bluuebunny/crossref_metadata_2025 huggingface dataset:
Diphtheria toxin- and Pseudomonas A toxin-mediated apoptosis. ADP ribosylation of elongation factor-2 is required for DNA fragmentation and cell lysis and synergy with tumor necrosis factor-alpha.
Authors: Morimoto H , Bonavida B
Published: January 2023

We have reported that diphtheria toxin (DTX) mediates target cell lysis and intranucleosomal DNA fragmentation (apoptosis) and also synergizes with TNF-alpha. In this paper, we examined which step in the pathway of DTX-mediated inhibition of protein synthesis was important for induction of cytolytic activity and for synergy. Using a DTX-sensitive tumor cell line, we first examined the activity of the mutant CRM 197, which does not catalyze the ADP ribosylation of elongation factor-2 (EF-2). CRM 197 was not cytolytic for target cells and did not mediate intranucleosomal DNA fragmentation of viable cells. The failure of CRM 197 to mediate target cell lysis suggested that the catalytic activity of DTX is prerequisite for target cell lysis. This was corroborated by demonstrating that MeSAdo, which blocks the biosynthesis of diphthamide, inhibited DTX-mediated protein synthesis inhibition and also blocked target cell lysis. Furthermore, the addition of nicotinamide, which competes with NAD+ on the DTX action site of EF-2, also blocked DTX-mediated lysis. These findings suggest that ADP-ribosylation of EF-2 may be a necessary step in the pathway leading to target cell lysis. In contrast to the sensitive line, the SKOV-3 tumor cell line is sensitive to protein synthesis inhibition by DTX but is not susceptible to cytolysis and apoptosis by DTX. Thus, protein synthesis inhibition by DTX is not sufficient to mediate target cell lysis. The synergy in cytotoxicity obtained with the combination of DTX and TNF-alpha was examined in order to determine the pathway mediated by DTX in synergy. Like the direct lysis by DTX, synergy was significantly reduced by MeSAdo and by nicotinamide. Furthermore, synergy was not observed with combination of CRM 197 and TNF-alpha. These results demonstrate that, in synergy, DTX may utilize the same pathway required for its cytolytic activity. Pseudomonas aeruginosa exotoxin shared most the properties shown for DTX. Altogether, these findings demonstrate that DTX-mediated apoptosis is initiated at a step beyond the ADP ribosylation of EF-2.
Mapping the major human T helper epitopes of tetanus toxin. The emerging picture.
Authors: Reece J C , Geysen H M , Rodda S J
Published: January 2023

Progress on the mapping of Th epitopes of tetanus toxin (tt) has been slow due to reliance on studies of clones. In this paper, human Th cell epitopes of tt were mapped using proliferation tests on PBMC in response to synthetic peptides. PBMC from nine donors were tested over the entire set of tt homologous overlapping dodecapeptides. The 1304 peptides were initially tested as 66 pools, each containing an average of 20 peptides. PBMC from individual donors responded to as few as 1 and as many as 17 of the 66 peptide pools. The sequences responsible for proliferation were identified for the two most frequently recognized pools, and for another two pools within a major immunodominant region. Three new epitope sequences were mapped in detail and based on their recognition by most individuals are likely to be promiscuous. A cocktail of peptides including the newly identified Th cell epitopes was able to induce proliferation in PBMC from 24 of 31 tetanus toxoid (TT)-responsive donors. This cocktail is a chemically defined reagent that can be used to quantitate in vitro Ag-specific Th cells in PBMC from most subjects, and may thus be useful for serial measurements of specific immunity such as in subjects undergoing immunotherapy or immunosuppressive treatment.
Inducible nitric oxide synthase from a rat alveolar macrophage cell line is inhibited by nitric oxide.
Authors: Griscavage J M , Rogers N E , Sherman M P , Ignarro L J
Published: January 2023

The objective of this study was to determine whether inducible nitric oxide (NO) synthase from a rat alveolar macrophage cell line (NR8383) activated by LPS plus IFN-gamma could be regulated by NO, one of the two products of the enzymatic reaction. This study was based on previous observations in this laboratory that NO is a negative feedback modulator of constitutive NO synthase from rat cerebellum. NO synthase activity was determined by monitoring the formation of 3H-L-citrulline from 3H-L-arginine in the presence of added cofactors. NO synthase catalyzed the conversion of L-arginine to equimolar quantities of NO and L-citrulline. NO and S-nitrosothiols inhibited NO synthase activity and this effect was enhanced by superoxide dismutase and attenuated by oxyhemoglobin. Nitrite and nitrate, the oxidation products of NO, as well as L-citrulline, the amino acid end-product, produced no significant effects on NO synthase activity. The inhibitory effect of NO on NO synthase appeared to be partially reversible upon addition of oxyhemoglobin. The inhibitory effect of NO was mimicked by other heme ligands including carbon monoxide, cyanide, and manganese-protoporphyrin IX. These observations indicate that (1) enzyme-bound heme plays a mechanistic role in the catalytic conversion of L-arginine to NO plus L-citrulline; (2) NO may function as a negative feedback modulator of inducible NO synthase by interacting with enzyme-bound heme; and (3) negative feedback modulation by NO may represent a mechanism by which the potentially toxic L-arginine-NO pathway in activated alveolar macrophages is turned off.
Tyrosine phosphorylation of a gamma-chain homodimer associated with Fc gamma RIII (CD16) in cultured human monocytes.
Authors: Masuda M , Verhoeven A J , Roos D
Published: January 2023

