bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of macrophage growth by advanced glycation end products of the Maillard reaction.
Authors: Yui S , Sasaki T , Araki N , Horiuchi S , Yamazaki M
Published: December 2022

We previously showed that negatively-charged phospholipids, acetylated low density lipoprotein (LDL) and oxidized LDL, which are known to be taken up by macrophages via the scavenger receptor for modified LDLs, were able to induce the growth of peripheral macrophages in vitro. The present study demonstrated the macrophage growth-stimulating activity of the ligands for another macrophage receptor for advanced glycation end products (AGE) of the Maillard reaction. Experiments with murine macrophages showed that significant cell growth was induced by AGE-modified proteins such as bovine serum albumin (AGE-BSA) and AGE-human hemoglobin. The capacity of AGE-BSA to induce macrophage growth was completely blocked when cells were cultured with the mAb against granulocyte/macrophage CSF (GM-CSF). Moreover, PCR analysis revealed that AGE-BSA induced the expression of mRNA of GM-CSF. These data suggest that the macrophage AGE receptor may play an active role not only in phagocytic elimination of AGE-proteins but also induction of the growth of macrophages.
Two neutralizing domains in the V3 region in the envelope glycoprotein gp125 of HIV type 2.
Authors: Björling E , Chiodi F , Utter G , Norrby E
Published: December 2022

The purposes of this study were to map the targets for neutralizing Abs in the HIV-2 glycoproteins with particular emphasis on the role of the V3 region. Sera obtained from guinea pig immunized with peptides representing five immunogenic regions of the envelope proteins were used in cross-neutralization experiments with nine HIV-2 isolates. Broad cross-neutralizing activity was elicited by immunization with two peptides representing the central and COOH-terminal portions of the HIV-2 V3 loop. Murine mAbs were established from animals immunized with two corresponding overlapping peptides. Six mAbs showed neutralizing activity against the homologous virus isolate SBL-6669. Peptide absorption experiments were performed to define the target regions for human neutralizing Abs in the HIV-2 envelope glycoproteins. A significant blocking of neutralizing activity of five HIV-2 Ab-positive sera was seen in the presence of two peptides corresponding to the V3 region. Two overlapping deletion sets of peptides, representing amino acids Ser311 and Arg337, were used to identify the role of individual HIV-2 V3 amino acids in the binding of polyclonal and mAbs. Two distinct antigenic sites were identified in this region, the first corresponding to a region including the conserved motif Phe-His-Ser (amino acid 315-317) and the second in proximity of the COOH-terminal cysteine Trp-Cys-Arg (amino acid 329-331). Potentially these two sites can interact to represent a single discontinuous antigenic site. Taken together, these results indicate that V3 is an important neutralizing domain of HIV-2.
Chronic treatment of C3H-lpr/lpr and C3H-gld/gld mice with anti-CD8 monoclonal antibody prevents the accumulation of double negative T cells but not autoantibody production.
Authors: Giese T , Davidson W F
Published: December 2022

Mice homozygous for lpr or gld develop autoimmunity and progressive lymphoproliferative disease characterized by the accumulation of an unusual population of functionally impaired B220+, TCR-alpha/beta +, CD4-, CD8- double negative (DN) T cells. Although these cells are thymus derived and appear to have undergone thymic negative selection, the identity of their immediate precursors and the mechanisms leading to their accumulation are poorly understood. Here we investigated the role of CD8+ T cells in the development of lymphoproliferative disease and autoantibody production. We showed that treatment of C3H-Ipr or C3H-gld mice with anti-CD8 mAb beginning at 3 wk of age and continuing to 15 wk of age caused a dramatic reduction in lymphadenopathy. the change in lymph node size resulted predominantly from a very significant decrease in both the proportions and the total numbers of B220+ DN T cells. The proportions of these cells were reduced up to 20-fold and the total numbers per LN up to 400-fold. Although chronic treatment with anti-CD8 mAb had the most profound effects on B220+ DN T cells, it also decreased the numbers of CD4+ T cells, CD4+B220+ T cells, and B cells in Ipr and gld LN up to fivefold. In contrast to its impact on lymphoproliferative disease, anti-CD8 mAb therapy had no significant effect on B cell hyperactivity. Comparisons of serum Ig and autoantibody levels in CD8+ T cell-depleted and control mAb-treated Ipr and gld mice revealed no changes in the elevated concentrations of serum IgM or total IgG and no significant reduction in the levels of circulating autoantibodies specific for thymocytes or dsDNA. The presence of active germinal centers and accumulations of plasma cells in the LN and spleen of anti-CD8 mAb-treated Ipr and gld mice provided further evidence for sustained B cell activation. These results suggest that in Ipr and gld mice, CD8+ T cells play a crucial role in the accumulation of B220+ DN T cells and also may contribute to the characteristic increase in the numbers of B cells and CD4+ T cells in these mice, but have no significant effect on B cell hyperactivity or autoantibody production.
Cytokine-enhanced NK cell-mediated cytotoxicity. Positive modulatory effects of IL-2 and IL-12 on stimulus-dependent granule exocytosis.
Authors: Bonnema J D , Rivlin K A , Ting A T , Schoon R A , Abraham R T , Leibson P J
Published: December 2022

