bluuebunny/crossref_metadata_2025 huggingface dataset:
Conservation of Structure and Function Between Human and Murine IL-16
Authors: Keane Joseph , Nicoll John , Kim Sue , Wu David M. H. , Cruikshank William W. , Brazer William , Natke Barbara , Zhang Yujun , Center David M. , Kornfeld Hardy
Published: January 2023

IL-16 is a proinflammatory cytokine that signals via CD4, inducing chemotactic and immunomodulatory responses of CD4+ lymphocytes, monocytes, and eosinophils. Comparative analysis of murine and human IL-16 homologs could reveal conserved structures that would help to identify key functional regions of these cytokines. To that end, we cloned the murine IL-16 cDNA and found a high degree of amino acid similarity comparing the predicted murine and human IL-16 precursor proteins (pro-IL-16). The highest similarity (82.1%) was found in the C-terminal region, which is cleaved from pro-IL-16 to yield biologically active IL-16. Chemotaxis experiments with IL-16 of murine and human origin, using murine splenocytes or human T lymphocytes as targets, showed cross-species stimulation of motility. Synthetic oligopeptides and anti-peptide Ab were produced, based on the sequences of three predicted hydrophilic domains of IL-16 potentially presented in exposed positions. None of these peptides had intrinsic IL-16 bioactivity, but one (corresponding to a hydrophilic C-terminal domain of IL-16) partially displaced binding of OKT4 mAb to human lymphocytes. This peptide, and its cognate Ab, also inhibited IL-16 chemoattractant activity for human and murine cells. These studies demonstrate a high degree of structural and functional similarity between human and murine IL-16 and suggest that amino acids in the C terminus are critical for its chemoattractant function. The data suggest cross-species conservation of IL-16 receptor structures as well. Inhibitory peptides may be useful in disease states where the proinflammatory functions of IL-16 are detrimental to the host.
Influenza A Virus-Induced IFN-α/β and IL-18 Synergistically Enhance IFN-γ Gene Expression in Human T Cells
Authors: Sareneva Timo , Matikainen Sampsa , Kurimoto Masashi , Julkunen Ilkka
Published: January 2023

T cells contribute significantly the to host’s early defense against viral and bacterial infections and are essential for clearance of the pathogen. IFN-γ, a product of activated T and NK cells, has, in addition to its direct antimicrobial activity, a major role in activating cell-mediated immunity. Here we report that cytokines secreted by influenza A virus-infected macrophages are able to induce IFN-γ synthesis in human T cells. Influenza A virus-infected human peripheral macrophages secreted IFN-α/β, TNF-α, IL-1β, and a recently identified cytokine, IL-18 (or IFN-γ-inducing factor), whereas the production of IL-12 was not detected. Supernatants collected from virus-infected macrophages induced rapid IFN-γ mRNA expression and protein production in T cells. This was down-regulated by the addition of neutralizing anti-IFN-α/β Abs, whereas neutralizing anti-IL-12 Abs had no effect on IFN-γ gene expression. Exogenously added IFN-α/β also rapidly stimulated the synthesis of IFN-γ mRNA in T cells independently of protein synthesis. IL-18 synergized with IFN-α to up-regulate IFN-γ gene expression and protein production. The data suggest that IFN-α/β and IL-18 produced by macrophages during virus infection may act together to induce IFN-γ synthesis and, consequently, may play an important role for both of these cytokines in the development of Th1-type immune responses.
Cutting Edge Commentary: Differential TCR Signaling and the Generation of Memory T Cells
Authors: Farber Donna L.
Published: January 2023

There are currently two models for the generation of memory T cells: 1) memory T cells arise directly from activated effector T cells that have reverted to the resting state via an unknown mechanism; and 2) memory T cells are generated directly from naive T cells, bypassing an effector stage. I discuss here how recent results on the activation and signaling requirements of naive vs memory CD4 T cells favor the second model and how differential signaling of naive T cells may direct their developmental outcome.
Intercellular Adhesion Molecule-1 and Leukocyte Function-Associated Antigen-3 Provide Costimulation for Superantigen-Induced T Lymphocyte Proliferation in the Absence of a Specific Presenting Molecule
Authors: Lamphear James G. , Stevens Kristin Reda , Rich Robert R.
Published: January 2023

Bacterial superantigens can bind TCR in the absence of MHC class II molecules and activate T lymphocytes when cocultured with certain class II-deficient accessory cells. It has not been determined, however, whether these accessory cells provide direct costimulation to the T cell or serve to present superantigens via a nonconventional ligand. We have identified a human adenocarcinoma cell line, SW480, that assists in the activation of human T cells by the staphylococcal enterotoxins B (SEB), C1 (SEC1), and D (SED), but not SEA, SEC2, SEC3, or SEE. SW480 cells did not express class II molecules, and anti-class II mAbs did not inhibit T cell proliferation, supporting the hypothesis that class II is not absolutely required for enterotoxin-mediated T cell activation. The TCR Vβ profile of T cells stimulated by SEB plus SW480 cells was similar to that of T cells stimulated by SEB plus class II+ APC, indicating that TCR-SEB interactions were preserved in the absence of class II molecules. Binding studies failed to detect specific association of SEB with SW480 cells, suggesting that SW480 cells do not express receptors for enterotoxin. SEB coupled to beads, however, stimulated T cell proliferation, but only in the presence of SW480 cells. SW480 cells express both ICAM-1 and LFA-3 molecules, and the addition of Abs to these receptors inhibited T cell proliferation. These findings support a model in which certain enterotoxins engage the TCR independent of MHC class II or other specific presenting molecules and induce T cell proliferation with signals provided by nonconventional accessory cells.
Induction of Tyrosine Phosphorylation in Human MHC Class II-Positive Antigen-Presenting Cells by Stimulation with Contact Sensitizers
Authors: Kühn Ulrich , Brand Pia , Willemsen Judith , Jonuleit Helmut , Enk Alexander H. , van Brandwijk-Petershans Renate , Saloga Joachim , Knop Jürgen , Becker Detlef
Published: January 2023

