bluuebunny/crossref_metadata_2025 huggingface dataset:
Transgene CD23 expression on lymphoid cells modulates IgE and IgG1 responses.
Authors: Texido G , Eibel H , Le Gros G , van der Putten H
Published: December 2022

Transmembrane (m) and soluble (s) forms of CD23 perform activities related to various immune functions. Abnormal expression patterns of CD23 on lymphoid cells have been associated with certain pathologic conditions. To explore the effects of CD23 when it is overexpressed, on lymphoid cell development and immune function in vivo, transgenic mice were generated. These mice overexpressed either mCD23 or a 38-kDa molecular form of sCD23. Transgene expression under the control of Thy-1-regulatory sequences and the mouse Ig heavy chain enhancer (E mu) was prominent in thymus, spleen, bone marrow, and lymph nodes. Cells that expressed the transgenes included most thymocytes, peripheral CD4+ and CD8+ T cells, IgM+/highIgD-/low immature B cells, and IgMlowIgDhigh mature B cells. To resolve the expression pattern in the B cell lineage unambiguously, we used mice that carried a transgene and a disrupted endogenous CD23 gene simultaneously. Neither mCD23 nor sCD23 overexpression caused significant alterations in lymphoid cell maturation. In addition, basal serum levels of IgE and IgG1 proved to be normal. In three different experimental immune response paradigms, mCD23 transgenic, but not sCD23 and nontransgenic mice proved to be impaired in increasing serum levels of polyclonal IgE up to expected levels. In addition, mCD23 transgenic mice showed below normal increases of serum IgG1 levels in two of the three immune responses. In the presence of activated T cells and appropriate lymphokines, B cells from mCD23 mice secreted normal amounts of IgE and IgG1 in vitro, which suggests that there was no serious impairment of the T-B cell contact required for Ig production. In addition, there is no evidence for a significant role of mCD23 in IgE clearance. Therefore, we discuss alternative mechanisms by which mCD23+ B and/or T cells influence Ig production.
Trimeric structure of human proliferating cell nuclear antigen. Implications for enzymatic function and autoantibody recognition.
Authors: Brand S R , Bernstein R M , Mathews M B
Published: December 2022

The proliferating cell nuclear Ag (PCNA) is a DNA replication factor postulated to function as a sliding clamp around DNA. PCNA is also a target for autoimmunity in systemic lupus erythematosus. The autoantigenicity of PCNA is highly conformation-dependent, and reaction with most anti-PCNA sera requires a nearly full-length PCNA molecule. Here we describe the use of gel filtration and glycerol gradient sedimentation to analyze the native structure and size of PCNA. PCNA from three sources was studied (PCNA from HeLa cells, PCNA purified after its overexpression in bacteria, and PCNA produced in the wheat germ cell-free translation system) as well as mutant forms of PCNA translated in vitro. In each case, full-length PCNA behaved as a trimer. Analysis of mutant proteins revealed a correlation between the trimeric form and binding to the common type of human anti-PCNA autoantibody, suggesting that the Abs are specific for the active form of the protein. These findings are consistent with the idea that autoantibodies are generated as a response to native Ag and provide experimental support for the hypothesis that PCNA serves its processive function in DNA replication as a trimeric ring structure.
Characterization of peptide binding to the murine MHC class I H-2Kk molecule. Sequencing of the bound peptides and direct binding of synthetic peptides to isolated class I molecules.
Authors: Brown E L , Wooters J L , Ferenz C R , O'Brien C M , Hewick R M , Herrmann S H
Published: December 2022

Peptides that are bound by the murine class I MHC molecule H-2Kk have been isolated and sequenced. The initial step in the fractionation was affinity column isolation of the peptide-class I complex from either RDM-4 or x5563 tumor cell lines. Acid denaturation of the complex followed by HPLC fractionation of the peptides allowed us to sequence individual peptides, as well as pools of peptides. To date, a total of 10 sequences have been characterized, and all were 8 mers. The sequences were variable except for glutamic acid in the second position (P2) and isoleucine in the eighth (P8), which were highly conserved. To further study peptide binding to H-2Kk, a competitive binding assay consisting of the immobilized histocompatibility protein and a biotinylated self-peptide for signal generation was developed. A complete set of single-alanine variants for this one self-peptide was tested in the assay, demonstrating that substitution at P2 and P8 markedly decreased the affinity for the class I molecule; alanine at position 3 had an intermediate effect on binding. A comparison of the identified self-peptides for binding to H-2Kk showed that they differed in affinity by more than one order of magnitude. Influenza virus nucleoprotein peptide, SDY EGR LI, associated with the plate-bound class I molecule, and the resulting MHC-peptide complex could trigger TNF release by influenza-primed CTLs. This result demonstrated the functional activity of the plate-bound H-2Kk-peptide complex.
Identification of a novel HLA-B27 subtype by restriction analysis of a cytotoxic gamma delta T cell clone.
Authors: Del Porto P , D'Amato M , Fiorillo M T , Tuosto L , Piccolella E , Sorrentino R
Published: December 2022

Seven HLA-B27 alleles are known, which share the same allospecificity, but differ by one to six amino acid substitutions. Herein, we describe a novel HLA-B27 allele, provisionally named B27-ci, which is expressed by an individual from whom a B27-restricted gamma delta T cell clone has been derived. This clone recognizes B cell lines from the proband and all of the other B27-positive members of the family, but does not lyse B cell lines that express other HLA-B27 alleles. The amino acid sequence deduced from three B27-ci cDNA clones was found to differ from the B*2705 sequence by one amino acid substitution (Asp to His) in position 116 of the alpha 2 domain. This position has been shown to lie in the floor of the F pocket, where it plays a key role in determining the nature of the amino acid side chain that will fit into this pocket. Moreover, the fact that the clone described here possesses a TCR-gamma delta indicates that this subset of cells not only can be HLA-restricted, but also can finely discriminate among classical class I molecules.
Insight into the mechanism of TCR-V beta 8+/CD8+ T cell-mediated MOPC-315 tumor eradication.
Authors: Mokyr M B , Prokhorova A , Rubin M , Bluestone J A
Published: December 2022

