bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of Extracellular Signal-Regulated Kinase Module in HIV-Mediated CD4 Signals Controlling Activation of Nuclear Factor-κB and AP-1 Transcription Factors
Authors: Briant Laurence , Robert-Hebmann Véronique , Sivan Virginie , Brunet Anne , Pouysségur Jacques , Devaux Christian
Published: December 2022

Although the molecular mechanisms by which the HIV-1 triggers either T cell activation, anergy, or apoptosis remain poorly understood, it is well established that the interaction of HIV-1 envelope glycoproteins with cell surface CD4 delivers signals to the target cell, resulting in activation of transcription factors such as NF-κB and AP-1. In this study, we report the first evidence indicating that kinases MEK-1 (MAP kinase/Erk kinase) and ERK-1 (extracellular signal-regulated kinase) act as intermediates in the cascade of events that regulate NF-κB and AP-1 activation upon HIV-1 binding to cell surface CD4. We found that CEM cells transfected with dominant negative forms of MEK-1 or ERK-1 do not display NF-κB activation after HIV-1 binding to CD4. In contrast, NF-κB activation was observed in these cells after PMA stimulation. Although the different cell lines studied expressed similar amounts of CD4 and p56lck, HIV-1 replication and HIV-1-induced apoptosis were slightly delayed in cells expressing dominant negative forms of MEK-1 or ERK-1 compared with parental CEM cells and cells expressing a constitutively active mutant form of MEK-1 or wild-type ERK-1. In light of recently published data, we propose that a positive signal initiated following oligomerization of CD4 by the virus is likely to involve a recruitment of active forms of p56lck, Raf-1, MEK-1, and ERK-1, before AP-1 and NF-κB activation.
RANTES Activation of Phospholipase D in Jurkat T Cells: Requirement of GTP-Binding Proteins ARF and RhoA
Authors: Bacon Kevin B. , Schall Thomas J. , Dairaghi Daniel J.
Published: December 2022

The chemokine RANTES is a potent agonist of T cell activation. In an investigation of signal-transduction events activated by this chemokine, we have shown that RANTES stimulates dose-dependent phospholipase D (PLD) activity in Jurkat cells. Equilibrium-binding analyses using 125I-labeled RANTES indicated the presence of a receptor for RANTES on these cells, which has a Kd of 0.1 nM, is expressed at approximately 600 sites per cell, and a binding specificity that was not comparable with that of any of the known chemokine receptors, since 125I-labeled RANTES was displaced by macrophage-inflammatory protein-1β (but not macrophage-inflammatory protein-1α), monocyte-chemotactic protein-1 (MCP-1), MCP-3, MCP-4, and eotaxin. RANTES-induced PLD activation was augmented by GTPγS, but not GDPβS, and inhibited by the protein kinase C inhibitor bisindolylmaleimide, as well as the fungal metabolite brefeldin A, and C3 exoenzyme (Clostridium botulinum), implicating the activation of RhoA. RANTES also induced GTP-GDP exchange of immunoprecipitated RhoA. RANTES-stimulated PLD activity was dependent on an ADP-ribosylation factor(s), as assessed by inhibition studies using a synthetic inhibitory peptide of the N-terminal 16 amino acids of ADP-ribosylation factor 1. These studies indicate the potential existence of a novel receptor-mediated mechanism for activation of T cells by the chemokine RANTES.
Induction of a Selective and Persistent Extravasation of Neutrophils into the Peritoneal Cavity by Tryptase Mouse Mast Cell Protease 6
Authors: Huang Chifu , Friend Daniel S. , Qiu Wen-Tao , Wong Guang W. , Morales Guillermo , Hunt John , Stevens Richard L.
Published: December 2022

Recombinant mouse mast cell protease 6 (mMCP-6) was generated to study the role of this tryptase in inflammatory reactions. Seven to forty-eight hours after the i.p. injection of recombinant mMCP-6 into BALB/c, mast cell-deficient WCB6F1-Sl/Sld, C5-deficient, or mMCP-5-null mice, the number of neutrophils in the peritoneal cavity of each animal increased significantly by >50-fold. The failure of the closely related recombinant tryptase mMCP-7 to induce a comparable peritonitis indicates that the substrate specificities of the two tryptases are very different. Unlike most forms of acute inflammation, the mMCP-6-mediated peritonitis was relatively long lasting and neutrophil specific. Mouse MCP-6 did not induce neutrophil chemotaxis directly in an in vitro assay, but did promote chemotaxis of the leukocyte in the presence of endothelial cells. Mouse MCP-6 did not induce cultured human endothelial cells to express TNF-α, RANTES, IL-1α, or IL-6. However, the tryptase induced endothelial cells to express large amounts of IL-8 continually over a 40-h period. Neither enzymatically active mMCP-7 nor enzymatically inactive pro-mMCP-6 was able to induce endothelial cells to increase their expression of IL-8. Although the mechanism by which mMCP-6 induces neutrophil accumulation in tissues remains to be determined, the finding that mMCP-6 induces cultured human endothelial cells to selectively release large amounts of IL-8 raises the possibility that this tryptase regulates the steady state levels of neutrophil-specific chemokines in vivo during mast cell-mediated inflammatory events.
C1qRP, the C1q Receptor That Enhances Phagocytosis, Is Detected Specifically in Human Cells of Myeloid Lineage, Endothelial Cells, and Platelets
Authors: Nepomuceno Ronald R. , Tenner Andrea J.
Published: December 2022