NK cells are a subpopulation of lymphocytes that kill virally infected cells and tumor cells without previous sensitization. Although exposure to distinct cytokines, including IL-2 and IL-12, can enhance these cytotoxic responses, the mechanism of this lymphokine-augmented killing remains unclear. Inasmuch as the cytotoxic event is a multistep process, there are many potential targets for lymphokine regulation. We focused on whether selected lymphokines directly modulate the intracellular signaling pathways critical for NK cell secretory function. In our experimental model, homogeneous, cloned human CD16+/CD3- NK cells were pretreated with either IL-2 or IL-12 and then stimulated with direct pharmacologic activators of the secretory response (e.g., PMA and ionomycin for intact cells or GTP gamma S for streptolysin-O permeabilized cells). Previous exposure of the cells to IL-2 or IL-12 enhanced the stimulus-induced release of granule-derived proteins (hexosaminidase and serine proteases) in a cytokine concentration- and time-dependent fashion. Furthermore, the cytokines increased the efficacies without changing the potencies of the secretagogues used in these studies. These results suggest that IL-2 and IL-12 augment NK cell-mediated cytotoxicity by increasing the maximal level of granule exocytosis evoked by Ca2+ and/or G protein-dependent intracellular signaling pathways.
Effects of IL-12 on helper T cell-dependent immune responses in vivo.
Authors: McKnight A J , Zimmer G J , Fogelman I , Wolf S F , Abbas A K
Published: December 2022

The differentiation of naive T cells to the Th1 or Th2 subset of effector cells is regulated by the cytokines to which T cells are exposed at the time of antigenic stimulation. Mice immunized with a hapten-protein conjugate and treated systemically with IL-12 show a marked inhibition of IL-4-secreting cells and suppressed production of anti-hapten IgG1 Ab, as well as a marked enhancement of IFN-gamma-producing T cells. Thus, IL-12 is a potent inhibitor of Th2 differentiation and an inducer of Th1 development in vivo. The IFN-gamma-inducing effect of IL-12 is partially blocked by pretreatment of mice with anti-asialo GM1 (anti-NK cell) Ab, but anti-asialo GM1 does not abrogate any of the other systemic actions of IL-12. Furthermore, none of the effects of IL-12 is prevented by treating mice with neutralizing anti-IFN-gamma Abs. These results suggest that the effects of IL-12 may be mediated both via NK cell stimulation and by directly altering the differentiation patterns of T cells. Finally, a neutralizing anti-IL-12 Ab significantly inhibits Th1 responses to Ag, indicating that IL-12 may play an obligatory role in Ag-induced Th1 differentiation in vivo.
Properties of a novel gene isolated from a Hodgkin's disease cell line that is expressed early during lymphoid cell activation.
Authors: Bennett J S , Tredway T L , Dizikes G J , Nawrocki J F
Published: December 2022

We have isolated a novel 667-bp cDNA clone, designated epag, from a Hodgkin's-disease cell line-derived library that is expressed in association with T cell activation and which is not related to any known gene family. By using reverse transcription/PCR, we have demonstrated that epag mRNA is expressed as early as 1 h after stimulation of normal PBMCs with anti-CD3. The levels of mRNA peaked by 4 h, and no expression was detectable by 12 h postactivation or in resting cells incubated in culture without activation. Expression of epag was also detected in PMA- and PHA-stimulated, but not in nonstimulated Jurkat cells, and overall its expression in transformed cell lines of hemopoietic origin is highly restricted. Sequence analysis of multiple independent cDNA clones showed that epag expressed in the Hodgkin's-disease cell line L428 is identical to the gene expressed in normal activated PBMC. Epag expression was detected by reverse transcription/PCR in RNA preparations made from various normal nonlymphoid tissues. Computer analysis of the sequence identified an open reading frame encoding a putative protein of 13.2 kDa initiating at a CUG translational codon. In vitro translation and Western blot analysis with anti-peptide serum supported this analysis. We hypothesize that epag functions as an early signal that helps mediate the activation of T cells.
Expansion and contraction of rhesus macaque DRB regions by duplication and deletion.
Authors: Slierendregt B L , Otting N , van Besouw N , Jonker M , Bontrop R E
Published: December 2022

Previous sequence analysis of the rhesus macaque MHC (MhcMamu) class II DRB region has allowed the detection of at least 34 alleles belonging to different lineages. In this communication, 36 new Mamu-DRB alleles are reported. The gene content of the DRB region has been determined for several homozygous animals of consanguineous origin. As in other primates, the number of DRB genes present per haplotype is not constant, varying from two to six genes in rhesus macaques. Six major groups of DRB haplotypes have been defined in our rhesus macaque colony. Two haplotype groups were found to carry, as well as other Mamu-DRB genes, two genes that cluster into distinct HLA-DRB1 lineages. In one of these two groups, a haplotype harbors another two sets of DRB alleles that belong to the Mhc-DRB6 and -DRB*W6 lineages, respectively. Such a haplotype was probably generated by duplication, and our data suggest that after this particular expansion of the DR region, one of the duplicated Mamu-DRB6 alleles was the target of an Alu insertion. Although certain transspecies allelic lineages are evolutionarily stable, and have been conserved for at least 36 million years, the rhesus macaque class II haplotypes differ significantly from those found in humans, chimpanzees, and gorillas. Mhc-DRB regions are therefore comparatively unstable over longer evolutionary time spans, with regard to both the number of genes and the gene content, and must have been subjected to expansion and contraction.
A 100-kDa protein in the C4-activating component of Ra-reactive factor is a new serine protease having module organization similar to C1r and C1s.
Authors: Takayama Y , Takada F , Takahashi A , Kawakami M
Published: December 2022