We have previously shown that depletion of TCR-V beta 8+ T cells by treatment with mAb reduces the curative effectiveness of low dose melphalan (L-phenylalanine mustard; L-PAM) for mice bearing a large MOPC-315 tumor and extensive metastases. Here we show that V beta 8+/CD8+ T cell lines derived from mice that are in the process of immune-mediated eradication of a large MOPC-315 tumor as a consequence of low dose L-PAM therapy (L-PAM TuB mice) are capable of mediating tumor eradication in vivo upon adoptive transfer. Analysis of the possible mechanisms through which these cell lines bring about tumor eradication revealed that the V beta 8+/CD8+ cells can exert in vitro a potent lytic activity and secrete large amounts of IFN-gamma. Both of these activities can be triggered by the MOPC-315 but not the MOPC-104E plasmacytoma and are restricted by the MHC class I H-2Kd molecule. In vivo neutralization of IFN-gamma by treatment with mAb was found to cause a noticeable delay in tumor rejection in mice subjected to adoptive chemoimmunotherapy with low dose L-PAM and V beta 8+/CD8+ cells; however, all tumors did regress after initial growth. Thus, the V beta 8+/CD8+ cells use an IFN-gamma-dependent mechanism for the realization of their in vivo tumor-eradicating immunity. However, an IFN-gamma-independent mechanism, most likely involving direct V beta 8+/CD8+ CTL activity, is apparently also used.
Role of sialic acid in the resistance of Trypanosoma cruzi trypomastigotes to complement.
Authors: Tomlinson S , Pontes de Carvalho L C , Vandekerckhove F , Nussenzweig V
Published: December 2022

Trypomastigotes of Trypanosoma cruzi, mammalian infective forms of the parasite, express an unusual cell surface trans-sialidase. This enzyme enables the parasite to rapidly sialylate its surface when supplied with alpha(2,3)-linked sialic acid from glycoconjugates in serum or on cell surfaces. Here we used a novel fluorescence-based, trypomastigote lysis assay to evaluate the role of sialic acid on the parasite's plasma membrane in providing protection against the complement cascade. Trypomastigotes were desialylated, and sialic acid removal was confirmed by a chemical assay and also by flow cytometry with the use of a mAb that recognizes a T. cruzi-sialylated epitope. Compared with sialylated trypomastigotes, which were completely refractory to lysis by human serum, only about 5% of the desialylated trypomastigotes were lysed by complement. However, further analysis revealed that the desialylated parasites had been resialylated during exposure to serum complement. Next we incubated desialylated trypomastigotes with samples of desialylated human serum. Although the sialidase-treated serum retained its full hemolytic activity, lysis of trypomastigotes increased only from 5 to 24%. This increase correlated with an enhanced deposition of complement protein C3 on the parasite surface. The ratio of C3b to lytically inactive iC3b was increased for desialylated, compared with sialylated, parasites. We conclude that although parasite sialic acid promotes C3b cleavage into iC3b, this mechanism alone does not account for the robust resistance of these parasites to complement lysis.
Identification of immunodominant epitopes in Trypanosoma cruzi trypomastigote surface antigen-1 protein that mask protective epitopes.
Authors: Wrightsman R A , Dawson B D , Fouts D L , Manning J E
Published: December 2022

The gene that encodes trypomastigote surface Ag-1 (TSA-1), a major surface Ag of the bloodstream trypomastigote stage of Trypanosoma cruzi, was expressed in a baculovirus expression system. To determine the epitope(s) in TSA-1 that was recognized during T. cruzi infection and after immunization with TSA-1, subregions of the TSA-1 gene were expressed in a bacterial expression system. As seen by Western blotting, both mice and rabbits immunized with recombinant TSA-1 protein, as well as T. cruzi-infected mice, developed strong immune responses to the carboxyl-proximal region of TSA-1, but show no reaction to the amino-proximal portion of TSA-1. When mice were immunized with either recombinant TSA-1 protein or the carboxyl-proximal region of TSA-1, they did not survive challenge with 10(3) bloodstream trypomastigotes. However, 70% of the mice immunized with the amino-proximal portion of TSA-1 survived challenge with 10(3) bloodstream trypomastigotes. Thus, the immune responses elicited by recombinant TSA-1 or the carboxyl-proximal portion of TSA-1 are nonprotective during T. cruzi infection. In contrast, vaccination with the amino proximal region of TSA-1 elicits a protective immune response. These results suggest that responses to immunodominant epitope(s) within the carboxyl-proximal portion of TSA-1 mask epitopes within the amino-proximal portion that are capable of stimulating host-protective immune responses. It is suggested that immunodominant regions in surface molecules such as TSA-1 may provide a mechanism for the parasite to evade the host immune response by directing the response away from epitopes that have the potential to elicit a reaction that is damaging to the parasite.
Monocyte chemotactic protein-2, monocyte chemotactic protein-3, and fibroblast-induced cytokine. Three new chemokines induce chemotaxis and activation of basophils.
Authors: Alam R , Forsythe P , Stafford S , Heinrich J , Bravo R , Proost P , Van Damme J
Published: December 2022

