bluuebunny/crossref_metadata_2025 huggingface dataset:
Macrophage Inflammatory Protein-2 and KC Induce Chemokine Production by Mouse Astrocytes
Authors: Luo Yi , Fischer Falko R. , Hancock Wayne W. , Dorf Martin E.
Published: April 2014

Astrocytes are specialized cells of the CNS that are implicated in the pathogenesis of multiple sclerosis and experimental allergic encephalomyelitis. In acute and relapsing-remitting experimental allergic encephalomyelitis, the neutrophil chemoattractant CXC chemokines macrophage-inflammatory protein (MIP)-2 and KC are associated with reactive astrocytes in the parenchyma. In vitro treatment of primary astrocyte cultures with nanomolar concentrations of MIP-2 or KC markedly up-regulated expression of the monocyte/T cell chemoattractants monocyte chemoattractant protein-1, inflammatory protein-10, and RANTES by a mechanism that includes stabilization of mRNA. Production of TNF-α and IL-6 transcripts were also noted, as was autocrine induction of MIP-2 and KC message. In addition, low levels of MIP-1α and MIP-1β were induced following treatment with MIP-2 or KC. These effects are specific to astrocytes as MIP-2 treatment of microglial cells failed to elicit chemokine production. The astrocyte chemokine receptor for MIP-2 has 2.5 nM affinity for ligand. Astrocytes from CXCR2-deficient mice still respond to KC and MIP-2, indicating the presence of an alternative or novel high affinity receptor for these ligands. We propose that this KC/MIP-2 chemokine cascade may contribute to the persistence of mononuclear cell infiltration in demyelinating autoimmune diseases.
Studies on a Mechanism by Which Cytosolic Phospholipase A2 Regulates the Expression and Function of Type IIA Secretory Phospholipase A2
Authors: Kuwata Hiroshi , Yamamoto Shinji , Miyazaki Yoshitaka , Shimbara Satoko , Nakatani Yoshihito , Suzuki Hiroshi , Ueda Natsuo , Yamamoto Shozo , Murakami Makoto , Kudo Ichiro
Published: April 2014

Although it has been proposed that arachidonate release by several secretory phospholipase A2 (sPLA2) isozymes is modulated by cytosolic PLA2 (cPLA2), the cellular component(s) that intermediates between these two signaling PLA2s remains unknown. Here we provide evidence that 12- or 15-lipoxygenase (12/15-LOX), which lies downstream of cPLA2, plays a pivotal role in cytokine-induced gene expression and function of sPLA2-IIA. The sPLA2-IIA expression and associated PGE2 generation induced by cytokines in rat fibroblastic 3Y1 cells were markedly attenuated by antioxidants that possess 12/15-LOX inhibitory activity. 3Y1 cells expressed 12/15-LOX endogenously, and forcible overexpression of 12/15-LOX in these cells greatly enhanced cytokine-induced expression of sPLA2-IIA, with a concomitant increase in delayed PG generation. Moreover, studies using 293 cells stably transfected with sPLA2-IIA revealed that stimulus-dependent hydrolysis of membrane phospholipids by sPLA2-IIA was enhanced by overexpression of 12/15-LOX. These results indicate that the product(s) generated by the cPLA2-12/15-LOX pathway following cell activation may play two roles: enhancement of sPLA2-IIA gene expression and membrane sensitization that leads to accelerated sPLA2-IIA-mediated hydrolysis.
Endogenous and Exogenous IL-6 Inhibit Aeroallergen-Induced Th2 Inflammation
Authors: Wang Jingming , Homer Robert J. , Chen Qingsheng , Elias Jack A.
Published: April 2014

Chronic Th2-dominated inflammation and exaggerated IL-6 production are characteristic features of the asthmatic airway. To understand the processes that are responsible for the chronicity of this response and the role(s) of IL-6 in the regulation of airway Th2 inflammation, we compared the responses induced by OVA in sensitized wild-type mice, IL-6 deficient (−/−) mice, and transgenic mice in which IL-6 was overexpressed in the airway (CC10-IL-6 mice). When compared with wild-type mice, IL-6−/− mice manifest exaggerated inflammation and eosinophilia, increased levels of IL-4, IL-5, and IL-13 protein and mRNA, exaggerated levels of eotaxin, JE/monocyte chemoattractant protein-1, macrophage inflammatory protein-1α and -2, and mRNA, increased bronchoalveolar lavage (BAL) TGF-β1, and exaggerated airway responses to aerosolized methacholine. In contrast, CC10-IL-6 mice, on both C57BL/6 and BALB/c backgrounds, manifest diminished inflammation and eosinophilia, decreased levels of IL-4, IL-5, and IL-13 protein and mRNA, and decreased levels of bronchoalveolar lavage TGF-β1. IL-6 also decreased the expression of endothelial VCAM-1 and airway responsiveness to methacholine in these animals. These alterations in the IL-6−/− and CC10-IL-6 mice were not associated with significant decreases or increases in the levels of IFN-γ, respectively. These studies demonstrate that endogenous and exogenous IL-6 inhibit aeroallergen-induced Th2 inflammation and that this inhibition is not mediated by regulatory effects of IFN-γ. IL-6 may be an important anti-inflammatory, counterregulatory, and healing cytokine in the airway.
Peptide Dose, MHC Affinity, and Target Self-Antigen Expression Are Critical for Effective Immunotherapy of Nonobese Diabetic Mouse Prediabetes
Authors: Winer Shawn , Gunaratnam Lakshman , Astsatourov Igor , Cheung Roy K. , Kubiak Violetta , Karges Wolfram , Hammond-McKibben Denise , Gaedigk Roger , Graziano Daniel , Trucco Massimo , Becker Dorothy J. , Dosch H.-Michael
Published: April 2014

