation of APCs by Pam3Cys resulted in the induction of Th2-associated effector molecules like IL-13, and IL-1β, GM-CSF and up-regulation of B7RP-1, but low levels of Th1-associated cytokines (IL-12, IFNα, IL-18, IL-27). Accordingly, TLR2 ligands aggravated experimental asthma. These data indicate that the type of TLR stimulation during the initial phase of immune activation determines the polarization of the adaptive immune response and may play a role in the initiation of Th2-mediated immune disorders, such as asthma.
Depletion of CXCR2 Inhibits Tumor Growth and Angiogenesis in a Murine Model of Lung Cancer
Authors: Keane Michael P. , Belperio John A. , Xue Ying Y. , Burdick Marie D. , Strieter Robert M.
Published: April 2014

The Glu-Leu-Arg+ (ELR+) CXC chemokines are potent promoters of angiogenesis and have been demonstrated to induce a significant portion of nonsmall cell lung cancer-derived angiogenic activity and support tumorigenesis. ELR+ CXC chemokines share a common chemokine receptor, CXCR2. We hypothesized that CXCR2 mediates the proangiogenic effects of ELR+ CXC chemokines during tumorigenesis. To test this postulate, we used syngeneic murine Lewis lung cancer (LLC; 3LL, H-2b) heterotopic and orthotopic tumor model systems in C57BL/6 mice replete (CXCR2+/+) and deficient in CXCR2 (CXCR2−/−). We first demonstrated a correlation of the expression of endogenous ELR+ CXC chemokines with tumor growth and metastatic potential of LLC tumors. Next, we found that LLC primary tumors were significantly reduced in growth in CXCR2−/− mice. Moreover, we found a marked reduction in the spontaneous metastases of heterotopic tumors to the lungs of CXCR2−/− mice. Morphometric analysis of the primary tumors in CXCR2−/− mice demonstrated increased necrosis and reduced vascular density. These findings were further confirmed in CXCR2+/+ mice using specific neutralizing Abs to CXCR2. The results of these studies support the notion that CXCR2 mediates the angiogenic activity of ELR+ CXC chemokines in a preclinical model of lung cancer.
Lassa Virus Infection of Human Dendritic Cells and Macrophages Is Productive but Fails to Activate Cells
Authors: Baize Sylvain , Kaplon Jérôme , Faure Caroline , Pannetier Delphine , Georges-Courbot Marie-Claude , Deubel Vincent
Published: April 2014

Lassa fever is a hemorrhagic fever caused by Lassa virus (LV), an old-world Arenavirus. Little is known about the immune responses that occur during the disease, but protection seems to be linked to the induction of cellular responses specific for viral glycoproteins. Conversely, severe Lassa fever may be associated with immunosuppression. We studied the infection of human dendritic cells (DC) and macrophages (MP) by LV. Both these cell types are susceptible to LV infection. Viral nucleoprotein was detected in DC and MP, and high and moderate viral titers were obtained with culture supernatants of DC and MP, respectively. LV did not induce apoptosis in DC and MP. These cells were not activated by LV infection. No change was observed in the expression of surface molecules involved in activation, costimulation, adhesion, and Ag presentation following LV infection, or in the functional properties of DC. Inflammatory cytokine production was not detected at the mRNA or protein level after LV infection of DC and MP. Thus, MP, and particularly DC, are crucial targets for LV and are probably involved in the early replication of LV from the initial site of infection. The lack of activation and maturation of cells following infection may be associated with the immunosuppression observed in severe LV infection.
Analysis of Autoreactive CD4 T Cells in Experimental Autoimmune Encephalomyelitis after Primary and Secondary Challenge Using MHC Class II Tetramers
Authors: Bischof Felix , Hofmann Matthias , Schumacher Ton N. M. , Vyth-Dreese Florry A. , Weissert Robert , Schild Hansjörg , Kruisbeek Ada M. , Melms Arthur
Published: April 2014