Cytokine-dependent mediator release from basophils and mast cells may play an important role in the pathogenesis of allergic and inflammatory conditions. Many C-C chemokines have been found to activate basophils and mast cells. We investigated the effect of three newly identified C-C chemokines, monocyte chemotactic protein-2 and -3 (MCP-2, MCP-3) and fibroblast-induced cytokine (FIC) on basophils and mast cells. We found that all three cytokines induced histamine secretion from basophils in a dose-dependent manner. The secretion of histamine was a Ca(2+)-dependent process. MCP-3 was the most potent activator of basophils. MCP-3 and FIC activated basophils from all study subjects, whereas the histamine release by MCP-2 was donor-dependent. The histamine-releasing activity of MCP-2, MCP-3, and FIC was compared with that of MCP-1, RANTES, and macrophage inflammatory protein-1 alpha using basophils from 10 donors. MCP-1 was the most potent among all the C-C chemokines. However, MCP-3 was nearly as potent. MCP-2, MCP-3, and FIC induced significant chemotaxis of basophils. None of the cytokines activated mouse peritoneal mast cells. The synthesis of mRNA for MCP-3 was investigated by reverse-transcription PCR using allergen-stimulated PBMC and bronchoalveolar lavage cells. Both MNC and bronchoalveolar lavage cells expressed mRNA for MCP-3. The results of this study indicate that MCP-2, MCP-3, and FIC are novel histamine-releasing factors.
Apoptotic neutrophils are phagocytosed by fibroblasts with participation of the fibroblast vitronectin receptor and involvement of a mannose/fucose-specific lectin.
Authors: Hall S E , Savill J S , Henson P M , Haslett C
Published: December 2022

The fate of neutrophils (PMNs) at sites of inflammation is important to our understanding of many disease processes. Previously, it had been widely assumed that extravasated PMNs inevitably disintegrated before their fragments were removed by local phagocytes, but we have recently described an alternative process whereby senescent PMNs undergo apoptosis (programmed cell death). This process leads to macrophage (Mphi) ingestion of the intact cell by a novel phagocytic recognition process. In this study, we show that monolayers of fibroblasts also can selectively phagocytose apoptotic PMNs and that the recognition of apoptotic PMNs by fibroblasts involves two distinct mechanisms: one uses the vitronectin receptor, as in Mphi ingestion of PMNs; the other uses a mannose/fucose-specific lectin, which plays no part in Mphi phagocytosis of apoptotic PMNs. The direct interactions between PMNs and fibroblasts demonstrated herein may have implications for our understanding of the relationship between inflammation and scarring in many diseases.
Inhibition of human peripheral blood lymphocyte function by protoporphyrin and longwave ultraviolet light.
Authors: Barrett K E , Yen A , Bigby T D , Montisano D , Gigli I
Published: December 2022

Modulation of immunologic effector cells by exogenous photoactive substances has been advanced as an underlying mechanism for the efficacy of various photochemotherapeutic regimens. It is also possible that endogenous photosensitizers, such as protoporphyrin, could similarly modify the function of immune cell types. We examined the effects of protoporphyrin plus longwave UV light on the ability of human PBL to proliferate in response to mitogens. Noncytotoxic dosages of protoporphyrin plus UV light suppressed PHA-stimulated proliferation of both PBMC and enriched T cells. CD8+ cells were more sensitive to this inhibitory effect than CD4+ cells. The inhibitory effect was also observed when proliferation was induced by the combination of a phorbol ester and ionomycin. Inhibition of PBMC proliferation was associated with inhibition of IL-2 secretion but proliferation was not restored with exogenous IL-2. Instead, the effect of protoporphyrin plus UV light may be on IL-2R. Cells treated with protoporphyrin and UV light did not display the increase in CD25 and the beta-chain of the IL-2R induced by PHA in control cells. In contrast to the effects of protoporphyrin and UV light on IL-2 and IL-2R alpha-chain protein expression, the accumulation of mRNA for these proteins induced by PHA was unaffected. None of the effects of protoporphyrin plus UV light on lymphocytes were observed in control experiments where cells were treated with either protoporphyrin or UV light alone. We conclude that biologically relevant dosages of protoporphyrin and UV light modify the function of circulating lymphocytes.
Idiotype- and anti-idiotype-reactive T lymphocytes in myasthenia gravis. Evidence for the involvement of different subpopulations of T helper lymphocytes.
Authors: Yi Q , Lefvert A K
Published: December 2022

Patients with myasthenia gravis have peripheral blood T cells that are stimulated to secrete IFN-gamma and IL-2 by human monoclonal anti-acetylcholine receptor and anti-idiotypic Abs. The human CD4+ Th cells may be subdivided into different subsets according to the pattern of cytokine production. To elucidate the subgroup of T cells involved in myasthenia gravis, we have analyzed IL-4-, IFN-gamma-, and IL-2-secretion patterns induced by two human disease-specific mAbs, one idiotypic and one anti-idiotypic Ab. T cells stimulated to secrete one or more of the cytokines were found in 33 of 34 patients. The idiotypic Ab stimulated IL-4 secretion in 19 of 34 patients, IFN-gamma secretion in 26 of 34 patients, and IL-2 secretion in 25 of 34. The anti-idiotypic Ab induced IL-4 secretion in 9 of 34 patients, IFN-gamma secretion in 29 of 34 patients, and IL-2 secretion in 28 of 34 patients. These T cell responses were MHC class II-restricted. According to the cytokine-secretion patterns, Id-reactive T cells might correspond to both Th1/Th2 and/or Th0 cells. The anti-idiotypic Ab more frequently induced a Th1-type response. This study indicates that different subsets of T lymphocytes are involved in the idiotypic network in myasthenia gravis, and that these functionally different T cells may participate in the immunoregulation of the disease.
The effect of mutant beta 2-microglobulins on the conformation of HLA-B27 detected by antibody and by CTL.
Authors: Fukazawa T , Hermann E , Edidin M , Wen J , Huang F , Kellner H , Floege J , Farahmandian D , Williams K M , Yu D T
Published: December 2022