The complement component C1q can interact with a variety of different cells, resulting in multiple functional consequences depending on the cell type. mAbs R3 and R139, which recognize a 126,000 Mr (reduced) cell surface protein, are able to abrogate the C1q-mediated enhancement of monocyte phagocytosis. The cDNA encoding this C1q receptor that modulates phagocytosis, C1qRP, has recently been cloned. Using a DNA probe based on the coding region of the receptor, Northern blot and RT-PCR analysis of RNA isolated from different cell types showed C1qRP expression in cells of myeloid origin and in endothelial cells, but not in cells of lymphoid origin nor in the HeLa epithelial-like cell line or iliac artery smooth muscle cells. FACS analysis of cell surface expression of C1qRP, as detected by mAb R139 and R3, corresponded in all cases to the mRNA levels detected. Using the anti-C1qRP mAb, the 126,000 Mr receptor was also detected in lysates of human platelets. Interestingly, C1qRP is not expressed by the promyelocytic leukemia cell line HL-60, and differentiation of these cells with various chemical compounds did not induce C1qRP expression. It has been reported that C1q can induce specific receptor-mediated responses in fibroblasts. However, RNA and cell surface expression analysis for C1qRP indicate that this particular C1q receptor is not expressed by either human gingival or human skin fibroblasts. These data demonstrate selective expression of C1qRP in specific cell types and support the hypothesis that there is more than one C1q receptor mediating the diverse responses triggered by C1q.
IL-6-Deficient Mice Are Resistant to Experimental Autoimmune Encephalomyelitis: Roles of IL-6 in the Activation and Differentiation of Autoreactive T Cells
Authors: Samoilova Elena B. , Horton Jennifer L. , Hilliard Brendan , Liu Tzu-Shang T. , Chen Youhai
Published: December 2022

Although autoreactive T cells recognizing self myelin Ags are present in most individuals, autoimmune disease of the central nervous system is a relatively rare medical condition. Development of autoimmune disease may require not only the presence of autoreactive T cells but also that autoreactive T cells become activated. Activation of T cells may require a minimum of two signals: an Ag-specific signal delivered by MHC-peptide complex and a second signal delivered by costimulatory molecules or cytokines. Although in vitro studies have suggested that cytokines, especially proinflammatory cytokines such as IL-1, IL-6, and TNF are involved in T cell activation, their precise roles in vivo are not clear. To determine the roles of proinflammatory cytokines in T cell activation in vivo and in the development of autoimmune disease, we have studied experimental autoimmune encephalomyelitis (EAE) in mice deficient in IL-6. We found that IL-6-deficient mice were completely resistant to EAE induced by myelin oligodendrocyte glycoprotein (MOG), whereas IL-6-competent control mice developed EAE characterized by focal inflammation and demyelination in the central nervous system and deficiency in neurologic functions. Furthermore, we established that the resistance to EAE in IL-6-deficient mice was associated with a deficiency of MOG-specific T cells to differentiate into either Th1 or Th2 type effector cells in vivo. These results strongly suggest that IL-6 plays a crucial role in the activation and differentiation of autoreactive T cells in vivo and that blocking IL-6 function can be an effective means to prevent EAE.
Nitric Oxide Synthase Plays a Signaling Role in TCR-Triggered Apoptotic Death
Authors: Williams Mark S. , Noguchi Soichi , Henkart Pierre A. , Osawa Yoichi
Published: December 2022

A functional role for stimulated nitric oxide (NO) production was tested in the TCR-triggered death of mature T lymphocytes. In purified peripheral human T cell blasts or the 2B4 murine T cell hybridoma, apoptotic cell death induced by immobilized anti-CD3 was blocked by inhibitors of NO synthase (NOS) in a stereospecific and concentration-dependent manner. This effect appeared to be selective since apoptotic death induced by anti-Fas Ab or the steroid dexamethasone was not affected by NOS inhibitors. TCR-stimulated expression of functional Fas ligand was attenuated in a stereospecific manner by NOS inhibitors, but these compounds did not inhibit TCR-stimulated IL-2 secretion or CD69 surface expression. Nitrosylated tyrosines, a stable marker for NO generation, were immunochemically detected in T cells using flow cytometry. TCR signals induced NO production, as measured by an increase in nitrotyrosine-specific staining. NOS enzymatic activity was detected in lysates of 2B4 cells, and Western blot analysis suggests that the activity is due to expression of the neuronal isoform of NOS. Thus, T cells have the capacity to generate NO upon Ag signaling, which may affect signal transduction, Fas ligand surface expression, and apoptotic cell death of mature T lymphocytes.
Selection and Function of CD4+ T Lymphocytes in Transgenic Mice Expressing Mutant MHC Class II Molecules Deficient in Their Interaction with CD4
Authors: Gilfillan Susan , Shen Xiaoli , König3 Rolf
Published: December 2022

Interactions of the T cell coreceptors, CD4 and CD8, with MHC molecules participate in regulating thymocyte development and T lymphocyte activation and differentiation to memory T cells. However, the exact roles of these interactions in normal T cell development and function remain unclear. CD4 interacts with class II MHC7 molecules via several noncontiguous regions in both the class II MHC α- and β-chains. We have introduced a double mutation that disrupts interaction with CD4 into the I-Aβk gene and used this construct to generate transgenic mice expressing only mutant class II MHC. Although CD4+ thymocytes matured to the single-positive stage in these mice, their frequency was reduced by threefold compared with that of wild-type transgenics. Positive selection of CD4+ T cells in the mutant transgenic mice may have been mediated by TCRs with a higher than usual affinity for class II MHC/Ag complexes. In Aβk mutant transgenics, peripheral CD4+ lymphocytes promoted B cell differentiation to plasma cells. These CD4+ T cells also secreted IFN-γ in response to various stimuli (e.g., protein Ag, bacterial superantigen, and alloantigen), but were deficient in IL-2 secretion. Interactions between CD4 and class II MHC molecules appeared to regulate lymphokine production, with a strong bias toward IFN-γ and against IL-2 in the absence of these interactions. Our results have implications for the manipulation of T cell-dependent immune responses.
Terminal Deoxynucleotidyl Transferase Expression During Neonatal Life Alters DH Reading Frame Usage and Ig-Receptor-Dependent Selection of V Regions
Authors: Marshall Aaron J. , Doyen Noelle , Bentolila Laurent A. , Paige Christopher J. , Wu Gillian E.
Published: December 2022