Cross-reactive T cells that recognize both Tep69 (dominant nonobese diabetic (NOD) T cell epitope in ICA69 (islet cell autoantigen of 69 kDa)) and ABBOS (dominant NOD T cell epitope in BSA) are routinely generated during human and NOD mouse prediabetes. Here we analyzed how systemic administration of these mimicry peptides affects progressive autoimmunity in adoptively transferred and cyclophosphamide-accelerated NOD mouse diabetes. These models were chosen to approximate mid to late stage prediabetes, the typical status of probands in human intervention trials. Unexpectedly, high dose (100 μg) i.v. ABBOS prevented, while Tep69 exacerbated, disease in both study models. Peptide effects required cognate recognition of endogenous self-Ag, because both treatments were ineffective in ICA69null NOD congenic mice adoptively transferred with wild-type, diabetic splenocytes. The affinity of ABBOS for NOD I-Ag7 was orders of magnitude higher than that of Tep69. This explained 1) the expansion of the mimicry T cell pool following i.v. Tep69, 2) the long-term unresponsiveness of these cells after i.v. ABBOS, and 3) precipitation of the disease after low dose i.v. ABBOS. Disease precipitation and prevention in mid to late stage prediabetes are thus governed by affinity profiles and doses of therapeutic peptides. ABBOS or ABBOS analogues with even higher MHC affinity may be candidates for experimental intervention strategies in human prediabetes, but the dose translation from NOD mice to humans requires caution.
B Lymphoblastoid Cell Lines as Efficient APC to Elicit CD8+ T Cell Responses Against a Cytomegalovirus Antigen
Authors: Sun Qi , Burton Robert L. , Dai Li-Jun , Britt William J. , Lucas Kenneth G.
Published: April 2014

Potent and readily accessible APC are critical for development of immunotherapy protocols to treat viral disease and cancer. We have shown that B lymphoblastoid cell lines (BLCL) that stably express CMV phosphoprotein 65 (BLCLpp65), as a result of retroviral transduction, can be used to generate ex vivo CTL cultures that possess cytotoxicity against CMV and EBV. In this report, we demonstrate that the EBV-specific cytotoxicity in the BLCLpp65-primed culture had a spectrum of EBV-Ag recognition similar to that of the BLCL-primed counterpart, suggesting that retroviral transduction and expression of the CMV Ag would not compromise the Ag-presenting capacity of BLCL. In addition, BLCLpp65 appeared to present multiple natural pp65 epitopes, because pp65-specific CTL, which recognized different CMV clinical isolates, were generated in BLCLpp65-primed cultures from individuals with various HLA backgrounds. Consistent with a polyclonal expansion of virus-specific CTL, T cell lines established from the BLCLpp65-primed CTL cultures expressed different TCR-Vβ. Although most of the virus-specific T cell isolates were CD8+, EBV-specific CD4+ lines were also established from BLCLpp65-primed cultures. Western blot analysis revealed that the CD8+ lines, but not the CD4+ line, expressed granzyme B, consistent with features of classic CTL. Thus, our results suggested that BLCL stably expressing a foreign Ag might be used as a practical APC to elicit CD8+ T cell responses.
Impairment of STAT Activation by IL-12 in a Patient with Atypical Mycobacterial and Staphylococcal Infections
Authors: Gollob Jared A. , Veenstra Korina G. , Jyonouchi Harumi , Kelly Anne M. , Ferrieri Patricia , Panka David J. , Altare Frédéric , Fieschi Claire , Casanova Jean-Laurent , Frank David A. , Mier James W.
Published: April 2014

IL-12 plays a pivotal role in the stimulation of immune responses against intracellular infections. This role is manifested in the increased susceptibility to atypical mycobacterial and salmonella infections among individuals whose lymphocytes lack expression of IL-12Rβ1. Here, we report on a patient with Mycobacterium avium infection, recurrent Staphylococcus aureus sinusitis, and multiple adverse drug reactions whose T cells were unable to produce IFN-γ or proliferate in response to IL-12 despite the expression of wild-type IL-12Rβ1 and IL-12Rβ2. The defect in these functional responses to IL-12 was selective, as cytolytic activity induced by IL-12 was intact, and lymphocytes were responsive to stimulation by IL-2. An examination of cytokine signaling revealed that STAT4 and extracellular regulated kinase 1 (ERK1) activation by IL-12 was intact, whereas the activation of STAT1, -3, and -5 by IL-12 was lost. This impairment of STAT activation was specific for IL-12, as STAT activation by IL-2, IL-15, and IFN-γ was unaffected. These findings demonstrate that the activation of STAT4 alone is not sufficient for IL-12-induced IFN-γ production and proliferation and suggest that other STATs play a role in these responses to IL-12. While the etiology of the impaired IL-12 signaling in this patient has not yet been elucidated, the absence of mutations in IL-12Rβ1 or IL-12Rβ2 and the preservation of STAT4 activation raise the possibility that there may be a mutation in an as yet undiscovered component of the IL-12 signaling complex that is normally required for the recruitment and activation of STAT1, -3, and -5.
Spontaneous Production of C-C Chemokines by Individuals Infected with Human T Lymphotropic Virus Type II (HTLV-II) Alone and HTLV-II/HIV-1 Coinfected Individuals
Authors: Lewis Martha J. , Gautier Virginie W. , Wang Xue-Ping , Kaplan Mark H. , Hall William W.
Published: April 2014