Ra-reactive factor (RaRF), a C-dependent bactericidal factor in mice, is composed of one polysaccharide-binding component and one C4/C2-activating component. The former is an oligomer of 28-kDa protein corresponding to the mannose-binding protein of mice. The 100-kDa protein, P100, has been shown to be present in the C4/C2-activating component. This protein generates 29- and 70-kDa polypeptide chains when reduced. In this study, we determined the nucleotide sequence of cDNA coding for P100. cDNAs were prepared by reverse transcription PCR and cassette-ligation-mediated PCR on mRNA from BALB/c mouse liver, using primers synthesized by reference to the sequence determined in a previous study. The results of cDNA sequencing indicate that the precursor protein of P100 containing a 24-residue signal peptide consists of 704 amino acid residues. Taking the results of the previous electrophoretic study into consideration, it is thought that the cleavage of mature P100 protein generates a 29-kDa chain of 251 residues and a 70-kDa chain of 429 residues. Although homology in the amino acid sequence of P100 with that of human C1r and C1s subcomponents of C was less than 40%, a striking similarity in domain organization was found among these proteins, indicating that P100 is a new C4-activating serine protease structurally similar to C1r and C1s. Northern hybridization showed that the liver was the primary site of the expression of the P100 gene.
Sensitivity of human leukemia cells in exponential or stationary growth phase to anti-CD5 immunotoxins. Role of intracellular processing events.
Authors: Chignola R , Anselmi C , Franceschi A , Pasti M , Candiani C , Tridente G , Colombatti M
Published: December 2022

We have assayed the sensitivity of Jurkat cells in different growth phases to an anti-CD5-ricin A chain (ST.1-RTA) immunotoxins (IT). Jurkat cells proliferated exponentially until a stationary growth phase was reached. Proliferating and stationary cells displayed marked differences in sensitivity to ST.1-RTA treatment; the time required to kill one log of target cells (T10) was 70 h in proliferating and 12 h in stationary cells, respectively. Differences in sensitivity to IT treatment were greatly diminished by the addition of the IT enhancer monensin (T10 = 4.9 and 3.5 h in proliferating and stationary cells, respectively). Binding and internalization studies carried out with fluoresceinated ST.1 mAb revealed that the higher sensitivity of stationary cells to ST.1-RTA treatment was not due to an increased uptake or to faster internalization kinetics of IT molecules in this cell population; rather, our data indicated that a different intracellular routing of IT molecules took place in the two cell populations. Mathematical modeling of experimental data allowed us to calculate the efficiency of the intracellular transport of IT molecules toward a subcellular compartment facilitating toxin translocation to the cell cytosol. The IT intracellular processing in stationary cells was 5.5-fold more efficient than in proliferating cells. This value strictly correlated with the higher sensitivity of the stationary cell population to ST.1-RTA treatment.
Regulation of expression of the IL-1 receptor antagonist (IL-1ra) gene by products of the human cytomegalovirus immediate early genes.
Authors: Kline J N , Geist L J , Monick M M , Stinski M F , Hunninghake G W
Published: December 2022

Human cytomegalovirus (HCMV), which is a major cause of morbidity and mortality in immunosuppressed patients, can itself alter immune function. It has previously been shown that HCMV immediate early (IE) gene products regulate the IL-1 beta promoter. The purpose of these studies was to determine whether HCMV IE gene products regulate expression of the IL-1 receptor antagonist (IL-1ra) gene. THP-1 cells, a myelomonocytic cell line, were transfected with a plasmid containing one or more of the HCMV IE genes downstream of the HCMV major immediate early promoter, or with a control plasmid. IL-1 beta and IL-1ra protein secretion was evaluated by ELISA, and expression of the mRNA for the cytokines was examined by means of Northern blot analysis. The HCMV IE1+2 gene products were found to increase expression of the mRNA for both IL-1 beta and IL-1ra; however, only the IL-1ra protein was released in increased amounts. The individual HCMV IE gene products had different effects on expression of the IL-1ra gene; the HCMV IE1 gene product down-regulated expression of the IL-1ra gene, whereas the IE2 gene product up-regulated expression of the IL-1ra gene. Thus, HCMV IE gene products can either up-regulate or down-regulate expression of the IL-1ra gene, depending on which IE genes are expressed in monocytes-macrophages. This study adds to the understanding of how HCMV can alter immune function during both active and latent infection.
The role of T cell activation in anti-CD3 x antitumor bispecific antibody therapy.
Authors: Weiner G J , Kostelny S A , Hillstrom J R , Cole M S , Link B K , Wang S L , Tso J Y
Published: December 2022

Anti-CD3 x antitumor bispecific Ab can retarget T cell mediated lysis in an MHC-independent fashion and prevent tumor growth in animal models. Two bispecific Ab preparations that differ in the presence or absence of Fc were compared in the 38C13 immunocompetent murine lymphoma model to evaluate how functional Fc and T cell activation impact on response to bispecific Ab therapy. Bispecific (bs) IgG contained functional Fc and was purified from hybrid-hybridoma Ab product. Bsf(ab')2 lacked functional Fc, and was genetically constructed using the leucine zipper technique. In vitro, bsF(ab')2 induced tumor cell lysis by activated T cells more effectively than bsIgG. However, bsF(ab')2 failed to induce T cell activation in the absence of tumor cells, and did so more slowly than bsIgG when tumor cells were present. In vivo, bsIgG induced nonspecific T cell activation whereas bsF(ab')2 did not. In therapy experiments, bsIgG inhibited tumor growth in mice although a single dose of bsF(ab')2 had minimal antitumor effect. BsF(ab')2 was capable of preventing tumor growth and improving survival when mice were also treated with T cell activators (IL-2 or staphylococcal enterotoxin B), or given repeated bsF(ab')2 doses. We conclude that therapeutic response to bispecific Ab was not dependent on functional Fc, but did require T cell activation. The use of bifunctional constructs that lack functional Fc therefore allows for separate manipulation of T cell retargeting and T cell activation and deserves further evaluation as a potential immunotherapy for malignancy.
Platelet-activating factor antagonist TCV-309 attenuates the induction of the cytokine network in experimental endotoxemia in chimpanzees.
Authors: Kuipers B , van der Poll T , Levi M , van Deventer S J , ten Cate H , Imai Y , Hack C E , ten Cate J W
Published: December 2022