During neonatal life, Ig diversity is limited in many respects. The absence of terminal deoxynucleotidyl transferase (TdT) expression with the consequent lack of nontemplated addition during the neonatal period, coupled with the predominant usage of a single DH reading frame (RF), leads to severe limitations of diversity in the CDR3 region of Ig heavy (H) chains. The neonatal Ig H chain repertoire is also characterized by restricted VH usage, with predominant expression of certain VH segments, such as VH81x, that are rarely evident during adult life. In this report, we examine the effect of enforced TdT expression on the neonatal repertoire of VH81xDJH rearrangements. We find that TdT synthesis abrogates DH RF bias during the fetal/neonatal period through a Ig-receptor-independent mechanism. These findings suggest that DH RF bias during neonatal life is determined largely by homology-directed joining. We also find that TdT synthesis alters the selection of productively rearranged VH81xDJH alleles in the neonatal spleen through a Ig-receptor-dependent mechanism. Analysis of predicted CDR3 amino acid sequences indicates that positive selection of VH81x-encoded H chains is correlated with the presence of a consensus sequence immediately adjacent to the VH segment. These data support the hypothesis that the CDR3 region is critical in determining the ability of VH81x-encoded H chains to form functional receptors that support positive selection of B lymphocytes. Together, our results demonstrate that TdT can indirectly influence the Ig repertoire by influencing both receptor-dependent and receptor-independent selection processes.
Two Families of GTPases Dominate the Complex Cellular Response to IFN-γ
Authors: Boehm Ulrich , Guethlein Lisbeth , Klamp Thorsten , Ozbek Kural , Schaub Annette , Fütterer Agnes , Pfeffer Klaus , Howard Jonathan C.
Published: December 2022

IFN-γ induces a number of cellular programs functional in innate and adaptive resistance to infectious pathogens. It has recently become clear that the complete cellular response to IFN-γ is extraordinarily complex, with >500 genes (i.e., ∼0.5% of the genome) activated. We made suppression-subtractive hybridization differential libraries from IFN-γ-stimulated primary mouse embryonic fibroblasts and from a mouse macrophage cell line, ANA-1, in each case with reference to unstimulated cells. Of ∼250 clones sequenced at random from the two libraries, >35% were representatives of one or the other of two small unrelated families of GTPases, the 65-kDa and 47-kDa families. These families dominate the IFN-γ-induced response in both cell types. We report here the full-length sequences of one new 65-kDa and two new 47-kDa family members. The 65-kDa family members are under transcriptional control of IRF-1, whereas the 47-kDa family members are inducible in embryonic fibroblasts from IRF-1−/− mice. Members of both GTPase families are strongly up-regulated in livers of wild-type mice infected with the pathogenic bacterium, Listeria monocytogenes, but not in IFN-γR0/0 mice. These GTPases appear to be dedicated to the IFN-γ response, since resting levels are negligible and since neither family shows any significant relationship to any other described family of GTPases. Understanding the role of these GTPases in IFN-γ-mediated resistance against pathogens is the task for the future.
Marsupial Light Chains: Complexity and Conservation of λ in the OpossumMonodelphis domestica
Authors: Lucero Julie E. , Rosenberg George H. , Miller Robert D.
Published: December 2022

The Igλ chains in the South American opossum, Monodelphis domestica, were analyzed at the expressed cDNA and genomic organization level, the first described for a nonplacental mammal. The Vλ segment repertoire in the opossum was found to be comprised of at least three diverse Vλ families. Each of these families appears to be related to distinct Vλ families present in placental mammals, suggesting the divergence of these genes before the separation of metatherians and eutherians more than 100 million years ago. Based on framework and constant region sequences from full-length cDNAs and intron sequences from genomic clones, it appears that there are multiple functional Jλ-Cλ pairs in the opossum locus. The opossum Jλ-Cλ sequences are phylogenetically clustered, suggesting that these gene duplications are more recent and species specific. Sequence analysis of a large set of functional, expressed Vλ-Jλ recombinations is consistent with an unbiased, highly diverse λ light chain repertoire in the adult opossum. Overall, the complexity of the Igλ locus appears to be greater than that found in the Ig heavy chain locus in the opossum, and light chains are therefore likely to contribute significantly to Ig diversity in this species.
MEK and ERK Activation in Ras-Disabled RBL-2H3 Mast Cells and Novel Roles for Geranylgeranylated and Farnesylated Proteins in FcεRI-Mediated Signaling
Authors: Graham Timothy E. , Pfeiffer Janet R. , Lee Rebecca J. , Kusewitt Donna F. , Martinez A. Marina , Foutz Terry , Wilson Bridget S. , Oliver Janet M.
Published: December 2022

