bluuebunny/crossref_metadata_2025 huggingface dataset:
A Cytokine Cascade Including Prostaglandin E2, IL-4, and IL-10 Is Responsible for UV-Induced Systemic Immune Suppression
Authors: Shreedhar Vijay , Giese Todd , Sung Victor W. , Ullrich Stephen E.
Published: January 2023

Even though all of the energy contained with the UV wavelengths of solar radiation is absorbed within the epidermis and upper layers of the dermis, UV irradiation can suppress immune responses to Ag introduced at distant nonirradiated sites. In addition, data from a number of laboratories have suggested that one consequence of UV exposure is suppressed Th1 cell activation with normal or enhanced Th2 cell activation, resulting in a shift to a Th2-like phenotype. Cytokines secreted by UV-irradiated keratinoctyes, particularly IL-10, have been shown to play a major role in the induction of systemic immune suppression and differential activation of T helper cell subsets. Although IL-10 can influence Th1 cell activation by altering Ag presentation and suppressing IFN-γ secretion, the major signal for the development of a Th2 response is IL-4. Here we tested the hypothesis that UV irradiation induces IL-4 secretion. UV irradiation induced serum IL-4 in a dose-dependent fashion. Injecting UV-irradiated mice with anti-IL-4 blocked immune suppression. We could find no evidence, however, supporting secretion of IL-4 by UV-irradiated keratinocytes. Rather, we suggest that prostaglandins released by irradiated keratinocytes induce serum IL-4 since treating UV-irradiated mice with a cyclooxygenase-2 inhibitor blocked its production. Moreover, we found that treating UV-irradiated mice with anti-IL-4 suppressed serum IL-10 levels. In addition, injecting normal mice with PGE2 induced serum IL-4 and IL-10. We suggest that UV exposure activates a cytokine cascade (PGE2 → IL-4 → IL-10) that ultimately results in systemic immune suppression.
Differential Requirement for p56lck in Fetal and Adult Thymopoiesis
Authors: Molina Thierry J. , Perrot Jean-Yves , Penninger Josef , Ramos Amélia , Audouin Josée , Briand Pascale , Mak Tak W. , Diebold Jacques
Published: January 2023

The protein tyrosine kinase p56lck is critical for the generation of mature thymocytes in adult mice. However its requirement during the maturation of thymocytes from the fetal to the adult stage has not been clearly defined. We analyzed prenatal and postnatal thymocyte maturation in mice deficient for p56lck (lck−/−). Before birth, lck appears to play a crucial role in the expansion and proliferation of CD4+CD8+ double positive thymocytes, whereas proliferation and absolute numbers of CD4−CD8− double negative thymocyte precursors remained within the normal range until the end of the second week postnatal. Three weeks after birth, the total numbers of double negative and immature single positive thymocytes underwent a dramatic reduction that correlated with a decrease in the double positive population. This ontogenic defect was associated with a significant decrease in the proliferation rates of thymocyte precursors. Our data suggest that signaling via p56lck kinase is differentially required within a given phenotypically defined thymocyte subpopulation, depending on its stage of thymocyte maturation.
Characterization of Oct2 from the Channel Catfish: Functional Preference for a Variant Octamer Motif
Authors: Ross David A. , Magor Bradley G. , Middleton Darlene L. , Wilson Melanie R. , Miller Norman W. , Clem L. William , Warr Gregory W.
Published: January 2023

The Ig heavy chain enhancer of the channel catfish (Ictalurus punctatus) has an unusual position and structure, being found in the 3′ region of the μ gene and containing eight functional octamer motifs of consensus (ATGCAAAT) and variant sequences. The presence of multiple octamer motifs suggests that an Oct2 homologue may play an important role in driving expression of the Ig heavy chain locus in a teleost fish. To test this hypothesis, two catfish Oct2 cDNAs (α and β) were cloned by screening a catfish B cell cDNA library. Catfish Oct2 α and β isoforms are derived by alternative RNA splicing; as determined by Southern analysis, Oct2 is a single copy gene. In comparisons with mammalian Oct2, the catfish Oct2 isoforms show high sequence conservation in their N-terminal regions and POU domains, but extensive divergence in their C-terminal regions. Catfish Oct2 α and β are tissue restricted, bind both consensus and variant octamer motifs, and activate transcription in both catfish and murine cells. In contrast, mouse Oct2 activated transcription in mouse but not catfish cells. Catfish Oct2 β is a more potent transcriptional activator than Oct2 α. In transient expression assays, catfish Oct2 β showed a marked preference for the octamer variant, ATGtAAAT, which occurs twice in the catfish enhancer. Mouse Oct2 also showed increased activity with the variant octamer when tested in mouse B cells. Gel-shift analysis competition assays indicated that catfish Oct2 binds the consensus octamer motif with an apparently higher affinity than it does the variant motif.
Molecular Cloning and Functional Characterization of the Pig Analogue of CD59: Relevance to Xenotransplantation
Authors: Hinchliffe Stewart J. , Rushmere Neil K. , Hanna S. Melanie , Morgan B. Paul
Published: January 2023