The efficient expression of transmembrane-anchored Fc gamma RIIIa requires the presence of other peptides, such as the gamma-chain of the IgE receptor I or the zeta-chain of the TCR. We found that Fc gamma RIIIa in cultured human monocytes is specifically associated with the gamma-chain homodimer, and that the gamma-chains in this complex are phosphorylated on tyrosine residues. Anti-Fc gamma RIII immunoprecipitates, which were prepared from 1% digitonin lysates of cultured human monocytes, incorporated phosphate into a homodimer consisting of two 14-kDa polypeptides when incubated with [gamma-32P]ATP. Identity of this co-associated structure of Fc gamma RIIIa as the gamma-chain dimer was confirmed by elution of the protein from the anti-Fc gamma RIII immunoabsorbent with 1% Nonidet P-40 detergent and reimmunoprecipitation with anti-gamma-chain antibody. Phosphoamino acid analysis showed that the gamma-chain exclusively contained phosphotyrosine. The gamma-chain was also phosphorylated when electropermeabilized cells were activated by cross-linking Fc gamma RIIIa. The gamma-chain may play an important role in signal transduction via Fc gamma RIIIa in human macrophages.
Heterogeneity of human sperm surface antigens identified by indirect immunoprecipitation of antisperm antibody bound to biotinylated sperm.
Authors: D'Cruz O J , Haas G G , Lambert H
Published: January 2023

Target Ag recognized by clinically important C-fixing anti-sperm antibodies (ASA) were identified by indirect immunoprecipitation after incubation of biotinylated, motile sperm with ASA-positive sera from autoimmune, isoimmune, and vasectomized patients. This method assessed native protein thus permitting analysis of conformation-dependent membrane Ag that would be missed by previously reported Ag identification techniques. Sera from 35 ASA-positive and 10 ASA-negative patients were exposed to capacitated, surface-biotinylated motile sperm. After detergent-extraction of the membrane fraction, the immune complexes were precipitated with protein G-agarose. The Ag were eluted under reducing conditions, electrophoresed on SDS-10% PAGE, electroblotted onto nitrocellulose membranes, and visualized by the biotin/avidin-peroxidase detection method. By this method, only high titered ASA-positive sera demonstrated variable reactivity restricted to a total of eight to nine protein bands. Individual sera revealed a differential reactivity to a constant set of protein bands (63/61, 58/56, 29, and 21/19 kDa) and a preferential reactivity to either 45-, 43-, or 41-kDa protein Ag that were patient specific. None of these bands were precipitable by ASA-negative sera. ASA directed against the 63/61-kDa and 58/56-kDa polypeptides were found in 85% of immunoprecipitable sera; the 29-kDa was detectable with 70% of sera. Immunoreactivity to the 45-kDa to 41-kDa bands was detectable with 30% of the immunoprecipitable sera. Although the pattern of Ag recognized by ASA from autoimmune, isoimmune, and vasectomized patients was similar, our results indicate that in addition to the recognition of common human sperm surface determinants, distinct sperm surface epitopes are also recognized as foreign by the patient's immune system. Sera that immunoprecipitated the predominant monomeric 29-kDa or the dimeric 58/56-kDa Ag when preincubated with capacitated sperm markedly inhibited sperm binding and penetration to the human zona when compared with ASA-negative serum controls. It is suggested that C-fixing ASA directed to these surface determinants may account for some of the clinical manifestations of ASA-mediated infertility.
Selective expression of integrin alpha 4 beta 7 on a subset of human CD4+ memory T cells with Hallmarks of gut-trophism.
Authors: Schweighoffer T , Tanaka Y , Tidswell M , Erle D J , Horgan K J , Luce G E , Lazarovits A I , Buck D , Shaw S
Published: January 2023

Human memory CD4+ T lymphocytes are heterogenous in expression of integrins; one subset has the unexpected phenotype beta 1 low alpha 4 high. We demonstrate that this subset is unique among CD4+ cells in expression of high levels of alpha 4 beta 7, detected by a distinctive mAb Act-1. alpha 4 beta 7 is involved in binding to both fibronectin and vascular cell adhesion molecule-1; Act-1 blocks cell binding to the former and augments binding to the latter. Act-1 expression marks a subset of memory cells that, unlike the predominant circulating memory cell, has up-regulated beta 7 rather than beta 1. Their phenotype is distinct from that described for skin-homing T cells and is fully consistent with that described for gut-homing T cells. Differential adhesion capacity of this subset is verified by selective binding to FN and vascular cell adhesion molecule-1 in a beta 1-independent fashion. Thus, alpha 4 beta 7 detected on this subset of circulating normal T cells fits the expectations for a gut-homing receptor.
Identification of Caucasian CD4 T cell epitopes on the circumsporozoite protein of Plasmodium vivax. T cell memory.
Authors: Bilsborough J , Carlisle M , Good M F
Published: January 2023

We have identified a population of Caucasians with a defined past history of infection with Plasmodium vivax malaria. Using purified synthetic peptides overlapping the sequence of the circumsporozoite protein, we determined the percentage of individuals whose T cells proliferated or secreted IFN-gamma in response to peptide stimulation, for both this population and a population of nonmalaria-exposed control individuals. A number of peptides were recognized by both groups, but 11 peptides were uniquely recognized by the exposed population, and thus represented malaria-specific T cell epitopes. CD4 T cells were found to be responsible for the proliferative response. Humans last exposed to vivax sporozoites as long ago as 49 yr responded as well or better to these malaria-specific epitopes as individuals exposed within the previous month. Since such malaria-induced memory response may not be a feature of Plasmodium falciparum infections, and since P. falciparum does not have a persisting hypnozoite stage, our data argue that the persistence of T cell memory to vivax epitopes may result from antigenic persistence in the liver.
Characterization of a (+)-azidophenazocine-sensitive sigma receptor on splenic lymphocytes.
Authors: Garza H H , Mayo S , Bowen W D , DeCosta B R , Carr D J
Published: January 2023