To investigate the immunological features of human T lymphotropic virus type II (HTLV-II) infection and specific mechanisms whereby HTLV-II might influence the progression of HIV-1 disease in coinfected individuals, we have analyzed the production of the C-C chemokines RANTES and macrophage inflammatory proteins 1α and 1β (MIP-1α and MIP-1β) by PBMCs from HTLV-II-infected and HTLV-II/HIV-1-coinfected individuals. We observed spontaneous production of significant levels of MIP-1α and -1β and, to a lesser extent, RANTES, from individuals infected with HTLV-II alone or with concomitant HIV-1 infection. Spontaneous C-C chemokine production was not observed in PBMCs from uninfected or HIV-1-infected individuals. Although HTLV-II is known to preferentially infect CD8+ lymphocytes in vivo, we observed that whereas RANTES was produced exclusively by the CD8+-enriched fraction, MIP-1α and -1β were produced by both the CD8+-enriched and CD8+-depleted fractions of HTLV-II-infected PBMCs. RT-PCR demonstrated active expression of the HTLV-II regulatory protein Tax in the infected CD8+ T lymphocyte population, and it was further shown that Tax transactivates the promoters of MIP-1β and RANTES. Therefore, it appears that HTLV-II stimulates the production of C-C chemokines both directly at a transcriptional level via the viral transactivator Tax and also indirectly. Although the HTLV-II-infected individuals in this study are all virtually asymptomatic, they certainly display an abnormal immune phenotype. Moreover, our findings suggest that HTLV-II, via chemokine production, would be expected to alter the progression of HIV-1 infection in coinfected individuals.
Generation of Tumor-Reactive CTL Against the Tumor-Associated Antigen HER2 Using Retrovirally Transduced Dendritic Cells Derived from CD34+ Hemopoietic Progenitor Cells
Authors: Meyer zum Büschenfelde Christian , Nicklisch Nicole , Rose-John Stefan , Peschel Christian , Bernhard Helga
Published: April 2014

Ag-specific CD8+ CTL are crucial for effective tumor rejection. Attempts to treat human malignancies by adoptive transfer of tumor-reactive CTL have been limited due to the difficulty of generating and expanding autologous CTL with defined Ag specificity. The current study examined whether human CTL can be generated against the tumor-associated Ag HER2 using autologous dendritic cells (DC) that had been genetically engineered to express HER2. DC progenitors were expanded by culturing CD34+ hemopoietic progenitor cells in the presence of the designer cytokine HyperIL-6. Proliferating precursor cells were infected by a retroviral vector encoding the HER2 Ag and further differentiated into CD83+ DC expressing high levels of MHC, adhesion, and costimulatory molecules. Retroviral transduction of DC resulted in the expression of the HER2 molecule with a transduction efficiency of 15%. HER2-transduced DC correctly processed and presented the Ag, because HLA-A*0201-positive DC served as targets for CTL recognizing the HLA-A*0201-binding immunodominant peptide HER2369–377. HER2-transduced DC were used as professional APCs for stimulating autologous T lymphocytes. Following repetitive stimulation, a HER2-specific, HLA-A*0201-restricted CTL line was generated that was capable of lysing HLA-A*0201-matched tumor cells overexpressing HER2. A CD8+ T cell clone could be generated that displayed the same specificity pattern as the parenteral CTL line. The ability to generate and expand HER2-specific, MHC class I-restricted CTL clones using HER2-transduced autologous DC in vitro facilitates the development of adoptive T cell transfer for patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.
Cutting Edge: B Cell Linker Protein Is Dispensable for the Allelic Exclusion of Immunoglobulin Heavy Chain Locus But Required for the Persistence of CD5+ B Cells
Authors: Xu Shengli , Wong Siew-Cheng , Lam Kong-Peng
Published: April 2014

The pre-B cell receptor (pre-BCR) and the BCR are required for B lymphopoiesis and for the allelic exclusion of Ig genes. Mice lacking B cell linker (BLNK) protein that is a component of the BCR signaling pathway have impaired B cell development. In this report, we show that allelic exclusion is intact in BLNK−/− mice harboring a VH12 transgene. This differs from mice lacking the tyrosine kinase Syk that is upstream of BLNK in BCR signaling and contrasts with mice lacking SLP-76 that is the equivalent adaptor molecule in TCR-signal transduction. We also show that, whereas most wild-type VH12-expressing B cells are CD5+, the majority of the splenic VH12-expressing BLNK−/− B cells are CD5−. A small population of VH12-expressing, BLNK−/− CD5+ B cells is detectable in the peritoneal cavity of younger but not older mice. This suggests that BLNK deficiency affects not only the generation but also the persistence of B-1 cells.
A Subset of Cytolytic Dendritic Cells in Rat
Authors: Trinité Benjamin , Voisine Cécile , Yagita Hideo , Josien Régis
Published: April 2014

Dendritic cells (DCs) are a rare population of leukocytes specialized in Ag processing and presentation to T cells. We have previously shown that cultured rat splenic DCs exhibit a cytotoxic activity against selected target cells. In this study, we analyzed this function in DCs freshly prepared from lymphoid organs using the DC-specific OX62 mAb and magnetic beads. Freshly extracted splenic DCs, but not lymph node and thymic DCs, exhibited a strong and moderate cytotoxic activity against YAC-1 and K562 target cells, respectively. FACS analyses showed that spleen contained a minor subset (10–15%) of CD4+ and class IIint DCs that also expressed the OX41 Ag and the lymphoid-related Ags CD5 and CD90 (Thy-1) and a major (80–85%) subset of CD4−/OX41−/CD5− and class IIint DCs. The cytotoxic activity of splenic DCs was strictly restricted to the CD4− DCs, a subset poorly represented in LN and thymus. Contrasting with our previous report using cultured splenic DCs, freshly isolated splenic DCs killed YAC-1 cells using a Ca2+-independent mechanism, but this function did not appear mediated by Fas ligand, TNF-related apoptosis-inducing ligand, or TNF-α. Therefore, rat DCs contain a subset of naturally cytolytic cells that could play a role in both innate and acquired immune responses. Together with our previous report, these data suggest that rat DCs can use two mechanisms of cytotoxicity depending on their maturation/activation state.
Supra-Agonist Peptides Enhance the Reactivation of Memory CTL Responses
Authors: Micheletti Fabiola , Canella Alessandro , Vertuani Simona , Marastoni Mauro , Tosi Lara , Volinia Stefano , Traniello Serena , Gavioli Riccardo
Published: April 2014