To investigate the intracellular signaling mechanisms involved in the activation of APC by contact sensitizers, we studied the induction of tyrosine phosphorylation by these agents. Selective analysis of phosphotyrosine (p-tyr) in human Langerhans cells and different mononuclear cell types was achieved using a multicolor flow-cytometric technique. Stimulation with contact sensitizers revealed a distinct increase in p-tyr exclusively for MHC class II-positive cells. For different haptens, irritants, as well as activators of distinct signal transduction pathways, it was demonstrated that only strong sensitizers or the protein tyrosine phosphatase inhibitor sodium orthovanadate or cross-linking of MHC class II molecules were able to induce formation of p-tyr in human blood-derived dendritic cells serving as model for the dendritic cell family. This event required physiologic cell culture conditions and was blocked by specific inhibitors of protein tyrosine kinases. No evidence for the inhibition of protein tyrosine phosphatases by haptens was found. Western blot analysis of monocyte-enriched populations revealed an augmented phosphorylation of distinct proteins after hapten stimulation partly resembling the pattern noticed after cross-linking of HLA-DR molecules. In dendritic cells generated from mononuclear progenitors, the protein tyrosine kinase inhibitor genistein was able to block tyrosine phosphorylation as well as production of IL-1β mRNA transcripts. Our data underline the unique capacity of haptens to activate APC and the important role of tyrosine phosphorylation for this process.
Adenovector-Mediated Expression of Human Thrombopoietin cDNA in Immune-Compromised Mice: Insights into the Pathophysiology of Osteomyelofibrosis
Authors: Frey Beat M. , Rafii Shahin , Teterson Michael , Eaton Dan , Crystal Ronald G. , Moore Malcolm A.S.
Published: January 2023

Thrombopoietin (TPO) cDNA can be effectively delivered in vivo by adenovectors. Immune normal mice (BALB/c) and syngeneic mice with variable degrees of immune dysfunction nu, SCID, and NOD-SCID) were treated with an adenovirus vector expressing the human TPO cDNA (AdTPO). Platelet peaks were significantly higher in SCID and NOD-SCID mice compared with BALB/c and nu mice. Human plasma TPO concentration correlated with the platelet counts. SCID and NOD-SCID mice exhibited also granulocytosis and increased numbers of hemopoietic progenitors in bone marrow. Following platelet peak, BALB/c mice developed autoantibodies against murine TPO leading to thrombocytopenia and depletion of megakaryocytes and hemopoietic progenitors in bone marrow. AdTPO-treated SCID mice developed osteomyelofibrosis and extramedullary/extrasplenal hemopoiesis. In contrast, NOD-SCID mice with a similar magnitude of TPO overexpression did not show fibrotic changes in bone marrow. We conclude, first, that a chronic high level of TPO overexpression stimulates megakaryocytopoiesis and myelopoiesis leading to thrombocytosis and granulocytosis. Second, increased megakaryocytopoiesis is not sufficient for development of secondary osteomyelofibrosis. The functionally deficient monocytes and macrophages of NOD-SCID mice probably prevented fibrotic marrow changes. Third, immune deficiency enhances expression of adenovirally mediated transgenes, and fourth, xenogeneic transgene delivered by adenovector to a host with normal immune functions may induce loss of immune tolerance and autoimmune phenomenon.
A Periarteriolar Lymphoid Sheath-Associated B Cell Focus Response Is Not Observed During the Development of the Anti-Arsonate Germinal Center Reaction
Authors: Vora Kalpit A. , Tumas-Brundage Kathleen M. , Manser Tim
Published: January 2023

The behavior of p-azophenylarsonate (Ars)-specific B cell clones during the primary T cell-dependent splenic response of A/J mice was investigated using an immunohistochemical approach. The earliest Ars-specific B cells were observed as isolated cells in the red pulp by day 3 after immunization with Ars-keyhole limpet hemocyanin, (KLH) and at day 6, large clusters of Ars-specific B cells were first detected in germinal centers, which continued to be observed for an additional 8 to 15 days. Surprisingly, no Ars-specific B cell foci were observed in or near the CD4 T cell-rich periarteriolar lymphoid sheath (PALS) during the entire primary response. Nevertheless, A/J mice immunized with (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma globulin (NP-CGG) or Ars-CGG mounted robust splenic (4-hydroxy-3-nitrophenyl)acetyl or CGG-specific PALS-associated focus reactions, respectively. In contrast, no Ars-specific PALS B cell foci were detected in A/J mice immunized with Ars-CGG. These data add to a growing body of evidence indicating that B cell proliferation and differentiation in CD4 T cell-rich microenvironments are not prerequisites for the GC reaction. Taken together with previous results obtained using other model Ags, the data suggest that the specificity of the B cell Ag receptor may strongly influence the lymphoid microenvironment in which a B cell clone first undergoes Ag-driven clonal expansion and differentiation.
Aberrant Intermolecular Disulfide Bonding in a Mutant HLA-DM Molecule: Implications for Assembly, Maturation, and Function
Authors: Busch Robert , Doebele Robert C. , von Scheven Emily , Fahrni Jimothy , Mellins Elizabeth D.
Published: January 2023