The arthritis-predisposing HLA-B27 consists of a heavy chain, a small peptide, and the monomorphic beta 2-microglobulin (beta 2-m). CTLs and a mAb, Ye-2, which recognize the complex with specificities both for the heavy chain and for the peptide, are available. The beta 2-m is in noncovalent association with the heavy chain at multiple points and is exchangeable with free beta 2-m outside of the complex. The purpose of our experiments was to test whether mutant beta 2-m capable of modulating HLA-B27 activity could be created. Eighteen recombinant mutants of the human beta 2-m were experimentally generated. In 14 of these, mutations were at or near residues that are either contact residues or interface residues with the heavy chain. Relative to the parent beta 2-m, two-thirds of the mutants showed reduced ability to exchange into HLA-B27 complexes. However, at least four of them induced more than 80% decrease in Ye-2 Ab reactivity. Two mutants were able to induce a minor decrease in susceptibility to lysis by four CTL clones. One of the CTL clones was autoreactive. Two of the CTL clones were specific for HLA-B27 cells experimentally infected with arthritis-causing Yersinia enterocolitica. These results indicate that certain beta 2-m residues play an indirect role in peptide presentation, although they are not directly associated with the peptide residues.
Evidence for glycosphingolipid modification of the type 1 IFN receptor.
Authors: Ghislain J , Lingwood C A , Fish E N
Published: December 2022

P1-determinant glycolipids that include two membrane glycosphingolipids, globotriaosylceramide (Gal alpha 1-4Gal beta 1-4Glc-ceramide, Gb3) and galabiosylceramide (Gal alpha 1-4Gal-ceramide, Gb2) are receptors for an Escherichia coli-derived subunit toxin, verotoxin (VT-1). Studies with Daudi cells and glycosphingolipid-deficient Daudi mutants and U937 cells identified that the presence of Gb2/Gb3 correlates with IFN-alpha sensitivity. Comparison of amino acid sequences between VT-1 and the extracellular region of the 63-kDa IFN-alpha beta receptor (IFNAR) peptide reveals regions of identity, specifically in those domains in the VT-1 B subunit, that have been implicated as Gb2/Gb3 binding sites. In direct ligand binding studies, we show that membrane Gb2/Gb3 content affects the binding capacity of cells for IFN-alpha, although IFNAR cell surface expression is unaffected. Binding of IFN-alpha to the receptor leads to kinase-associated phosphorylation of the latent transcription factor, ISGF3, which activates transcription by binding to IFN-stimulated regulatory elements in IFN-sensitive genes. Electrophoretic mobility band shift assays indicated that U-937 and Daudi mutant cells, deficient in Gb2 and Gb3, exhibited reduced nuclear factor binding to the human 2-5A synthetase IFN-stimulated regulatory element when compared with wild-type Daudi cells, after exposure to IFN-alpha. Moreover, when Daudi cells were treated with a ceramide analogue, 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol, Gb2 and Gb3 synthesis was inhibited and a concomitant reduction in IFN-induced ISGF3 activation was noted. IFNAR cell surface expression was unaffected by 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol treatment. By using a fusion protein of the extracellular domain of IFNAR linked at the carboxyl terminus to the Fc portion of IgG1, we demonstrate that IFNAR is able to bind preferentially to Gb2. These results suggest that an association of IFNAR with membrane Gal alpha 1-4Gal containing glycolipids facilitates receptor-mediated signaling.
Activation of human monocytes via a non-neurokinin substance P receptor that is coupled to Gi protein, calcium, phospholipase D, MAP kinase, and IL-6 production.
Authors: Kavelaars A , Broeke D , Jeurissen F , Kardux J , Meijer A , Franklin R , Gelfand E W , Heijnen C J
Published: December 2022

Substance P (SP) is a tachykinin involved in the regulation of inflammatory processes. Tachykinins bind to three subtypes of neurokinin (NK) receptors. However, recently we demonstrated that monocytes express a SP binding site that is not one of the known NK receptors. Activation of this SP receptor leads to the stimulation of MAP kinase in monocytes. In the present paper we show that this novel SP binding site is coupled to a GTP binding protein of the Gi alpha 1/2 subclass. Triggering of the SP receptor leads to a rapid rise in cytosolic calcium. In a more sustained way, SP stimulates phospholipase D (PLD) activity in human monocytes. The effects of SP on calcium, PLD, and MAP kinase activity can be blocked by pretreatment of the cells with pertussis toxin, which is in agreement with receptor coupling to Gi. At a functional level, stimulation of the non-NK SP receptor on monocytes results in the induction of IL-6 production. We show here that the order of potency for activation of monocytes by various ligands is directly related to the Ki for displacement of labeled SP by these ligands. Therefore, our data strongly suggest that the effects of SP are mediated via the novel SP receptor we recently described.
Prevention of autoimmune type I diabetes in biobreeding (BB) rats by a newly established, autoreactive T cell line from acutely diabetic BB rats.
Authors: Nagata M , Yoon J W
Published: December 2022