In this work, we report the cloning of the cDNA for the porcine analogue of human CD59. Degenerate primers, derived from the N-terminal sequence of pig erythrocyte CD59, were used to obtain the corresponding cDNA sequence. From this sequence, gene-specific primers were designed and used to amplify the 3′ and 5′ ends of the cDNA using the rapid amplification of cDNA ends (RACE) method. The complete 768-bp cDNA so obtained consisted of a 84-bp 5′ untranslated region, a 26-amino-acid NH2-signal peptide, a 98-amino-acid coding region, including putative N-glycosylation sites and a glycosylphosphatidylinositol-anchoring signal, and a 312-bp 3′ untranslated region. The mature protein sequence was 48% identical to human CD59 at the amino acid level. Northern blot analysis revealed several distinct CD59 transcripts, and a variability in expression levels of the different transcripts in the panel of tissues screened. Stable expression of pig CD59 in a CD59-negative human cell line conferred protection against lysis by complement from pig and several other species. Separate expression of pig and human CD59 at similar levels in the same cell line allowed a direct functional comparison between these two analogues. Pig CD59 and human CD59 showed similar activity in inhibiting lysis by complement from all species tested; in particular, expressed pig CD59 efficiently inhibited lysis by human complement. The relevance of these data to current work in the engineering of pig organs for xenotransplantation is discussed.
Dendritic Cells Express Multiple Chemokine Receptors Used as Coreceptors for HIV Entry
Authors: Rubbert Andrea , Combadiere Christophe , Ostrowski Mario , Arthos James , Dybul Mark , Machado Elizabeth , Cohn Michael A. , Hoxie James A. , Murphy Philip M. , Fauci Anthony S. , Weissman Drew
Published: January 2023

Cells of the dendritic lineage are thought to be among the first cells infected after mucosal exposure to HIV. In this study, we have identified the presence of multiple chemokine receptors on dendritic cells (DC) that may function as coreceptors for HIV entry. DC effectively used CCR5 for entry of macrophage (M)-tropic isolates. CCR3, the eotaxin receptor, initially identified on eosinophils, is expressed on DC and may be used as an entry coreceptor by certain dual-tropic strains. CXCR4 was not expressed on DC, although SDF-1 induced a calcium flux and DC could be infected by T cell line (T)-tropic HIV. Our findings provide evidence for the presence of a non-CXCR4 SDF-1 receptor on DC that is used mainly by T-tropic strains of HIV. DC from individuals homozygous for a 32-bp deletion of the CCR5 gene are also infectable with M-tropic strains of HIV-1, and this infection is inhibited by stromal cell-derived factor (SDF)1, suggesting that this receptor can also be used by M-tropic HIV for entry. Delineation of the spectrum of coreceptor usage on DC may offer new approaches to interfere with the initiation and propagation of HIV infection.
Mice Lacking IL-12 Develop Polarized Th1 Cells During Viral Infection
Authors: Schijns Virgil E. C. J. , Haagmans Bart L. , Wierda Christel M. H. , Kruithof Boudewijn , Heijnen Ingmar A. F. M. , Alber Gottfried , Horzinek Marian C.
Published: January 2023

Studies in IL-12-deficient mice established the necessity for IL-12 to generate a Th1 cytokine response that is often required for elimination of intracellular pathogens. In this study, we demonstrate that mice with a targeted disruption of the IL-12p40 and/or p35 gene effectively control liver damage induced by mouse hepatitis virus (MHV) infection, similar to wild-type animals. In contrast, MHV-infected IFN-γ receptor-deficient (IFN-γR−/−) mice showed an increased susceptibility to coronaviral hepatitis. Surprisingly, MHV-infected mice lacking IL-12 produced a polarized Th1-type cytokine response, as evidenced by high IFN-γ and nondetectable IL-4 production by CD4+ splenocytes and normal virus-specific serum IgG2a/IgG1 ratios. The virus-induced type 1 cytokine secretion pattern was not reversed in IL-12-deficient mice by in vivo neutralization of IFN-γ nor in IFN-γR−/− mice receiving IL-12-neutralizing Abs. In IL-12-deficient mice, Th1-type responses were also generated upon immunization with inactivated MHV. In contrast, following immunization with keyhole limpet hemocyanin, mice lacking IL-12 mounted strongly reduced specific IgG2a and increased IgE responses, indicative of a type 2-dominated cytokine pattern. These findings demonstrate that following a virus infection, IL-12 is not essential for the generation of polarized T cell type 1 cytokine expression and associated immune responses, which is in marked contrast to nonviral systems. Our data suggest that viruses may selectively induce IFN-γ production and Th1-type immune reactions even in the absence of IL-12.
The TCR-Binding Region of the HLA Class I α2 Domain Signals Rapid Fas-Independent Cell Death: A Direct Pathway for T Cell-Mediated Killing of Target Cells?
Authors: Pettersen Rolf D. , Gaudernack Gustav , Olafsen Mette Kløvstad , Lie Sverre O. , Hestdal Kjetil
Published: January 2023

TCR binding to an MHC class I/peptide complex is a central event in CTL-mediated elimination of target cells. In this study, we demonstrate that specific activation of the TCR-binding region of the HLA-A2 class I α2 domain induces apoptotic cell death. mAbs to this region rapidly induced apoptosis of HLA-A2-expressing Jurkat E11 cells, as determined by morphologic changes, phosphatidylserine exposure on the cell surface, and propidium iodide uptake. In contrast, apoptosis was not induced following culture with mAbs directed to other regions of the class I molecule. Death signaling by class I molecules is apparently dependent on coreceptor activation, as apoptosis is also signaled by HLA-A2 molecules, where the intracytoplasmic residues were deleted. HLA class I α2-mediated cell death appeared to proceed independent of the Fas pathway. Compared with apoptotic signaling by Fas ligation, HLA class I α2-mediated responses displayed a faster time course and could be observed within 30 min. Furthermore, class I α2-induced cell death did not involve observable DNA fragmentation. The apoptotic response was not affected significantly by peptide inhibitors of IL-1β converting enzyme (ICE)-like proteases and CPP32. Taken together, activation of the TCR-binding domain of the class I α2 helix may result in apoptotic signaling apparently dependent on a novel death pathway. Thus, target HLA class I molecules may directly signal apoptotic cell death following proper ligation by the TCR.
Evidence for Sulfate Modification of H-2Dd on N-Linked Carbohydrate(s): Possible Involvement in Ly-49A Interaction
Authors: Chang Chew Shun , Kane Kevin P.
Published: January 2023

