bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and Characterization of Epitopes Recognized by T Lymphocytes and Autoantibodies from Patients with Herpes Gestationis
Authors: Lin M.-S. , Gharia M. A. , Swartz S. J. , Diaz L. A. , Giudice G. J.
Published: January 2023

Autoantibodies associated with herpes gestationis (HG), a pregnancy-associated autoimmune skin disease, target the hemidesmosomal protein BP180. It was shown that the major noncollagenous stretch of the BP180 ectodomain (NC16A) harbors epitopes recognized by HG sera. Furthermore, Abs reactive with the homologous domain of murine BP180 are known to trigger a cutaneous blistering disease in mice by passive transfer experiments. The present study was aimed at characterizing the T cell responses and specificities of autoantibodies from two HG patients. Using immunoblotting and T cell proliferation assays, we have identified a 14-amino-acid stretch of the BP180 ectodomain (MCW-1; aa 507–520) that is recognized by both T cells and autoantibodies produced by the HG patients. The neonate born to one of these HG patients showed no signs of skin disease and had no detectable T cell response to the BP180 Ag, but did have a low titer of circulating anti-BP180 autoantibodies, presumably of maternal origin. BP180-specific T cell lines and clones developed from an HG patient specifically reacted with the MCW-1 epitope. The proliferative responses of these clones were restricted to HLA-DR, but not -DQ or -DP. These Ag-specific T cells expressed α/β TCRs and a CD4 memory T cell phenotype and secreted IFN-γ and IL-2, but not IL-4 or IL-6, suggesting that they are Th1-type lymphocytes. Further characterization of these Ag-specific T cells and autoantibodies will aid in elucidating the autoimmune mechanism(s) leading to the development of HG.
Glutamate Augments Retrovirus-Induced Immunodeficiency Through Chronic Stimulation of the Hypothalamic-Pituitary- Adrenal Axis
Authors: Espey Michael Graham , Basile Anthony S.
Published: January 2023

The mechanisms for activating the hypothalamic-pituitary-adrenal (HPA) axis and the roles glucocorticoids play in the pathogenesis of chronic infectious disease are largely undefined. Using the LP-BM5 model of retrovirus-induced immunodeficiency, we found alterations in HPA axis function, manifested as an increase in circulating levels of adrenocorticotropic hormone and corticosterone, beginning after only 3 mo of infection. These changes occurred contemporaneously with a shift in the profile of circulating cytokines from a Th1-dominant (IFN-γ) to Th2-dominant (IL-4, IL-10) phenotype. No significant changes in either circulating IL-1β, IL-6, or TNF-α levels were observed in infected mice. Administering the N-methyl-d-aspartate receptor antagonist MK-801 to infected mice normalized plasma adrenocorticotropic hormone and corticosterone levels, indicating that glutamate was a major activator of the HPA axis. Moreover, MK-801 treatment of late-stage mice also reversed the type 1 to type 2 cytokine shift to a degree comparable or superior to treatment with the glucocorticoid receptor antagonist RU-486. These findings indicate that HPA axis activation during LP-BM5 retrovirus infection is mediated by the chronic hyperactivation of glutamatergic pathways in the hypothalamus. Through this mechanism, the degree of peripheral immunodeficiency observed in the late-stage disease is profoundly augmented.
Mutations That Cause the Wiskott-Aldrich Syndrome Impair the Interaction of Wiskott-Aldrich Syndrome Protein (WASP) with WASP Interacting Protein
Authors: Stewart Donn M. , Tian Lan , Nelson David L.
Published: January 2023

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, eczema, immune deficiency, and a proclivity toward lymphoid malignancy. Lymphocytes of affected individuals show defects of activation, motility, and cytoskeletal structure. The disease gene encodes a 502-amino acid protein named the WAS protein (WASP). Studies have identified a number of important interactions that place WASP in a role of integrating signaling pathways with cytoskeletal function. We performed a two-hybrid screen to identify proteins interacting with WASP and cloned a proline-rich protein as a specific WASP interactor. Our clone of this protein, termed WASP interacting protein (WIP) by others, shows a difference in seven amino acid residues, compared with the previously published sequence revealing an additional profilin binding motif. Deletion mutant analysis reveals that WASP residues 101–151 are necessary for WASP-WIP interaction. Point mutant analyses in the two-hybrid system and in vitro show impairment of WASP-WIP interaction with three WASP missense mutants known to cause WAS. We conclude that impaired WASP-WIP interaction may contribute to WAS.
Cutting Edge: A Short Polypeptide Domain of HIV-1-Tat Protein Mediates Pathogenesis
Authors: Boykins Robert A. , Mahieux Renaud , Shankavaram Uma T. , Gho Yong Song , Lee Sherwin F. , Hewlett Indira K. , Wahl Larry M. , Kleinman Hynda K. , Brady John N. , Yamada Kenneth M. , Dhawan Subhash
Published: January 2023

HIV-1 encodes the transactivating protein Tat, which is essential for virus replication and progression of HIV disease. However, Tat has multiple domains, and consequently the molecular mechanisms by which it acts remain unclear. In this report, we provide evidence that cellular activation by Tat involves a short core domain, Tat21–40, containing only 20 aa including seven cysteine residues highly conserved in most HIV-1 subtypes. Effective induction by Tat21–40 of both NF-κB-mediated HIV replication and TAR-dependent transactivation of HIV-long terminal repeat indicates that this short sequence is sufficient to promote HIV infection. Moreover, Tat21–40 possesses potent angiogenic activity, further underscoring its role in HIV pathogenesis. These data provide the first demonstration that a 20-residue core domain sequence of Tat is sufficient to transactivate, induce HIV replication, and trigger angiogenesis. This short peptide sequence provides a potential novel therapeutic target for disrupting the functions of Tat and inhibiting progression of HIV disease.
CD30 Overexpression Enhances Negative Selection in the Thymus and Mediates Programmed Cell Death Via a Bcl-2-Sensitive Pathway
Authors: Chiarle Roberto , Podda Antonello , Prolla Gabriel , Podack Eckhard R. , Thorbecke G. Jeanette , Inghirami Giorgio
Published: January 2023