Single amino acid substitutions at TCR contacts may transform a natural peptide Ag in CTL ligands with partial agonist, antagonist, or null activity. We obtained peptide variants by changing nonanchor amino acid residues involved in MHC class I binding. These peptides were derived from a subdominant HLA-A2-presented, latent membrane protein 2-derived epitope expressed in EBV-infected cells and in EBV-associated tumors. We found that small structural changes produced ligands with vastly different activities. In particular, the variants that associated more stably to HLA-A2/molecules did not activate any CTL function, behaving as null ligands. Interestingly, T cell stimulations performed with the combination of null ligands and the natural epitope produced significantly higher specific CTL reactivation than reactivation of CTLs induced by the wild-type epitope alone. In addition, these particular variants activated memory CTL responses in the presence of concentrations of natural epitope that per se did not induce T cell responses. We show here that null ligands increased ZAP-70 tyrosine kinase activation induced by the natural epitope. Our results demonstrate for the first time that particular peptide variants, apparently behaving as null ligands, interact with the TCR, showing a supra-agonist activity. These variant peptides did not affect the effector T cell functions activated by the natural epitope. Supra-agonist peptides represent the counterpart of antagonists and may have important applications in the development of therapeutic peptides.
Possible Involvement of Cyclophilin B and Caspase-Activated Deoxyribonuclease in the Induction of Chromosomal DNA Degradation in TCR-Stimulated Thymocytes
Authors: Nagata Takuya , Kishi Hiroyuki , Liu Qing Li , Yoshino Tomoyasu , Matsuda Tadashi , Jin Zhe Xiong , Murayama Kimie , Tsukada Kazuhiro , Muraguchi Atsushi
Published: April 2014

TCR engagement of immature CD4+CD8+ thymocytes induces clonal maturation (positive selection) as well as clonal deletion (negative selection) in the thymus. However, the cell death execution events of thymocytes during the negative selection process remain obscure. Using a cell-free system, we identified two different DNase activities in the cytosol of in vivo anti-TCR-stimulated murine thymocytes: one that induced chromosomal DNA fragmentation, which was inhibited by an inhibitor of caspase-activated DNase, and another that induced plasmid DNA degradation, which was not inhibited by an inhibitor of caspase-activated DNase. We purified the protein to homogeneity that induced plasmid DNA degradation from the cytosol of anti-CD3-stimulated thymocytes and found that it is identical with cyclophilin B (Cyp B), which was reported to locate in endoplasmic reticulum. Ab against Cyp B specifically inhibited the DNA degradation activity in the cytosol of anti-CD3-stimulated thymocytes. Furthermore, recombinant Cyp B induced DNA degradation of naked nuclei, but did not induce internucleosomal DNA fragmentation. Finally, we demonstrated that TCR engagement of a murine T cell line (EL4) with anti-CD3/CD28 resulted in the release of Cyp B from the microsome fraction to the cytosol/nuclear fraction. Our data strongly suggest that both active caspase-activated DNase and Cyp B may participate in the induction of chromosomal DNA degradation during cell death execution of TCR-stimulated thymocytes.
Arsenite Induces Apoptosis of Murine T Lymphocytes Through Membrane Raft-Linked Signaling for Activation of c-Jun Amino-Terminal Kinase
Authors: Hossain Khaled , Akhand Anwarul A. , Kato Masashi , Du Jun , Takeda Kozue , Wu Jianghong , Takeuchi Kei , Liu Wei , Suzuki Haruhiko , Nakashima Izumi
Published: April 2014

Because of its dual roles in acute toxicity and in therapeutic application in cancer treatment, arsenic has recently attracted a renewed attention. In this study, we report NaAsO2-induced signal cascades from the cell surface to the nucleus of murine thymic T lymphocytes that involve membrane rafts as an initial signal transducer. NaAsO2 induced apoptosis through fragmentation of DNA, activation of caspase, and reciprocal regulation of Bcl-2/Bax with the concomitant reduction of membrane potential. We demonstrated that NaAsO2-induced caspase activation is dependent on curcumin-sensitive c-Jun amino-terminal kinase and barely dependent on SB203580-sensitive p38 kinase or PD98059-sensitive extracellular signal-regulated kinase. Additionally, staurosporine, which severely inhibited the activation of mitogen-activated protein (MAP) family kinases and c-Jun, partially blocked the NaAsO2-mediated signal for poly(ADP-ribose) polymerase (PARP) degradation. Potentially as the initial cell surface event for intracellular signaling, NaAsO2 induced aggregation of GPI-anchored protein Thy-1 and superoxide production. This Thy-1 aggregation and subsequent activation of MAP family kinase and c-Jun and the degradation of PARP induced by NaAsO2 were all inhibited by DTT, suggesting the requirement of interaction between arsenic and protein sulfhydryl groups for those effects. β cyclodextrin, which sequestrates cholesterol from the membrane rafts, inhibited NaAsO2-induced activation of protein tyrosine kinases and MAP family kinases, degradation of PARP, and production of superoxide. In addition, β cyclodextrin dispersed NaAsO2-induced Thy-1 clustering. These results suggest that a membrane raft integrity-dependent cell surface event is a prerequisite for NaAsO2-induced protein tyrosine kinase/c-Jun amino-terminal kinase activation, superoxide production, and downstream caspase activation.
The Expression of p18INK4 and p27kip1 Cyclin-Dependent Kinase Inhibitors Is Regulated Differently During Human B Cell Differentiation
Authors: Schrantz Nicolas , Beney Georges Edouard , Auffredou Marie Thérèse , Bourgeade Marie Françoise , Leca Gérald , Vazquez Aimé
Published: April 2014