A study was undertaken to structurally define and functionally assess sigma receptors on splenocytes using the highly selective sigma ligand (+)-azidophenazocine. Radioreceptor assays under reduced lighting show (+)-azidophenazocine can effectively block the binding of sigma ligands [3H]haloperidol (IC50 = 30 nM, Ki = 19.0 nM) and [3H](+)-pentazocine (IC50 = 40 nM, Ki = 350 nM), but not the dopamine (D2) ligand [3H]spiperone (IC50 > 5 microM) to splenic lymphocytes. [3H](+)-1-Propyl-3-(3-hydroxyphenyl)piperidine ([3H](+)-PPP) sites (Kd = 40.8 nM, Bmax = 2.32 pmol/mg) were also present on these lymphocytes. Additional studies using [3H](+)-azidophenazocine indicated the presence of saturable sites (Kd = 29.7 nM, Bmax = 760 fmol/mg) on splenic lymphocytes. There are no significant differences in affinity between sites found on T-enriched (Kd = 59 +/- 47 nM) and B-enriched lymphocytes (Kd = 23 +/- 5 nM). Photoaffinity labeling studies of splenocyte membranes with [3H](+)-azidophenazocine revealed a protein migrating at an apparent m.w. of 57 kDa under reducing and nonreducing conditions on SDS-PAGE. The labeling was specific because pretreatment with unlabeled haloperidol, (+)-PPP, 1,3 di(2-tolyl)guanidine, (+)-pentazocine, and (+)-azidophenazocine before cross-linking competed away > 75% of the radioactivity associated with the protein, whereas (-)-pentazocine and naloxone were significantly less effective. This data together with the observation that both (+)-azidophenazocine or haloperidol inhibit Con A-induced production of IFN by splenocytes, indicates that lymphocytes possess a biologically relevant sigma receptor.
Antioxidant treatment of thymic organ cultures decreases NF-kappa B and TCF1(alpha) transcription factor activities and inhibits alpha beta T cell development.
Authors: Ivanov V , Merkenschlager M , Ceredig R
Published: January 2023

Using electrophoretic mobility shift assays (EMSA), we have recently shown that nuclear extracts of 14-day mouse fetal thymocytes contain abundant NF-kappa B transcription factor activity. To determine the functional role of NF-kappa B in early thymocyte development, we have exposed fetal thymus organ cultures to inhibitors of NF-kappa B activation, namely the antioxidants N-acetyl-L-cysteine and butylated hydroxyanisole. Both compounds caused a dose-dependent arrest of thymocyte differentiation toward alpha beta, but not gamma delta, T cells. This was associated with a profound decrease in nuclear content of NF-kappa B and TCF1(alpha) transcription factor activity, as determined by EMSA. In contrast, NF-Y was affected less strongly, and cyclic AMP-response-element-binding protein levels remained essentially unchanged by antioxidants. To test the idea that alpha beta T cell development is correlated with NF-kappa B and TCF1(alpha) activity, we conducted additional experiments in a submersion culture system in which the generation of alpha beta T cells can be manipulated. Standard submersion culture supports gamma delta but alpha beta T cell development. Under these conditions, EMSA showed that transcription factor activities were similar to those seen in the presence of antioxidants. Importantly, when the generation of alpha beta T cells in submersion culture was restored by elevating oxygen concentrations, there was a dramatic increase in TCF1(alpha) activity, and both NF-kappa B and NF-Y returned to control levels. Taken together, these results strongly suggest that NF-kappa B and TCF1(alpha), presumably in concert with other transcription factors, play an important role in the development of alpha beta T cells.
Spontaneous and IL-2-induced anti-leukemic and anti-host effects against tumor- and host-specific alloantigens.
Authors: Cohen P , Vourka-Karussis U , Weiss L , Slavin S
Published: January 2023

The graft-vs-leukemia (GVL) effect and its connection to graft-vs-host disease (GVHD) were studied in F1 hybrid mice after parental bone marrow transplantation. (BALB/c x C57BL/6) F1 mice (F1) were sublethally and lethally irradiated and inoculated with 10(4) BCL1 cells, a murine leukemia of BALB/c (BALB) origin. Subsequently mice were transplanted with F1 (syngeneic), BALB (allogeneic to the host but syngeneic to the tumor), C57BL/6 (B6) (allogeneic to both host and tumor) spleen cells, or spleen cells mixed with syngeneic (F1) bone marrow cells, respectively. Fresh BALB (H-2d) and B6 (H-2b) spleen cells, which induce equally strong GVHD in the F1 recipient against H-2b and H-2d alloantigens, respectively, were assayed for their ability to induce GVL effects on BCL1 (H-2d) cells with and without in vivo administration of human rIL-2. In order to increase the GVL effects, spleen cells were incubated for 4 days with rIL-2 to generate IL-2-activated killer cells. The results of adoptive transfer experiments showed that only immunocompetent B6 cells led to consistent eradication of leukemia. Neither BALB nor syngeneic F1 cells induced curative GVL effects regardless of whether spleen cells were further stimulated with rIL-2 in vitro, in vivo, or both. Our data support the theory that GVL effects are caused by T cell-dependent immune responses of allogeneic T cells and not by MHC-nonrestricted NK or rIL-2-activated NK cells, independently of the GVHD process itself. Hence, recognition and killing of tumor cells is most probably a MHC-restricted T cell-dependent process.
Regulation of adhesion of CD4+ T lymphocytes to intact or heparinase-treated subendothelial extracellular matrix by diffusible or anchored RANTES and MIP-1 beta.
Authors: Gilat D , Hershkoviz R , Mekori Y A , Vlodavsky I , Lider O
Published: January 2023