The biological function of CD30 in the thymus has been only partially elucidated, although recent data indicate that it may be involved in negative selection. Because CD30 is expressed only by a small subpopulation of medullary thymocytes, we generated transgenic (Tg) mice overexpressing CD30 in T lymphocytes to further address its role in T cell development. CD30 Tg mice have normal thymic size with a normal number and subset distribution of thymocytes. In vitro, in the absence of CD30 ligation, thymocytes of CD30 Tg mice have normal survival and responses to apoptotic stimuli such as radiation, dexamethasone, and Fas. However, in contrast to controls, CD30 Tg thymocytes are induced to undergo programmed cell death (PCD) upon cross-linking of CD30, and the simultaneous engagement of TCR and CD30 results in a synergistic increase in thymic PCD. CD30-mediated PCD requires caspase 1 and caspase 3, is not associated with the activation of NF-κB or c-Jun, but is totally prevented by Bcl-2. Furthermore, CD30 overexpression enhances the deletion of CD4+/CD8+ thymocytes induced by staphylococcal enterotoxin B superantigen and specific peptide. These findings suggest that CD30 may act as a costimulatory molecule in thymic negative selection.
Development of T Lymphocytes at Extrathymic Sites
Authors: Antica Mariastefania , Scollay Roland
Published: January 2023

T lymphocytes expressing both CD4 and CD8 are the predominant cell type in the thymic cortex but are extremely rare outside the thymus of normal mice. In this article, we show that if precursor thymocytes (CD4−CD8−) from fetal or adult donors are injected i.v. into irradiated recipients, some of these cells will lodge in lymph nodes and develop into both CD4+CD8+ (double-positive) and CD4+ or CD8+ (single-positive) cells. This phenomenon also occurred in thymectomized recipients, strongly suggesting it is genuine extrathymic development. Prethymic precursors (e.g., fetal liver), were unable to use the lymph node for T cell development, without thymic processing. The data suggest that given unusual circumstances (irradiation or thymectomy and availability of appropriate precursors), the lymph nodes can support T cell development.
Cutting Edge: Expression of the NF of Activated T Cells in Eosinophils: Regulation by IL-4 and IL-5
Authors: Jinquan Tan , Quan Sha , Jacobi Henrik H. , Reimert Claus M. , Millner Anders , Hansen Jens B. , Thygesen Charlotte , Ryder Lars P. , Madsen Hans O. , Malling Hans-Jørgen , Poulsen Lars K.
Published: January 2023

We report that NF-AT1 and NF-AT4 are expressed cytoplasmically in resting eosinophils, whereas NF-AT2 and NF-AT3 have not been seen. Likewise, NF-AT1 mRNA and NF-AT4 mRNA have been detected in resting eosinophils, and their levels can be significantly up-regulated by the Th2-associated cytokines IL-4 and IL-5. There is no detectable NF-AT protein expression in the nuclei of resting eosinophils. However NF-ATs appear in the nuclei of IL-4-, IL-5-, or ionomycin-stimulated eosinophils. Only NF-AT1 and NF-AT4, but not NF-AT2 and NF-AT3, have translocated into the nuclei in IL-4- or IL-5-stimulated eosinophils. These findings delineate a novel pathway in the cytokine network in which Th2 lymphocytes “control” eosinophils via the release of IL-4 and IL-5, and activation of NF-AT in eosinophils. The findings also suggest that a later feedback “talking” may exist between eosinophils and Th2 lymphocytes.
Diversity of NK Cell Receptor Repertoire in Adult and Neonatal Mice
Authors: Kubota Akira , Kubota Satoko , Lohwasser Stefan , Mager Dixie L. , Takei Fumio
Published: January 2023

Murine NK cytotoxicity is regulated by two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2. We developed a single-cell RT-PCR method to analyze expression of all known Ly49 and NKG2A genes in individual NK cells and determined the receptor repertoires of NK cells from adult and neonatal (1-wk-old) C57BL/6 mice. In adult mouse NK cells, up to six different receptors were coexpressed in random combinations. Of 62 NK cells examined, 42 different patterns of receptor expression were observed. Most of them expressed at least one Ly49, whereas NKG2A was detected in 32% of the cells. Over 75% of them expressed Ly49C, I, or NKG2A, which are thought to recognize self-class I MHC (H-2b). Coexpression of multiple Ly49 receptors and NKG2A was stochastic. In contrast, very few neonatal NK cells expressed any Ly49, but almost 60% of them expressed NKG2A. These results demonstrate that adult NK cells are quite heterogeneous and have diverse receptor repertoires. They also suggest that the expression of NKG2A precedes Ly49 expression in NK cell ontogeny, and NKG2A is a major inhibitory receptor in neonatal NK cells.
Genetic Immunization of Mice Against Listeria monocytogenes Using Plasmid DNA Encoding Listeriolysin O
Authors: Cornell Kenneth A. , Bouwer H. G. Archie , Hinrichs David J. , Barry Ronald A.
Published: January 2023