Platelet-activating factor (PAF) has been postulated to play a role in the pathogenesis of sepsis. Additionally, in vitro studies have revealed tight interactions between PAF and the cytokine network, and PAF is considered to be an important stimulator of neutrophil functions. To assess the intermediate role of PAF in the induction of cytokines and neutrophil degranulation in endotoxemia in vivo, 12 healthy adult chimpanzees were i.v. injected with a bolus dose of Escherichia coli endotoxin (4 ng/kg); four animals received endotoxin alone, whereas the other chimpanzees were infused with the specific and potent PAF antagonist TCV-309 (bolus of 100 micrograms/kg, followed by either 100 micrograms/kg/h (n = 4) or 500 micrograms/kg/h (n = 4) for 5 h). At both doses TCV-309 significantly inhibited the endotoxin-induced rise in cytokine levels. Peak TNF concentrations after injection of endotoxin alone were 366 +/- 96 pg/ml, vs 105 +/- 47 and 115 +/- 56 pg/ml after administration of endotoxin together with the lower or higher dose of TCV-309, respectively (p < 0.05). TCV-309 also reduced the appearance of soluble TNFRs. Maximal levels of the type I soluble TNFR were diminished from 2.53 +/- 0.27 ng/ml (endotoxin alone) to 1.69 +/- 0.36 ng/ml (high dose TCV-309; p < 0.05); peak values of the type II soluble TNFR were diminished from 8.62 +/- 1.19 ng/ml to 5.76 +/- 0.92 ng/ml (p < 0.05). Furthermore, TCV-309 attenuated the endotoxin-induced release of IL-6 (160 +/- 82 pg/ml after endotoxin alone, vs 63 +/- 30 pg/ml in the low dose TCV-309 group (p < 0.05) and 65 +/- 29 pg/ml in the high dose group (p = 0.07) as well as that of IL-8 (279 +/- 168, vs 71 +/- 15 and 46 +/- 17 pg/ml, respectively; both p < 0.05). TCV-309 tended to reduce the endotoxin-provoked rise in serum IL-1R antagonist levels. In contrast, TCV-309 did not affect the neutrophilic leukocytosis elicited by endotoxin, nor did it inhibit endotoxin-induced neutrophil degranulation, as monitored by the plasma levels of elastase-alpha 1-antitrypsin complexes. We conclude that PAF plays a role, either directly or indirectly, in the stimulation of the cytokine network and in the shedding of soluble TNFR in endotoxemia. PAF does not seem to be an important intermediate factor in endotoxin-induced neutrophilia or neutrophil degranulation.
Simian immunodeficiency virus as a model for vaccination against HIV. Induction in rhesus macaques of GAG- or NEF-specific cytotoxic T lymphocytes by lipopeptides.
Authors: Bourgault I , Chirat F , Tartar A , Lévy J P , Guillet J G , Venet A
Published: December 2022

The protection against infection by HIV probably requires the induction of both neutralizing Abs and CTL responses. Vaccination by attenuated HIV is hardly acceptable and the use of viral genes inserted in recombinant living vectors needs further development, especially with respect to safety. The peptidic vaccination is a promising approach but free peptides are usually poorly immunogenic. Because potent immune responses have been obtained in mice with modified peptides such as lipopeptides, we have designed a study to assess the immunogenicity of lipopeptides in nonhuman primates. Seven lipopeptides were synthesized, derived from known immunogenic regions of the simian immunodeficiency virus (SIV) NEF and GAG proteins. Twelve rhesus macaques, randomly chosen and not selected on their MHC basis, were immunized subcutaneously with the seven lipopeptides in IFA. An MHC class I-restricted and CD(8+)-mediated CTL response has been observed in seven macaques directed against one or two of the synthetic immunizing peptides in each case. These CTLs were able to lyse autologous target cells infected with a recombinant vaccinia virus expressing the SIV nef or gag genes, suggesting that they recognized the naturally processed peptides. These activities are detectable in peripheral blood cells for at least 10 mo after the last immunization. Abs against the immunizing peptides have also been observed in all cases. This study demonstrates that lipopeptides can generate cytotoxic and humoral immune responses in a large number of unselected animals and this approach may thus be worth considering in the vaccination against HIV.
Purification and characterization of Yersinia enterocolitica envelope proteins which induce antibodies that react with human thyrotropin receptor.
Authors: Luo G , Seetharamaiah G S , Niesel D W , Zhang H , Peterson J W , Prabhakar B S , Klimpel G R
Published: December 2022