Cell cycle progression is under the control of cyclin-dependent kinases (cdks), the activity of which is dependent on the expression of specific cdk inhibitors. In this paper we report that the two cdk inhibitors, p27Kip1 and p18INK4c, are differently expressed and control different steps of human B lymphocyte activation. Resting B cells contain large amounts of p27Kip1 and no p18INK4c. In vitro stimulation by Staphylococcus aureus Cowan 1 strain or CD40 ligand associated with IL-10 and IL-2 induces a rapid decrease in p27Kip1 expression combined with cell cycle entry and progression. In contrast, in vitro Ig production correlates with specific expression of p18INK4c and early G1 arrest. This G1 arrest is associated with inhibition of cyclin D3/cdk6-mediated retinoblastoma protein phosphorylation by p18INK4c. A similar contrasting pattern of p18INK4c and p27Kip1 expression is observed both in B cells activated in vivo and in various leukemic cells. Expression of p18INK4c was also detected in various Ig-secreting cell lines in which both maximum Ig secretion and specific p18INK4c expression were observed during the G1 phase. Our study shows that p27Kip1 and p18INK4c have different roles in B cell activation; p27Kip1 is involved in the control of cell cycle entry, and p18INK4c is involved in the subsequent early G1 arrest necessary for terminal B lymphocyte differentiation.
Identification of Five Different Patr Class I Molecules That Bind HLA Supertype Peptides and Definition of Their Peptide Binding Motifs
Authors: McKinney Denise M. , Erickson Ann L. , Walker Christopher M. , Thimme Robert , Chisari Francis V. , Sidney John , Sette Alessandro
Published: April 2014

We have sequenced the Pan troglodytes class I (Patr) molecules from three common chimpanzees and expressed them as single molecules in a class I-deficient cell line. These lines were utilized to obtain purified class I molecules to define the peptide binding motifs associated with five different Patr molecules. Based on these experiments, as well as analysis of the predicted structure of the B and F polymorphic MHC pockets, we classified five Patr molecules (Patr-A*0101, Patr-B*0901, Patr-B*0701, Patr-A*0602, and Patr-B*1301) within previously defined supertype specificities associated with HLA class I molecules (HLA-A3, -B7, -A1, and -A24 supertypes). The overlap in the binding repertoire between specific HLA and Patr class I molecules was in the range of 33 to 92%, depending on the particular Patr molecule as assessed by the binding of HIV-, hepatitis B virus-, and hepatitis C virus-derived epitopes. Finally, live cell binding assays of nine chimpanzee-derived B cell lines demonstrated that HLA supertype peptides bound to Patr class I molecules with frequencies in the 20–50% range.
Molecular Cloning, Characterization, and Expression of TNF cDNA and Gene from Japanese FlounderParalychthys olivaceus
Authors: Hirono Ikuo , Nam Bo-Hye , Kurobe Tomofumi , Aoki Takashi
Published: April 2014

We cloned a cDNA and the gene for Japanese flounder TNF. The TNF cDNA consisted of 1217 bp, which encoded 225 amino acid residues. The identities between Japanese flounder TNF and members of the mammalian TNF family were ∼20–30%. The positions of cysteine residues that are important for disulfide bonds were conserved with respect to those in mammalian TNF-α. The Japanese flounder TNF gene has a length of ∼2 kbp and consists of four exons and three introns. The positions of the exon-intron junction positions of Japanese flounder TNF gene are similar to those of human TNF-α. However, the length of the first intron of Japanese flounder is much shorter than that of the human TNF-α gene. There are simple CA or AT dinucleotide repeats in the 5′-upstream and 3′-downstream regions of the Japanese flounder TNF gene. Southern blot hybridization indicted that Japanese flounder TNF exists as a single copy. Expression of Japanese flounder TNF mRNA is greatly induced after stimulation of PBLs with LPS, Con A, or PMA. These results indicated that Japanese flounder TNF is more like mammalian TNF-α than mammalian lymphotoxin-α, with respect to its gene structure, length of amino acid sequence, number and position of cysteine residues, and regulation of gene expression.
Heavy Chain Revision in MRL Mice: A Potential Mechanism for the Development of Autoreactive B Cell Precursors
Authors: Klonowski Kimberly D. , Monestier Marc
Published: April 2014

Abs reactive to DNA and DNA/histone complexes are distinguished by the presence of positively charged amino acids, such as arginine, in the heavy chain complementarity-determining region 3. The presence of these amino acids partly results from atypical VH-D-JH rearrangements such as D-D fusions and D inversions. Previous results in our laboratory demonstrated that newborn autoimmune MRL/MpJ-+/+ mice undergo these unusual recombinations more frequently when compared with normal C3H/HeJ controls. In addition, the heavy chain junctions in newborn MRL mice demonstrated a preferred usage of VH-proximal D genes and distal JH genes suggestive of secondary gene rearrangements. In this study we explore the possibility that adult MRL B220+IgM− pre B cells, which have not yet undergone Ag selection, exhibit similar rearrangement patterns. Indeed, MRL pre-B cells possessed more atypical rearrangements (D-D fusions) than those of C3H/HeJ mice. However, the biased use of upstream D genes and downstream JH genes observed in the newborn MRL mice was not present in the pre-B cell library. These results suggest that the heavy chain rearrangement process persists later during B cell life in lupus-prone mice and lead us to propose a model of heavy chain receptor revision in the periphery of autoimmune mice.
Induction of Vigorous Helper and Cytotoxic T Cell as well as B Cell Responses by Dendritic Cells Expressing a Modified Antigen Targeting Receptor-Mediated Internalization Pathway
Authors: You Zhaoyang , Huang Xue F. , Hester Jenny , Rollins Lisa , Rooney Cliona , Chen Si-Yi
Published: April 2014