The development of protective immunity against many intracellular bacterial pathogens commonly requires sublethal infection with viable forms of the bacteria. Such infection results in the in vivo activation of specific cell-mediated immune responses, and both CD4+ and CD8+ T lymphocytes may function in the induction of this protective immunity. In rodent models of experimental infection with Listeria monocytogenes, the expression of protective immunity can be mediated solely by the immune CD8+ T cell subset. One major target Ag of Listeria-immune CD8+ T cells is the secreted bacterial hemolysin, listeriolysin O (LLO). In an attempt to generate a subunit vaccine in this experimental disease model, eukaryotic plasmid DNA expression vectors containing genes encoding either the wild-type or modified forms of recombinant LLO were generated and used for genetic vaccination of naive mice. Results of these studies indicate that the intramuscular immunization of mice with specifically designed plasmid DNA constructs encoding recombinant forms of LLO stimulates peptide-specific CD8+ immune T cells that exhibit in vitro cytotoxic activity. More importantly, such immunization can provide protective immunity against a subsequent challenge with viable L. monocytogenes, demonstrating that this experimental approach may have direct application in prevention of acute disease caused by intracellular bacterial pathogens.
Schistosome-Infected IL-4 Receptor Knockout (KO) Mice, in Contrast to IL-4 KO Mice, Fail to Develop Granulomatous Pathology While Maintaining the Same Lymphokine Expression Profile
Authors: Jankovic Dragana , Kullberg Marika C. , Noben-Trauth Nancy , Caspar Patricia , Ward Jerrold M. , Cheever Allen W. , Paul William E. , Sher Alan
Published: January 2023

Th2 lymphocytes have been postulated to play a major role in the immunopathology induced by Schistosoma mansoni infection. Nevertheless, infected IL-4 knockout (KO) and wild-type (wt) mice develop egg granulomas comparable in size. To further investigate the function of the Th2 response in egg pathology we studied IL-4Rα-deficient mice, which are nonresponsive to both IL-4 and IL-13. In striking contrast to IL-4 KO animals, infected IL-4Rα KO mice developed only minimal hepatic granulomas and fibrosis despite the presence of CD3+ T cells in the residual egg lesions. Moreover, liver lymphokine mRNA levels in these animals and IL-4 KO mice were equivalent. In addition, infected IL-4Rα-deficient, IL-4-deficient, and wt animals developed similar egg Ag-specific IgG Ab titers, arguing that CD4-dependent Th activity is intact in KO mice. As expected, IFN-γ secretion was strongly up-regulated in mesenteric lymph node cultures from both groups of deficient animals, a change reflected in increased serum IgG2a and IgG2b Ab levels. Surprisingly, Th2 cytokine production in infected IL-4Rα KO mice was not abolished but was only reduced and resembled that previously documented in IL-4 KO animals. This residual Th2 response is likely to explain the ability of IL-4 KO mice to generate egg granulomas, which cannot be formed in IL-4Rα-deficient animals because of their lack of responsiveness to the same cytokine ligands. Taken together, these findings argue that tissue pathology in schistosomiasis requires, in addition to egg-specific CD4+ lymphocytes, a previously unrecognized IL-4Rα+ non-T cell effector population.
Pulmonary Surfactant Protein A Modulates the Cellular Response to Smooth and Rough Lipopolysaccharides by Interaction with CD14
Authors: Sano Hitomi , Sohma Hitoshi , Muta Tatsushi , Nomura Shin-ichi , Voelker Dennis R. , Kuroki Yoshio
Published: January 2023

Pulmonary surfactant protein A (SP-A) plays an important part in Ab-independent host defense mechanisms of the lung. In this study we investigated how SP-A interacts with distinct serotypes of bacterial LPS and modulates LPS-elicited cellular responses. SP-A bound to rough forms but not to smooth forms of LPS. In the macrophage-like cell line U937, SP-A inhibited mRNA expression and secretion of TNF-α induced by smooth LPS, but rough LPS-induced TNF-α expression was unaffected by SP-A. When U937 cells and rat alveolar macrophages were preincubated with SP-A, smooth LPS failed to induce TNF-α secretion, whereas rough LPS-induced TNF-α secretion was modestly increased. To clarify the mechanism by which SP-A modulates LPS-elicited cellular responses, we further examined the interaction of SP-A with CD14, which is known as a major LPS receptor. Western blot analysis revealed that CD14 was one of the SP-A binding proteins isolated from solubilized U937 cells. In addition, SP-A directly bound to recombinant soluble CD14 (rsCD14). When rsCD14 was preincubated with SP-A, the binding of rsCD14 to smooth LPS was significantly reduced but the association of rsCD14 with rough LPS was augmented. These results demonstrate the different actions of SP-A upon distinct serotypes of LPS and indicate that the direct interaction of SP-A with CD14 constitutes a likely mechanism by which SP-A modulates LPS-elicited cellular responses.
Decreased Expression of Th2 Type Cytokine mRNA Contributes to the Lack of Allergic Bronchial Inflammation in Aged Rats
Authors: Ide Kyotaro , Hayakawa Hiroshi , Yagi Takeshi , Sato Atsuhiko , Koide Yukio , Yoshida Atsushi , Uchijima Masato , Suda Takafumi , Chida Kingo , Nakamura Hirotoshi
Published: January 2023

Sensitized Brown Norway rats are known to develop eosinophilic bronchial inflammation and airway hyperresponsiveness after Ag exposure. However, we have previously observed that sensitized aged rats of the same strain failed to develop such allergic inflammation. In the present study, we investigated age-associated changes of cytokine mRNA expression in bronchoalveolar lavage (BAL) cells. Both young (8- to 10-wk-old) and aged (100- to 120-wk-old) Brown Norway rats were sensitized with OVA, and BAL was performed 24 h after OVA inhalation challenge. Semiquantitative RT-PCR analysis of BAL cells showed that the cells from aged rats preferentially expressed Th1 type cytokine (IFN-γ) mRNA, whereas cells from young animals expressed more Th2 type cytokine mRNAs including those for IL-4 and IL-5. Decreased expression of Th2 type cytokine transcripts in aged animals was further confirmed by quantitative analysis, competitive RT-PCR of BAL cells, and in situ hybridization. The age-associated changes of cytokine profile were not restricted to BAL cells but were a general feature of lymphocytes, as shown by examination of popliteal lymph nodes draining the site of sensitization. These findings suggest that decreased allergic inflammation in aged animals is attributable to age-dependent impairment of Th2 generation in response to Ag.
Skin Homing (Cutaneous Lymphocyte-Associated Antigen-Positive) CD8+ T Cells Respond to Superantigen and Contribute to Eosinophilia and IgE Production in Atopic Dermatitis
Authors: Akdis Mübeccel , Simon Hans-Uwe , Weigl Lorenz , Kreyden Oliver , Blaser Kurt , Akdis Cezmi A.
Published: January 2023