An autoreactive T cell line, BNY-2, was established from lymphocytes isolated from the islets of acutely diabetic BioBreeding (BB) rats after continuous stimulation of the isolated lymphocytes by irradiated lymph node cells. Flow cytometric analysis showed that BNY-2 cells were positive for CD4 and CD5 and were negative for CD8 and RT6, indicating that these cells were phenotypically Th cells rather than cytotoxic T cells. mAbs against rat MHC class II Ags (anti-RT1.D) blocked the proliferation response of BNY-2 cells, suggesting that these cells recognize the MHC class II Du molecule. When splenic T lymphocytes from diabetes-prone BB rats were stimulated by Con A in the presence of irradiated BNY-2 cells or irradiated lymph node cells, the BNY-2 cells had a significant suppressive effect on splenic T cell proliferation, whereas lymph node cells had no effect. When we injected diabetes-prone BB rats i.v. at 30 and 60 days of age with activated BNY-2 cells, the incidence of diabetes was significantly reduced compared with that seen in saline-injected control rats (diabetic incidences were 12 and 80%, respectively). On the basis of these observations, we conclude that autoreactive BNY-2 T cells, established from the pancreatic islets of acutely diabetic BB rats, can prevent the development of autoimmune diabetes in the diabetes-prone BB rat by an immunosuppressive effect.
Clonotype analysis of peripheral blood T cells and autoantigen-reactive T cells from patients with mixed connective tissue disease.
Authors: Okubo M , Kurokawa M , Ohto H , Nishimaki T , Nishioka K , Kasukawa R , Yamamoto K
Published: December 2022

T cells that recognize autoantigens may play a central role in the autoimmune response. We have previously shown that autoantigen-reactive T cells (CD4+) to U1-small nuclear ribonucleoprotein A were found in the PBMC of patients with systemic lupus erythematosus or mixed connective tissue disease. To reveal clonotypes of such T cells that spread to the periphery, T cell clonal accumulations and their TCR-beta chain variable gene usages in fresh PBMC from eight patients with mixed connective tissue disease were analyzed by a new method of single strand conformation polymorphism applying to TCR gene detection. The results revealed that the numbers of accumulated T cell clones (mean: 24.3 clones) were greater than the numbers of clones detected in healthy donors (mean: 4.0 clones). Frequently used V beta genes in these accumulated T cell clones were V beta 1, 3, 4, 5.2, 14, and 16. After the stimulation for these samples with the soluble recombinant U1-small nuclear ribonucleoprotein A protein, proliferative T cell responses were observed. We found that T cell clones expressing more restricted TCR V beta genes (1, 3, 5.2, and 14 families) accumulated in vitro. These results suggest that autoantigen-reactive oligoclonal T cell accumulations are present in the peripheral blood from systemic autoimmune disease patients.
A region of the third variable loop of HIV-1 gp120 is recognized by HLA-B7-restricted CTLs from two acute seroconversion patients.
Authors: Safrit J T , Lee A Y , Andrews C A , Koup R A
Published: December 2022

HIV-1 envelope-specific CTL clones were isolated from the peripheral blood of two patients from within weeks of seroconversion. These clones were CD8+ and restricted by the HLA-B7 molecule. The minimum epitope recognized by the clones was determined to be the 30-amino acid (aa) sequence RPNNNTRKSI within the third variable (V3) loop of the envelope glycoprotein gp120. The aa sequence of this epitope is consistent with the motif found in naturally processed peptides eluted from HLA-B7 molecules. This region of the V3 loop is reasonably well conserved among clade B and some nonclade B isolates of HIV-1, especially at the anchor residues that determine binding to the HLA-B7 molecule. Using peptides based upon virus sequences present within each patient, we determined that autologous viruses were recognized by the clones, and we detected no escape variants from the initial clonal response during the acute phase of infection. Interestingly, a serine to arginine change at position 9 of the epitope abrogated clone recognition in one of the patients. This aa change is one factor that has been associated with a change from a nonsyncytium-inducing to a syncytium-inducing phenotype of HIV-1, raising the possibility that in HLA-B7-expressing patients, escape from this clonal CTL response and a change in viral phenotype may be linked. This study demonstrates that human CTL can be generated against sequences within the third variable loop of HIV-1 gp120. Because multiple vaccine strategies are based upon the V3 loop of HIV-1 gp120, this defined epitope can be exploited in determining the ability of certain vaccines to stimulate a CTL response in a select population of individuals.
Endothelial cell-induced resistance to cyclosporin A in human peripheral blood T cells requires contact-dependent interactions involving CD2 but not CD28.
Authors: Karmann K , Pober J S , Hughes C C
Published: December 2022

The ability of cultured human endothelial cells (EC) to activate resting T cells depends on signals provided by EC that augment T cell IL-2 synthesis. EC-mediated augmentation of IL-2 secretion by PHA-activated T cells is resistant to inhibition by cyclosporin A (CsA) or by ascomycin. Specifically, the 50% inhibitory dose of these drugs is increased over 100-fold in the presence of EC. Although rapamycin also inhibits IL-2 secretion, there is no effect of EC on the sensitivity of T cells to this drug. Resistance to CsA requires cell contact between the EC and T cells and develops 8 h after initiation of coculture. Experiments with blocking Abs implicate T cell CD2 and its endothelial cell ligands, both LFA-3 and CD59, but not T cell CD28 and its putative ligands, in EC-induced resistance to CsA. However, CD2 signals are not sufficient to induce resistance to CsA, and other EC signals remain to be identified. These observations suggest that interactions of graft EC with host T cells may contribute to CsA-resistant reactions in vivo.
IL-9 production of naive CD4+ T cells depends on IL-2, is synergistically enhanced by a combination of TGF-beta and IL-4, and is inhibited by IFN-gamma.
Authors: Schmitt E , Germann T , Goedert S , Hoehn P , Huels C , Koelsch S , Kühn R , Müller W , Palm N , Rüde E
Published: December 2022