Graves' disease is an autoimmune disease mediated by autoantibodies to the thyrotropin receptor (TSHR). Several studies have suggested that the development of Graves' disease may be linked to infection with the enteric pathogen Yersinia enterocolitica. Using the purified recombinant extracellular domain of human TSHR (ETSHR), we have recently shown that immunization of mice with Y. enterocolitica results in the production of antibodies capable of reacting with the ETSHR. In this study, we identify two low molecular weight (5.5 kDa and 8 kDa) envelope proteins of Yersinia containing epitopes that are crossreactive with the TSHR. Identification of these crossreactive envelope proteins was achieved by Western blotting using affinity-purified anti-Y. enterocolitica antibodies that specifically react with the TSHR and, conversely, for envelope proteins of Yersinia. Confirmation that these Yersinia proteins contained crossreactive epitopes with the ETSHR was obtained by immunizing mice with partially purified envelope proteins, which resulted in the production of Abs that recognized the ETSHR. Further, some of the cross-reactive envelope proteins were purified with SDS-PAGE and HPLC. The crossreactive envelope proteins were shown to be chromosomally encoded, exposed on the surface of bacteria, and produced by virulent as well as avirulent strains of Yersinia (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica VW+, and Y. enterocolitica VW-). These results identify for the first time the Yersinia envelope proteins that are crossreactive with the ETSHR. Availability of these proteins will allow future studies to determine whether patients with Graves' disease have a unique immune response against these proteins when compared with healthy individuals.
Thyroglobulin-induced murine thyroiditis assessed by intrathyroidal T cell receptor sequencing.
Authors: Matsuoka N , Unger P , Ben-Nun A , Graves P , Davies T F
Published: December 2022

Thyroid glands from CBA/J (H-2k) mice, with human thyroglobulin (Tg)-induced thyroiditis, were removed and total cellular RNA was prepared and reverse transcribed as target cDNA for PCRs to detect 17 murine T cell receptor (mTCR) V beta families. Southern analysis of the resulting PCR fragments showed a marked bias in the intrathyroidal expression of mTCR V beta gene families with only 10 detectable V beta gene families of the 17 tested. Four Tg-specific CBA/J T cell lines also showed a biased use of mTCR V beta gene families with only 7 V beta gene families detected of the 17 tested. Sequence analysis showed evidence of oligoclonal expansion of the intrathyroidal T cell populations, and some of the CDR3 sequences found in the thyroid infiltrates were identical, or similar, to examples of the patterns of CDR3 sequences seen in the Tg-specific lines. Overall, however, there was marked N-D-N sequence variation in both the lines and thyroid infiltrates. These data indicated that 1) mTcR V gene analysis of intact thyroid tissue reflected the thyroid Ag-specific T cells infiltrating the thyroid gland; 2) confirmed the phenomenon of oligoclonal expansion in early thyroiditis; 3) showed that certain V beta gene families may be overemployed in the T cell recognition of Tg in the context of H-2k; and 4) revealed, by the varied patterns of the CDR3 sequences, that the V gene itself may be more important in thyroid Ag recognition than N-D-N region motifs.
Predominant TCR-alpha beta variable and joining gene expression by muscle-infiltrating lymphocytes in the idiopathic inflammatory myopathies.
Authors: O'Hanlon T P , Dalakas M C , Plotz P H , Miller F W
Published: December 2022

The idiopathic inflammatory myopathies (IIM) are a heterogeneous group of diseases in which autoreactive T cells are thought to play a pathogenetic role. We have determined the pattern of TCR-alpha beta gene expression by muscle-infiltrating lymphocytes within clinically and serologically defined groups of IIM patients. We utilized the PCR to study TCR V gene expression in muscle biopsies from nine polymyositis (PM) and eight dermatomyositis (DM) patients, all of whom had autoantibodies directed against histidyl-transfer RNA synthetase (anti-Jo-1 autoantibodies). While the TCR repertoire in DM patients was generally polyclonal, an oligoclonal profile characterized PM patients. Certain V gene families were predominantly expressed; V alpha 1 and V beta 6 gene families were detected in 82 and 91% of PM biopsies, respectively. TCR expression was characterized further by analyzing J gene usage from four PM patients expressing the V beta 6 gene. Sequence analysis of 40 independent recombinants (10 per patient) identified only seven V beta 6 clonotypes and restricted usage of the related J beta 2.1, -2.3, and -2.7 genes. These data, describing predominant TCR V and J gene usage by muscle-infiltrating lymphocytes in myositis patients, suggest that Ag-driven T cell responses may play a primary role in mediating some forms of the IIM.
Nucleotide sequence analysis of natural and combinatorial anti-PDC-E2 antibodies in patients with primary biliary cirrhosis. Recapitulating immune selection with molecular biology.
Authors: Pascual V , Cha S , Gershwin M E , Capra J D , Leung P S
Published: December 2022

We have analyzed at the nucleotide level the variable region gene sequences of five human mAbs and five recombinant Fab fragments derived from the mesenteric lymph nodes of patients with primary biliary cirrhosis. Both mAbs and Fabs were monospecific for dihydrolipoamide acetyltransferase, the E2 subunit of the pyruvate dehydrogenase complex, which has been shown to be the major autoantigen of primary biliary cirrhosis. We found that although the mAbs, mainly of the IgM isotype, were encoded by a diverse array of VH and VL gene segments either as direct copies of germline genes or somatically mutated, the recombinant IgG Fabs expressed clonally related heavy chains displaying a high number of somatic mutations that very likely occurred in the context of Ag selection. Combinatorial pairing of clonally related heavy chains with highly homologous light chains suggests that the IgG anti-pyruvate dehydrogenase complex repertoire of primary biliary cirrhosis patients is the result of the clonal expansion of a restricted set of B cells.
Ionizing radiation and autoimmunity. Induction of autoimmune disease in mice by high dose fractionated total lymphoid irradiation and its prevention by inoculating normal T cells.
Authors: Sakaguchi N , Miyai K , Sakaguchi S
Published: December 2022