Cross-linking the high affinity IgE receptor FcεRI of basophils and mast cells activates receptor-associated protein-tyrosine kinases and stimulates a signaling cascade leading to secretion, ruffling, spreading, and cytokine production. Previous evidence that the pan-prenylation inhibitor lovastatin blocks Ag-stimulated Ca2+ influx, secretion, and membrane/cytoskeletal responses implicated isoprenylated proteins in the FcεRI-coupled signaling cascade but could not distinguish between contributions of C15 (farnesylated) and C20 (geranylgeranylated) species. Here we establish concentrations of lovastatin and the farnesyl-specific inhibitor BZA-5B that inhibit the farnesylation and Ag-induced activation of Ras species in RBL-2H3 cells (H-Ras, K-RasA, and K-RasB). These inhibitors have little effect on tyrosine kinase activation, which initiates FcεRI signaling. Although Ras is disabled, only lovastatin substantially blocks Raf-1 activation, and neither inhibitor affects mitogen-activated protein kinase kinase/extracellular signal regulated kinase kinase (MEK) or ERK1/ERK2 activation. Thus, the pathway to FcεRI-mediated MEK/ERK and ERK activation can apparently bypass Ras and Raf-1. Predictably, only lovastatin inhibits Ag-induced ruffling, spreading, and secretion, previously linked to geranylgeranylated Rho and Rab family members. Additionally, only lovastatin inhibits phospholipase Cγ-mediated inositol (1,4,5) trisphosphate production, sustained Ca2+ influx, and Ca2+-dependent IL-4 production, suggesting novel roles for geranylgeranylated (lovastatin-sensitive, BZA-5B-insensitive) proteins in FcεRI signal propagation. Remarkably, BZA-5B concentrations too low to inactivate Ras reduce the lag time to Ag-induced Ca2+ stores release and enhance secretion. These results link a non-Ras farnesylated protein(s) to the negative regulation of Ca2+ release from intracellular stores and secretion. We identified no clear role for Ras in FcεRI-coupled signaling but suggest its involvement in mast cell growth regulation based on the inhibition of cell proliferation by both BZA-5B and lovastatin.
Coelomocytes Express SpBf, a Homologue of Factor B, the Second Component in the Sea Urchin Complement System
Authors: Smith L. Courtney , Shih Chi-Schin , Dachenhausen Sebastian G.
Published: December 2022

A homologue of factor B, SpBf, has been cloned and sequenced from an LPS-activated coelomocyte cDNA library from the purple sea urchin, Strongylocentrotus purpuratus. The deduced amino acid sequence and domain structure show significant similarity to the vertebrate Bf/C2 family proteins. SpBf is a mosaic protein, composed of five short consensus repeats, a von Willebrand Factor domain, and a serine protease domain. It has a deduced molecular mass of 91 kDa, with a conserved cleavage site for a putative factor D protease. It has ten consensus recognition sites for N-linked glycosylation. Amino acids involved in both Mg2+ binding and in serine protease activity in the vertebrate C2/Bf proteins are conserved in SpBf. Phylogenetic analysis of SpBf indicates that it is the most ancient member of the vertebrate Bf/C2 family. Additional phylogenetic analysis of the SCRs indicates that five SCRs in SpBf may be ancestral to three SCRs, which is the typical pattern in the vertebrate Bf/C2 proteins. RNA gel blots show that SpBf transcripts are 5.5 kb and are specifically expressed in coelomocytes. Genome blots suggest that the SpBf gene (Sp152) is single copy gene per haploid genome. This is the second complement component to be identified from the sea urchin, and, with the sea urchin C3 homologue, these two components may be part of a simple complement system that is homologous to the alternative pathway in higher vertebrates.
Alteration of Intracellular Calcium Flux and Impairment of Nuclear Factor-AT Translocation in T Cells During AcuteToxoplasma gondiiInfection in Mice
Authors: Haque Sakhina , Dumon Henri , Haque Azizul , Kasper Lloyd H.
Published: December 2022

Down-regulation of host immune response to Toxoplasma gondii is associated with the expression of specific cytokines, in particular IL-10, and the induction of CD4+ T cell anergy. In the present study we report that the expression of both CD4 and CD2 antigen is down-regulated during the acute phase of infection. A decrease in the expression of CD2 was apparent during the acute phase of T. gondii infection in three genetically distinct strains of mice, CBA/J, C57BL/6, and BALB/c. The lymphoproliferative response induced by cross-linked anti-CD3 mAb or by Con A was markedly depressed. This suppressed response was associated with a reduction in the influx of Ca2+. We have examined whether lymphocytes from T. gondii mice maintain NF-AT transcription factors in the nucleus where they participate in the Ca2+-dependent induction of genes required for lymphocyte activation and proliferation. Immunofluorescence with confocal microscopy using an Ab to NF-ATc demonstrates a decrease in translocation of NF-ATc in T lymphocytes from acutely infected mice. Together, these results suggest that the defect in T cell expansion that occurs during acute murine toxoplasmosis is related to reduced activity of NF-AT, a calcium-dependent transcription factor required for T cell proliferation.
Astrocyte-Derived Monocyte-Chemoattractant Protein-1 Directs the Transmigration of Leukocytes Across a Model of the Human Blood-Brain Barrier
Authors: Weiss Jonathan M. , Downie Sherry A. , Lyman William D. , Berman Joan W.
Published: December 2022

The migration of leukocytes across the blood-brain barrier (BBB) into the central nervous system is critical in the pathogenesis of central nervous system inflammatory diseases. The production of chemokines, such as monocyte-chemoattractant protein-1 (MCP-1), by endothelial cells (EC) and astrocytes may initiate and amplify this process. Using a coculture of human EC and astrocytes to model the BBB, we demonstrated that exogenous MCP-1 induces the transmigration of monocytes in a dose-dependent manner. TNF-α, IFN-γ, or IL-1β treatment of cocultures also induced significant migration of monocytes that correlates with the induction of MCP-1 protein. TGF-β, previously shown to induce MCP-1 expression in astrocytes, but not in EC, caused migration of monocytes across cocultures, but not across EC grown alone. Monocytes and lymphocytes transmigrated across cytokine-treated cocultures in greater numbers than across EC alone. Astrocytes were the main source of cytokine-induced MCP-1, supporting a role for astrocytes in facilitating leukocyte transmigration. A blocking Ab to MCP-1 inhibited MCP-1- and cytokine-induced transmigration of monocytes by 85–90%. Cytokine treatment of cocultures also resulted in the transmigration of activated, CD69-positive lymphocytes. The MCP-1-mediated transmigration of monocytes across cocultures was blocked using an Ab to ICAM-1 and inhibited by 55% using an Ab to E-selectin. These data suggest a central role for astrocyte-derived MCP-1 in directing the migration of monocytes and lymphocytes across the BBB.
Peptide Modification or Blocking of CD8, Resulting in Weak TCR Signaling, Can Activate CTL for Fas- But Not Perforin-Dependent Cytotoxicity or Cytokine Production
Authors: Kessler Benedikt , Hudrisier Denis , Schroeter Michael , Tschopp Jürg , Cerottini Jean-Charles , Luescher Immanuel F.
Published: December 2022