Dense CD4+ T cells isolated from naive mice produce only trace amounts of IL-9 when stimulated by immobilized anti-CD3 in combination with anti-CD28 Abs. In this situation, IL-9 production is significantly stimulated by TGF-beta and further enhanced by the addition of IL-4, which, by itself, has only a minimal influence. IFN-gamma was found to inhibit the enhancing effect of IL-4. However, increasing amounts of IL-4 in the presence of a constant concentration of IFN-gamma could overcome the inhibitory activity of IFN-gamma. The application of CD4+ T cells isolated from IL-2 knockout mice unequivocally revealed that IL-2 is essential for the production of IL-9 by T cells. In addition, the use of T cells from IL-4 knockout mice elucidated that the basic (IL-2 + TGF-beta) mediated IL-9 production is independent of IL-4. Therefore, our results demonstrate that optimal IL-9 production of naive dense CD4+ T cells is positively regulated at different levels: 1) by IL-2, which is essential for IL-9 secretion; 2) followed by TGF-beta, which promotes a considerable increase in IL-9 production above the level induced by IL-2; and 3) finally, by IL-4, which requires the presence of IL-2 and TGF-beta to strongly enhance the production of IL-9. IFN-gamma inhibits the production of IL-9 mainly at the level of IL-4 by neutralizing the effect of this cytokine.
Cytokine regulation by platelet-activating factor in a human B cell line.
Authors: Smith C S , Parker L , Shearer W T
Published: December 2022

TNF-alpha is an inflammatory cytokine produced by B cells that also impacts B cell differentiation and Ig secretion. We have investigated the regulation of the TNF-alpha gene in a human B cell line in response to platelet-activating factor (PAF), a potent phospholipid often produced in inflammatory reactions. We report here that PAF increases the RNA levels for the TNF-alpha gene in the human B cell line Ramos. This is mediated by an increase in the transcription rate and a longer half-life of the TNF-alpha transcripts. Induction of the TNF-alpha gene is rapid in these cells and independent of new protein synthesis. Despite increasing RNA for the TNF-alpha gene, PAF increases TNF-alpha protein levels only in the presence of a costimulus, such as PMA. We hypothesize that PAF acts at the transcriptional level to increase TNF-alpha RNA, but a post-transcriptional block acts to regulate protein production. We also report that PAF enhances PMA-induced TNF-alpha production from human peripheral B cells. This increase is maximal at 0.1 microM PAF and is blocked by PAF receptor antagonists. The ability of PAF to modulate cytokine production from human B cells provides further evidence that PAF has a role in B cell biology and suggests it may be important in conditions involving B cell-derived cytokines.
Macrophage-inactivating IL-13 suppresses experimental autoimmune encephalomyelitis in rats.
Authors: Cash E , Minty A , Ferrara P , Caput D , Fradelizi D , Rott O
Published: December 2022

Experimental autoimmune encephalomyelitis (EAE) is initiated by myelin basic protein (MBP)-specific CD4+ T cells of the Th1 phenotype that subsequently trigger the invasion of monocytes/macrophages into the brain. In this study, we evaluated the potential of human recombinant (hr) IL-13 to exert a protective effect on the development of EAE in Lewis rats. hrIL-13 is found to be a potent in vitro modulator of various rat macrophage functions, including an inhibition of the production of the proinflammatory cytokines IL-1 beta and TNF, and a simultaneous enhancement of MHC class II and CD4 receptor expression. Furthermore, hrIL-13 displayed a slight, but highly reproducible, inhibitory effect on the in vitro proliferative responses of encephalitogenic MBP-specific T cells stimulated in the presence of thymic APCs. Upon in vivo application of hrIL-13-secreting vector cells into MBP-immunized animals, the cytokine was capable of markedly suppressing the development of EAE, as assessed by a reduction of the mean duration, severity, and incidence of disease. This suppression of disease coincided with an only minimal reduction of MBP-directed T cell autoreactivity and no alteration in MBP-specific autoantibody production. We infer from these results that a strictly Th1-initiated immune disease can be attenuated efficiently by the administration of a cytokine that primarily targets cells of the macrophage/monocyte lineage and seems to exert no undesirable general suppression on either T cell or B cell immunoreactivity in vivo.
A humanized, bispecific immunoadhesin-antibody that retargets CD3+ effectors to kill HIV-1-infected cells.
Authors: Chamow S M , Zhang D Z , Tan X Y , Mhatre S M , Marsters S A , Peers D H , Byrn R A , Ashkenazi A , Junghans R P
Published: December 2022