Murine class I molecules are ligands for Ly-49 molecules, a family of regulatory receptors expressed on murine NK cells. Since soluble sulfated mono- and polysaccharides interfere with the interaction of Ly-49A, a C-type lectin, and its class I ligand, Dd, it is possible that the oligosaccharides on class I molecules are sulfated and participate in Ly-49A binding. In this report, we show that H-2Dd expressed by activated T cells and various tumor cell lines is sulfated, as demonstrated by immunoprecipitation of Dd following Na235SO4 labeling. The 35SO4−2 label on Dd expressed by a representative tumor cell, NZB1.1, is removed by peptide N-glycosidase F, but is resistant to endoglycosidase H treatment, indicating that the sulfate group is located on mature N-linked oligosaccharides. Two-dimensional SDS-PAGE analysis revealed that all major mature glycosylation variants of the Dd expressed by NZB1.1 are sulfated. Sodium chlorate, a potent inhibitor of ATP-sulfurylase, which prevents the formation of the sulfate donor, 3′-phosphoadenosine 5′-phosphosulfate, inhibited metabolic sulfation of Dd. NZB1.1 binds isolated Ly-49A immobilized on solid phase through an interaction by cell surface Dd, since cell adhesion was blocked by Abs directed against Dd or Ly-49A. Treatment of the Dd-expressing NZB1.1 tumor cells with sodium chlorate reduced their ability to bind immobilized Ly-49A, particularly when Ly-49A density was limiting. These results provide evidence for sulfation of H-2Dd oligosaccharide moieties, and suggest a role for this posttranslational modification in the interaction of Dd with Ly-49A.
Down-Regulation of CXCR2 Expression on Human Polymorphonuclear Leukocytes by TNF-α
Authors: Asagoe Kohsuke , Yamamoto Kokichi , Takahashi Atsushi , Suzuki Kazuo , Maeda Akinori , Nohgawa Masaharu , Harakawa Nari , Takano Kuniko , Mukaida Naofumi , Matsushima Kouji , Okuma Minoru , Sasada Masataka
Published: January 2023

TNF-α is implicated in the initiation of cytokine cascades in various inflammatory settings. To assess the interactions of multiple cytokines at the level of inflammatory effector cells, we examined the effects of TNF-α on the expression of two IL-8Rs (CXCR1 and CXCR2) on polymorphonuclear leukocytes (PMNs). TNF-α decreased the surface expression of CXCR2 in a dose- and time-dependent manner. In contrast, CXCR1 expression was not affected by TNF-α. The release of CXCR2 into the supernatant of TNF-α-treated PMNs was detected by immunoblotting and immuno-slot-blot analyses, suggesting that the down-regulation of CXCR2 was caused mainly by shedding from the cell surface. The CXCR2 down-regulation was inhibited by PMSF and aprotinin, supporting the hypothesis that the shedding was mediated by serine protease(s). The intracellular Ca2+ mobilization and chemotaxis in response to IL-8 were suppressed by the pretreatment of PMNs with TNF-α, indicating that the decrease in CXCR2 was reflected in the decreased functional responses to IL-8. In contrast, the O2− release, which is mediated by CXCR1, was not suppressed by TNF-α. The treatment of whole blood with TNF-α also caused a significant reduction in CXCR2 and markedly suppressed intracellular Ca2+ mobilization and chemotaxis in response to IL-8, while enhancing the O2− release. These findings suggest that TNF-α down-regulates CXCR2 expression on PMNs and modulates IL-8-induced biologic responses, leading to the intravascular retention of PMNs with an enhanced production of reactive oxygen metabolites.
Involvement of CD14 and Complement Receptors CR3 and CR4 in Nuclear Factor-κB Activation and TNF Production Induced by Lipopolysaccharide and Group B Streptococcal Cell Walls
Authors: Medvedev Andrei E. , Flo Trude , Ingalls Robin R. , Golenbock Douglas T. , Teti Giuseppe , Vogel Stefanie N. , Espevik Terje
Published: January 2023

This study was undertaken to evaluate the role of CD14 and complement receptors type 3 (CR3) and 4 (CR4) in mediating TNF release and NF-κB activation induced by LPS and cell wall preparations from group B streptococci type III (GBS). LPS and GBS caused TNF secretion from human monocytes in a CD14-dependent manner, and soluble CD14, LPS binding protein, or their combination potentiated both LPS- and GBS-induced activities. Blocking of either CD14 or CD18, the common β-subunit of CR3 and CR4, decreased GBS-induced TNF release, while LPS-mediated TNF production was inhibited by anti-CD14 mAb only. Chinese hamster ovary cell transfectants (CHO) that express human CD14 (CHO/CD14) responded to both LPS and GBS with NF-κB translocation, which was inhibited by anti-CD14 mAb and enhanced by LPS binding protein. While LPS showed fast kinetics of NF-κB activation in CHO/CD14 cells, a slower NF-κB response was induced by GBS. LPS also activated NF-κB in CHO cells transfected with either human CR3 or CR4 cDNA, although responses were delayed and weaker than those of CHO/CD14 cells. In contrast to LPS, GBS failed to induce NF-κB in CHO/CR3 or CHO/CR4 cells. Both C3H/OuJ (Lpsn) and C3H/HeJ (Lpsd) mouse peritoneal macrophages responded to GBS with TNF production and NF-κB translocation, whereas LPS was active only in C3H/OuJ macrophages. Thus, LPS and GBS differentially involve CD14 and CR3 or CR4 for signaling NF-κB activation in CHO cells and TNF release in human monocytes, and engage a different set of receptors and/or intracellular signaling pathways in mouse macrophages.
Cutting Edge: The Expression In Vivo of a Second Isoform of pTα: Implications for the Mechanism of pTα Action
Authors: Barber Domingo F. , Passoni Lorena , Wen Li , Geng Liping , Hayday Adrian C.
Published: January 2023