In allergic inflammations of the skin, activation of CD4+ T cells was demonstrated to play an important role; however, a minor role for CD8+ T cells is implied. In the present study, we compared cutaneous lymphocyte-associated Ag (CLA)-expressing CD4+ and CD8+ subsets, which were isolated from peripheral blood and lesional skin biopsies in atopic dermatitis (AD) patients. We demonstrated that CD8+CLA+ T cells proliferate in response to superantigen and are as potent as CD4+CLA+ T cells in IgE induction and support of eosinophil survival. In atopic skin inflammation, the existence of high numbers of CD4+ and CD8+ T cells was demonstrated by immunohistochemistry and by culturing T cells from skin biopsies. In peripheral blood, both CD4+ and CD8+ subsets of CLA+CD45RO+ T cells were in an activated state in AD. The in vivo-activated CLA+ T cells of both subsets spontaneously released an IL-5- and IL-13-dominated Th2 type cytokine pattern. This was confirmed by intracytoplasmic cytokine staining immediately after isolation of the cells from peripheral blood. In consequence, both CD4+ and CD8+, CLA+ memory/effector T cells induced IgE production by B cells mainly by IL-13, and enhanced eosinophil survival in vitro by delaying eosinophil apoptosis, mainly by IL-5. These results indicate that in addition to the CD4+ subset, the CD8+CLA+ memory/effector T cells are capable of responding to superantigenic stimulation and play an important role in the pathogenesis of AD.
Induced Expression of B7-1 on Myeloma Cells Following Retroviral Gene Transfer Results in Tumor-Specific Recognition by Cytotoxic T Cells
Authors: Tarte Karin , Zhang Xue-Guang , Legouffe Eric , Hertog Catherine , Mehtali Majid , Rossi Jean-François , Klein Bernard
Published: January 2023

The aim of this study was to evaluate whether tumor cells from patients with multiple myeloma activate allogeneic and autologous T cells. Results showed that myeloma cells expressed few B7-2 and no B7-1 in six cell lines and primary cells from 11 patients. They expressed substantial levels of HLA class I, CD40, and a set of adhesion molecules. In accordance with the low density of B7 molecules on these cells, they were poor allogeneic CD8+ T cell stimulators. Neither IFN-γ plus TNF-α nor CD40 stimulation significantly induced B7-1 or up-regulated B7-2 on human myeloma cell line or primary myeloma cells from six of seven patients. However, such induction was found on autologous bone-marrow nontumoral cells and on autologous dendritic cells following CD40 stimulation. High B7-1 expression was stably obtained on human myeloma cell line using transduction with a B7-1 retrovirus, enabling these cells to stimulate allogeneic CD8+, though not CD4+, T cell proliferation. For one patient with advanced disease, B7-1 gene transfer made it possible to amplify autologous cytotoxic T cells that killed autologous myeloma cells in an HLA class I-restricted manner, but not autologous PHA blasts. These results suggest that B7-1 gene transfer could be a promising immunotherapeutic approach in multiple myeloma.
Impaired Ability of MHC Class II−/− Dendritic Cells to Provide Tumor Protection is Rescued by CD40 Ligation
Authors: Hermans Ian F. , Ritchie David S. , Daish Angela , Yang Jianping , Kehry Marilyn R. , Ronchese Franca
Published: January 2023

The contribution of CD4+ T cells to dendritic cell (DC) activation and to the induction of CD8+ T cell responses in vivo was investigated using a model of antitumor immune responses. Immunization with peptide-loaded MHC class II-deficient (MHC class II−/−) DC induced the activation of Ag-specific CD8+ T cells and their accumulation in the lymph nodes and spleens of immunized mice. The accumulation induced by MHC class II−/− DC immunization was lower than the accumulation observed after immunization with MHC class II+/+ DC. Similarly, immunization with peptide-loaded, MHC class II−/− DC induced some degree of protection against tumor challenge, but this protection was lower than the protection achieved after immunization with MHC class II+/+ DC. Incubation with a membrane-associated form of CD40 ligand resulted in the up-regulation of costimulatory molecules on MHC class II−/− DC and fully rescued their ability to induce antitumor immunity. We conclude that CD4+ T cells play a critical role in the generation of antitumor immune responses through their capacity to induce the activation of DC via CD40/CD40 ligand interaction, and thus maximize CD8+ T cell responses.
Calcium Mobilization in Human Myeloid Cells Results in Acquisition of Individual Dendritic Cell-Like Characteristics Through Discrete Signaling Pathways
Authors: Koski Gary K. , Schwartz Gretchen N. , Weng David E. , Czerniecki Brian J. , Carter Charles , Gress Ronald E. , Cohen Peter A.
Published: January 2023