HIV infection depletes the immune system of the coordinating functions of CD4+ T cells and APCs, whereas the population of CD8+ CTLs remains largely intact: functional but undirected. We have developed a humanized bispecific immunoadhesin-antibody (BIA) that redirects these remaining T cells to kill HIV-infected cells. This BIA expresses effector cell retargeting via a targeting activity that exploits the natural affinity of CD4 for gp120, and a recruiting activity that employs an anti-CD3 moiety to engage CTLs. The resultant molecule is 97% human in origin. In functional tests, this BIA mediated killing of HIV-infected cells using either pure CTL preparations, or whole PBL fractions that additionally include Fc gamma receptor-bearing large granular lymphocyte effectors. In contrast, a human anti-gp120 Ab induced target lysis via Ab-dependent cellular cytotoxicity (ADCC) only with large granular lymphocyte-containing fractions and not with CTLs. ADCC with this Ab was blocked in human serum, whereas BIA-mediated effector cell retargeting lysis of HIV-infected cells by CTLs was preserved. The affinity of the BIA for HIV-gp120 on infected cells and for CD3 epsilon on CTLs was derived in a flow cytometric Scatchard procedure. Relative to the bivalent parent molecules, CD4/gp120 affinity on cells was unchanged in the BIA (Ka 7 x 10(7) M-1), whereas the anti-CD3 affinity was diminished 50-fold (Ka 2 x 10(6) M-1 vs 1 x 10(8) M-1). Physical association of CD3+ effectors and gp120-expressing targets was confirmed by fluorescence microscopy and was dependent upon the presence of BIA and expression of target gp120. The unimpaired cytocidal activity of the BIA in the presence of serum highlights a potentially important advantage of this type of construct over native Abs for HIV-directed therapy.
T cell repertoire in patients with B chronic lymphocytic leukemia. Evidence for multiple in vivo T cell clonal expansions.
Authors: Farace F , Orlanducci F , Dietrich P Y , Gaudin C , Angevin E , Courtier M H , Bayle C , Hercend T , Triebel F
Published: December 2022

To characterize circulating T cell subpopulations in B chronic lymphocytic leukemia patients, TCR V alpha and V beta gene-segment use was analyzed by PCR using a panel of V gene-segment subfamily-specific oligonucleotide primers (V alpha 1-29/V beta 1-24). Virtually all V alpha and V beta subfamily specificities were expressed in these patients (nine stage A and four stage C), and the mean values obtained for each specificity were similar to those of a group of 13 healthy donors. Nonetheless, individual analysis revealed that unique V alpha or V beta gene-segment transcripts were overrepresented in patients compared with the control group. Overrepresentation of some TCR V beta chains was also detected by cytofluorometric analysis using a panel of 18 anti-V beta-specific mAbs. To further characterize these T cell subpopulations, we sequenced five different V beta-C beta PCR products in two selected stage A patients and found highly predominant recurrent transcripts in each of the five V beta specificities (50% to 100% of the analyzed sequences with identical V(D)J regions). These results were confirmed on bulk cDNA (i.e., without cloning) and extended to other V beta specificities (up to nine clonal expansions of 24 V beta specificities in one patient) and two other patients using a PCR-based method that determines V(D)J junction size patterns. Finally, it was observed that a V beta 19+ T cell subpopulation was clonally expanded in one patient to up to 30% of circulating T cells. This V beta 19+ CD8+ T cell clone was shown to specifically recognize the autologous tumor cells in vitro, as determined in cytokine release assays. Together, these results support the view that multiple expansions of unique T cell clones may derive in vivo from B chronic lymphocytic leukemia tumor-associated Ag stimulation.
TCR expression of activated T cell clones in the lungs of patients with pulmonary sarcoidosis.
Authors: Forrester J M , Wang Y , Ricalton N , Fitzgerald J E , Loveless J , Newman L S , King T E , Kotzin B L
Published: December 2022

Sarcoidosis is a systemic granulomatous disease of unknown etiology in which CD4+ T cells seem to be critically involved. In the lungs of patients with pulmonary disease, CD4+ T cells accumulate in large numbers, and a subset of these cells is activated. By using both quantitative PCR and anti-V beta mAbs, we analyzed the TCR repertoire of total and activated bronchoalveolar lavage T cells, the latter subset being defined by the ability to proliferate in short-term culture supplemented with IL-2. Overall, there was little difference when TCR V beta expression of freshly isolated lung and peripheral blood cells was compared in individual patients. Some individuals did demonstrate a modest increase in a few V beta-expressing subsets. However, after 1 to 2 wk of in vitro growth in IL-2-supplemented media, bronchoalveolar lavage cells from most patients, but not from any healthy individuals, demonstrated a selective expansion of particular V beta-expressing subsets. Interestingly, different V beta-bearing subsets were expanded in different patients. Junctional region sequencing indicated that the proliferating T cells in culture were strikingly oligoclonal and were derived from T cell clones already selectively expanded in vivo. These results provide evidence for a disease process that involves recognition of local Ag(s) by specific subsets of CD4+ T cells. Analysis of the Ag specificity of these IL-2-expanded populations is likely to provide insight into the pathogenesis of this disease.
Humanized antibody directed to the IL-2 receptor beta-chain prolongs primate cardiac allograft survival.
Authors: Tinubu S A , Hakimi J , Kondas J A , Bailon P , Familletti P C , Spence C , Crittenden M D , Parenteau G L , Dirbas F M , Tsudo M
Published: December 2022