This study describes a form of partial agonism for a CD8+CTL clone, S15, in which perforin-dependent killing and IFN-γ production were lost but Fas (APO1 or CD95)-dependent cytotoxicity preserved. Cloned S15 CTL are H-2Kd restricted and specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252–260 (SYIPSAEKI). The presence of a photoactivatable group in the epitope permitted assessment of TCR-ligand binding by TCR photoaffinity labeling. Selective activation of Fas-dependent killing was observed for a peptide-derivative variant containing a modified photoreactive group. A similar functional response was obtained after binding of the wild-type peptide derivative upon blocking of CD8 participation in TCR-ligand binding. The epitope modification or blocking of CD8 resulted in an ≥8-fold decrease in TCR-ligand binding. In both cases, phosphorylation of ζ-chain and ZAP-70, as well as calcium mobilization were reduced close to background levels, indicating that activation of Fas-dependent cytotoxicity required weaker TCR signaling than activation of perforin-dependent killing or IFN-γ production. Consistent with this, we observed that depletion of the protein tyrosine kinase p56lck by preincubation of S15 CTL with herbimycin A severely impaired perforin- but not Fas-dependent cytotoxicity. Together with the observation that S15 CTL constitutively express Fas ligand, these results indicate that TCR signaling too weak to elicit perforin-dependent cytotoxicity or cytokine production can induce Fas-dependent cytotoxicity, possibly by translocation of preformed Fas ligand to the cell surface.
Combination Chemotherapy and IL-15 Administration Induce Permanent Tumor Regression in a Mouse Lung Tumor Model: NK and T Cell-Mediated Effects Antagonized by B Cells
Authors: Chapoval Andrei I. , Fuller Jane A. , Kremlev Sergey G. , Kamdar Sonya J. , Evans Robert
Published: December 2022

Previous studies have demonstrated that IL-15 administration after cyclophosphamide (CY) injection of C57BL/6J mice bearing the i.m. 76-9 rhabdomyosarcoma resulted in a significant prolongation of life. In the present study, we investigated the immune response against the 76-9 experimental lung metastases after CY + IL-15 therapy. Administration of CY + IL-15, but not IL-15 alone, induced prolongation of life and cures in 32% of mice bearing established experimental pulmonary metastases of 76-9 tumor. The CY + IL-15 therapy resulted in increased levels of NK1.1+/LGL-1+ cells, and CD8+/CD44+ T cells in PBL. In vitro cytotoxic assay of PBL indicated the induction of lymphokine-activated killer cell activity, but no evident tumor-specific class I-restricted lytic activity. Survival studies showed that the presence of NK and T lymphocytes is necessary for successful CY + IL-15 therapy. Experiments using knockout mice implied that either αβ or γδ T cells were required for an antitumor effect induced by CY + IL-15 therapy. However, mice lacking in both αβ and γδ T cells failed to respond to combination therapy. Cured B6 and αβ or γδ T cell-deficient mice were immune to rechallenge with 76-9, but not B16LM tumor. B cell-deficient mice showed a significant improvement in the survival rate both after CY and combination CY + IL-15 therapy compared with normal B6 mice. Overall, the data suggest that the interaction of NK cells with tumor-specific αβ or γδ T lymphocytes is necessary for successful therapy, while B cells appear to suppress the antitumor effects of CY + IL-15 therapy.
Identification of New Melanoma Epitopes on Melanosomal Proteins Recognized by Tumor Infiltrating T Lymphocytes Restricted by HLA-A1, -A2, and -A3 Alleles
Authors: Kawakami Yutaka , Robbins Paul F. , Wang Xiang , Tupesis Janis P. , Parkhurst Maria R. , Kang Xiaoqiang , Sakaguchi Kazuyasu , Appella Ettore , Rosenberg Steven A.
Published: December 2022

To isolate melanoma Ags recognized by T cells, cDNA libraries made from melanoma cell lines were screened with four CTLs derived from tumor infiltrating lymphocytes (TIL) that were able to recognize melanoma cells in a HLA-A1, -A2, or -A3 restricted manner. Although cDNAs encoding the previously identified melanoma Ags, tyrosinase and gp100, were isolated, these TIL were found to recognize previously unidentified peptides. An HLA-A1-restricted CTL, TIL1388, was found to recognize a tyrosinase peptide (SSDYVIPIGTY), and an HLA-A3-restricted CTL, TIL1351, recognized a gp100 peptide (LIYRRRLMK). CTL clones isolated from the HLA-A2-restricted TIL1383 recognized a gp100 peptide (RLMKQDFSV). HLA-A2-restricted CTL, TIL1200, recognized a gp100 peptide (RLPRIFCSC). Replacement of either cysteine residue with α-amino butyric acid in the gp100 peptide, RLPRIFCSC, enhanced CTL recognition, suggesting that the peptide epitope naturally presented on the tumor cell surface may contain reduced cysteine residues. Oxidation of these cysteines might have occurred during the course of the synthesis or pulsing of the peptide in culture. These modifications may have important implications for the development of efficient peptide-based vaccines. These newly identified peptide epitopes can extend the ability to perform immunotherapy using synthetic peptides to a broader population of patients, especially those expressing HLA-A1 or HLA-A3 for whom only a few melanoma epitopes have previously been identified.
Genetic Determinants of Autoimmune Disease and Coronary Vasculitis in the MRL-lpr/lprMouse Model of Systemic Lupus Erythematosus
Authors: Gu Lingjie , Weinreb Ari , Wang Xu-Ping , Zack Debra J. , Qiao Jian-Hua , Weisbart Richard , Lusis Aldons J.
Published: December 2022