Chemokines, a superfamily of 8- to 11-kDa mediators of inflammation, affect the attachment of immune cells to vascular endothelia by binding to cell surface glycosaminoglycans. We analyzed whether chemokines are also involved in interactions between CD4+ T lymphocytes and the subendothelial extracellular matrix (ECM). Soluble mediators, such as MIP-1 beta and RANTES, induced the binding of resting human CD4+ T cells to ECM in an integrin-dependent manner. Both MIP-1 beta and RANTES bound to intact ECM and retained their adhesive properties, and moreover, ECM-bound RANTES and MIP-1 beta prolonged the time course of interactions between the CD4+ T cells and the ECM. Because the adhesive effect of these chemokines was restricted by an inhibitor of GTP-binding proteins, the adhesive effect of ECM-bound RANTES and MIP-1 beta, which requires an intact cytoskeleton, seems to involve activation of a G protein-linked receptor. MIP-1 beta and RANTES exert their pro-adhesive effects through interactions with glycosaminoglycans, because heparinase-treated ECM did not bound chemokines and because the chemokines ability to induce T cell adhesion was abrogated if: 1) either of the chemokines is pretreaed with heparin or heparan-sulfate (HS), 2) HS is removed from intact ECM by heparinase, an HS-specific endoglycosidase, or 3) the ECM-bound chemokines are released by pretreatment with heparinase. Hence, the adhesive effects of immobilized chemokines is not restricted to T cells interacting with endothelial cells, but also affects the migration of immune cells which reside and function in the context of ECM.
Cross-linking of CD53 promotes activation of resting human B lymphocytes.
Authors: Rasmussen A M , Blomhoff H K , Stokke T , Horejsi V , Smeland E B
Published: January 2023

The CD53 pan-leukocyte glycoprotein is a member of the recently described tetraspan family of cell membrane proteins. The structure and functional characteristics of these molecules indicate that they may play important roles in transmembrane signaling in different cells. Recently, it was reported that cross-linking of CD53 on human B cells led to an increase in cytoplasmic calcium fluxes. In the present study, we wished to further explore the possible role of CD53 in functional B cell responses. Cross-linking of CD53 with the use of the mAb MEM-53 and a polyclonal sheep anti-mouse Ig promoted activation of resting B cells into the G1 phase of the cell cycle as judged by increased expression of the early activation Ag CD69, increases in cellular volume, RNA synthesis, and c-myc protein levels, and enhanced binding of 7-aminoactinomycin D. In contrast, MEM-53 alone had no detectable effects. Cross-linking of anti-CD53 induced negligible S phase entry in the absence of other stimuli. However, cytokines, in particular IL-2 and IL-4, potentiated the DNA synthesis induced by cross-linking of CD53. Furthermore, cross-linking of the CD53 Ag induced Ig production in the presence of T cell supernatant. Taken together, the data suggest that CD53 plays an important functional role in B cell activation and differentiation.
Differential transcriptional activities of HLA-DR genes in the various haplotypes.
Authors: Louis P , Vincent R , Cavadore P , Clot J , Eliaou J F
Published: January 2023

The regulation of HLA class II genes is of particular interest with regard to the modulation of the immune response. The polymorphism of their coding regions is directly involved in the specificity of the Ag presentation, and their level of expression affects the extent of T cell activation. Previously, we have described an allelic polymorphism in the proximal promoter regions of HLA-DRB genes. The aim of this study was to compare the transcriptional activities of the promoters of the DRB genes and DRB1 alleles in a transient expression system. We have demonstrated a marked difference in their promoter strengths, as determined by their relative abilities to initiate transcription of the chloramphenicol acetyltransferase reporter gene in human B cell lines. The polymorphism of the promoter regions has been mapped to the regulatory boxes, and, by using gel retardation experiments, we found a differential ability of the nuclear proteins to bind to the partially conserved X box regions. Taken together, our results demonstrate the functional consequences of the allelic polymorphism of the proximal promoter regions of the DRB genes. These findings strongly suggest the existence, for the HLA-DR genes, of an interdependence between the polymorphism of the coding regions, which directly affects the capacity of peptide binding, and the polymorphism of the regulatory regions, which influences the transcriptional activities of the promoters.
Two forms of the mb-1 gene transcript in cattle.
Authors: Youn H Y , Goitsuka R , Okuda M , Watari T , Tsujimoto H , Hasegawa A
Published: January 2023

We have isolated two forms of bovine mb-1 cDNA clones from a cDNA library of a bovine leukemia virus-infected B-lymphoblastoid cell line. The nucleotide sequence analysis of these cDNA clones indicated that the shorter form of cDNA had a 119-bp deletion that precisely corresponded to the exon III domain of the human and murine mb-1 genes. This deletion would result in a frame shift, producing a premature translation termination leading to the lack of the entire transmembrane and cytoplasmic domains of the normal mb-1 molecule. The existence of two species of mRNA transcripts corresponding to the isolated cDNA clones were confirmed in all of the bovine leukemia cell lines examined by PCR with the use of the primers flanking the deletion site. Furthermore, the shorter mb-1 transcript was induced in normal PBMC by stimulation with mitogens or culture supernatants of bovine leukemia virus-infected cell lines. These findings indicate the presence of the truncated mb-1 gene product which may have some function different from that of the normal mb-1 gene product.
Elevated expression of Th1 cytokines and nitric oxide synthase in the lungs of vaccinated mice after challenge infection with Schistosoma mansoni.
Authors: Wynn T A , Oswald I P , Eltoum I A , Caspar P , Lowenstein C J , Lewis F A , James S L , Sher A
Published: January 2023