HLA-DM (abbreviated DM) is an MHC-encoded glycoprotein that catalyzes the selective release of peptides, including class II-associated invariant chain peptides, from MHC class II molecules. To perform its function, DM must assemble in the endoplasmic reticulum (ER), travel to endosomes, and interact productively with class II molecules. We have described previously an EBV-transformed B cell line, 7.12.6, which displays a partial Ag presentation defect and expresses a mutated DM β-chain with Cys79 replaced by Tyr. In this study, we show that HLA-DR molecules in 7.12.6 have a defect in peptide loading and accumulate class II-associated invariant chain peptides (CLIP). Peptide loading is restored by transfection of wild-type DMB. The mutant DM molecules exit the ER slowly and are degraded rapidly, resulting in greatly reduced levels of mutant DM in post-Golgi compartments. Whereas wild-type DM forms noncovalent αβ dimers, such dimers form inefficiently in 7.12.6; many mutant DM β-chains instead form a disulfide-bonded dimer with DM α. Homodimers of DM β are also detected in 7.12.6 and in the α-chain defective mutant, 2.2.93. We conclude that during folding of wild-type DM, the native conformation is stabilized by a conserved disulfide bond involving Cys79β and by noncovalent contacts with DM α. Without these interactions, DM β can form malfolded structures containing interchain disulfide bonds; malfolding is correlated with ER retention and accelerated degradation.
Lack of Local Suppression in Orally Tolerant CD8-Deficient Mice Reveals a Critical Regulatory Role of CD8+ T Cells in the Normal Gut Mucosa
Authors: Grdic Dubravka , Hörnquist Elisabeth , Kjerrulf Martin , Lycke Nils Y.
Published: January 2023

We found that feeding keyhole limpet hemocyanin (KLH) to CD8-deficient (CD8−/−) mice induced oral tolerance that was comparable in both magnitude and quality to that induced in wild-type (wt) mice. The tolerance was dose dependent, and only higher doses of KLH caused significant reduction in specific Ab and T cell responses. Both Th1 and Th2 CD4+ T cell functions were affected. Feeding KLH together with cholera toxin (CT) adjuvant, however, abrogated the induction of oral tolerance equally well in CD8−/− and wt mice. On the contrary, CT adjuvant was unable to abrogate already established oral tolerance in both CD8−/− and wt mice. Most importantly, whereas Ag feeding induced hyporesponsiveness in systemic as well as in local gut IgA responses in wt mice, a lack of local suppression was evident in orally tolerant CD8−/− mice following oral immunizations. Thus, contrary to the situation in wt mice, Ag feeding induces systemic, but not local, gut IgA hyporesponsiveness in CD8−/− mice, suggesting that CD8+ T cells in the normal gut mucosa exert an important down-regulatory function. In wt mice the local suppression extended to an unrelated Ag, OVA, given together with KLH and CT adjuvant, i.e., bystander suppression. Based on these results we propose that tolerance induced by feeding Ag is highly compartmentalized, requiring CD8+ T cells for local suppression of IgA responses, whereas systemic tolerance may affect CD4+ T cells of both Th1 and Th2 types independently of CD8+ T cells. Finally, the adjuvant effect of CT abrogates induction, but not established, oral tolerance through a mechanism that does not require CD8+ T cells.
Degenerate Recognition of Alloantigenic Peptides on a Positive-Selecting Class I Molecule
Authors: Tallquist Michelle D. , Weaver Arthur J. , Pease Larry R.
Published: January 2023

The well-defined 2C T cell was used to investigate alloreactive degeneracy. A panel of class I molecules that are known ligands for the 2C TCR were sensitized with three known peptide ligands, p2Ca (LSPFPFDL), dEV-8 (EQYKFYSV), and SIYR-8 (SIYRYYGL). The peptide p2Ca was originally identified as the allopeptide seen in the Ld class I molecule by 2C T cells, 2C recognizes the dEV-8 peptide as the ligand in the Kbm3 class I molecule, and SIYR-8 was recently identified as a peptide ligand for 2C in the context of the Kb class I molecule. Strong recognition of all three Ag-presenting molecules occurred in the context of their respective allopeptides, but 2C recognized all three peptides to a measurable extent in the context of Kb. Molecular modeling of these Kb/peptide complexes revealed a high degree of similarity between dEV-8 and SIYR-8, but very little conformational similarity of either of these peptides with p2Ca. Furthermore, the structural changes in the mutant Kbm3 binding site resulted in generalized changes in the conformation of each of five bound peptides compared with those of the same peptides bound to Kb. The finding that degenerate recognition occurs on Kb, the restriction element responsible for selecting 2C T cells, suggests a unique relationship between a TCR and the Ag-presenting molecule that mediates its positive selection.
In Vitro and In Vivo Mechanisms of Action of the Antiproliferative and Immunosuppressive Agent, Brequinar Sodium
Authors: Xu Xiulong , Williams James W. , Shen Jikun , Gong Haihua , Yin Deng-Ping , Blinder Leonard , Elder Robert T. , Sankary Howard , Finnegan Alison , Chong Anita S.-F.
Published: January 2023

Intracellular pyrimidine nucleotides (PyN) can be synthesized de novo from glutamine, CO2, and ATP, or they can be salvaged from preformed pyrimidine nucleosides. The antiproliferative and immunosuppressive activities of brequinar sodium (BQR) are thought to be due to the inhibition of the activity of dihydroorotate dehydrogenase, which results in a suppression of de novo pyrimidine synthesis. Here we describe the effects of the pyrimidine nucleoSide, uridine, on the antiproliferative and immunosuppressive activities of BQR. In vitro reduction of PyN levels in Con A-stimulated T cells and inhibition of cell proliferation by low concentrations of BQR (≤65 μM) are reversed by uridine. However, uridine is unable to reverse the effects of high concentrations of BQR (≥65 μM). The ability of BQR to induce anemia in BALB/c mice is prevented by the coadministration of uridine. In contrast, the immunosuppressive activity of BQR is unaffected by similar doses of uridine. PyN levels in the bone marrow, but not in the spleen, are depressed in mice treated with BQR. These observations suggest that the induction of anemia by BQR is due to depletion of intracellular PyN in hemopoietic stem cells located in the bone marrow. They also suggest that the mechanism of immunosuppression by BQR may be only marginally dependent on depletion of intracellular PyN in lymphocytes located in the periphery. We report a novel activity of BQR: inhibition of tyrosine phosphorylation, and hypothesize that the immunosuppressive activity may be due, in part, to this unsuspected ability of BQR to inhibit tyrosine phosphorylation in lymphocytes.
CpG DNA Is a Potent Enhancer of Specific Immunity in Mice Immunized with Recombinant Hepatitis B Surface Antigen
Authors: Davis Heather L. , Weeranta Risini , Waldschmidt Thomas J. , Tygrett Lorraine , Schorr Joachim , Krieg Arthur M.
Published: January 2023