A second isoform of pTα, “pTαb,” is derived from the pTα locus by tissue-specific, alternative splicing. pTαb is coexpressed in the thymus with the previously characterized form of pTα (which we term pTαa) and is also expressed in peripheral cells without pTαa. While pTαa acts to retain most TCR β-chains intracellularly, pTαb permits higher levels of cell surface TCRβ expression and facilitates signaling from a CD3-TCRβ complex.
A Role for Heat Shock Protein 27 in CTL-Mediated Cell Death
Authors: Beresford Paul J. , Jaju Madhuri , Friedman Rachel S. , Yoon Margaret J. , Lieberman Judy
Published: January 2023

CTL exocytosis of granules containing perforin and granzyme proteases induces apoptotic cell death. Either granzyme A or B can act with perforin to trigger apoptosis. Granzyme B activates a ubiquitous apoptotic cascade induced by caspase cleavage, but the granzyme A pathway is largely unknown. Using affinity chromatography with recombinant mutant inactive granzyme A, we previously isolated two granzyme A-binding proteins, PHAP (putative HLA-associated protein) I and II. PHAP II, a substrate of granzyme A, is degraded within minutes of CTL attack. Two additional cytoplasmic proteins of 27 and 53 kDa bind strongly to the mutant granzyme A column, requiring 6 M urea to elute. Sequencing identified these as the monomer and dimer of hsp27, a small heat shock protein up-regulated by stress and cellular activation. Hsp27 coprecipitates with granzyme A from cytoplasmic lysates and is not a substrate of the enzyme. Hsp27 translocates to the detergent-insoluble fraction of target cells and relocalizes from diffuse cytoplasmic staining to long filamentous fibers, especially concentrated in a perinuclear region, within minutes of CTL attack. Hsp27 may participate in morphologic changes during granule-mediated lysis. Low or absent levels of hsp27 expression in T lymphocytes, even after heat shock, may play a role in CTL resistance to granule-mediated lysis.
CD4+ T Cells Mature in the Absence of MHC Class I and Class II Expression in Ly-6A.2 Transgenic Mice
Authors: Henderson S. Christine , Berezovskaya Alla , English Andrea , Palliser Deborah , Rock Kenneth L. , Bamezai Anil
Published: January 2023

The TCRs expressed on T lymphocytes recognize foreign peptides bound to MHC molecules. This reactivity is the basis of specific immune response to the foreign Ag. How such specificities are generated in the thymus is still being debated. Signals generated through TCR upon interaction with self MHC-peptide complexes are critical for maturation of the CD4+ helper and CD8+ cytotoxic subsets. We have observed maturation of CD4+ but not CD8+ T cells in Ly-6A.2 transgenic MHC null mice. Since there can be no interactions with MHC molecules in these mice, these CD4+ cells must express the T cell repertoire that exists before positive and negative selection. Interestingly, despite an absence of selection by MHC molecules, the CD4+ cells that mature recognize MHC molecules at a frequency as high as in CD4+ cells in normal mice. These results demonstrate that: 1) the germline sequences encoding TCRs are biased toward reactivity to MHC molecules; and 2) CD4+ cells as opposed to CD8+ cells have distinct lineage commitment signals. These results also suggest that signals originating from Ly-6 can promote or substitute for signals generated from TCR that are required for positive selection. Moreover, this animal model offers a system to study T cell development in the thymus that can provide insights into mechanisms of lineage commitment in developing T cells.
A Synthetic CD4-CDR3 Peptide Analog Enhances Skin Allograft Survival Across a MHC Class II Barrier
Authors: Koch Ute , Choksi Swati , Marcucci Lisa , Korngold Robert
Published: January 2023

The efficacy of a synthetic peptide analogue (rD-mPGPtide), mimicking the CDR3 region in the first domain of the CD4 surface molecule, was investigated in a murine model for CD4+ T cell-mediated skin allograft rejection. A single injection of rD-mPGPtide shortly before transplantation exhibited significantly prolonged graft survival in the B6 anti-B6.C-H2bm12 MHC class II-disparate strain combination. Long-term graft survival (>100 days) was achieved when thymectomized adult recipient mice were transplanted along with rD-mPGPtide treatment. The peptide also affected secondary rechallenge responses with MHC class II allografts. In addition, the inhibitory effect of the rD-mPGPtide in this transplantation model was directed against CD4+ T cells and was exclusively specific toward donor alloantigen. In vitro analysis of CD4+ T cells isolated from the draining lymph nodes of rD-mPGPtide-treated recipients indicated a 450-fold decrease in precursor frequency in response to donor allostimulation compared with the untreated control group. There was also significant down-regulation of the frequency of IL-2-, IFN-γ-, and IL-4-producing CD4+ T cells upon in vitro allogeneic restimulation of host cells 4 days posttransplantation. However, these same CD4+ T cells maintained the capacity to produce normal cytokine levels upon third-party allostimulation. Thus, these studies demonstrate that a CD4-CDR3 peptide analogue can specifically and effectively prolong skin graft survival across MHC class II barriers.
Experimental Autoimmune Encephalomyelitis Induced with a Combination of Myelin Basic Protein and Myelin Oligodendrocyte Glycoprotein Is Ameliorated by Administration of a Single Myelin Basic Protein Peptide
Authors: Leadbetter Elizabeth A. , Bourque Cheryl R. , Devaux Brigitte , Olson Carl D. , Sunshine Geoffrey H. , Hirani Shirish , Wallner Barbara P. , Smilek Dawn E. , Happ Mary Pat
Published: January 2023