Efficient Ag presentation is essential to induce effective cellular and humoral immune responses. Thus, one central goal of current immunotherapy and vaccine development is to enhance Ag presentation to induce potent and broad immune responses. Here, a novel Ag presentation strategy is developed by transducing dendritic cells (DCs) to produce an Ag for presentation as an exogenous Ag to efficiently induce both humoral and cellular immunity. The principle of this strategy is illustrated by genetically modifying DCs to secrete a model hepatitis B virus Ag fused with a cell-binding domain and to process the fusion Ag as an exogenous Ag after receptor-mediated internalization for MHC class I and II presentation. Vigorous Ag-specific CD4+ helper and CD8+ cytotoxic T cell, as well as B cell, responses were induced by the transduced DCs in mouse models. Thus, this novel strategy uses a receptor-mediated internalization process to efficiently induce all arms of the adaptive immunity and may provide a powerful means to develop potent vaccines and immunotherapies.
The Soluble Endothelial Protein C Receptor Binds to Activated Neutrophils: Involvement of Proteinase-3 and CD11b/CD18
Authors: Kurosawa Shinichiro , Esmon Charles T. , Stearns-Kurosawa Deborah J.
Published: April 2014

The protein C pathway is a primary regulator of blood coagulation and a critical component of the host response to inflammatory stimuli. The most recent member of this pathway is the endothelial protein C receptor (EPCR), a type I transmembrane protein with homology to CD1d/MHC class I proteins. EPCR accelerates formation of activated protein C, a potent anticoagulant and antiinflammatory agent. The current study demonstrates that soluble EPCR binds to PMA-activated neutrophils. Using affinity chromatography, binding studies with purified components, and/or blockade with specific Abs, it was found that soluble EPCR binds to proteinase-3 (PR3), a neutrophil granule proteinase. Furthermore, soluble EPCR binding to neutrophils was partially dependent on Mac-1 (CD11b/CD18), a β2 integrin involved in neutrophil signaling, and cell-cell adhesion events. PR3 is involved in multiple diverse processes, including hemopoietic proliferation, antibacterial activity, and autoimmune-mediated vasculitis. The observation that soluble EPCR binds to activated neutrophils via PR3 and a β2 integrin suggests that there may be a link between the protein C anticoagulant pathway and neutrophil functions.
Extracellular ATP and TNF-α Synergize in the Activation and Maturation of Human Dendritic Cells
Authors: Schnurr Max , Then Florian , Galambos Peter , Scholz Christoph , Siegmund Britta , Endres Stefan , Eigler Andreas
Published: April 2014

Extracellular ATP mediates numerous biological activities by interacting with plasma membrane P2 purinergic receptors. Recently, P2 receptors have been described on dendritic cells (DC), but their functional role remains unclear. Proposed functions include improved Ag presentation, cytokine production, chemotaxis, and induction of apoptosis. We investigated the effects of ATP and of other P2 receptor agonists on endocytosis, phenotype, IL-12 secretion, and T cell stimulatory capacity of human monocyte-derived DC. We found that in the presence of extracellular ATP, DC transiently increase their endocytotic activity. Subsequently, DC up-regulate CD86, CD54, and MHC-II; secrete IL-12; and exhibit an improved stimulatory capacity for allogeneic T cells. These effects were more pronounced when chemically modified ATP derivatives with agonistic activity on P2 receptors, which are resistent to degradation by ectonucleotidases, were applied. Furthermore, ATP and TNF-α synergized in the activation of DC. Stimulated with a combination of ATP and TNF-α, DC expressed the maturation marker CD83, secreted large amounts of IL-12, and were potent stimulators of T cells. In the presence of the P2 receptor antagonist suramin, the effects of ATP were completely abolished. Our results suggest that extracellular ATP may play an important immunomodulatory role by activating DC and by skewing the immune reaction toward a Th1 response through the induction of IL-12 secretion.
In Vitro and In Vivo Induction of a Th Cell Response Toward Peptides of the Melanoma-Associated Glycoprotein 100 Protein Selected by the TEPITOPE Program
Authors: Cochlovius Björn , Stassar Marike , Christ Oliver , Raddrizzani Laura , Hammer Jürgen , Mytilineos Ioannis , Zöller Margot
Published: April 2014

The melanoma-associated Ag glycoprotein 100 was analyzed by the T cell epitope prediction software TEPITOPE. Seven HLA-DR promiscuous peptides predicted with a stringent threshold were used to load dendritic cells (DC), and induction of a proliferative response was monitored. PBMC of all nine donors including two patients with malignant melanoma responded to at least one of the peptides. The proliferative response was defined as a Th response by the selective expansion of CD4+ cells, up-regulation of CD25 and CD40L, and IL-2 and IFN-γ expression. Peptide-loaded DC also initiated a T helper response in vivo (i.e., tumor growth in the SCID mouse was significantly retarded by the transfer of PBMC together with peptide-loaded DC). Because the use of the TEPITOPE program allows for a prediction of T cell epitopes; because the predicted peptides can be rapidly confirmed by inducing a Th response in the individual patient; and because application of peptide-loaded DC suffices for the in vivo activation of helper cells, vaccination with MHC class II-binding peptides of tumor-associated Ags becomes a feasible and likely powerful tool in the immunotherapy of cancer.
Multiepitopic HLA-A*0201-Restricted Immune Response Against Hepatitis B Surface Antigen After DNA-Based Immunization
Authors: Loirat Delphine , Lemonnier François A. , Michel Marie-Louise
Published: April 2014