MRL-lpr/lpr (MRL/lpr) mice are a model of human autoimmune disease. They exhibit a number of characteristics of systemic lupus erythematosus, including anti-DNA Abs, anti-cardiolipin Abs, immune complex-mediated vasculitis, lymphadenopathy, and severe glomerulonephritis. Although the autoimmune disorder is mediated primarily by mutation of the Fas gene (lpr), which interferes with lymphocyte apoptosis, MRL/lpr mice also have other predisposing genetic factors. In an effort to identify these additional factors, we have applied quantitative trait locus (QTL) mapping using an intercross between MRL/lpr mice and the nonautoimmune inbred strain BALB/cJ. A complete linkage map spanning the entire genome was constructed for 189 intercross progeny, and genetic loci contributing to features of the autoimmunity were identified using statistical analytic procedures. As expected, the primary genetic determinant of autoimmune disease in this cross was the Fas gene on mouse chromosome 19, exhibiting a lod score of 60. In addition, two novel loci, one on chromosome 2 (lod score, 4.3) and one on chromosome 11 (lod score, 3.1), were found to contribute to levels of anti-DNA Abs. Interestingly, the chromosome 19 and chromosome 11 QTLs, but not the chromosome 2 QTL, also exhibited associations with anti-cardiolipin Abs (lod scores, 38.4 and 2.6). We further examined the effects of these QTLs on the development of coronary vasculitis in the F2 mice. Our results indicate that the QTLs on chromosomes 11 and 19 also control the development of vasculitis, demonstrating common genetic determinants of autoantibody levels and vasculitis.
Prevention of Experimental Allergic Encephalomyelitis via Inhibition of IL-12 Signaling and IL-12-Mediated Th1 Differentiation: An Effect of the Novel Anti-Inflammatory Drug Lisofylline
Authors: Bright John J. , Du Caigan , Coon Michael , Sriram Subramaniam , Klaus Stephen J.
Published: December 2022

Experimental allergic encephalomyelitis (EAE) is an inflammatory, CD4+ Th1-mediated autoimmune disease, which serves as a model for multiple sclerosis. We examined the effect of a novel anti-inflammatory drug, lisofylline (LSF), on EAE induced either by injection of mouse spinal cord homogenate or following transfer of myelin basic protein-reactive T cells. Orally administered LSF significantly inhibited EAE in both cases, decreasing peak clinical scores by >70% and >80%, respectively. In addition, analysis of representative spinal cord sections from LSF-treated mice showed complete lack of demyelination and lymphocyte infiltration. The reduction in EAE correlated with the inhibition of Th1 differentiation by LSF in vivo, as indicated by a reduction in T cell IFN-γ production ex vivo after Ag restimulation. The inhibition of Th1 differentiation in vivo is consistent with a block in IL-12 receptor signaling, because LSF blocked IL-12-driven Th1 differentiation and T cell proliferation in vitro, yet had no effect on IL-12 secretion from APCs ex vivo or in vitro.
Molecular and Immunologic Characterization of a Highly Cross-Reactive Two EF-Hand Calcium-Binding Alder Pollen Allergen, Aln g 4: Structural Basis for Calcium-Modulated IgE Recognition
Authors: Hayek Brigitte , Vangelista Luca , Pastore Annalisa , Sperr Wolfgang R. , Valent Peter , Vrtala Susanne , Niederberger Verena , Twardosz Anna , Kraft Dietrich , Valenta Rudolf
Published: December 2022

Serum IgE was used to isolate a cDNA coding for a 9.4-kDa two EF-hand calcium-binding allergen, Aln g 4, from a λgt11 expression cDNA library constructed from alder (Alnus glutinosa) pollen. rAln g 4 was overexpressed in Escherichia coliand purified to homogeneity. It reacted with serum IgE from 18% of pollen-allergic patients (n = 122); shared IgE epitopes with homologous allergens present in tree, grass, and weed pollens; and thus belongs to a family of highly cross-reactive pollen allergens. Exposure of two E. coli-expressed rAln g 4 fragments comprising amino acids 1–41 and 42–85 to patients’ IgE Abs, as well as to a rabbit antiserum raised against purified rAln g 4, indicated that most of the B cell epitopes reside in the N-terminal portion of the protein. IgE recognition of Aln g 4 was strongly modulated by the presence or absence of calcium. Circular dichroism analysis of rAln g 4 revealed that the protein consisted mostly of α helical secondary structure and possessed a remarkable thermal stability and refolding capacity, a property that was greatly reduced after calcium depletion. Circular dichroism analysis of the calcium-bound and apo form of rAln g 4 indicated that calcium-induced modulation of IgE binding could be due to changes in the protein conformation. Purified rAln g 4 elicited dose-dependent basophil histamine release and immediate type skin reactions in sensitized patients. It may hence be useful for allergy diagnosis and for specific immunotherapy.
Perforin/Fas-Ligand Double Deficiency Is Associated with Macrophage Expansion and Severe Pancreatitis
Authors: Spielman Julie , Lee Richard K. , Podack Eckhard R.
Published: December 2022