We have shown previously that calcium ionophore (CI) treatment of various myeloid origin cells results in rapid acquisition of properties associated with mature, activated dendritic cells. These properties include increased CD83 and costimulatory molecule expression, tendencies to form dendritic processes, loss of CD14 expression by monocytes, and typically an enhanced capacity to sensitize T lymphocytes to Ag. We here analyze the intracellular signaling pathways by which CI induces acquisition of such properties. Thapsigargin, which raises intracellular Ca2+ levels by antagonizing its sequestration, induced immunophenotypic and morphologic changes that paralleled CI treatment. CI-induced activation was broadly attenuated by the Ca2+ chelating compound EGTA and by calmodulin antagonists trifluoperazine dimaleate and W-7. However, antagonists of signaling pathways downstream to calmodulin displayed more selective inhibitory effects. Calcineurin antagonists cyclosporin A and the FK-506 analogue, ascomycin, diminished costimulatory molecule and CD83 expression, as well as formation of dendritic processes in CI-treated myeloid cells, and strongly attenuated the T cell allosensitizing capacity of CI-treated HL-60 cells. These calcineurin antagonists displayed minimal effect on CI-induced CD14 down-regulation in monocytes. In contrast, the calmodulin-dependent protein kinase antagonists, K252a and KT5926, while displaying only modest effects on CI-induced costimulatory molecule and CD83 expression, strongly blocked CD14 down-regulation. These results are consistent with a Ca2+-dependent mechanism for CI-induced differentiation of myeloid cells, and indicate that multiple discrete signaling pathways downstream to calcium mobilization and calmodulin activation may be essential in regulating this process.
A Novel Function of Vα14+CD4+NKT Cells: Stimulation of IL-12 Production by Antigen-Presenting Cells in the Innate Immune System
Authors: Tomura Michio , Yu Wen-Gong , Ahn Hyun-Jong , Yamashita Motozo , Yang Yi-Fu , Ono Shiro , Hamaoka Toshiyuki , Kawano Tetsu , Taniguchi Masaru , Koezuka Yasuhiko , Fujiwara Hiromi
Published: January 2023

The balance between Th1 and Th2 development is determined by IL-4 and IL-12. While the role for CD4+ NK1.1+ T (NKT) cells in influencing this balance has been recognized based on their capacity to produce IL-4, it is unknown how IL-12 is produced in the innate immune system in which they participate. This study demonstrates that Ag-activated CD4+ NKT cells express CD40 ligand (CD40L) (CD154), which engages CD40 on APC and stimulates them to produce IL-12. Culture of B cell-depleted spleen cells from C57BL/6 mice with α-galactosylceramide (α-GalCer) capable of selectively stimulating Vα14/Jα281+ NKT cells resulted in the production of IL-12 together with IFN-γ and IL-4. α-GalCer-induced IL-12 production occurred in I-Abβ-deficient mice, but not in β2-microglobulin-deficient and Vα14/Jα281 TCR-deficient mice, and was inhibited by anti-CD40L mAb. Of CD4+ and CD4− NKT cells, the capacity to express CD40L/CD154 and trigger IL-12 production following α-GalCer stimulation was exhibited preferentially by the CD4+ NKT subset. IL-12 production was also observed in α-GalCer-treated mice. Production of IL-12 preceded IFN-γ production, and IL-12 was required for IFN-γ, but not IL-4, production. A stimulatory/inhibitory relationship existed between IL-12 and IL-4 production. These results illustrate a novel function of CD4+ NKT cells that could be involved in the regulation of Th1 vs Th2 development.
Inhibition of Signaling Through the B Cell Antigen Receptor by the Protooncogene Product, c-Cbl, Requires Syk Tyrosine 317 and the c-Cbl Phosphotyrosine-Binding Domain
Authors: Yankee Thomas M. , Keshvara Lakhu M. , Sawasdikosol Sansana , Harrison Marietta L. , Geahlen Robert L.
Published: January 2023

The Syk protein-tyrosine kinase couples the B cell Ag receptor (BCR) to intracellular biochemical pathways. Syk becomes phosphorylated on multiple tyrosine residues upon receptor cross-linking. Tyrosine 317 is a site of phosphorylation located within the linker region of Syk that separates the amino-terminal, tandem pair of SH2 domains from the carboxyl-terminal catalytic domain. The amino acid sequence surrounding phosphotyrosine 317 matches the consensus sequence for recognition by the phosphotyrosine-binding (PTB) domain of the protooncogene product, c-Cbl. The overexpression of c-Cbl in DT40 B cells inhibits Ag receptor-mediated activation of the NF-AT transcription factor. The ability of overexpressed c-Cbl to inhibit signaling requires both Syk tyrosine 317 and a functional c-Cbl PTB domain. Mutant forms of Syk lacking tyrosine 317 exhibit an enhanced ability to couple the BCR to pathways leading to the activation of both NF-AT and Elk-1. Coimmunoprecipitation experiments indicate that Syk phosphotyrosine 317 and the c-Cbl PTB domain enhance, but are not required for, all interactions between these two proteins. In unstimulated cells, c-Cbl and Syk can be isolated in a complex that also contains tubulin. A mutant form of Syk lacking tyrosine at position 317 exhibits an enhanced ability to interact with a diphosphopeptide modeled on the immunoreceptor tyrosine-based activation motif of the CD79a component of the Ag receptor. These studies indicate that c-Cbl may contribute to the regulation of BCR signaling by modulating the ability of Syk to associate with the BCR and couple the receptor to intracellular signaling pathways.
Tyrosine Phosphorylation of Shc in Response to B Cell Antigen Receptor Engagement Depends on the SHIP Inositol Phosphatase
Authors: Ingham Robert J. , Okada Hidetaka , Dang-Lawson May , Dinglasan Jason , van der Geer Peter , Kurosaki Tomohiro , Gold Michael R.
Published: January 2023