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODN) cause B cell proliferation and Ig secretion, monocyte cytokine secretion, and activation of NK cell lytic activity and IFN-γ secretion in vivo and in vitro. The potent immune activation by CpG ODN suggests possible utility for enhancing immune responses to vaccines. Mice immunized with recombinant hepatitis B virus surface Ag and a CpG ODN as an immune enhancer have titers of Abs against HBsAg (anti-HBs) that are five times higher than those of mice immunized with HBsAg and the standard adjuvant, aluminum hydroxide (alum). Ab titers in mice immunized with HBsAg and both CpG ODN plus alum were 35 times higher than the titers in mice immunized with alum alone, indicating a strong synergistic interaction between the CpG ODN and alum. ODN without CpG motifs had little or no immune-enhancing activity at the doses used herein. Alum induces a Th2 humoral response (mostly IgG1) and no CTL. In contrast, CpG ODN gives a strong Th1 response with predominantly IgG2a Abs and CTL, even when mixed with alum. In vitro studies to determine possible mechanisms of CpG immune-enhancing effects show that CpG ODN induce expression of costimulatory molecules on Ag-presenting cells and drive B cell isotype switching in the appropriate cytokine milieu. These studies demonstrate that CpG ODN are promising new immune enhancers for vaccination applications.
Recognition of an Antigenic Peptide Derived from Tyrosinase-Related Protein-2 by CTL in the Context of HLA-A31 and -A33
Authors: Wang Rong-Fu , Johnston Samuel L. , Southwood Scott , Sette Alessandro , Rosenberg Steven A.
Published: January 2023

Tumor-infiltrating lymphocytes (TILs) derived from tumor-bearing patients recognize tumor-associated Ags presented by MHC class I molecules. The infusion of TIL586 along with IL-2 into the autologous patient with metastatic melanoma resulted in the objective regression of tumor. Two T cell epitopes derived from tumor Ags, tyrosinase-related protein (TRP)-1 and TRP-2, were shown to be recognized by HLA-A31 restricted TIL586 and its T cell clones. In this study we tested the hypothesis that these two peptides can be recognized by CTL from non-HLA-A31 patients with melanoma. It was found that both peptides were capable of binding to HLA-A3, -A11, -A31, -A33, and -A68 of the HLA-A3 supertype. Importantly, we found that HLA-A33-positive TIL1244 and its T cell clones can recognize TRP197–205 presented by both HLA-A31 and -A33 molecules, suggesting that a single TCR can recognize peptide/A31 and peptide/A33 complexes. However, peptide titration experiments showed that the affinity of TCR receptor to peptide/A33 could be higher than that to the peptide/A31. These studies have important implications for the development of peptide-based cancer vaccines.
Prostaglandin E2 Modulation of p59fyn Tyrosine Kinase in T Lymphocytes During Sepsis
Authors: Choudhry Mashkoor A. , Uddin Shahab , Sayeed Mohammed M.
Published: January 2023

Prostaglandin E2 (PGE2) has been implicated in the suppression of T cell IL-2 production and proliferation during burn and sepsis. The present study evaluated the potential intracellular mechanism of suppressed T cell responses by assessing the activation of p59fyn kinase in T cells from septic rats as well as the T cells incubated with PGE2. p59fyn is known to regulate T cell functions. Sepsis was induced in rats by implanting fecal pellets containing Escherichia coli (150 CFU) and Bacteroides fragilis (104 CFU) into the abdominal cavity. For the assessment of PGE2 role in sepsis, a group of septic rats were treated with indomethacin, which inhibits endogenous PGE2 synthesis. As assessed by immunoblotting or in vitro kinase assay, a more than 40% inhibition of p59fyn phosphorylation and kinase activity was observed in septic rat T cells compared with the T cells from sterile or control rats. A similar inhibition in p59fyn phosphorylation and kinase activity was observed in PGE2-treated T cells compared with the T cells incubated in the absence of PGE2. The septic-related suppression in p59fyn phosphorylation and kinase activity in T cells was prevented in rats treated with indomethacin. We observed that the inhibition in p59fyn activation in septic or PGE2-treated T cells was due primarily to a suppression in p59fyn phosphorylation and not due to alterations in p59fyn protein expression. These findings suggest that PGE2 released during sepsis could contribute to the sepsis-related suppression in T cell proliferation by attenuating p59fyn phosphorylation and its kinase activity.
Human Melanoma Patients Recognize an HLA-A1-Restricted CTL Epitope from Tyrosinase Containing Two Cysteine Residues: Implications for Tumor Vaccine Development
Authors: Kittlesen David J. , Thompson Lee W. , Gulden Pamela H. , Skipper Jonathan C. A. , Colella Teresa A. , Shabanowitz Jeffrey A. , Hunt Donald F. , Engelhard Victor H. , Slingluff Craig L.
Published: January 2023