Multiple sclerosis is an autoimmune disease of the central nervous system in which T cell reactivity to several myelin proteins, including myelin basic protein (MBP), proteolipid protein, and myelin oligodendrocyte glycoprotein (MOG), has been implicated in the perpetuation of the disease state. Experimental autoimmune encephalomyelitis (EAE) is used commonly as a model in which potential therapies for multiple sclerosis are evaluated. The ability of T cell epitope-containing peptides to down-regulate the disease course is well documented for both MBP- and proteolipid protein-induced EAE, and recently has been shown for MOG-induced EAE. In this study, we describe a novel EAE model, in which development of severe disease symptoms in (PL/J × SJL)F1 mice is dependent on reactivity to two different immunizing Ags, MBP and MOG. The disease is often fatal, with a relapsing/progressive course in survivors, and is more severe than would be predicted by immunization with either Ag alone. The MOG plus MBP disease can be treated postinduction with a combination of the MOG 41–60 peptide (identified as the major therapeutic MOG epitope for this strain) and the MBP Ac1–11[4Y] peptide. A significant treatment effect can also be obtained by administration of the MBP peptide alone, but this effect is strictly dose dependent. This MBP peptide does not treat the disease induced only with MOG. These results suggest that peptide immunotherapy can provide an effective means of mitigating disease in this model, even when the treatment is targeted to only one component epitope or one component protein Ag of a diverse autoimmune response.
Pentoxifylline Inhibits Adhesion and Activation of Human T Lymphocytes
Authors: González-Amaro Roberto , Portales-Pérez Diana , Baranda Lourdes , Redondo Juan M. , Martínez-Martínez Sara , Yáñez-Mó María , García-Vicuña Rosario , Cabañas Carlos , Sánchez-Madrid Francisco
Published: January 2023

We have herein studied the effect of pentoxifylline (PTX) on the adhesion and activation of human T lymphocytes. We found that PTX inhibited the adhesion of T cells to the β1 and β2 integrin ligands VCAM-1 and ICAM-1; this inhibitory activity was dose dependent, with a maximal effect from 12 to 24 h. We also found that PTX was able to interfere with the activation of β1 integrins induced by intracellular signals; however, the conformational change of β1 integrins induced by extracellular stimuli (e.g., activating mAbs, or Mn2+) was not significantly affected by this drug. In addition, the homotypic aggregation of T cells induced by anti-β1 and -β2 integrin chain mAbs was also inhibited by PTX. PTX had a significant inhibitory effect on the T lymphocyte expression of the activation Ags CD25 (IL-2Rα-chain), CD69 (activation-inducer molecule), and CD98 (4F2) induced by PHA. Accordingly, PTX also interfered with early cell activation events such as the rise in intracellular Ca2+ and the activation of the Na+/H+ antiporter induced by PHA and phorbol esters, respectively. Furthermore, this drug inhibited both the cell cycle progression and cell proliferation of T cells induced through the CD3/TCR complex. However, this drug did not show any effect on the cell activation/proliferation induced by PMA plus ionomycin. Our results indicate that PTX interferes efficiently with the activation and cell adhesion of human T lymphocytes. These effects may be of relevance for the clinical uses of this drug.
Cutting Edge: Ly-49D and Ly-49H Associate with Mouse DAP12 and Form Activating Receptors
Authors: Smith Kathleen M. , Wu Jun , Bakker Alexander B. H. , Phillips Joseph H. , Lanier Lewis L.
Published: January 2023

Several members of the Ly-49 receptor family inhibit NK cell-mediated lysis of targets expressing appropriate MHC class I molecules. Ly-49D and Ly-49H, two Ly-49 molecules that lack immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their cytoplasmic domains, associate with mouse DAP12, a molecule that possesses an immunoreceptor tyrosine-based activation motif (ITAM). Cotransfection of either Ly-49D or Ly-49H with DAP12 induces surface expression of both Ly-49 and DAP12. The Ly-49/DAP12 complex was coimmunoprecipitated from the transfected cells, demonstrating a physical association of DAP12 with Ly-49D or Ly-49H in the plasma membrane. Stimulation of transfectants with Abs recognizing either Ly-49D or Ly-49H results in cellular activation, as assessed by induction of tyrosine phosphorylation of multiple cellular substrates.
H2-M3 Presents a Nonformylated Viral Epitope to CTLs Generated In Vitro
Authors: Byers Derek E. , Fischer Lindahl Kirsten
Published: January 2023

Most CTL responses to epitopes from influenza virus are restricted by MHC class Ia molecules. However, a synthetic peptide corresponding to residues 173 to 190 of influenza A/JAP/305/57 hemagglutinin (HA) can induce, in vitro, a CTL response to peptide presented by a mouse class Ib molecule encoded by a gene telomeric to H2-Q. Here, we identify the molecule as H2-M3 and show that the last five residues of HA173–190, MLIIW, is the minimal epitope for CTL recognition. Cells that express M3wt, from C57BL/6 or BALB/c mice, are sensitized by both MLIIW and the longer peptide HA173–190, whereas cells that express M3f, from A.CA or B10.M mice, are sensitized only by MLIIW; a single amino acid change at residue 31 (V→M) of M3 accounts for this difference. Although M3-restricted CTLs preferably recognize N-formylated epitopes, i.e., those of mitochondrial or prokaryotic origin, our findings show that M3-restricted primary CTL responses can be generated in vitro against nonformylated epitopes.
In Vivo Persistence of CD8 Polarized T Cell Subsets Producing Type 1 or Type 2 Cytokines
Authors: Cerwenka Adelheid , Carter Laura L. , Reome Joyce B. , Swain Susan L. , Dutton Richard W.
Published: January 2023