CTL together with anti-envelope Abs represent major effectors for viral clearance during hepatitis B virus (HBV) infection. The induction of strong cytotoxic and Ab responses against the envelope proteins after DNA-based immunization has been proposed as a promising therapeutic approach to mediate viral clearance in chronically infected patients. Here, we studied the CTL responses against previously described hepatitis B surface Ag (HBsAg)-HLA-A*0201-restricted epitopes after DNA-based immunization in HLA-A*0201 transgenic mice. The animal model used was Human Human Db (HHD) mice, which are deficient for mouse MHC class I molecules (β2-microglobulin−/− Db−/−) and transgenic for a chimeric HLA-A*0201/Db molecule covalently bound to the human β2-microglobulin (HHD+/+). Immunization of these mice with a DNA vector encoding the small and the middle HBV envelope proteins carrying HBsAg induced CTL responses against several epitopes in each animal. This study performed on a large number of animals described dominant epitopes with specific CTL induced in all animals and others with a weaker frequency of recognition. These results confirmed the relevance of the HHD transgenic mouse model in the assessment of vaccine constructs for human use. Moreover, genetic immunization of HLA-A2 transgenic mice generates IFN-γ-secreting CD8+ T lymphocytes specific for endogenously processed peptides and with recognition specificities similar to those described during self-limited infection in humans. This suggests that responses induced by DNA immunization could have the same immune potential as those developing during natural HBV infection in human patients.
Cutting Edge: Resistance to Apoptosis and Continuous Proliferation of Dendritic Cells Deficient for TNF Receptor-1
Authors: Funk Jens Oliver , Walczak Henning , Voigtländer Constanze , Berchtold Susanne , Baumeister Thomas , Rauch Pia , Rössner Susanne , Steinkasserer Alexander , Schuler Gerold , Lutz Manfred B.
Published: April 2014

The individual roles of the two TNFRs on dendritic cells (DC) are poorly understood. Investigating bone marrow-derived DC from TNFR-deficient mice, we found that cultures from TNFR1−/− mice continue to form proliferating clusters for 6–9 mo. In contrast, DC derived from wild-type, TNFR2−/−, or TNFR1/2−/− mice survived for only 3–4 wk. DC obtained from these TNFR1−/− long term cultures (LTC) mice show an unusual mixed immature/mature phenotype. The continuous proliferation of the LTC is GM-CSF dependent and correlates with decreased protein levels of the cyclin-dependent kinase inhibitors p27KIP1 and p21CIP1. Prolonged survival of TNFR1−/− DC appears to be independent from NF-κB and Bcl-2 pathways and is rather enabled by the down-regulation of CD95, resulting in the resistance to CD95 ligand-induced apoptosis. These data point to proapoptotic signals mediated via TNFR1 and antiapoptotic signals mediated via TNFR2 in DC.
The Ability of B Cells and Dendritic Cells to Present Antigen Increases During Ontogeny
Authors: Muthukkumar Subramanian , Goldstein Julia , Stein Kathryn E.
Published: April 2014

The immune response to polysaccharide (PS) Ags in mice is delayed during ontogeny even when administered in a thymus-dependent (TD) form. In this study, Neisseria meningitidis group C PS-tetanus toxoid conjugate (MCPS-TT) vaccine was used to examine whether the delay in the development of Ab responses to TD PS conjugate vaccines in neonatal mice is due to defective Ag presentation. The results show that B cells and dendritic cells (DC) from 3- and 7-day-old mice were severely defective in presenting TT and MCPS-TT to Ag-specific T cell clones. The ability of these cells to present Ag reaches adult levels by 4 wk. The development of anti-MCPS and anti-TT Abs in neonatal mice parallels the functional ability of their APC to present Ag. DC from neonatal mice expressed very low levels of MHC class II, costimulatory molecules B7.1, B7.2, and CD11c but high levels of monocyte-specific markers F4/80 and CD11b and granulocyte marker, Ly6G. Significant changes in the expression of these markers were observed as the age of the mice increased. MHC class II, B7.1 and B7.2, and CD11c all increased with age, reaching adult levels between 3 and 4 wk, concurrent with the function of APC. These results demonstrate that one reason neonates fail to produce high titers of anti-PS Abs even when immunized in a TD form is that their B cells and DC are not fully functional.
Changing Patterns of Dominant TCR Usage with Maturation of an EBV-Specific Cytotoxic T Cell Response
Authors: Annels Nicola E. , Callan Margaret F. C. , Tan Linda , Rickinson Alan B.
Published: April 2014

Infection with EBV provides a unique opportunity to follow the human CD8+ T cell response to a persistent, genetically stable agent from the primary phase, as seen in infectious mononucleosis (IM) patients, into long-term memory. This study focuses on the response to an immunodominant HLA-A2.01-restricted epitope, GLCTLVAML, from the EBV-lytic cycle Ag BMLF1. TCR analysis of the highly amplified primary response to this epitope revealed markedly oligoclonal receptor usage among in vitro-derived clones, with similar clonotypes dominant in all three IM patients studied. Direct staining of IM T cell preparations with the A2.01/GLCTLVAML tetramer linked this oligoclonal epitope-specific response with appropriate Vβ subset expansions in the patients’ blood. These patients were studied again >2 years later, at which time TCR analysis of in vitro-reactivated clones suggested that rare clonotypes within the primary response had now come to dominate memory. Five additional A2.01-positive IM patients were studied prospectively for Vβ subset representation within primary and memory epitope-specific populations as identified by tetramer staining. In each case, the primary response contained large Vβ2, Vβ16, or Vβ22 components, and in three of five cases the originally dominant Vβ was represented very poorly, if at all, in memory. We conclude 1) that an EBV epitope-specific primary response large enough to account for up to 10% CD8+ T cells in IM blood may nevertheless be dominated by just a few highly expanded clonotypes, and 2) that with persistent viral challenge such dominant T cell clonotypes may be lost and replaced by others in memory.
Defining the Requirements for Peptide Recognition in Gene Therapy-Induced T Cell Tolerance
Authors: Bagley Jessamyn , Wu Yin , Sachs David H. , Iacomini John
Published: April 2014