To identify shared epitopes for melanoma-reactive CTL restricted by MHC molecules other than HLA-A*0201, six human melanoma patient CTL lines expressing HLA-A1 were screened for reactivity against the melanocyte differentiation proteins Pmel-17/gp100, MART-1/Melan-A, and tyrosinase, expressed via recombinant vaccinia virus vectors. CTL from five of the six patients recognized epitopes from tyrosinase, and recognition of HLA-A1+ target cells was strongly correlated with tyrosinase expression. Restriction by HLA-A1 was further demonstrated for two of those tyrosinase-reactive CTL lines. Screening of 119 synthetic tyrosinase peptides with the HLA-A1 binding motif demonstrated that nonamer, decamer, and dodecamer peptides containing the sequence KCDICTDEY (residues 243–251) all reconstituted the CTL epitope in vitro. Epitope reconstitution in vitro required high concentrations of these peptides, which was hypothesized to be a result of spontaneous modification of cysteine residues, interfering with MHC binding. Substitution of serine or alanine for the more N-terminal cysteine prevented modification at that residue and permitted target cell sensitization at peptide concentrations 2 to 3 orders of magnitude lower than that required for the wild-type peptide. Because spontaneous modification of sulfhydryl groups may also occur in vivo, tumor vaccines using this or other cysteine-containing peptides may be improved by amino acid substitutions at cysteine residues.
Analysis of the Multiple Interactions Between IL-12 and the High Affinity IL-12 Receptor Complex
Authors: Presky David H. , Minetti Lisa J. , Gillessen Silke , Wilkinson Victoria L. , Wu Chang-You , Gubler Ueli , Chizzonite Richard , Gately Maurice K.
Published: January 2023

IL-12 is a heterodimeric cytokine, composed of a p40 and a p35 subunit, that exerts its biological effects by binding to specific cell surface receptors. Two IL-12R proteins, designated human IL-12 (huIL-12) receptor β1 (huIL-12Rβ1) and huIL-12Rβ2, have been previously identified. These IL-12R individually bind huIL-12 with low affinity and in combination bind huIL-12 with high affinity and confer IL-12 responsiveness. In this study the interactions of huIL-12 with these two identified human IL-12R protein subunits are examined. The heterodimer-specific anti-huIL-12 mAb 20C2, which neutralizes huIL-12 bioactivity but does not block 125I-huIL-12 binding to huIL-12Rβ1, blocked binding of huIL-12 to huIL-12Rβ2. In contrast, anti-huIL-12Rβ1 mAb 2B10 and mouse IL-12 p40 subunit homodimer (mo(p40)2) blocked 125I-huIL-12 binding to huIL-12Rβ1, but not to huIL-12Rβ2. Therefore, two classes of IL-12 inhibitors can be identified that differ in their ability to block huIL-12 interaction with either huIL-12Rβ1 or huIL-12Rβ2. Both mo(p40)2 and 20C2 blocked high affinity binding to huIL-12Rβ1/β2-cotransfected COS-7 cells, although, as previously reported, mo(p40)2 does not block high affinity binding to IL-12R on PHA-activated human lymphoblasts. Furthermore, these two classes of IL-12 inhibitors synergistically decreased huIL-12-stimulated proliferation and IFN-γ production. Therefore, IL-12, in binding to the high affinity IL-12R, interacts with IL-12Rβ1 primarily via regions on the IL-12 p40 subunit and with IL-12Rβ2 via 20C2-reactive, heterodimer-specific regions of IL-12 to which the p35 and p40 subunits both contribute.
Maternal Ig Mediates Neonatal Tolerance in Rheumatoid Factor Transgenic Mice but Tolerance Breaks Down in Adult Mice
Authors: Wang Haowei , Shlomchik Mark J.
Published: January 2023

We have recently demonstrated that B cell deletion occurs in the bone marrow of IgHa high affinity anti-IgG2aa (RF) transgenic mice. Here we demonstrate via genetic crosses that the source of IgG2a is the mother, thus establishing a transplacental mechanism that ensures tolerance to developmentally expressed Ags. Since maternal IgG can mediate tolerance in young mice, whether tolerance is maintained or, instead, autoimmunity ensues after weaning was investigated. We find that deletion remits abruptly in these RF transgenic mice beginning at 2 to 3 wk postweaning, and some degree of autoreactivity can be observed thereafter for weeks to months. The mechanism of sustained expression of autoreactive RF B cells in normal mice is unclear as yet, but a plausible mechanism is that once self-reactive cells are present, the antibody they secrete markedly reduces the autoantigen levels, presumably allowing further development, rather than deletion, of newly arising B lineage cells. The phenotype of these RF transgenic mice suggests a positive feedback mechanism that tends to perpetuate autoimmunity once it has been established. If such a mechanism were to exist in autoimmune animals, it could have important implications for the establishment and maintenance of B and T cell tolerance in chronic autoimmune diseases.
Redundancy of C/EBPα, -β, and -δ in Supporting the Lipopolysaccharide-Induced Transcription of IL-6 and Monocyte Chemoattractant Protein-1
Authors: Hu Hsien-Ming , Baer Mark , Williams Simon C. , Johnson Peter F. , Schwartz Richard C.
Published: January 2023

C/EBPα, -β, and -δ are members of the CCAAT/enhancer binding protein family of transcriptional regulators. All three of these factors are expressed by bone marrow-derived macrophages, with the DNA binding activity of C/EBPβ and -δ increased by treatment with LPS while that of C/EBPα is decreased. We have ectopically expressed each C/EBP protein in P388 lymphoblasts. The expression of any of these transcription factors is sufficient to confer the LPS-inducible expression of IL-6 and monocyte chemoattractant protein-1 to lymphoblasts, which normally lack C/EBP factors and do not display LPS induction of proinflammatory cytokines. Thus, the activities of C/EBPα, -β, and -δ are redundant in regard to the expression of IL-6 and monocyte chemoattractant protein-1. Since C/EBPβ-deficient mice have been reported to be largely normal in their expression of proinflammatory cytokines, it is likely that the lack of C/EBPβ is compensated for by the induction of C/EBPδ upon LPS treatment.
Induction of CD8+ T Cell Responses to Dominant and Subdominant Epitopes and Protective Immunity to Sendai Virus Infection by DNA Vaccination
Authors: Chen Yongjin , Webster Robert G. , Woodland David L.
Published: January 2023