We report that perforin/Fas-ligand double-deficient mice die early of severe pancreatitis. Female mice, in addition, are infertile and suffer from hysterosalpingitis. Tissue destruction is accompanied by infiltration with Mac-1 (CD11b)-positive monocytes/macrophages, Mac-1-positive T cells, and expansion of CD8+ T cells. In vivo inactivation of monocytes/macrophages by carrageenan reverses disease progression and restores fertility of female mice. Perforin/Fas-ligand double-deficient CD4+ or CD8+ CTL are unable to lyse cognate-activated macrophages, and therefore are unable to mediate negative feedback regulation by lysis of APCs, thereby preventing further T cell activation. These studies demonstrate a novel role for perforin in homeostatic regulation of the immune response.
Cutting Edge: Cytokines Regulate Expression and Function of the HIV Coreceptor CXCR4 on Human Mature Dendritic Cells
Authors: Zoeteweij J. Paul , Golding Hana , Mostowski Howard , Blauvelt Andrew
Published: December 2022

HIV-infected dendritic cells (DC) efficiently transmit infection to CD4+ T cells during the process of T cell activation. To further understand interactions between DC and HIV, cytokine regulation of HIV coreceptors on cultured Langerhans cells (cLC, as prototypes of mature DC) was studied. Expression of cell surface CXCR4 on cLC was up-regulated by IL-4 and TGF-β1 and inhibited by IFN-α, IFN-β, and IFN-γ, whereas cytokines did not appreciably regulate CCR5. Changes in cell surface CXCR4 expression on cLC correlated with T cell-tropic (X4)-HIV envelope-mediated syncytium formation and X4-HIV infection levels. A relative increase in the ratio of type 2/type 1 cytokine production, which can occur in HIV disease, may up-regulate CXCR4 expression on mature DC and promote infection by X4 viruses. Importantly, these findings suggest that cytokine dysregulation may be linked to the emergence of X4-HIV strains as HIV-infected individuals progress to AIDS.
Expansion by Self Antigen Is Necessary for the Induction of Experimental Autoimmune Encephalomyelitis by T Cells Primed with a Cross-Reactive Environmental Antigen
Authors: Carrizosa Ana M. , Nicholson Lindsay B. , Farzan Michael , Southwood Scott , Sette Alessandro , Sobel Raymond A. , Kuchroo Vijay K.
Published: December 2022

Cross-reactivity with environmental antigens has been postulated as a mechanism responsible for the induction of autoimmune disease. Experimental autoimmune encephalomyelitis is a T cell-mediated autoimmune disease model inducible in susceptible strains of laboratory animals by immunization with protein constituents of myelin. We used myelin proteolipid protein (PLP) peptide 139–151 and its analogues to define motifs to search a protein database for structural homologues of PLP139–151 and identified five peptides derived from microbial Ags that elicit immune responses that cross-react with this self peptide. Exposure of naive SJL mice to the cross-reactive environmental peptides alone was insufficient to induce autoimmune disease even when animals were treated with Ag-nonspecific stimuli (superantigen or LPS). However, immunization of SJL mice with suboptimal doses of PLP139–151 after priming with cross-reactive environmental peptides consistently induced experimental autoimmune encephalomyelitis. Furthermore, T cell lines from mice immunized with cross-reactive environmental peptides and restimulated in vitro with PLP139–151 could induce disease upon transfer into naive recipients. These data suggest that expansion by self Ag is required to break the threshold to autoimmune disease in animals primed with cross-reactive peptides.
Roles of Chemokines and Receptor Polarization in NK-Target Cell Interactions
Authors: Nieto Marta , Navarro Francisco , Perez-Villar Juan José , del Pozo Miguel Angel , González-Amaro Roberto , Mellado Mario , Frade José M. R. , Martínez-A Carlos , López-Botet Miguel , Sánchez-Madrid Francisco
Published: December 2022

We report that the ability of NK cells to produce chemokines is increased in NK-target cell conjugates. The chemokines produced play a critical role in the polarization and recruitment of NK cells as well as in the NK effector-target cell conjugate formation. Chemokines induce the formation of two specialized regions in the NK cell: the advancing front or leading edge, where chemokine receptors CCR2 and CCR5 cluster, which might guide the cells toward the chemotactic source, and the uropod, where adhesion molecules ICAM-1 and -3 are redistributed. NK cell polarity was intrinsically involved in conjugate formation. The redistribution of both adhesion receptors and CCR was preserved during the formation of NK-target cell conjugates. Time-lapse videomicroscopy studies of the formation of effector-target conjugates showed that morphologic poles are also functionally distinct; while the binding to target cells was preferentially mediated through the leading edge, the uropod was found at the rear of migrating NK cells and recruited additional NK cells to the vicinity of K562 target cells. Inhibition of cell polarization and adhesion receptor redistribution blocked the formation of NK-K562 cell conjugates and the cytotoxic activity of NK cells. We discuss the implication of NK-cell polarization in the development of cytotoxic responses.
C4d DNA Sequences of Two Infrequent Human Allotypes (C4A13 AND C4B12) and the Presence of Signal Sequences Enhancing Recombination
Authors: Martínez-Quiles Narcisa , Paz-Artal Estela , Moreno-Pelayo Miguel A. , Longás Javier , Ferre-López Sergio , Rosal Manuel , Arnaiz-Villena Antonio
Published: December 2022