Ionizing radiation can functionally alter the immune system and break self-tolerance. High dose (42.5 Gy), fractionated (2.5 Gy 17 times) total lymphoid irradiation (TLI) on mice caused various organ-specific autoimmune diseases, such as gastritis, thyroiditis, and orchitis, depending on the radiation dosages, the extent of lymphoid irradiation, and the genetic background of the mouse strains. Radiation-induced tissue damage is not the primary cause of the autoimmune disease because irradiation of the target organs alone failed to elicit the autoimmunity and shielding of the organs from irradiation was unable to prevent it. In contrast, irradiation of both the thymus and the peripheral lymphoid organs/tissues was required for efficient induction of autoimmune disease by TLI. TLI eliminated the majority of mature thymocytes and the peripheral T cells for 1 mo, and inoculation of spleen cell, thymocyte, or bone marrow cell suspensions (prepared from syngeneic nonirradiated mice) within 2 wk after TLI effectively prevented the autoimmune development. Depletion of T cells from the inocula abrogated the preventive activity. CD4+ T cells mediated the autoimmune prevention but CD8+ T cells did not. CD4+ T cells also appeared to mediate the TLI-induced autoimmune disease because CD4+ T cells from disease-bearing TLI mice adoptively transferred the autoimmune disease to syngeneic naive mice. Taken together, these results indicate that high dose, fractionated ionizing radiation on the lymphoid organs/tissues can cause autoimmune disease by affecting the T cell immune system, rather than the target self-Ags, presumably by altering T cell-dependent control of self-reactive T cells.
T cell receptor V beta 15 dominates the antiacetylcholine receptor response in Lewis rat T cell lines.
Authors: Smith L R , Kono D H , Asthana D , Balderas R S , Fujii Y , Lindstrom J , Theofilopoulos A N
Published: December 2022

Ten Lewis rat T cell lines responsive to Torpedo californica acetylcholine receptor (AChR) were assayed for TCR V beta usage. All lines were CD4+, OX-22-, and exhibited reactivity to one or more AChR chains. Several different V beta s were expressed by these lines, but V beta 15 was dominant in 5 of 10 lines. Unique CDR3 sequences were observed among the 10 lines, although three of the V beta 15 rearrangements used J beta 1.4. These data suggest that V beta 15+ T cells are selected in the in vitro response to the antigenically complex AChR in the Lewis rat.
High resolution epitope mapping of an anti-DNA autoantibody using model DNA ligands.
Authors: Swanson P C , Cooper B C , Glick G D
Published: December 2022

Although Ab-DNA complexes are involved in the pathogenesis of SLE, the mechanism by which anti-DNA recognize their DNA targets remains elusive. We have designed and synthesized small test DNA ligands that model ssDNA and dsDNA determinants commonly recognized by anti-DNA autoantibodies. These ligands were used in DNA footprinting assays to conduct a high resolution epitope-mapping study of anti-ssDNA BV04-01, which is typical of anti-ssDNA expressed in (NZB x NZW)F1 mice. We find that BV04-01 recognizes the ssDNA, but not dsDNA, epitopes presented on the test ligands, and makes specific base and backbone contacts with the DNA. Moreover, BV04-01 binding induces conformational changes in both the single-stranded and duplex region of the ligands. The epitope-mapping data is used in conjunction with computational molecular modeling to construct a three-dimensional model of a BV04-01-ligand complex. The salient aspects of this model are consistent with many features observed in the crystal structure of BV04-01 liganded to d(pT)3. This work extends previous anti-DNA binding studies and attempts to provide a unified approach that draws upon the merits of many precedented experimental methods to assemble three-dimensional structures of anti-DNA-DNA complexes. Analysis of these structures may ultimately help guide the development of molecules designed to inhibit anti-DNA binding in SLE.
Expression of functional trk tyrosine kinase receptors after T cell activation.
Authors: Ehrhard P B , Erb P , Graumann U , Schmutz B , Otten U
Published: December 2022

Increasing evidence indicates that nerve growth factor (NGF), in addition to its neurotrophic actions, exerts specific effects on cells of the immune system. This report show that the CD4-positive T cell line 9/6 expresses trk protooncogene, the signal transducing receptor unit for NGF, after TCR-mediated activation by Ag and APC. This receptor is of functional importance because interaction of NGF with Ag-stimulated 9/6 T cells induced the transcriptional activation of the c-fos gene, a hallmark of the biochemical response to NGF. Our findings that neither mitogen nor Ag stimulation induced the expression of the low affinity NGF receptor in 9/6 T cells indicate that trk alone is sufficient to mediate biologic activity of NGF in T lymphocytes.
c-kit expression by B cell precursors in mouse bone marrow. Stimulation of B cell genesis by in vivo treatment with anti-c-kit antibody.
Authors: Rico-Vargas S A , Weiskopf B , Nishikawa S , Osmond D G
Published: December 2022

To examine the in vivo role of c-kit receptor in B lymphopoiesis we have evaluated precursor B cell populations expressing c-kit in mouse bone marrow and the effects on B cell genesis of administering a neutralizing anti-c-kit mAb, ACK2. Double immunofluorescence labeling and mitotic arrest were used to examine bone marrow cells from BALB/c mice. Almost one-half of TdT+ cells and one-quarter of B220+ cells coexpressed c-kit, mainly at low intensities, and were actively proliferating in vivo. c-kit+ cells that lacked B lineage markers expressed c-kit in high intensities and entered mitosis at an exceptionally rapid rate. In ACK2-treated mice, erythroid and granulocytic cells were almost completely absent from the bone marrow, whereas, in contrast, B lymphopoiesis was stimulated. Pre-B cells expressing cytoplasmic mu-chains; as well as TdT+B220+ cells before mu expression, were increased two- to fourfold in number and production rate. IgM-bearing B lymphocytes were increased in bone marrow and spleen. The results demonstrate that many early precursor B cells in mouse bone marrow constitutively express c-kit receptor. The failure of ACK2 treatment to block B lymphopoiesis, however, suggests that c-kit receptor function is not essential for precursor B cell development in vivo, but can be replaced by alternative signaling systems. The stimulation of B cell genesis by ACK2 treatment may reflect a conferred advantage in the competition for microenvironmental factors which underlies the balance between B lymphopoiesis and other hemopoietic lineages in vivo.
Cyclosporin A inhibits positive selection and delays negative selection in alpha beta TCR transgenic mice.
Authors: Urdahl K B , Pardoll D M , Jenkins M K
Published: December 2022