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is primarily mediated by CD4 T cells specific for Ags in the CNS. Using MHC class II tetramers, we assessed expansion and phenotypic differentiation of polyclonal self-reactive CD4 T cells during EAE after primary and secondary challenge with the specific Ag. After EAE induction in SJL mice with proteolipid protein 139–151, CNS-specific T cells up-regulated activation markers and expanded in the draining lymph nodes and in the spleen. Less than 20% of total autoreactive T cells entered the CNS simultaneously with Th cells of other specificities. Almost all tetramer-positive cells in the CNS were activated and phenotypically distinct from the large peripheral pool. When EAE was induced in Ag-experienced mice, disease symptoms developed earlier and persisted longer; autoreactive T cells were more rapidly activated and invaded the CNS earlier. In striking contrast to specific CTLs that respond after secondary viral challenge, the absolute numbers of autoreactive CD4 T cells were not increased, indicating that the accelerated autoreactivity in Ag-experienced mice is not related to higher frequencies of autoreactive CD4 T cells.
Spontaneous Organ-Specific Th2-Mediated Autoimmunity in TCR Transgenic Mice
Authors: Candon Sophie , McHugh Rebecca S. , Foucras Gilles , Natarajan Kannan , Shevach Ethan M. , Margulies David H.
Published: April 2014

CD4+ T cells that lead to autoimmune gastritis (AIG) in BALB/c mice are either Th1 or Th2 cells. To test whether the phenotype of disease is related to the particular TCR expressed by the pathogenic cell, we have generated several lines of TCR transgenic mice using receptors cloned from pathogenic Th1 or Th2 cells. We previously described spontaneous inflammatory AIG in A23 mice, caused by the transgenic expression of the TCR from a Th1 clone, TXA23. In this study we describe the generation of A51 mouse lines, transgenic for the TCR of a CD4+ self-reactive Th2 clone, TXA51. A proportion of A51 mice spontaneously develop AIG by 10 wk of age, with a disease characterized by eosinophilic infiltration of the gastric mucosa and Th2 differentiation of transgenic T cells in the gastric lymph node. The Th2 phenotype of this autoimmune response seems to be related to a low availability of MHC class II-self peptide complexes. This in vivo model of spontaneous Th2-mediated, organ-specific autoimmunity provides a unique example in which the clonotypic TCR conveys the Th2 disease phenotype.
A Regulatory Role for CD37 in T Cell Proliferation
Authors: van Spriel Annemiek B. , Puls Kirsten L. , Sofi Mariam , Pouniotis Dodie , Hochrein Hubertus , Orinska Zane , Knobeloch Klaus-Peter , Plebanski Magdalena , Wright Mark D.
Published: April 2014

CD37 is a leukocyte-specific protein belonging to the tetraspanin superfamily. Previously thought to be predominantly a B cell molecule, CD37 is shown in this study to regulate T cell proliferation. CD37-deficient (CD37−/−) T cells were notably hyperproliferative in MLR, in response to Con A, or CD3-TCR engagement particularly in the absence of CD28 costimulation. Hyperproliferation was not due to differences in memory to naive T cell ratios in CD37−/− mice, apoptosis, or TCR down-modulation. Division cycle analyses revealed CD37−/− T cells to enter first division earlier than wild-type T cells. Importantly, proliferation of CD37−/− T cells was preceded by enhanced early IL-2 production. We hypothesized CD37 to be involved in TCR signaling and this was supported by the observation that CD4/CD8-associated p56Lck kinase activity was increased in CD37−/− T cells. Remarkably, CD37 cross-linking on human T cells transduced signals that led to complete inhibition of CD3-induced proliferation. In the presence of CD28 costimulation, CD37 engagement still significantly reduced proliferation. Taken together, these results demonstrate a regulatory role for CD37 in T cell proliferation by influencing early events of TCR signaling.