Tyrosine phosphorylation of Shc in response to B cell Ag receptor (BCR) engagement creates binding sites for the Src homology 2 (SH2) domain of Grb2. This facilitates the recruitment of both Grb2 · Sos complexes and Grb2 · SHIP complexes to the plasma membrane where Sos can activate Ras and SH2 domain-containing inositol phosphatase (SHIP) can dephosphorylate phosphatidylinositol 3,4,5-trisphosphate. Given the importance of Shc phosphorylation, we investigated the mechanism by which the BCR stimulates this response. We found that both the SH2 domain and phosphotyrosine-binding (PTB) domain of Shc are important for BCR-induced tyrosine phosphorylation of Shc and the subsequent binding of Grb2 to Shc. The unexpected finding that the PTB domain of Shc is required for Shc phosphorylation was investigated further. Because the major ligand for the Shc PTB domain is SHIP, we asked whether the interaction of Shc with SHIP was required for BCR-induced tyrosine phosphorylation of Shc. Using SHIP-deficient DT40 cells, we show that SHIP is necessary for the BCR to induce significant levels of Shc tyrosine phosphorylation. BCR-induced tyrosine phosphorylation of Shc could be restored in the these cells by expressing wild-type SHIP but not by expressing a mutant form of SHIP that cannot bind to Shc. This suggests that BCR-induced tyrosine phosphorylation of Shc may depend on the binding of SHIP to the Shc PTB domain. Thus, we have described a novel role for SHIP in BCR signaling, promoting the tyrosine phosphorylation of Shc.
A Long-Term Memory Obtained by Genetic Immunization Results in Full Protection from a Mammary Adenocarcinoma Expressing an EBV Gene
Authors: Charo Jehad , Ciupitu Anne-Marie T. , Le Chevalier de Préville Alain , Trivedi Pankaj , Klein George , Hinkula Jorma , Kiessling Rolf
Published: January 2023

We have tested the capability of a plasmid DNA (pDNA) expressing the EBV nuclear Ag-4 (EBNA-4) to evoke a T cell response-associated protective immune response against a tumor expressing this gene. We have found that ACA mice immunized with EBNA-4-expressing plasmid were partially protected against syngeneic mammary carcinoma line (S6C) expressing EBNA-4 (S6C-E4). This protection was enhanced by coimmunizing mice with EBNA-4- and GM-CSF-expressing plasmids, and a full protection was achieved by coimmunizing mice with EBNA-4- and IFN-γ-expressing plasmids. Furthermore, mice that have rejected the EBNA-4-positive tumor were also resistant against a subsequent challenge with the original nontransfected tumor line. We then checked for the ability of pDNA immunization to provide a protective long-term memory response. We indeed found that even after 3 mo from the last immunization, full protection was obtained by this method, as compared with full tumor outgrowth in the control-immunized group. These findings support the concept that a nonviral, pDNA-based vaccination strategy is useful to fully protect from the outgrowth of tumors expressing this EBV gene.
Circulating CD2+ Monocytes Are Dendritic Cells
Authors: Crawford Keith , Gabuzda Dana , Pantazopoulos Vassilios , Xu Jianhua , Clement Chris , Reinherz Ellis , Alper Chester A.
Published: January 2023

Low levels of CD2 have been described on subsets of monocytes, macrophages, and dendritic cells. CD2 is expressed on about one-third of circulating monocytes, at levels one-half log lower than on T or NK cells, representing 2–4% of PBMC. FACS analysis of CD2+ and CD2− monocytes revealed no significant difference in the expression of adhesion molecules (CD11a/b/c), class II Ags (HLA-DR, -DQ, -DP), myeloid Ags (CD13, CD14, CD33), or costimulatory molecules (CD80, CD86). Freshly isolated CD2+ and CD2− monocytes were morphologically indistinguishable by phase contrast microscopy. However, scanning electron microscopy revealed large prominent ruffles on CD2+ monocytes in contrast to small knob-like projections on CD2− monocytes. After 2 days of culture, the CD2+ monocytes largely lost CD14 expression and developed distinct dendrites, whereas the CD2− monocytes retained surface CD14 and remained round or oval. Freshly isolated CD2+ monocytes were more potent inducers of the allogeneic MLR and more efficiently induced proliferation of naive T cells in the presence of HIV-1 gp120 than did CD2− monocytes. After culture in the presence of GM/CSF and IL-4, CD2+ monocytes were up to 40-fold more potent than monocyte-derived dendritic cells or CD2− monocytes at inducing allogeneic T cell proliferation. These findings suggest that circulating CD2+ and CD2− monocytes are dendritic cells and the precursors of macrophages, respectively. Thus, dendritic cells are far more abundant in the blood than previously thought, and they and precursors of macrophages exist in the circulation as phenotypically, morphologically, and functionally distinct monocyte populations.
The CXC Chemokine Stromal Cell-Derived Factor Activates a Gi-Coupled Phosphoinositide 3-Kinase in T Lymphocytes
Authors: Sotsios Yannis , Whittaker Gillian C. , Westwick John , Ward Stephen G.
Published: January 2023

The cellular effects of stromal cell-derived factor-1 (SDF-1) are mediated primarily by binding to the CXC chemokine receptor-4. We report in this study that SDF-1 and its peptide analogues induce a concentration- and time-dependent accumulation of phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns(3,4,5)P3) in Jurkat cells. This SDF-1-stimulated generation of D-3 phosphoinositide lipids was inhibited by pretreatment of the cells with an SDF-1 peptide antagonist or an anti-CXCR4 Ab. In addition, the phosphoinositide 3 (PI 3)-kinase inhibitors wortmannin and LY294002, as well as the Gi protein inhibitor pertussis toxin, also inhibited the SDF-1-stimulated accumulation of PtdIns(3,4,5)P3. The effects of SDF-1 on D-3 phosphoinositide lipid accumulation correlated well with activation of the known PI 3-kinase effector protein kinase B, which was also inhibited by wortmannin and pertussis toxin. Concentrations of PI 3-kinase inhibitors, sufficient to inhibit PtdIns(3,4,5)P3 accumulation, also inhibited chemotaxis of Jurkat and peripheral blood-derived T lymphocytes in response to SDF-1. In contrast, SDF-1-stimulated actin polymerization was only partially inhibited by PI 3-kinase inhibitors, suggesting that while chemotaxis is fully dependent on PI 3-kinase activation, actin polymerization requires additional biochemical inputs. Finally, SDF-1-stimulated extracellular signal-related kinase (ERK)-1/2 mitogen-activated protein kinase activation was inhibited by PI 3-kinase inhibitors. In addition, the mitogen-activated protein/ERK kinase inhibitor PD098059 partially attenuated chemotaxis in response to SDF-1. Hence, it appears that ERK1/2 activation is dependent on PI 3-kinase activation, and both biochemical events are involved in the regulation of SDF-1-stimulated chemotaxis.
Irradiation-Induced Rescue of Thymocyte Differentiation and V(D)J Recombination in Mice Lacking the Catalytic Subunit of DNA-Dependent Protein Kinase
Authors: Wang Chiyu , Bogue Molly A. , Nguyen Anthony P. , Roth David B.
Published: January 2023