While recent studies have demonstrated that DNA vaccination induces potent CD8+ T cell memory in vivo, it is unclear whether this memory is qualitatively and quantitatively comparable with that induced by natural viral infection. In the current studies, we have investigated the induction of CD8+ memory CTL responses to Sendai virus nucleoprotein (NP) in C57BL/6 mice following gene gun vaccination. The data demonstrate that this mode of vaccination induces potent long-lived memory CTL precursors (CTLp) specific for both the dominant (NP324–332/Kb) and the subdominant (NP324–332/Db) epitopes of NP. The frequencies of T cells specific for each of these epitopes in the spleen is about 1:2000 CD8+ T cells, similar to those induced by intranasal infection with Sendai virus. Moreover, the induction of memory CTLp by DNA vaccination is independent of MHC class II molecules or Ab, as is the case for memory CTLp induction by live Sendai virus infection. CTLp specific for both epitopes are capable of migrating to the lung following Sendai virus infection and express potent cytotoxic activity at the site of infection. Consistent with this activity, DNA vaccination with Sendai virus NP induced a substantial degree of Ab-independent protection from a challenge with a lethal dose of Sendai virus. Taken together, these data demonstrate that for the parameters tested, DNA vaccination is indistinguishable from live virus infection in terms of priming functional memory CTLp with broad specificity for both dominant and subdominant T cell epitopes.
Desensitization of Formyl Peptide Receptors Is Abolished in Calcium Ionophore-Primed Neutrophils: An Association of the Ligand-Receptor Complex to the Cytoskeleton Is Not Required for a Rapid Termination of the NADPH-Oxidase Response
Authors: Liu Li , Harbecke Olle , Elwing Hans , Follin Per , Karlsson Anna , Dahlgren Claes
Published: January 2023

Binding of ligands to N-formyl peptide chemoattractant receptors exposed on human neutrophils generates signals in the cells that induce an activation of the superoxide anion producing NADPH-oxidase. Ligand binding is followed by a rapid association of the ligand-receptor complex with the cytoskeleton, a process leading to desensitization of the cells with respect to NADPH-oxidase activation. We show that neutrophils that have experienced an intracellular calcium rise obtained through interaction with the calcium-specific ionophore ionomycin are “primed” with respect to the FMLP-induced production of superoxide anions. Mobilization of FMLP receptors from intracellular pools is one well-known mechanism behind the primed response. Based on our finding that ionomycin-treated neutrophils could not be desensitized, we suggest that the lack of association between the ligand-receptor complex and the cytoskeleton is an additional priming mechanism. Since in vivo-exudated neutrophils, which also had mobilized intracellular organelles, could be desensitized, we suggest that the abolished desensitization in ionomycin-treated neutrophils is not due to an inability of newly recruited receptors to couple to the cytoskeleton. We show that a rapid termination of FMLP-induced superoxide anion production is obtained in both desensitizable and nondesensitizable neutrophils, suggesting that the desensitization phenomenon is of limited importance in the oxidase termination process.
Increasing Immunogenicity of Antigens Fused to Ig-Binding Proteins by Cell Surface Targeting
Authors: Léonetti Michel , Thai Robert , Cotton Joël , Leroy Sandrine , Drevet Pascal , Ducancel Frédéric , Boulain Jean Claude , Ménez André
Published: January 2023

Fusion of antigenic proteins to Ig-binding proteins such as protein A from Staphylococcus aureus and its derived ZZ fragment is known to increase immunogenicity of the fused Ag in vivo. To shed light on the origin of this effect, we used snake toxins as Ags and observed that 1) fusion of toxins to ZZ enhanced their presentation to a toxin-specific T cell hybridoma (T1B2), using A20 B lymphoma cells, splenocytes, or peritoneal exudate cells as APCs; 2) this enhancement further increased when the number of fused Ig-binding domains varied from two with ZZ to five with protein A; and 3) the phenomenon vanished when the fusion protein was preincubated with an excess of free ZZ or when P388D1 monocytes cells were used as APCs. Therefore, ZZ-fused toxins are likely to be targeted to surface Igs of APCs by their ZZ moiety. Furthermore, ZZ-α and toxin α stimulated similar profiles of toxin-specific T cells in BALB/c mice, suggesting a comparable processing and presentation in vivo for both toxin forms. To improve the targeting efficiency, ZZ-α was noncovalently complexed to various Igs directed to different cell surface components of APCs. The resulting complexes were up to 103-fold more potent than the free toxin at stimulating T1B2. Also, they elicited both a T cell and an Ab response in BALB/c mice, without the need of any adjuvant. This simple approach may find practical applications by increasing the immunogenicity of recombinant proteins without the use of adjuvant.
Selective Inhibition of Expression of the Chemokine Receptor CCR2 in Human Monocytes by IFN-γ
Authors: Penton-Rol Giselle , Polentarutti Nadia , Luini Walter , Borsatti Alessandro , Mancinelli Roberta , Sica Antonio , Sozzani Silvano , Mantovani Alberto
Published: January 2023