Naive CD8 T cells can be polarized into effectors producing the type 1 cytokines IFN-γ and IL-2 or the type 2 cytokines IL-4, IL-5, and IL-10, respectively. To study whether the polarized cytokine phenotype of the effectors is stable, we generated highly cytotoxic hemagglutinin (HA) peptide-specific CD8 Tc1 and Tc2 (cytotoxic CD8 T cells producing type 1 or type 2 cytokines) effectors from Clone-4 TCR-transgenic mice, which were adoptively transferred into syngeneic adult thymectomized irradiated and bone marrow-reconstituted recipients. The highly activated blast-size, CD25+ Tc1 and Tc2 effectors gave rise to homogeneous resting CD25−CD44highLy6Chigh Ag-specific populations, which persisted for at least 13 wk after adoptive transfer. These memory CD8 T cells, recovered 13 wk after transfer of Tc1 or Tc2 effectors, still produced either the type 1 or type 2 cytokines, i.e., IFN-γ, or IL-4 and IL-5, respectively, upon restimulation with APCs loaded with the HA peptide, but not in the absence of Ag. The amounts of IL-2 detected in the supernatants of Tc1 and Tc2 memory populations were comparable to that in memory CD4 cells, and both Tc1 and Tc2 memory cells became cytotoxic upon restimulation. Thus, cytokine-polarized CD8 memory T cells are a source of a variety of cytokines, which were classically considered helper cytokines, opening new perspectives on their function as regulatory cells in an immune response.
Cutting Edge: CD19+ Pro-B Cells Can Give Rise to Dendritic Cells In Vitro
Authors: Björck Pia , Kincade Paul W.
Published: January 2023

Dendritic cells (DC) have the specific capacity of initiating primary T cell responses and ultimately derive from precursors in bone marrow. DC were originally thought to be only of myeloid origin, and myeloid precursor cells could be induced to differentiate into functional DC in response to granulocyte-macrophage (GM)-CSF. However, early CD4low precursor cells from the thymus can also develop into DC when cultured in IL-1β, IL-3, IL-7, TNF-α, stem cell factor, and Flt-3L. In that case, GM-CSF was not required. We now show that CD19+ pro-B cells develop into DC with T cell stimulatory properties when cultured under similar conditions. These pro-B cells acquired the DC-related markers CD11c and NLDC145/DEC205, along with CD80/B7-1, CD86/B7-2, and a high density of MHC class II Ags. The marrow-derived DC did not express CD4 or CD8α, which are markers related to thymic DC. These findings are consistent with a new pathway through which DC are generated from B lymphoid precursors.
Cutting Edge: A Role for p21ras/MAP Kinase in TCR-Mediated Activation of LFA-1
Authors: O’Rourke Anne M. , Shao Hui , Kaye Jonathan
Published: January 2023

LFA-1 is a β2 integrin that plays well-characterized roles in adhesion of T lymphocytes to APC, T cell-mediated cytolysis, and leukocyte-endothelial cell interactions. Although it is clear that LFA-1 must undergo affinity or avidity changes to bind its cellular ligand ICAM-1, the intracellular signaling pathways involved are not well characterized. Here, we show that the Ras-mitogen-activated protein kinase (MAPK) signaling pathway is also involved in TCR-activated LFA-1 adhesion. Expression of a dominant negative form of p21ras in a thymocyte cell line inhibits, while constitutively active p21ras both enhances and sustains, subsequent TCR-triggered adhesion to isolated ICAM-1. However, the Ras/MAPK pathway alone is not sufficient for activating T cell LFA-1, as inhibition of both downstream MAPK/extracellular regulated kinase kinase (MEK) activity and phosphatidylinositol 3-kinase activity is required for complete inhibition of adhesion.
Efficient Presentation of Multivalent Antigens Targeted to Various Cell Surface Molecules of Dendritic Cells and Surface Ig of Antigen-Specific B Cells
Authors: Serre Karine , Machy Patrick , Grivel Jean-Charles , Jolly Gilles , Brun Nicole , Barbet Jacques , Leserman Lee
Published: January 2023

To study the relation between the form of an Ag and the response to it, we compared presentation in vitro with hen egg lysozyme (HEL)-specific T cells from TCR transgenic mice of free HEL and liposome-encapsulated HEL by different APC. HEL-specific splenic B cells or bone marrow-derived dendritic cells were incubated with free HEL or HEL-containing liposomes targeted by Ab to either surface Ig, the Fc receptor, or MHC class I and II molecules. Ag presentation by HEL-specific B cells was at least 100-fold more efficient for HEL in surface Ig-targeted liposomes than free HEL taken up by the same receptor or HEL in liposomes targeted to class I or II molecules. Ag presentation by dendritic cells from Fc receptor-targeted vesicles was augmented 1,000–10,000-fold compared with free Ag or nontargeted liposomes, but presentation was also efficient when Ag was targeted to class I or II molecules. These results indicate that Ag-specific B cells and dendritic cells can be equally efficient in stimulating IL-2 production by Ag-specific T cells from unimmunized TCR transgenic mice when the Ag is multivalent and taken up by appropriate receptors. In contrast to B cells, which require engagement of surface Ig for optimal presentation, dendritic cells may present Ag by means of several different cell surface molecules.
Isolation and Quantitation of a Minor Determinant of Hen Egg White Lysozyme Bound to I-Ak by Using Peptide-Specific Immunoaffinity
Authors: Gugasyan Raffi , Vidavsky Ilan , Nelson Christopher A. , Gross Michael L. , Unanue Emil R.
Published: January 2023