Scid mice express a truncated form of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and are unable to properly rearrange their Ig and TCR genes, resulting in a severe combined immunodeficiency that is characterized by arrested differentiation of B and T lymphocytes. Treatment of scid mice with low doses of gamma irradiation rescues rearrangements at several TCR loci and promotes limited thymocyte differentiation. The machinery responsible for sensing DNA damage and the mechanism by which irradiation compensates for the scid defect in TCR recombination remain unknown. Because DNA-PKcs is present in scid thymocytes, it may mediate some or all of the irradiation effects. To test this hypothesis, we examined the effects of irradiation on DNA-PKcs-deficient (slip) mice. Our data provide the first evidence that DNA-PKcs is not required for limited rescue of thymocyte differentiation or TCR rearrangements.
Activation of CD40 Favors the Growth and Vascularization of Kaposi’s Sarcoma
Authors: Biancone Luigi , Cantaluppi Vincenzo , Boccellino Mariarosaria , Del Sorbo Lorenzo , Russo Simona , Albini Adriana , Stamenkovic Ivan , Camussi Giovanni
Published: January 2023

Although CD40 is expressed by several tumor lines and is up-regulated in tumor vascular endothelium, its role in tumor biology is still unclear. In the present study, we investigated the role of CD40 in the growth and vascularization of Kaposi’s sarcoma (KS). In vitro, stimulation of CD40 induced migration of KS cells and inhibited vincristine-induced apoptosis. Similarly, the CD40 engagement on endothelial cells resulted in cell contraction, migration, and prevention of serum withdrawal-apoptosis. To understand the biological relevance of CD40 in vivo, KS cells were engineered to express and release a soluble form of CD40 (KS-sCD40) able to disrupt CD40-CD154 interaction. SCID mice s.c. injected with KS-sCD40 cells developed tumors that were significantly smaller than those induced by control cells (KS-neo). In addition, KS-sCD40 tumors showed several areas of necrosis, diffuse presence of apoptotic cells, and poor vascularization. In contrast, KS-neo tumors showed few or absent areas of necrosis and apoptosis and intense vascularization. Moreover, anti-CD40 Abs stimulated neo-angiogenesis in a murine model in which s.c. implantation of Matrigel was used as a vehicle for the delivery of mediators. These observations provide demonstration that CD40 supports tumor cell survival, growth, and neo-vascularization of KS.
Engagement of α4β7 Integrins by Monoclonal Antibodies or Ligands Enhances Survival of Human Eosinophils In Vitro
Authors: Meerschaert JoAnn , Vrtis Rose F. , Shikama Yusuke , Sedgwick Julie B. , Busse William W. , Mosher Deane F.
Published: January 2023

Asthma is characterized by an airway inflammatory infiltrate that is rich in eosinophilic leukocytes. Cellular fibronectin and VCAM-1, ligands for α4 integrins, are enriched in the fluid of airways of allergic patients subjected to Ag challenge. We therefore hypothesized that ligands of α4 integrins can promote eosinophil survival independent of cell adhesion. Cellular fibronectin and VCAM-1 increased viability of human peripheral blood eosinophil in a dose- and time-dependant manner whether the ligand was coated on the culture well or added to the medium at the beginning of the assay. Eosinophils cultured with cellular fibronectin were not adherent to the bottom of culture wells after 3 days. Treatment with mAb Fib 30 to β7, but not mAb P4C10 or TS2/16 to β1, increased eosinophil survival. The increased survival of eosinophils incubated with Fib 30 was blocked by Fab fragments of another anti-β7 mAb, Fib 504. Eosinophils incubated with soluble cellular fibronectin or mAb Fib 30 for 6 h demonstrated a higher level of GM-CSF mRNA than eosinophils incubated with medium alone. Addition of neutralizing mAb to GM-CSF during incubation, but not mAbs to IL-3 or IL-5, reduced the enhancement of eosinophil survival by soluble cellular fibronectin or mAb Fib 30 to control levels. Thus, viability of eosinophils incubated with cellular fibronectin or VCAM-1 is due to engagement, probably followed by cross-linking, of α4β7 by soluble ligand (or mAb) that stimulates autocrine production of GM-CSF and promotes eosinophil survival.
On Isoagglutinin Reactions of Human Blood Other Than Those Defining the Blood Groups
Authors: Landsteiner K. , Levine Philip
Published: January 2023

A study was undertaken on the occurrence of atypical isoagglutinin reactions of human blood. Such a technic was purposely chosen as would detect sera of even slight activity. In this way rather numerous irregular reactions were found in a material obtained from patients with mental diseases.

Most of the reactions observed may be put under the heading of cold agglutination, that is, the sera are active only at low temperature and give very weak agglutination at room temperature (20°). A smaller number of sera (about 3 per cent) contained agglutinins of a type intermediate between cold agglutinins and typical isoagglutinins since their reactions were distinctly noticeable also under the usual conditions of blood grouping. In all of the cases with atypically reacting sera the blood cells showed no abnormal behavior and could readily be grouped. Also the grouping of the sera was not interfered with by the presence of the irregular agglutinins. Hence the irregularities are superimposed upon the much more prominent group properties, and are not in conflict with the scheme of the four blood groups, the usefulness of which cannot be questioned (cf. Thomsen (18)).