IFN-γ is a potent activator of mononuclear phagocyte function and promotes the development of Th1 responses. Moreover, it induces and modulates chemokine production in a variety of cell types, including mononuclear phagocytes. In the present study, we examined the effect of IFN-γ on the expression of CC chemokine receptors in human monocytes. IFN-γ selectively and rapidly inhibited expression of the monocyte chemotactic protein (MCP) receptor CCR2 with an ED50 of ∼50 U/ml. The effect was rapid (detectable after 1 h) and reversible. Other chemokine receptors (CCR1, CCR3, CCR4, and CCR5) were not substantially affected, and CXCR4 was reduced. IFN-γ acted in concert with LPS, TNF-α, and IL-1β in inhibiting CCR2 expression. IFN-γ-treated monocytes showed a shorter half-life of CCR2 mRNA compared with untreated cells, whereas the rate of nuclear transcription was unaffected. The inhibition of CCR2 mRNA expression by IFN-γ was associated with a lower number of surface receptors and lower chemotactic responsiveness. Thus, IFN-γ, an inducer of MCP-1 and MCP-3 in mononuclear phagocytes, selectively inhibits expression of the MCP receptor CCR2 in monocytes. These results are consistent with an emerging paradigm of divergent regulation by several agents of chemokine production and receptor expression in monocytes. The inhibition of MCP-1R expression may serve as a means of retaining mononuclear phagocytes at sites of inflammation and as a feedback mechanism in the regulation of recruitment from the blood.
Target Recognition of β2-Glycoprotein I (β2GPI)-Dependent Anticardiolipin Antibodies: Evidence for Involvement of the Fourth Domain of β2GPI in Antibody Binding
Authors: George Jacob , Gilburd Boris , Hojnik Maja , Levy Yair , Langevitz Pnina , Matsuura Eiji , Koike Takao , Shoenfeld Yehuda
Published: January 2023

β2-glycoprotein I (β2GPI) is an absolute requirement for the binding of autoimmune anticardiolipin Abs (aCL) to cardiolipin (CL). We evaluated the target recognition of human β2GPI by IgG derived from two patients with primary and two with secondary antiphospholipid syndrome. The total IgG serum fractions and β2GPI affinity-purified IgGs were assessed by using various domain-deleted mutants (DM) of human β2GPI (DMs: I-III, I-IV, II-V, III-V, IV-V, and V) and mouse mAbs against individual β2GPI domains. The four IgGs bound slightly to CL in the absence of β2GPI and showed increased binding in the β2GPI presence. Following affinity purification of the IgGs on a β2GPI column, reactivity toward CL was absent. DMs containing domain V inhibited the binding of biotinylated β2GPI to CL. The addition to CL-coated plates of DM V, but not the other DMs, reduced the binding of all four IgGs. The anti-β2GPI IgGs bound only to complete β2GPI and DM I-IV coated on the plates. The binding to plate-adsorbed β2GPI could be inhibited by complete β2GPI and DM I-IV, the latter being a more efficient inhibitor. Further, the human anti-β2GPI IgGs could compete with the binding to β2GPI of Cof-21 mouse mAb (directed at domain IV), but not with the two other mouse mAbs. The results suggest that some “autoimmune:” β2GPI-dependent anticardiolipin Abs recognize a β2GPI target that is distinct from the CL-binding site in domain V. The target site for some antiphospholipid syndrome IgGs appear to reside in domain IV of β2GPI.
Cutting Edge: Characterization of an Associated 16-kDa Tyrosine Phosphoprotein Required for Ly-49D Signal Transduction
Authors: Mason Llewellyn H. , Willette-Brown Jami , Anderson Stephen K. , Gosselin Pierre , Shores Elizabeth W. , Love Paul E. , Ortaldo John R. , McVicar Daniel W.
Published: January 2023

Ly-49D is an activating receptor on NK cells that does not become tyrosine phosphorylated upon activation. This report demonstrates that immunoprecipitation of Ly-49D, following pervanadate treatment or specific Ab cross-linking, coprecipitates a 16-kDa tyrosine-phosphorylated protein (pp16). Immunoblotting experiments and data from TCR-ζ/FcεRIγ double knockout mice confirm that pp16 is not TCR-ζ, TCR-η, or FcεRIγ. Association of pp16 with Ly-49D involves a transmembrane arginine since mutation to leucine (Ly-49DR54L) abolishes association with pp16 in transfected P815 cells. In addition, Ly-49DR54L transfectants fail to mediate Ca2+ mobilization following Ab cross-linking. Therefore, signaling through Ly-49D on NK cells depends on association with a distinct tyrosine phosphoprotein (pp16) in a manner analogous to that of TCR and FcR. Expression of this novel signaling peptide in both the NK and myeloid lineages indicates that pp16 is likely involved in the signal transduction cascade of additional receptor families.
Mechanisms of CD8β-Mediated T Cell Response Enhancement: Interaction with MHC Class I/β2-Microglobulin and Functional Coupling to TCR/CD3
Authors: Wheeler Christopher J. , Chen Jing-Yi , Potter Terry A. , Parnes Jane R.
Published: January 2023

CD8β expression results in enhanced IL-2 production and/or altered specificity in allogeneic MHC class I-restricted T cell hybridomas. Expression of chimeric CD8β-α molecules (extracellular CD8β, transmembrane and cytoplasmic CD8α) also results in enhancement of T hybridoma responses to alloantigen, suggesting that at least part of CD8β’s ability to influence responses similar to those of mature CD8+ T cells is mediated by its extracellular domain. Current data suggest that CD8β-mediated response enhancement proceeds through mechanisms similar to those mediated by CD8α, i.e., interacting with MHC class I and stabilizing CD8-associated Lck activity. In this study we present evidence that the extracellular portion of CD8β is capable of independent interaction with MHC class I/β2m dimers in the absence of CD8α. In addition, CD8β may enhance interaction with MHC class I/β2m when associated with CD8α. We also present evidence from T hybridoma responses suggesting that the extracellular portion of CD8β is uniquely capable of efficient interaction with the TCR/CD3 complex and may couple the TCR/CD3 complex to other surface components capable of enhancing TCR-mediated signals. This represents the first evidence that a critical coreceptor function can be preferentially associated with the CD8β subunit.
Oligoclonal Development of B Cells Bearing Discrete Ig Chains in Chicken Single Germinal Centers
Authors: Arakawa Hiroshi , Kuma Kei-ichi , Yasuda Masahiro , Furusawa Shuichi , Ekino Shigeo , Yamagishi Hideo
Published: January 2023