C57BL/6 mice were vaccinated with irradiated cercariae of Schistosoma mansoni, and, at various times after challenge infection, total lung mRNA was isolated to assess the induction of several cytokines that previously had been shown in in vitro studies to be involved in the activation of macrophages and/or endothelial cells for nitric oxide (NO) production and killing of schistosomula. Vaccinated mice demonstrated a highly significant increase in IFN-gamma mRNA upon subsequent infection when compared with infected nonvaccinated controls. A similar, although less dramatic, increase in two other macrophage-activating cytokines, TNF-alpha and IL-2, also was observed. In contrast, although the Th2 cytokines IL-4, IL-5, IL-10, and IL-13 were elevated in challenged vaccinated animals, only IL-10 and IL-13 showed increases that were significant with respect to the mRNA levels observed in challenged controls. Neutralization of IFN-gamma reduced immunity in vaccinated animals and resulted in decreased IFN-gamma, IL-2, IL-10, TNF-alpha, and IL-12 p40 but markedly increased IL-4, IL-5, and IL-13 mRNA expression and serum IgE levels. Pulmonary NO synthase expression was elevated in immunized mice at a time at which immune elimination of schistosomula is believed to occur. Moreover, suppression of NO synthase activity with the inhibitor aminoguanidine reduced immunity, as measured by a 32 to 33% increase in worm burden. Together, these data support previous in vitro studies that suggest a role for NO in schistosomulum killing. Furthermore, the observation that the down-regulatory cytokines IL-4, IL-10, and IL-13 are induced together with IFN-gamma may provide an explanation for the failure of this vaccine to provide complete protection.
Tolerance to a self-peptide from the third hypervariable region of HLA DRB1*0401 in rheumatoid arthritis patients and normal subjects.
Authors: Salvat S , Auger I , Rochelle L , Begovich A , Geburher L , Sette A , Roudier J
Published: January 2023

The third hypervariable region (HV3) of HLA-DRB1*0401 helps the development of severe rheumatoid arthritis by an unknown mechanism. To test whether the third hypervariable region of HLA-DRB1*0401 may shape the T cell repertoire, we studied proliferative responses to peptides encompassing the third hypervariable region of seven HLA-DRB1 alleles in normal subjects and patients with rheumatoid arthritis. We found that, in general, there is no tolerance to peptides from the third hypervariable region of self-HLA-DRB1 alleles. However, a peptide from the third hypervariable region of DRB1*0401 (DRB1*0401 HV3 peptide) is tolerated in people who express HLA-DRB1*0401. Indeed, PBMCs from people who express DRB1*0401 do not proliferate to DRB1*0401 HV3 peptide. Conversely, people who express DRB1*1501 respond to DRB1*0401 HV3 peptide. Finally, people who express both DRB1*0401 (nonresponder haplotype) and DRB1*1501 (high responder haplotype) do not respond to DRB1 HV3 peptide, thus demonstrating tolerance. Therefore, the third hypervariable region of HLA-DRB1*0401 shapes the T cell repertoire.
Activation-induced cell death in proliferating T cells is associated with altered tyrosine phosphorylation of TCR/CD3 subunits.
Authors: Orchansky P L , Teh H S
Published: January 2023

The first signaling event that occurs after stimulation of the TCR is an increase in the tyrosine phosphorylation of a number of proteins including the TCR-zeta chain and CD3 subunits. It is known that proliferating T cells respond differently to TCR stimulation when compared with naive T cells. Stimulation of the TCR on proliferating T cells has been shown to result in cell death by apoptosis, a process referred to as activation-induced cell death (AICD). In this study we have determined the pattern of tyrosine phosphorylation of TCR-zeta and the CD3 subunits of naive and proliferating CD4- CD8+ T cells that express a transgenic TCR that is specific for the male Ag presented by Db class I molecules after TCR stimulation. AICD in proliferating T cells was mediated by stimulation with anti-TCR/CD3, but not anti-Thy-1, anti-CD8, or anti-Db mAbs. When compared with naive T cells, tyrosine phosphorylation of TCR-zeta was dramatically decreased in proliferating cells. Furthermore, whereas CD3 delta, CD3 epsilon, and CD3 gamma of naive T cells were phosphorylated in a 1:2:1 ratio after TCR stimulation, these subunits in proliferating T cells were tyrosine phosphorylated in a 1:1:0 ratio after TCR stimulation. Altered phosphorylation of these subunits was not caused by the lack of synthesis or amount of these proteins in proliferating cells. This is the first study implicating altered tyrosine phosphorylation of TCR/CD3 subunits in AICD.
Production of IgE antibody and allergic sensitization of intestinal and peripheral tissues after oral immunization with protein Ag and cholera toxin.
Authors: Snider D P , Marshall J S , Perdue M H , Liang H
Published: January 2023

Cholera toxin (CTX) is a potent oral adjuvant for the induction of mucosal IgA Ab responses protein Ags. We examined the Ab responses and allergic sensitization of several strains of mice to protein Ags, administered orally with CTX. The mice made strong IgA and IgG1 serum Ab responses, but little IgG2a Ab to Ags such as hen egg lysozyme (HEL) and OVA. However, when given a subsequent i.p. challenge with Ag alone, the same mice had immediate hypersensitivity reactions that included respiratory distress and death. Within 10 min of i.p. challenge, immunized mice had high levels of plasma histamine and extensive degranulation of mast cells in target tissues. These mice had detectable serum IgE Ab. Ag administered orally with the B subunit (CTB) of CTX did not sensitize mice. Intestinal tissues taken from these mice had Ag-specific ion-secretory responses in vitro, typical of intestinal anaphylaxis. Ag given s.c. without adjuvant could also sensitize for systemic and intestinal anaphylaxis. Sensitization with HEL given s.c. was dose dependent and correlated with a critical amount of HEL in the circulation. HEL was detected in the circulation after oral immunization, but CTX did not increase the uptake of HEL. Thus, oral immunization with a protein Ag in the presence of CTX can sensitize an animal for systemic and intestinal anaphylaxis. These results suggest a cautious approach to the use of CTX as an adjuvant in oral vaccines, and provide a new model to study immediate hypersensitivity reactions to intestinal Ag.
Secretory vesicles are the intracellular reservoir of complement receptor 1 in human neutrophils.
Authors: Sengeløv H , Kjeldsen L , Kroeze W , Berger M , Borregaard N
Published: January 2023