With regard to the practice of transfusions, it may be stated that in several instances in which the serum of the recipient contained irregular agglutinins for the blood of the donor no untoward symptoms at all were observed.

Several sera of groups O, A, and B, described, had agglutinins which were similar with regard to their specificity.

In groups A and AB two types of agglutinins were found specific in general for cells of one or the other subgroup of group A; those agglutinating blood AA2 showed in addition a specific action on cells of group O (cf. Schiff (30)). The properties of these sera demonstrate again quite definitely the existence of subgroups in group A, a distinction hardly significant at present for practical purposes.

The results with the abnormal agglutinins of human sera give further support to the conclusion that there exists a great number of individual differences of human blood.
Virolysis of complement-resistant HIV-1 by antibodies in the plasmas from HIV-1-infected individuals (128.10)
Authors: Yu Qigui , Shikuma Cecilia , Shiramizu Bruce , Hu Ningjie
Published: January 2023

Background: Ab responses to HIV-1 Ags are generally vigorous at all stages of viral infection, but fail to contain viral replication through Ab-dependent complement-mediated virolysis (ADCMV). Human CD59, an inhibitor of the membrane-attack complex formation of complement on the surface of HIV-1 virions, provides viral resistance to ADCMV. Objective: to abrogate CD59 function for enhancing HIV-1 virolysis by Abs in plasma of HIV-1-infected individuals. Method: HIV-1 virions were pretreated with anti-human CD59 Ab, and then tested for sensitivity to ADCMV in the presence of plasma from HIV-1-infected individuals. Virolysis (%) was calculated by measuring the release of p24 from HIV-1 virions caused by complement activation compared to total p24 released by detergent. Results: HIV-1 virions prepared from OM10 cells, which express high levels of CD59, were resistant to ADCMV, but became sensitive to ADCMV when the virions were pretreated with anti-human CD59 Ab. In total, 16 plasma samples from different HIV-1-infected individuals at acute or chronic stages of infection were tested and the average % of virolysis was 32.7 % with blocking of CD59 function, which is significantly greater than the average % of virolysis without blocking of CD59 function (4.9%) (p<0.001). Conclusions: abrogating CD59 function could enhance HIV-1 virolysis by Abs in the plasmas from HIV-1-infected individuals.
Alloferon inhibits the activation of Kaposi's sarcoma-associated herpesvirus through suppression of nuclear factor-kappa B (NF-kB) activation (128.14)
Authors: Lee Na Eun , Kang Jae Seung , Kong Joo Myoung , Bae Se Yeon , Kim Hye Min , Maeng Hyung Gun , Hwang Young Il , Lee Wang Jae
Published: January 2023

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic herpesvirus that causes Kaposi's sarcoma and primary effusion lymphoma. KSHV maintains a latent phase in infected cells but undergoes lytic reactivation in response to various stimuli. Alloferon is an immunomodulating peptide originating from an insect which has been used clinically as antiviral drug in Russia and showed remarkable effect on the prevention of recurrent herpesvirus infections. However, its specific mechanism still remains unknown. The effects of alloferon on the regulation of viral activation and its specific mechanism in virus-infected cells are investigated. In this study, BCBL-1, a persistent KSHV-infected B lymphoma cell line is used, which is reactivated to lytic cycle by TPA treatment. The results tell that alloferon effectively suppressed the down-regulation of cell proliferation induced by TPA treatment, implying that the KSHV reactivation within BCBL-1 can be regulated by alloferon. The results showed that alloferon decreased the expression level of KSHV viral proteins, such as RTA, ORF45 and vIRF2. Furthermore, alloferon suppressed the increase of calcium influx in store-operated calcium entry (SOCE) induced by TPA treatment, which led us to investigate whether alloferon can regulate nuclear factor-kappa B (NF-kB) activity in BCBL-1, since NF-kB is activated by viral infection as well as by calcium influx in immune cells. NF-kB activation in BCBL-1 was effectively inhibited by alloferon. Taken together, it is suggested that alloferon might be used as an effective antiviral agent on KSHV infection through the down-regulation of NF-kB activity.
High levels of chronic immune activation in both CD8 and CD4 T cell compartments negatively influences HCV treatment outcome in HCV/HIV-1 co-infection (128.17)
Authors: Gonzalez Veronica D , Falconer Karolin , Blom Kim G , Reichard Olle , Weis Nina , Alaeus Annette , Sandberg Johan K
Published: January 2023

Chronic immune activation is strongly associated with Human Immunodeficiency virus 1 (HIV-1) disease progression. Here, we have investigated the state of T cell immune activation, as well as T cell responses to hepatitis C virus (HCV), in patients with HCV/HIV-1 co-infection on effective anti-retroviral therapy (ART). Co-infected subjects displayed elevated immune activation as determined by CD38 expression in both CD4 and CD8 T cells, as compared to mono-infected control groups. HCV-specific T cells in response to HCV antigens tended to be stronger in the co-infected patients with a more pronounced IFNγ profile by CD8 T cells and this group also had higher HCV loads at baseline. Patients who reached a sustained virological response to peg-interferon-α and ribavirin treatment displayed significantly lower pre-treatment levels of chronic T cell activation and a trend towards stronger HCV-specific T cell responses, as compared to patients who failed to clear. Data suggest a link between high levels of chronic immune activation, poor adaptive immune responses and HCV treatment failure in HCV/HIV-1 co-infected subjects. We suggest that the high level of chronic immune activation may provide a clue as to why patients co-infected with these viruses in general display faster viral disease progression.

This work was supported by the Swedish Research Council, SIDA and the Swedish National Board of Health and Welfare.