We report here the identification and quantitation of a minor epitope from hen egg white lysozyme (HEL) isolated from the class II MHC molecule I-Ak of APCs. We isolated and concentrated the peptides from the I-Ak extracts by a peptide-specific mAba, followed by their examination by electrospray mass spectrometry. This initial step improved the isolation, recovery, and quantitation and allowed us to identify 13 different minor peptides using the Ab specific for the HEL tryptic fragment 34–45. The HEL peptides varied on both the amino and carboxy termini. The shortest peptide was a 13-mer (residues 33–45), and the longest peptide was a 19-mer (residues 31–49). The two most abundant were 31–47 (1.3 pmol) and 31–46 (1 pmol), while the least abundant were 31–45 (40 fmol) and 32–45 (4 fmol). Only 0.3% of the total class II molecules were occupied by this family of HEL peptides. The amount of the 31–47 peptide, the predominant member of this series, was 22 times lower than that of 48–62, the major epitope of HEL. The 31–47 peptide bound about 20-fold weaker to I-Ak compared with the dominant 48–62 peptide. Thus, the lower abundance of the minor epitope correlated with its weaker binding strength.
LeIF: A Recombinant Leishmania Protein That Induces an IL-12-Mediated Th1 Cytokine Profile
Authors: Skeiky Yasir A. W. , Kennedy Mary , Kaufman David , Borges Monamaris M. , Guderian Jeffrey A. , Scholler John K. , Ovendale Pamela J. , Picha Kathleen S. , Morrissey Philip J. , Grabstein Kenneth H. , Campos-Neto Antonio , Reed Steven G.
Published: January 2023

We have evaluated the ability of the Leishmania protein LeIF to influence the Th1/Th2 cytokine responses and the generation of LeIF-specific T cell clones in the absence of adjuvant. We characterized LeIF-specific T cell responses in Leishmania major-infected and uninfected BALB/c mice. These mice develop a strong Th2 response during infection with L. major. When lymph node cells from infected BALB/c mice were stimulated in vitro with LeIF, only IFN-γ (and no detectable IL-4) was found in the culture supernatant. In addition, LeIF down-regulated Leishmania Ag-specific IL-4 production by lymph node cells from infected BALB/c mice. Subsequently, Th responses were evaluated in naive BALB/c mice following immunization with LeIF. T cell clones derived from mice immunized with LeIF preferentially secreted IFN-γ. Finally, to understand the basis for the preferential Th1 cytokine bias observed with LeIF, the ability of LeIF to influence the early cytokine profile was evaluated in splenocytes of SCID mice. We found that LeIF stimulated fresh spleen cells from naive SCID mice to secrete IFN-γ by IL-12/IL-18-dependent mechanisms. The N-terminal half of the molecule (amino acid residues 1–226) maintained the ability to stimulate IFN-γ from splenocytes of SCID mice. Finally, we also demonstrated that LeIF was able to provide partial protection of BALB/c mice against L. major. Thus, our results suggest the potential of LeIF as a Th1-type adjuvant and as a therapeutic and prophylactic vaccine Ag for leishmaniasis when used with other leishmanial Ags.
Differential Effects of CD28 Costimulation on HIV Production by CD4+ Cells
Authors: Barker Edward , Bossart Katharine N. , Levy Jay A.
Published: January 2023

We have observed that CD28 costimulation of CD4+ cells can have differential effects on HIV replication. Triggering the CD28 molecule on peripheral blood CD4+ cells during stimulation with anti-CD3 Abs enhances virus production following acute infection with HIV. Endogenous virus production in CD4+ cells from HIV-infected individuals is also increased by this procedure. The enhanced virus production occurs equally when anti-CD28 Abs and soluble forms of the natural ligands for CD28, CD80Ig, and CD86Ig are used to trigger CD28 on CD4+ cells during stimulation. This increased virus replication is observed only when the source of CD28 costimulation is removed immediately after stimulation and before infection. Continual exposure of CD4+ cells to anti-CD3 and CD28 Ab beads following acute infection prevents virus production. These findings may have relevance to therapeutic approaches aimed at inhibiting HIV replication by CD28 costimulation.
Soluble IL-6 Receptor Potentiates the Antagonistic Activity of Soluble gp130 on IL-6 Responses
Authors: Müller-Newen Gerhard , Küster Andrea , Hemmann Ulrike , Keul Radovan , Horsten Ursula , Martens Astrid , Graeve Lutz , Wijdenes John , Heinrich Peter C.
Published: January 2023

Soluble receptors for several cytokines have been detected in body fluids and are believed to modulate the cytokine response by binding the ligand and thereby reducing its bioavailability. In the case of IL-6, the situation is more complex. The receptor consists of two components, including a ligand-binding α-subunit (IL-6R, gp80, or CD126), which in its soluble (s) form (sIL-6R) acts agonistically by making the ligand accessible to the second subunit, the signal transducer gp130 (CD130). Soluble forms of both receptor subunits are present in human blood. Gel filtration of iodinated IL-6 that had been incubated with human serum revealed that IL-6 is partially trapped in IL-6/sIL-6R/sgp130 ternary complexes. sgp130 from human plasma was enriched by immunoaffinity chromatography and identified as a 100-kDa protein. Functionally equivalent rsgp130 was produced in baculovirus-infected insect cells to study its antagonistic potential on four different cell types. It was found that in situations in which cells lacking membrane-bound IL-6R were stimulated with IL-6/sIL-6R complexes, sgp130 was a much more potent antagonist than it was on IL-6R-positive cells stimulated with IL-6 alone. In the latter case, the neutralizing activity of sgp130 could be markedly enhanced by addition of sIL-6R. As a consequence of these findings, sIL-6R of human plasma must be regarded as an antagonistic molecule that enhances the inhibitory activity of sgp130. Furthermore, in combination with sIL-6R, sgp130 is a promising candidate for the development of IL-6 antagonists.
Treatment with Anti-Granulocyte Antibodies Inhibits the Effector Phase of Experimental Autoimmune Encephalomyelitis
Authors: McColl Shaun R. , Staykova Maria A. , Wozniak Andrzej , Fordham Sue , Bruce Joanna , Willenborg David O.
Published: January 2023