Expression of a retrovirally transduced MHC class I Ag, H-2Kb (Kb), in bone marrow-derived cells leads to specific prolongation of Kb disparate skin grafts. To examine the extent to which peptides derived from Kb contribute to the induction of tolerance, retroviruses carrying mutant Kb genes designed to enter separate pathways of Ag presentation were constructed. Thymectomized and CD8 T cell-depleted mice that had been irradiated and reconstituted with bone marrow cells expressing a secreted form of Kb showed prolongation of Kb disparate skin graft survival. Skin graft prolongation was not observed when similar experiments were performed using mice that were not CD8 T cell depleted. This suggests that hyporesponsiveness can be induced in CD4 T cells, but not CD8 T cells by Ags presented via the exogenous pathway of Ag processing. Modest prolongation of skin allografts was observed in mice reconstituted with bone marrow cells transduced with retroviruses carrying a gene encoding a mutant Kb molecule expressed only in the cytoplasm. Prolongation was also observed in similar experiments in mice that were thymectomized and CD4 T cell depleted following complete reconstitution, but not in mice that were reconstituted and then thymectomized and CD8 T cell depleted. Thus, hyporesponsiveness can be induced in a subset of CD8 T cells by recognition of peptides derived from Kb through both the direct and indirect pathways of Ag recognition, while CD4 T cell hyporesponsiveness to MHC class I disparate grafts occurs only through the indirect pathway of Ag recognition.
The Down-Regulation of HLA-DM Gene Expression in Rheumatoid Arthritis Is Not Related to Their Promoter Polymorphism
Authors: Louis-Plence Pascale , Kerlan-Candon Sophie , Morel Jacques , Combe Bernard , Clot Jacques , Pinet Valérie , Eliaou Jean-François
Published: April 2014

HLA-DM molecule, a class II-like heterodimer, is a critical factor of HLA class II-dependent Ag presentation. It acts as a molecular chaperone and also functions as a peptide editor favoring the presentation of high-stability peptides. Thus, it appears to skew the peptide repertoire presented to T cells. Variation in HLA-DM expression has considerable effect on Ag presentation and regulation of these genes is likely to be a prerequisite to prevent autoimmunity. In this study, rheumatoid arthritis (RA) was chosen as a model of human autoimmune disease since its genetic susceptibility is known to be associated with the HLA-DR and -DM components. We described a limited nucleotide polymorphism in the HLA-DM promoters with functional impact on basal transcriptional activity and IFN-γ induction as assessed in vitro. However, no difference of allele frequencies was found between controls and RA patients. Despite of this lack of association, expression of HLA-DM molecules was also investigated. Interestingly, an underexpression of HLA-DM transcripts and protein was shown in peripheral blood B cells from RA patients compared with controls or inflammatory arthritis patients. This underexpression does not affect HLA-DR genes and is responsible for a decrease of the DM:DR ratio in RA patients. This specific HLA-DM down-regulation is likely to have important consequences on Ag presentation and could participate in the autoimmune process in RA.
Graded Deletion and Virus-Induced Activation of Autoreactive CD4+ T Cells
Authors: Riley Michael P. , Cerasoli Douglas M. , Jordan Martha S. , Petrone Andria L. , Shih Fei F. , Caton Andrew J.
Published: April 2014

We have examined factors governing the negative selection of autoreactive CD4+ T cells in transgenic mice expressing low (HA12 mice) vs high (HA104 mice) amounts of the influenza virus hemagglutinin (HA). When mated with TS1 mice that express a transgenic TCR specific for the I-Ed-restricted determinant site 1 (S1) of HA, thymocytes expressing high levels of the clonotypic TCR were deleted in both HA-transgenic lineages. However, through allelic inclusion, thymocytes with lower levels of the clonotypic TCR evaded deletion in TS1 × HA12 and TS1 × HA104 mice to graded degrees. Moreover, in both lineages, peripheral CD4+ T cells could be activated by the S1 peptide in vitro, and by influenza virus in vivo. These findings indicate that allelic inclusion can allow autoreactive CD4+ thymocytes to evade thymic deletion to varying extents reflecting variation in the expression of the self peptide, and can provide a basis for the activation of autoreactive peripheral T cells by viruses bearing homologues of self peptides (“molecular mimicry”).
Direct NK Cell-Mediated Lysis of Syngenic Dorsal Root Ganglia Neurons In Vitro
Authors: Backström Eva , Chambers Benedict J. , Kristensson Krister , Ljunggren Hans-Gustaf
Published: April 2014

In contrast to extensive studies on the role of T and B lymphocytes in the pathogenesis of autoimmune diseases of the nervous system, little is known about NK cells and their potential role in the destruction of neural tissue. NK cells have been implicated in the selective death of sympathetic neurons resident in the superior cervical ganglia of rats after exposure to the drug guanethidine. This observation suggests that NK cells may function as principle effectors in immunological diseases of the nervous system. However, the direct mechanism of action of NK cells in this model is not known. In particular, it is not known whether NK cells can kill autologous neurons directly. The aim of the present study was to examine whether NK cells can kill directly dorsal root ganglia neurons cultured in vitro. We demonstrate that C57BL/6 (B6)-derived dorsal root ganglia neurons can be killed directly by syngenic IL-2-activated NK cells, and that this nerve cell lysis is dependent on the expression of perforin in the NK cells. NK cells were less effective in destroying neurons grown in the presence of glial cells. These observations indicate a potential role for NK cells in nerve cell degeneration in inflammatory diseases of the nervous system.
Anatomical Origin of Dendritic Cells Determines Their Life Span in Peripheral Lymph Nodes
Authors: Ruedl Christiane , Koebel Pascale , Bachmann Martin , Hess Michael , Karjalainen Klaus
Published: April 2014

Dendritic cells (DCs) exhibit considerable heterogeneity in their anatomical location, surface phenotype, and functional properties. In this study, we demonstrate that peripheral lymph nodes contain at least four major, functionally separable, and independently derived, DC subsets, which can be clearly demarcated by their CD11c, CD40, and CD8 expression pattern. Surprisingly, all DCs derived directly from the bone marrow, the myeloid- and the lymphoid-related subsets, turned over fast with t1/2 of a couple of days. In contrast, DCs exported from the skin, both dermal and epidermal, accumulated 3- to 4-fold slower, turnover that is dramatically increased by cutaneous inflammation.