IL-2Rs are expressed by T cells activated in response to foreign histocompatibility Ags but not by normal cells. This difference in IL-2R expression is exploited by blockade of IL-2Rs to achieve immunosuppression. High affinity IL-2Rs involve three subunits, IL-2R alpha, IL-2R beta, and IL-2R gamma. Murine Mik beta 1, a mAb that blocks IL-2 binding to IL-2R beta, was developed as an immunosuppressive agent. There was modest prolongation of cynomolgus cardiac allograft survival in animals treated with murine Mik beta 1 (mean survival 11.8 +/- 1.6 days compared with 8.2 +/- 0.4 days in untreated animals; p = 0.06). However, murine Mik beta 1 is ineffective in recruiting primate effector cells and is neutralized by monkey Abs directed toward the infused Ab. To circumvent these limitations, a humanized form of Mik beta 1, which is a largely human IgG1k Ab, except that murine hypervariable regions are retained, was developed. In vivo plasma survival of humanized Mik beta 1 was threefold longer than simultaneously administered murine Mik beta 1 (terminal t1/2, 104 +/- 10 h vs 37 +/- 2 h). Furthermore, humanized Mik beta 1 manifests Ab-dependent cellular cytotoxicity, an activity that is absent with the parental murine Mik beta 1. Graft survival was significantly prolonged by humanized Mik beta 1 treatment with survivals of 22, 22, 24, 27, 44, and > 300 days (p vs control < 0.01; p vs murine Mik beta 1 < 0.01). Survival was not prolonged further (p > 0.3) by the addition of humanized anti-Tac, which blocks interaction of IL-2 with IL-2R alpha subunits. There was no toxicity attributable to the use of Mik beta 1 Abs. Thus, humanized Mik beta 1 prolonged cardiac allograft survival in primates without toxicity and may be effective as an adjunct to standard immunosuppressive therapy.
Modulation of immunogenicity and antigenicity of proteins by maleylation to target scavenger receptors on macrophages.
Authors: Abraham R , Singh N , Mukhopadhyay A , Basu S K , Bal V , Rath S
Published: December 2022

We have maleylated proteins to target macrophage-specific scavenger receptors and have used this system to study changes in the epitopes and immunogenicity of such proteins. We show that maleylation of diphtheria toxoid (DT) induces targeting to macrophage scavenger receptors and enhances its immunogenicity. DT does not evoke detectable serum Ab responses upon injection as soluble protein. However, maleylated DT (mDT) does generate a significant Ab response. Furthermore, immunization with soluble mDT leads to a better T cell proliferative response in vitro than immunization with DT can generate, thereby demonstrating that maleylation leads to enhanced T cell immunogenicity in vivo. We also find that maleylation disrupts the native B cell epitopes of DT and creates new epitopes, because antisera to DT and mDT do not cross-react. At least some of the new epitopes generated are maleylation specific, because antisera against various maleylated proteins do cross-react. In contrast, maleylation does not significantly modify the repertoire of T cell epitopes generated from DT, because T cells generated by either DT or mDT immunization are cross-reactive, and both DT and mDT can stimulate T cells that are specific for single synthetic DT peptide. Maleylated proteins are better presented in vitro than are their native counterparts, and this enhancement of presentation is blocked by unrelated maleylated proteins. These results suggest that Ags targeted to scavenger receptors on macrophages by maleylation are better presented to T cells and are immunogenic in vivo without adjuvant.
Characterization of fibroblasts with a unique defect in processing antigens with disulfide bonds.
Authors: Merkel B J , Mandel R , Ryser H J , McCoy K L
Published: December 2022

A chinese hamster ovary (CHO) fibroblast, transfected with murine MHC class II genes, inefficiently stimulated CD4+ Th cells specific for OVA, hen egg lysozyme (HEL), and pork insulin which contain disulfide bonds. However, the fibroblasts elicited a T cell response to lambda repressor, which lacks disulfide bonds, and efficiently presented synthetic peptides. A somatic cell hybrid WALC, generated by fusing the hamster fibroblast with a murine L cell fibroblast, very efficiently processed OVA and HEL, suggesting that impaired processing was genetically complemented and was a recessive trait. The hamster fibroblasts were capable of processing two distinct denatured forms of OVA and carboxymethylated HEL, either as effectively or more efficiently than the B lymphoma cell. The CHO cells also displayed diminished disulfide reduction of an endocytosed [125I]tyramine linked to poly-(D-lysine) through a disulfide spacer compared with that of the cell hybrid, providing direct evidence for defective reductive cleavage by the CHO cells. Diminished aspartic acid-mediated proteolysis of Ag could not account for the phenotype, because cell lysates and separated organelles from the fibroblast possessed higher acidic aspartyl proteolytic activity than lysates and organelles from a B lymphoma cell. Thus, CHO cells exhibit a defect in processing Ag with disulfide bonds which is consistent with the impaired intracellular reduction of the disulfide bonds in endocytosed macromolecules.
Prostaglandin E2 promotes B lymphocyte Ig isotype switching to IgE.
Authors: Roper R L , Brown D M , Phipps R P
Published: December 2022

The mechanism by which PG of the E series (PGE) promote murine B lymphocyte IgE production was investigated. We previously reported that PGE, and other agents that increase intracellular cAMP, synergize with IL-4 and LPS to induce IgE and IgG1 production while inhibiting IgM and IgG3 synthesis. These data suggested that PGE may promote IL-4-induced class switching, but the mechanism by which PGE increases IgE synthesis remained obscure. We report here that 1) PGE increases (up to 14-fold) the number of splenic B cells secreting IgE, even though PGE mildly inhibits proliferation. 2) PGE acts on sorted surface IgM positive B cells, consistent with PGE acting on uncommitted B cells to promote class switching to IgE. 3) PGE synergizes with IL-4 to induce germline epsilon transcripts, demonstrating that PGE acts at the level of transcription in cells that have not yet switched to IgE. 4) In the presence of PGE, rearranged mature V(D)J epsilon mRNA transcripts can be detected earlier and at higher levels than with IL-4 and LPS alone. Taken together, these data provide strong evidence that PGE synergizes with IL-4 and LPS to direct isotype switching to the epsilon heavy chain gene in purified B lymphocytes. PGE is a potentially important in vivo immunoregulator, particularly with regard to IgE production and the genesis of allergy. In support of this hypothesis, there are numerous clinical conditions (hyper-IgE, trauma, sepsis, Hodgkin's lymphoma, arthritis) in which overproduction of PGE is coincident with elevated IgE titers.