Emerging data suggest that polymorphonuclear leukocytes (PMNLs) can play an important role in Ag-dependent immune responses. Therefore, we have assessed the involvement of these cells in the development of an organ-specific autoimmune disease, experimental autoimmune encephalomyelitis (EAE), in the mouse. Depletion of peripheral blood PMNLs beginning day 8 after immunization significantly delayed and in some cases totally prevented the development of clinical EAE in mice. Depletion of PMNLs beginning 1 day before sensitization and continuing until day 7 postimmunization had no effect on the subsequent development of EAE, suggesting that depletion alters the efferent but not the afferent arm of the immune response. In vitro studies showed that lymphoid cells from mice protected from EAE by PMNL depletion beginning on day 8 postsensitization proliferated in response to specific Ag to a level equal to cells from sensitized animals treated with control serum, again indicating that treatment was not affecting the afferent limb of the immune response. Further evidence that PMNL may be necessary in initiating the pathology of EAE was seen in passive transfer experiments where PMNL-depleted recipients of MBP-specific lymphoid effector cells developed EAE much less effectively than did animals treated with control Ab. Taken together, these data indicate that PMNLs play a critical role in the effector phase of the development of the clinicopathologic expression of EAE in mice.
Cutting Edge: Fc Receptor Type I for IgG on Macrophages and Complement Mediate the Inflammatory Response in Immune Complex Peritonitis
Authors: Heller Tanja , Gessner J. Engelbert , Schmidt Reinhold E. , Klos Andreas , Bautsch Wilfried , Köhl Jörg
Published: January 2023

The contributions of Fc receptors (FcRs) for IgG (FcγRs) and complement to immune complex (IC)-mediated peritonitis were evaluated in BALB/c-, C57BL/6-, FcRγ chain-, and FcR type III for IgG (FcγRIII)-deficient mice, backcrossed to the C57BL/6 background. In BALB/c mice, but not in C57BL/6 mice, neutrophil migration was markedly attenuated after complement depletion. In mice lacking FcRγ chain, neutrophil migration was abolished, whereas it was unaffected in FcγRIII-deficient mice. Huge amounts of TNF-α (TNF) were found in the peritoneal exudate of BALB/c and C57BL/6 mice but were absent in mice lacking FcRγ chain or FcγRIII. Surprisingly, a functional inhibition of TNF in BALB/c and C57BL/6 mice had no effect on neutrophil infiltration. These data provide evidence that in IC peritonitis, the activation of FcR type I for IgG on peritoneal macrophages and the activation of the complement cascade, but not the interaction of ICs with FcγRIII and the subsequent release of TNF, initiate the inflammatory response in BALB/c and C57BL/6 mice.
Differential Presentation of an Altered Peptide Within Fetal Central and Peripheral Organs Supports an Avidity Model for Thymic T Cell Development and Implies a Peripheral Readjustment for Activation
Authors: Legge Kevin L. , Min Booki , Pack Christopher , Caprio Jacque , Zaghouani Habib
Published: January 2023

Altered self peptides may drive T cell development by providing avidity of interactions low enough to potentiate positive selection but not powerful enough to trigger programmed cell death. Since the peptide repertoire in both central and peripheral organs is nearly the same, interactions of these peptides with T cells in the thymus would have to be different from those taking place in the periphery; otherwise, T cell development and maturation would result in either autoimmunity or T cell deficiency. Herein, a self and an altered self peptide were delivered to fetuses, and their presentation as well as the consequence of such presentation on T cell development were assessed. The results indicate that the self peptide was presented in both central and peripheral fetal organs and that such presentation abolished T cell responses to both peptides during adult life. However, the altered peptide, although presented in vivo as well as in vitro by splenic cells, was unable to stimulate a specific T cell clone when the presenting cells were of thymic origin and allowed offspring to be responsive to both peptides. These findings indicate that central and peripheral organs accommodate selection and peripheral survival of T cells by promoting differential altered peptide presentation.
Lymphoproliferative Disorder in CTLA-4 Knockout Mice Is Characterized by CD28-Regulated Activation of Th2 Responses
Authors: Khattri Roli , Auger Julie A. , Griffin Matthew D. , Sharpe Arlene H. , Bluestone Jeffrey A.
Published: January 2023

Mice lacking CTLA-4 die at an age of 2–3 wk due to massive lymphoproliferation, leading to lymphocytic infiltration and destruction of major organs. The onset of the lymphoproliferative disease can be delayed by treatment with murine CTLA4Ig (mCTLA4Ig), starting day 12 after birth. In this study, we have characterized the T cells present in CTLA-4-deficient mice before and after mCTLA4Ig treatment. The T cells present in CTLA-4-deficient mice express the activation markers, CD69 and IL-2R; down-regulate the lymphoid homing receptor, CD62L; proliferate spontaneously in vitro and cannot be costimulated with anti-CD28 mAb consistent with a hyperactivated state. The T cells from CTLA-4-deficient mice survive longer in culture correlating with higher expression of the survival factor, Bcl-xL, in these cells. Most significantly, the CD4+ T cell subset present in CTLA-4-deficient mice secretes high levels of IL-4 and IL-5 upon TCR activation. Treatment of CTLA-4-deficient mice treated with mCTLA4Ig reverses the activation and hyperproliferative phenotype of the CTLA-4-deficient T cells and restores the costimulatory activity of anti-CD28 mAb. Furthermore, T cells from mCTLA4Ig-treated mice are not skewed toward a Th2 cytokine phenotype. Thus, CTLA-4 regulates CD28-dependent peripheral activation of CD4+ T cells. This process results in apoptosis-resistant, CD4+ T cells with a predominantly Th2 phenotype that may be involved in the lethal phenotype in these animals.