The subcellular localization of complement receptor 1 (CR1) was investigated in human neutrophils. CR1 was located exclusively in the light membrane fractions containing secretory vesicles and plasma membranes in Percoll density gradients of unperturbed neutrophils. Separation of plasma membranes from secretory vesicles by high-voltage free-flow electrophoresis of the light membranes from the Percoll gradient revealed that more than 80% of the CR1 was located intracellularly in secretory vesicles. After weak stimulation of neutrophils, the increase in CR1 surface expression closely paralleled both surface increase of known secretory vesicle membrane Ags and the release of matrix proteins from secretory vesicles. More potent stimulation of the neutrophils did not enhance CR1 surface expression further, in agreement with the lack of CR1 in granules as demonstrated on Percoll gradient. CR3 (Mac-1), which has been shown to be located both in secretory vesicles and in neutrophil granules, was up-regulated in parallel with CR1 after weak stimuli, whereas a profound increase of CR3 was observed after more potent stimuli in accordance with granule mobilization. These results identify secretory vesicles as the reservoir of CR1, which translocates to the plasma membrane after weak stimulation, and underscore the functional significance of this recently identified organelle.
Immune-mediated brain atrophy. CD8+ T cells contribute to tissue destruction during borna disease.
Authors: Bilzer T , Stitz L
Published: January 2023

Borna disease (BD) is a virus-induced immunopathologic disease of the central nervous system in a variety of species from birds to primates and probably in humans. Severe inflammatory reactions lead to tissue destruction and finally to cortical brain atrophy. After experimental infection of the rat, intraparenchymal CD8+ T cells, MHC class I Ags on Borna disease virus (BDV)-infected neurons, and numerous nerve cell lesions were present. Treatment of BDV-infected rats with the mAb OX-8 directed against CD8+ cells inhibited the immunopathologic reactions and reduced MHC class I Ag expression. Neuronal lesions were minimal and no loss of brain substance could be observed. Because BDV has no acute cytopathic effects, we provide evidence that the presence of CD8+ T cells within the brain parenchyma and the expression of MHC class I Ags on neurons play a major role for immunopathologic brain tissue destruction and virus-infected neurons in vivo can be destroyed by T cell-mediated cytotoxicity.
LPS-induced expression of the human IL-1 receptor antagonist gene is controlled by multiple interacting promoter elements.
Authors: Smith M F , Eidlen D , Arend W P , Gutierrez-Hartmann A
Published: January 2023

Expression of the IL-1 receptor antagonist (IL-1ra) can be induced by treatment of monocytes and macrophages with LPS. We have previously demonstrated that the most proximal 294 bp of the human IL-1ra promoter are sufficient for full basal activity and LPS responsiveness. In the present study, we demonstrate the presence of one inhibitory and three positive-acting LPS response elements (LRE) within this proximal 294-bp IL-1ra promoter fragment. By both 5'-deletional analysis and heterologous promoter studies, an element between -294 and -250 was found to mask the LPS response. By 5'-deletional analysis and heterologous promoter experiments, two positive-acting LRE were identified between -250 and -200 (LRE3) and -200 and -148 (LRE2) which exhibited cooperativity in that neither element alone was active. Furthermore, LRE2 also cooperated with a more proximal site between -148 and -31 (LRE1), which also was not active alone. LRE1 was identified as an NF-kappa B-binding site. Site-directed mutagenesis of this site, located between -93 and -84, resulted in a > 50% decrease in the LPS responsiveness of the 294-bp promoter. By electrophoretic mobility shift assays, with or without specific antisera to members of the rel/NF-kappa B family, the complex binding to LRE1 was shown to contain primarily NF-kappa B1/p50 and lesser amounts of RelA/p65. These results indicate that the net activation of the human IL-1ra promoter in response to LPS involves the functional interaction of at least four cis-acting DNA elements within the proximal 294 bp.
Cellular interaction in germinal centers. Roles of CD40 ligand and B7-2 in established germinal centers.
Authors: Han S , Hathcock K , Zheng B , Kepler T B , Hodes R , Kelsoe G
Published: January 2023

Costimulatory interactions between T and B lymphocytes are crucial for T cell activation and B cell proliferation and differentiation. We have compared the roles of CD40L and B7-2 in the initiation and maturation of humoral immunity by administering anti-CD40 ligand (L) or anti-B7-2 Ab during the early (days -1 to 3) or late (days 6-10) phases of primary responses to thymus-dependent (Td) and -independent (Ti) Ags. Germinal center (GC) formation in response to a Td Ag was inhibited completely by the early administration of anti-CD40L or anti-B7-2 Abs. Later in the response, established GCs remained sensitive to anti-CD40L but were resistant to treatment with anti-B7-2. However, Ig hypermutation was reduced dramatically in GCs of anti-B7-2-treated mice and humoral memory was impaired. Early administration of anti-CD40L reduced serum Ab levels to approximately 10% of controls, whereas early treatment with anti-B7-2 reduced Ab production by only 50%. Later treatments with either Ab had no effect on Ab production. Response to a type II Ti Ag was more resistant than Td responses to interruption of costimulatory interactions. Our findings suggest that the costimulatory roles of CD40:CD40L and B7-2:CD28/CTLA-4 differ in the GC; administration of anti-CD40L abrogates an established GC reaction, whereas Ab to B7-2 suppresses Ig hypermutation and entry into the B cell memory compartment. Once B cells have entered the differentiation pathway to Ab production, neither CD40L nor B7-2 is necessary for their continued differentiation and persistence.
A novel cell surface antigen, immature thymocyte antigen-1, is involved in the differentiation of murine thymocytes.
Authors: Kishi H , Su D M , Muraguchi A , Watanabe T
Published: January 2023