Cyclosporin A (CsA) is an immunosuppressive drug that inhibits TCR-mediated signal transduction. This drug has two major effects on developing alpha beta thymocytes in normal mice: it blocks the development of most mature CD4+8- and CD4-8+ thymocytes and inhibits the deletion of some but not all self-specific thymocytes. The latter effect may explain how CsA treatment can paradoxically induce autoimmunity in certain situations. Here we investigated the effects of CsA on thymocyte development in transgenic mice that express on most of their T cells an alpha beta TCR specific for male H-Y Ag bound to H-2 Db class I molecules, a model system in which positive and negative selection have been clearly defined. Positive selection occurs in female mice, resulting in the development of mature CD4-CD8+ T cells, whereas negative selection occurs in male mice, resulting in the deletion of self-reactive CD4+8+ thymocytes. CsA blocked positive selection, as evidenced by the finding that most of the cells present in the thymuses of CsA-treated female mice were functionally immature precursors that expressed heat-stable Ag. In male mice, CsA delayed but did not prevent the deletion of most CD4+8+ thymocytes. A few transgenic thymocytes, however, were not deleted and achieved the TCR+, CD4-8+ phenotype of positively selected cells. Therefore, for a small subset of thymocytes, CsA may convert a normally negatively selecting TCR signal to a positively selecting one.
Functional analysis of two tetanus toxin universal T cell epitopes in their interaction with DR1101 and DR1104 alleles.
Authors: Valmori D , Sabbatini A , Lanzavecchia A , Corradin G , Matricardi P M
Published: December 2022

In this study we have investigated the interaction between two DR11 alleles (DB*1101 and DB*1104) and two previously described tetanus toxin (tt) universal T cell epitopes P2(tt830-843) and P30(tt947-967) by means of a functional cytotoxic competition assay. Both truncation analysis and single alanine substitution analysis were performed. In addition, the capacity of truncated and single alanine substituted peptides to be recognized by human T cell clones from donors bearing the DR1101 or DR1104 alleles was assessed. In the case of truncated peptides the same binding and recognition pattern was observed with both alleles. Longer peptides were better competitors and more potent stimulators, a result that should be taken into account when these peptides are used as immunogens. None of the single alanine substitutions could abrogate or strongly diminish the inhibitory capacity of the analogues tested indicating the lack of strong "anchor residues" present in P2 and P30 and implicated in DR binding. In addition, although the original peptide sequences were presented to specific T cell clones with comparable efficiency, some of the alanine single substituted peptides were better recognized in association with one of the alleles by clones derived from individuals bearing the homologous allele. The only exception was the tt951-967 analogue ttW955A, which was preferentially recognized in association with the DR1104 allele regardless of the clone tested. This suggests that, although it binds to both alleles with comparable efficiency, the MHC-peptide complex so formed is conformationally distinguishable by specific T cell clones.
In vitro and in vivo effects of lymphokine-activated killer cells upon preattachment ovine conceptuses.
Authors: Segerson E C , Gunsett F C
Published: December 2022

An investigation assessed the cytolytic effect of autologous ovine lymphokine-activated killer (LAK) cells upon preattachment ovine conceptuses. For an in vitro experiment, conceptuses were recovered from six ewes on each of days 14, 16, and 19 (estrus = day 0). Dissociated conceptus cells (5 x 10(4), considered target cells) were incubated with autologous LAK cells at varying E:T ratios (5:1-200:1) and cytotoxicity was assessed by 51Cr release. In vivo, control (saline), cultured PBL (1 x 10(7)) and LAK cells (1- and 4- x 10(7)) were infused into the lumen of the uterine horn ipsilateral to the corpus luteum of mated ewes on day 14. Conceptuses were recovered on day 19 and placed within one of three morphologic categories (intact, large fragments, or highly fragmented). The numbers of mononucleated and binucleated giant cells and remaining trophoblastic cells were then quantitated within sections of conceptus tissue. Mitotic activity was also recorded. At E:T ratios of 100:1 and 200:1, respectively, percent 51Cr release for LAK cell cultures was greater (p < 0.05) for day-16 and 19- than day-14 conceptus cells. The frequencies of highly fragmented conceptuses recovered from saline + cultured PBL and LAK cell-infused ewes were 0/10 and 8/13, respectively. Within tissue sections, the numbers of morphologically normal mononucleated giant and mitotic cells were less (p < 0.05) for LAK cell (4 x 10(7)) than for saline-infused ewes. Data from additional in vitro and in vivo experiments revealed that supernatants from LAK cell (4 x 10(7)) cultures failed (p > 0.05) to affect % 51Cr release from K-562 target cells, conceptus morphology, and numbers of conceptus cells. In conclusion, autologous LAK cells exerted in vitro and in vivo lytic damage upon preattachment ovine conceptuses. A temporal pattern was observed for the in vitro lytic responses.
Membrane vesiculation of Naegleria fowleri amoebae as a mechanism for resisting complement damage.
Authors: Toney D M , Marciano-Cabral F
Published: December 2022