Chicken single germinal centers enable us to analyze the postbursal diversifications of B cells due to their easy isolation. Germinal center formation has peaked by day 7 of primary responses and begins to wane 14 days after immunization. To detail the kinetics of Ig mutation and selection, we analyzed Ig light chain sequences recovered from single germinal centers at 7 and 11 days postimmunization with an artificial Ag. Our observations show that multiple, Ag-activated B cells migrating into single germinal centers are diversified by gene conversion in the very early phase of the germinal center reaction and are subsequently subjected to point mutations and selection for oligoclonality.
Presentation of Proteolipid Protein Epitopes and B7-1-Dependent Activation of Encephalitogenic T Cells by IFN-γ-Activated SJL/J Astrocytes
Authors: Tan Litjen , Gordon Kenneth B. , Mueller John P. , Matis Louis A. , Miller Stephen D.
Published: January 2023

There is controversy regarding the possible role of glial cells as APCs in the pathogenesis of central nervous system (CNS) demyelinating diseases such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Microglia have been clearly shown to present Ag in the CNS, and due to the proximity of activated astroglial cells to infiltrating T cells and macrophages in demyelinating lesions, it is also possible that astrocytes positively or negatively regulate disease initiation and/or progression. We examined the capacity of IFN-γ-treated astrocytes from EAE-susceptible SJL/J mice to process and present myelin epitopes. IFN-γ activation up-regulated ICAM-1, VCAM-1, MHC class II, invariant chain, H2-M, CD40, and B7-1 as determined by FACS and/or RT-PCR analyses. B7-2 expression was only marginally enhanced on SJL/J astrocytes. Consistent with the expression of these accessory molecules, IFN-γ-treated SJL/J astrocytes induced the B7-1-dependent activation of Th1 lines and lymph node T cells specific for the immunodominant encephalitogenic proteolipid protein (PLP) epitope (PLP139–151) as assessed by proliferation and activation for the adoptive transfer of EAE. Interestingly, IFN-γ-activated astrocytes efficiently processed and presented PLP139–151, but not the subdominant PLP178–191, PLP56–70, or PLP104–117 epitopes, from intact PLP and a recombinant variant fusion protein of PLP (MP4). The data are consistent with the hypothesis that astrocytes in the proinflammatory CNS environment have the capability of activating CNS-infiltrating encephalitogenic T cells specific for immunodominant epitopes on various myelin proteins that may be involved in either the initial or the relapsing stages of EAE.
The Glycosylphosphatidylinositol-Anchored Form and the Transmembrane Form of CD58 Associate with Protein Kinases
Authors: Itzhaky Dganit , Raz Nava , Hollander Nurit
Published: January 2023

The significance of the glycosylphosphatidylinositol (GPI) anchor is unknown. Since GPI-anchored proteins mediate signaling, it has been suggested that the GPI structure serves as a signal-transducing element. However, the division of signaling functions between transmembrane and GPI-anchored proteins is unclear. Studies of distinct membrane-anchored forms of the same protein may resolve this issue. The adhesion molecule CD58 is expressed on the cell surface in both a transmembrane and a GPI-anchored form and hence provides a useful model. We studied CD58 in the human B lymphoblastoid cell line JY. In addition to mediating adhesion, CD58 is involved in signal transduction. Incubation of JY cells with immobilized anti-CD58 Abs results in extensive tyrosine phosphorylation and in secretion of TNF-α. We demonstrate that CD58 is associated with protein kinase(s) and with several kinase substrates. We further demonstrate that both CD58 isoforms are involved. CD58 in JY variant cells, which express only the transmembrane form, as well as CD58 in JY variant cells, which express only the GPI-anchored form, are associated with kinase activity. This association results in a phosphorylation pattern that is common to the variant and to wild-type JY cells. Thus, these findings suggest that the capacity of GPI-anchored proteins to interact with kinases is not always dependent on the GPI anchor itself.
Selective Inhibition of IL-5 Receptor α-Chain Gene Transcription by IL-5, IL-3, and Granulocyte-Macrophage Colony-Stimulating Factor in Human Blood Eosinophils
Authors: Wang Peng , Wu Ping , Cheewatrakoolpong Boonlert , Myers Joyce G. , Egan Robert W. , Billah M. Motasim
Published: January 2023

High affinity receptor for IL-5 (IL-5R), a predominant eosinophil maturation factor, is composed of an IL-5-binding α-chain (IL-5Rα) and a signal-transducing β-chain that is shared by IL-3 and granulocyte-macrophage CSF (GM-CSF) receptors (IL-3R and GM-CSFR). By Northern blot analysis of mRNAs obtained from normal human blood eosinophils, we show in this report that the hematopoietic cytokines IL-5, IL-3, and GM-CSF down-regulate IL-5Rα mRNA while up-regulating α-chain mRNAs for both IL-3R and GM-CSFR as well as the β-chain mRNA. More detailed characterization reveals that the down-regulation of IL-5Rα mRNA is specific to IL-3, IL-5, and GM-CSF; occurs very rapidly (reaching maximum inhibition within 2 h); is cytokine dose dependent; and does not require protein synthesis. Nuclear run-on and mRNA stability experiments demonstrate that cytokine-induced inhibition of IL-5Rα mRNA accumulation occurs at the level of IL-5Rα gene transcription, whereas enhanced accumulation of mRNAs for IL-3Rα and the β-chain results from reduced mRNA degradation. We suggest from these experiments that in human blood eosinophils, IL-5Rα gene transcription and IL-5Rα mRNA metabolism can be regulated by mechanisms that are distinct from those used for IL-3Rα and GM-CSFRα.