A mAb, 1-23, which recognizes a novel cell surface Ag on immature murine thymocytes (designated as IMT-1 for immature thymocyte antigen-1) was prepared. IMT-1 was found to be expressed on 40 to 50% of CD4-8- double negative (DN), 5 to 10% of CD4-8+, and 5 to 20% of CD4+8+ double positive (DP) thymocytes in adult mice, but not expressed on CD4+8- thymocytes or peripheral T lymphocytes. It was not expressed on either nonlymphoid cell lines or thymic stromal cells. In subsets of DN thymocytes, IMT-1 was detected on 40 to 50% of the heat-stable Ag+, 15% of CD44+25+, 70% of CD44-25+, and 70% of CD44-25- populations, whereas it was not detected on heat-stable Ag- and CD44+25- populations. IMT-1+ thymocytes expressed low levels of or no CD3 molecules. In fetal thymuses, IMT-1 was expressed on a minor population of thymocytes at day 14.5 and 15.5 of gestation. However, at day 16.5 of gestation, a majority of DN as well as DP thymocytes became IMT-1-positive. Addition of the 1-23 Ab to the fetal thymus organ culture relatively increased CD8+ SP thymocytes. These results show that IMT-1 is expressed during the late DN stage as well as the early DP stage of thymocyte maturation and suggest the possible involvement of IMT-1 in thymocyte differentiation.
Clustering of diverse replicated sequences in the MHC. Evidence for en bloc duplication.
Authors: Leelayuwat C , Pinelli M , Dawkins R L
Published: January 2023

The MHC contains clusters of polymorphic duplicated genes and gene sequences. It has been thought that these duplicated genes and sequences have arisen from single gene duplications. We compared the cloned region between TNF and HLA-B with the region in close proximity to HLA-A using sequence analysis and DNA hybridization. The results indicate that several sequences existing in the region centromeric of HLA-B are also present in close proximity to HLA-A. These include sequences belonging to the P5, BAT1, and PERB11 gene families as well as HLA class I gene sequences. Interestingly, when the two regions of approximately 200 kilobases are compared, the replicated sequences are organized similarly but in an inverted fashion suggesting the existence of an historical inverted en bloc duplication. Thus, we propose that the origin of these MHC gene clusters involves several mechanisms. In addition to single gene replication, a long-range duplication of a genomic block must have occurred. It is possible that a block at the telomeric end of the MHC represents a basic functional genomic unit conserved and duplicated en bloc.
Rapid development of murine AIDS is dependent of signals provided by CD54 and CD11a.
Authors: Makino M , Yoshimatsu K , Azuma M , Okada Y , Hitoshi Y , Yagita H , Takatsu K , Komuro K
Published: January 2023

Murine AIDS (MAIDS) is induced by infection with the replication-defective virus (BM5def) component in the LP-BM5 murine leukemia virus (MuLV) mixture. The disease is characterized by polyclonally activated CD4+ T cells and B cells. It is known that BM5def is expressed at highest levels in B lymphocytes and that B cells serve as viral antigen-presenting cells. Full and sustained activation of CD4+ T cells against a conventional Ag usually requires both TCR and costimulating signals. Among various molecules known to provide costimulatory function, the expression of CD54 (ICAM-1) and CD11a/CD18 (LFA-1) on MAIDS B cells was increased, whereas that of CD2, heat-stable Ag (CD24), CD80 (B7-1), and CD86 (B7-2) was unchanged from normal. C57BL/6 mice depleted of both CD54 and CD11a expression as a result of chronic administration of mAb had developed no MAIDS at 4 wk and 8 wk after LP-BM5 MuLV infection. In addition, the proliferative response of B cells to mitogen was well conserved, whereas MAIDS-associated increases in serum Ig levels were inhibited. Replication of BM5def was suppressed markedly in infected mice treated with the CD54 and CD11a mAbs. These results suggest that the CD54/CD11a signal transduction pathway is a critical determinant of MAIDS development, and the lack of an immune response against viral Ag is enough to suppress BM5def replication and to prevent MAIDS.
A role for Ras signaling in coreceptor regulation during differentiation of a double-positive thymocyte cell line.
Authors: Shao H , Rubin E M , Chen L Y , Kaye J
Published: January 2023

Evidence suggests that the Ras/mitogen-activated protein kinase signaling pathway is required for positive selection of thymocytes. We have asked whether Ras activation is also sufficient to mediate changes in gene expression that are associated with positive selection. To accomplish this, we expressed a constitutively active form of Ras in the immature CD4+ 8+ DPK thymocyte cell line. DPK cells that express active Ras have reduced levels of CD8alpha and CD8beta at the level of cell surface protein and mRNA. These data provide evidence of a direct link between Ras signaling pathways and coreceptor regulation during positive selection. They also suggest that a sustained or potent Ras signal may play a critical role in directing thymocytes into the CD4 lineage. DPK cells that express active Ras, however, were not fully differentiated, indicating that Ras signaling provides only a partial signal for double-positive thymocyte differentiation.
Cyclic-AMP modulates downstream events in CD40-mediated signal transduction, but inhibition of protein kinase A has no direct effect on CD40 signaling.
Authors: Goldstein M D , Cochrane A , Watts T H
Published: January 2023