The DNA sequences of the polymorphic region (C4d) that belong to the infrequent complement C4 allotypes C4A13 and C4B12 have been obtained. In addition, C4A4 and C4B2 C4d sequences have been completed. C4A13 shows a new combination of amino acids at the following polymorphic positions: Asp1054, Pro1101, Cys1102, Leu1105, Asp1106, Asn1157, Ala1188, and Arg1191. These amino acids conform to the antigenic determinants Chido 1 and Rodgers 3; thus C4A13 is the only allele described thus far that carries both Ags. C4A13 and C4A4 carry the motif “ggctc∗” (∗ means “deletion”) at positions 14 to 19 in their intron 28; this motif had previously been reported only in C4B alleles. The C4B12 nucleotide sequence is analogous to C4B1b and C4B3 sequences, except for codon 1076, which is GCC in C4B1b and C4B3 and GGA in C4B12, which is coding for glycine in both cases. A recombination model for the generation of C4 alleles is formulated based on the analysis of these new sequences. One recombination would take place between positions 1157 and 1186 and would give rise to C4A13 and C4B5 or C4A3 (or C4A6) and C4B2; another one would occur between positions 1054 and 1076 and would generate C4A3 (or C4A6) and C4B12 or C4A2 and C4Bnew. Analysis of 1157 to 1186 and 1054 to 1076 fragments reveals the presence of putative sequence signals for recombination (similar to Escherichia coli χ recombination signal); the accumulation of such signals in fragments 1054 to 1076 supports the notion that a recombination hot spot for the C4 gene may exist and it also enhances new allele generation and intraspecies C4 gene homogenization.
Differential Transcriptional Regulation ofCD161and a Novel Gene,197/15a, by IL-2, IL-15, and IL-12 in NK and T Cells
Authors: Azzoni Livio , Zatsepina Olga , Abebe Bekele , Bennett Ian M. , Kanakaraj Palanisamy , Perussia Bice
Published: December 2022

Cytokine-mediated enhancement of spontaneous cytotoxicity depends, at least in part, on modulation of the expression of surface molecules responsible for recognition of target cell structures and triggering or inhibition of the cytotoxic machinery. We previously demonstrated that expression of transcription factors (e.g., Egr-1, JunB, and c-Fos) is differentially regulated by IL-2 and IL-12. Here we show that expression of CD161/NKR-P1A, a molecule involved in triggering cytotoxicity, is specifically up-regulated by IL-12. CD161 transcription, mRNA accumulation, and surface expression are increased by IL-12. Other cytokines sharing the IL-2R β- and/or common γ-chains (i.e., IL-15, IL-4, and IL-7) do not mediate these effects. In an effort to analyze the mechanisms by which IL-2, IL-12, and IL-15 differentially regulate gene transcription, we have isolated a novel gene, 197/15a, the expression of which in NK and T cells is down-regulated by IL-2 and IL-15, up-regulated by IL-12, and not affected by IL-4 and IL-7. IL-2 and IL-15 act, at least in part, repressing 197/15a transcription; their effect on 197/15a mRNA accumulation is partially independent of novel protein synthesis, likely not mediated by JunB, Bcl-2, or Bax, and requires the activity of rapamycin-sensitive molecule(s). The observation that IL-2 and IL-12 differentially modulate CD161 expression suggests the existence of cytokine-specific mechanisms of modulation of spontaneous cytotoxicity based on the regulation of expression of surface molecules involved in target cell recognition and/or triggering of the cytolytic machinery.
IFN-γ Is Required for IL-12 Responsiveness in Mice withCandida albicansInfection
Authors: Cenci Elio , Mencacci Antonella , Del Sero Giuseppe , Fé d’Ostiani Cristiana , Mosci Paolo , Bacci Angela , Montagnoli Claudia , Kopf Manfred , Romani Luigina
Published: December 2022

To elucidate the role of IFN-γ in antifungal CD4+ Th-dependent immunity, 129/Sv/Ev mice deficient for IFN-γ receptor (IFN-γR−/−) were assessed for susceptibility to gastrointestinal or systemic Candida albicans infection and for parameters of innate and adaptive T helper immunity. IFN-γR−/− mice failed to mount protective Th1-mediated acquired immunity upon mucosal immunization or in response to a live vaccine strain of the yeast. The impaired Th1-mediated resistance correlated with defective IL-12 responsiveness, but not IL-12 production, and occurred in the presence of an increased innate antifungal resistance. The development of nonprotective Th2 responses was observed in IFN-γR−/− mice upon mucosal infection and subsequent reinfection. However, under experimental conditions of Th2 cell activation, the occurrence of Th2 cell responses was similar in IFN-γR−/− and in IFN-γR+/+ mice. These results indicate the complex immunoregulatory role of IFN-γ in the induction of mucosal and nonmucosal anticandidal Th cell responses; IFN-γ is not essential for the occurrence of Th2 responses but is required for development of IL-12-dependent protective Th1-dependent immunity.
Characterization of a Peptide Analog of a Determinant of Type II Collagen That Suppresses Collagen-Induced Arthritis
Authors: Myers Linda K. , Tang Bo , Rosloniec E. F. , Stuart J. M. , Chiang T. M. , Kang A. H.
Published: December 2022

Immunization of susceptible strains of mice with type II collagen (CII) elicits an autoimmune arthritis known as collagen-induced arthritis (CIA). One analogue peptide of the immunodominant T cell determinant, A9 (CII245–270 (I260→A, A261→B, F263→N)), was previously shown to induce a profound suppression of CIA when coadministered at the time of immunization with CII. In the present study, A9 peptide was administered i.p., orally, intranasally, or i.v. 2 to 4 wk following CII immunization. We found that arthritis was significantly suppressed even when A9 was administered after disease was induced. To determine the mechanism of action of A9, cytokine responses to A9 and wild-type peptide A2 by CII-sensitized spleen cells were compared. An increase in IL-4 and IL-10, but not in IFN-γ, was found in A9 culture supernatants. Additionally, cells obtained from A9-immunized mice produced higher amounts of IL-4 and IL-10 when cultured with CII compared with cells obtained from mice immunized with A2, which produced predominantly IFN-γ. Suppression of arthritis could be transferred to naive mice using A9-immune splenocytes. Lastly, phosphorylation of TCRζ was not altered in the immunoprecipitates from the lysates of cells exposed to analogue peptides (A9 and A10) together with wild-type A2 in a T cell line and two I-Aq-restricted, CII-specific T hybridomas. We conclude that analogue peptide A9 is effective in suppressing established CIA by inducing T cells to produce a Th2 cytokine pattern in response to CII.