Pathogenic and nonpathogenic Naegleria amoebae activate the alternative C pathway; however, only pathogenic amoebae are resistant to C-mediated damage. The present study was undertaken to determine the mechanism by which highly pathogenic N. fowleri amoebae resist C-mediated damage. Nomarski optics microscopy and electron microscopy of Naegleria amoebae revealed membrane blebbing on the surface of C-resistant N. fowleri, but not on C-sensitive N. gruberi, in response to incubation in normal human serum diluted 1:4 to 1:16. Immunofluorescent staining of pathogenic amoebae, by using antiserum to human C proteins comprising the membrane attack complex, C5b through C9, and FITC-labeled goat anti-rabbit IgG, confirmed that the membrane attack complex was concentrated on the membrane blebs. Binding studies with the use of radioiodinated C9 demonstrated a decrease in the 125I-labeled C9 cpm associated with N. fowleri amoebae and an increase in the 125I-labeled C9 cpm associated with the released membrane vesicles after increasing incubation periods in normal human serum. Treatment of pathogenic, C-resistant N. fowleri with cytochalasin D or cytochalasin B to inhibit actin-dependent exocytic processes increased the susceptibility of the amoebae to C damage. In contrast, incubation of nonpathogenic, C-sensitive amoebae with cytochalasins did not alter their susceptibility to C lysis. These data indicate that pathogenic N. fowleri use membrane vesiculation to remove membrane-deposited C proteins, specifically the membrane attack complex (C5b-C9). The ability to remove surface-associated membrane attack complexes serves as one mechanism by which pathogenic N. fowleri resist C lysis.
Selective expansion of cytotoxic T lymphocytes with a CD4+CD56+ surface phenotype and a T helper type 1 profile of cytokine secretion in the liver of patients chronically infected with Hepatitis B virus.
Authors: Barnaba V , Franco A , Paroli M , Benvenuto R , De Petrillo G , Burgio V L , Santilio I , Balsano C , Bonavita M S , Cappelli G
Published: December 2022

Highly purified CD4+ T cells isolated from liver biopsies of patients with hepatitis B virus-induced CAH had a strong cytotoxic activity and were comprised of a substantial number of cells (25%-40%) expressing CD56 surface marker. These cells were absent in CD4+ T cells from the peripheral blood of CAH patients or normal controls and these suspensions did not have cytotoxic activity. CD4+CD56+ T cells were further characterized by studies at the clonal level. A total of 71 hepatitis B envelope antigen-specific CD4+ T cell clones was investigated (23 from liver biopsies, 48 from peripheral blood of patients or normal vaccinated individuals). A total of 16 out of 23 (69.5%) of the clones from liver biopsies, but only 4.1% (2 out of 48) of those from PBLs, expressed CD56. A clone was defined as CD56+ when 40% or more of the cells expressed the marker. Production of TNF-alpha, IL-4, IL-5, IL-2, and IFN-gamma was investigated in 15 CD4+CD56+ and in 18 CD4+CD56- T cell clones, which shared the same HLA restriction element (DR2w15) and the same fine specificity (peptide 193-207 of the S region). All of the clones from the two groups released TNF-alpha and IL-2. However, all of the CD4+CD56+ T cell clones produced IFN-gamma but not IL-4 and IL-5 (Th1-like cell clones). Fourteen of the CD4+CD56- clones released IFN-gamma, IL-4, and IL-5 (Th0-like cell clones); three produced IL-4 and IL-5 but not IFN-gamma (Th2-like cell clones); and only one had a Th1 cytokine secretion profile. Cell fractionating studies within single CD4+CD56+ T cell clones showed that cells expressing high density CD56 had a stronger cytotoxic activity and produced higher levels of IFN-gamma than cells with low density CD56, thus further supporting a correlation between CD56 expression and cell functions. The results indicate that: 1) in CAH patients, cytotoxic CD4+ T cells with a Th1 cytokine secretion profile are compartmentalized in the liver, 2) these cells may be identified by the expression of CD56, 3) the expansion of these cells may be facilitated by antigenic stimulation within the inflammatory environment of the liver, and 4) CD4+CD56+ cells may play a pathogenetic role in hepatitis B virus infection.
A novel subpopulation of primed T cells in the human fetus.
Authors: Byrne J A , Stankovic A K , Cooper M D
Published: December 2022

Virgin and memory T cells reciprocally express high levels of the RA or the RO isoforms of CD45, respectively. In an examination of T cell expression of these two CD45 isoforms during human development, the RO+RA- "memory" T cells were infrequent in the newborn blood and spleen, but comprised approximately half of the T cells in adult tissues. These anticipated findings probably reflect the immunologic naivete of the newborn. Surprisingly, however, RO+RA- T cells were relatively abundant in fetal spleen and in cord blood samples from premature births, comprising approximately 25% and 10% of the T cells in these tissues, respectively. This early peripheral wave of RO+RA- T cells was composed of polyclonal T cells in both the CD4 and CD8 subpopulations. The fetal RO+ cells of CD4+ phenotype frequently expressed the CD25-alpha chain subunits that characterize high affinity IL-2 receptors, and were able to proliferate in response to exogenous IL-2. In further contrast with their RO+ memory T cell counterparts in adults, the fetal T cells were unresponsive to mitogenic anti-CD2 and anti-CD3 antibodies. We conclude that the data suggest an embryonic population of autoreactive T cell clones with anergic characteristics.