The role of cAMP/protein kinase A (PKA) in CD40 signal transduction is controversial, with evidence both for and against its importance. In this study we have used a tetracycline-repressible expression system to reversibly express a dominant-negative form of the PKA regulatory subunit type I (PKA-R(G324D)) in a B lymphoma line, M12. Expression of PKA-R(G324D) in M12 lymphomas inhibits both cAMP-mediated growth inhibition and cAMP-mediated induction of B7-2. This inhibition is reversed by tetracycline treatment of the cells to turn off inhibitor expression. In contrast, the expression of the PKA-R(G324D) subunit has no effect on CD40-mediated growth inhibition in M12 cells, nor on CD40-mediated induction of B7-1, CD23, Fas, ICAM-1, or LFA-1. Thus, our data do not support a direct role for cAMP/PKA in CD40-mediated signal transduction. However, we do observe that cAMP can regulate CD40 signaling both positively and negatively. Cyclic-AMP synergizes with CD40-mediated B7-1 induction in M12 lymphomas, while inhibiting CD40-mediated CD23, Fas, and ICAM-1 induction.
Proteasome activity limits the assembly of MHC class I molecules after IFN-gamma stimulation.
Authors: Benham A M , Neefjes J J
Published: January 2023

For an effective CD8+ cytotoxic T cell response to occur during infection, MHC class I molecules must be loaded with antigenic peptides in the endoplasmic reticulum. The cytosolic factor responsible for peptide generation is believed to be the proteasome, with the TAP heterodimer mediating peptide transport into the endoplasmic reticulum. However, the rate-determining step(s) in this intracellular pathway of Ag presentation is currently unresolved. The availability of a specific and irreversible proteasome inhibitor called lactacystin has enabled us to determine the amount of proteasomes required for the peptide loading of MHC class I molecules in four cell types. In the absence of the IFN-gamma-inducible proteasome subunits LMP2 and LMP7, the trypsin-like (but not the chymotrypsin-like) activity of the proteasome is directly related to MHC class I peptide loading. However, IFN-gamma stimulation or assimilation of catalytic LMP2 and LMP7 subunits into proteasomes causes both chymotrypsin- and trypsin-like activities of the proteasome to become limiting for the loading of class I molecules. Our data suggest that upon full IFN-gamma stimulation, peptide supply by the proteasome is the limiting step in the assembly of MHC class I polypeptides. This mechanism may enable the cell to prevent competition between novel Ags and the pool of endogenous proteins for binding to MHC class I molecules.
A secreted/shed product of Helicobacter pylori activates transcription factor nuclear factor-kappa B.
Authors: Münzenmaier A , Lange C , Glocker E , Covacci A , Moran A , Bereswill S , Baeuerle P A , Kist M , Pahl H L
Published: January 2023

Helicobacter pylori is an etiologic agent in the development of chronic gastritis, duodenal ulceration, and gastric adenocarcinoma. Exposure of gastric epithelial cells to H. pylori induces secretion of the cytokine IL-8, which plays a pivotal role in the immunopathogenesis of H. pylori infections. Isolated Helicobacter strains differ in their virulence and in their ability to induce cytokine production. High degrees of virulence correlate with enhanced IL-8 production. However, the molecular mechanism of this variance in Helicobacter pathogenicity remains poorly understood. Here we show that H. pylori-mediated IL-8 secretion requires activation of the transcription factor nuclear factor-kappaB (NF-kappaB) in a gastric epithelial cell line. Several H. pylori strains which fail to induce IL-8 secretion do not activate NF-kappaB, while all IL-8-inducing strains activate the transcription factor. Moreover, the antioxidant curcumin, which inhibits NF-kappaB activation, also completely suppresses IL-8 induction by H. pylori. NF-kappaB activation is not mediated by LPSs, since purified H. pylori LPS had no effect on gastric epithelial cells. In contrast, both IL-8 secretion and NF-kappaB activation require a secreted H. pylori product, which is not secreted by strains mutated in picB/cagE, a recently identified putative transport protein.
Allergenic properties of ovomucoid in man.
Authors: Cooke S K , Sampson H A
Published: January 2023

Ovomucoid, the dominant allergen in hen's egg, is a highly glycosylated protein comprising 186 amino acids arranged in three tandem domains (Gal d 1.1, 1.2, and 1.3). The purpose of this study was to evaluate the allergenic properties of ovomucoid. The three ovomucoid domains were isolated and evaluated with sera from egg allergic patients to determine B cell domain specificity, B cell epitopes, and the relative importance of linear and conformational structures and carbohydrate chains to B cell epitopes. Peripheral blood T cells from egg allergic patients were used to evaluate T-dominant domains and reactivity to reduced and oxidized ovomucoid. There was significantly more IgE activity to the second ovomucoid domain (median percentage of ovomucoid-specific IgE: Gal d 1.2, 40%; Gal d 1.1, 23%; Gal d 1.3, 26%). Quantities of patient IgG Ab were comparable for all three domains. Five IgE and seven IgG binding regions were identified. IgE Ab binding to reduced ovomucoid and IgG binding to oxidized ovomucoid were significantly reduced compared with that to native ovomucoid (28 and 69%, respectively). Peripheral blood T cells of 21 of 33 patients reacted to Gal d 1.3, 18 of 33 reacted to Gal d 1.2, and 18 of 33 reacted to Gal d 1.1. T cell proliferation in vitro in response to reduced and oxidized ovomucoid were significantly greater than that in response to the native protein. These results indicate a dichotomy between T and B cell domain dominance, and the presence of both unique and common IgE and IgG epitopes. Furthermore, the results suggest that conformational B cell epitopes play a more significant role in ovomucoid allergenicity than previously appreciated, and that carbohydrate moieties have a minor effect on allergenicity.