bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombinant immunotoxins containing the VH or VL domain of monoclonal antibody B3 fused to Pseudomonas exotoxin.
Authors: Brinkmann U , Lee B K , Pastan I
Published: December 2022

We prepared recombinant immunotoxins in Escherichia coli in which the VH or VL domains of mAb B3 were fused to a truncated form of Pseudomonas exotoxin (PE) (PE38KDEL). mAb B3 binds to a carbohydrate Ag found on the surfaces of many types of cancers and only a few normal tissues. PE38KDEL is a 38-kDa form of PE (66 kDa) that is missing the cell-binding domain of PE and has the carboxyl end changed from REDLK to KDEL. We show that immunotoxins in which the H chain or the L chain V region is fused to PE38KDEL bind to and kill carcinoma cells containing the B3 Ag. B3 Ag-negative cells were not affected. The cytotoxicity of these molecules is between 20- and 100-fold less than B3(Fv)-immunotoxins, containing both the H and L chain V regions. The VL-containing toxin was more active than the VH-containing toxin, indicating that the L chain of mAb B3 probably contributes more to Ag-binding than the H chain. Refolding experiments show that B3(VL)-PE38KDEL aggregates less than the VH-derivative or than a single chain immunotoxin B3(Fv)-PE38KDEL, which contains both domains in a single chain form. Furthermore, in addition to monomers, active homodimers of B3(VH)- and B3(VL)-PE38KDEL were obtained from renaturation experiments. The VL-toxin dimers, which might have their binding regions arranged in a manner similar to Bence Jones proteins (L chain homodimers), were found to have almost the same cytotoxicity as the monomers, whereas the VH-toxin dimers had decreased cytotoxic activity.
VH gene expression and regulation in the mutant Alicia rabbit. Rescue of VHa2 allotype expression.
Authors: Chen H T , Alexander C B , Young-Cooper G O , Mage R G
Published: December 2022

Rabbits of the Alicia strain, derived from rabbits expressing the VHa2 allotype, have a mutation in the H chain locus that has a cis effect upon the expression of VHa2 and VHa- genes. A small deletion at the most J-proximal (3') end of the VH locus leads to low expression of all the genes on the entire chromosome in heterozygous ali mutants and altered relative expression of VH genes in homozygotes. To study VH gene expression and regulation, we used the polymerase chain reaction to amplify the VH genes expressed in spleens of young and adult wild-type and mutant Alicia rabbits. The cDNA from reverse transcription of splenic mRNA was amplified and polymerase chain reaction libraries were constructed and screened with oligonucleotides from framework regions 1 and 3, as well as JH. Thirty-three VH-positive clones were sequenced and analyzed. We found that in mutant Alicia rabbits, products of the first functional VH gene (VH4a2), (or VH4a2-like genes) were expressed in 2- to 8-wk-olds. Expression of both the VHx and VHy types of VHa- genes was also elevated but the relative proportions of VHx and VHy, especially VHx, decreased whereas the relative levels of expression of VH4a2 or VH4a2-like genes increased with age. Our results suggest that the appearance of sequences resembling that of the VH1a2, which is deleted in the mutant ali rabbits, could be caused by alterations of the sequences of the rearranged VH4a2 genes by gene conversions and/or rearrangement of upstream VH1a2-like genes later in development.
TNF-alpha response of human monocyte-derived macrophages to Mycobacterium avium, serovar 4, is of brief duration and protein kinase C dependent.
Authors: Gan H , Newman G , McCarthy P L , Remold H G
Published: December 2022

Human monocyte-derived macrophages (M phi) from the majority of normal donors respond to inoculation with Mycobacterium avium, serotype 4, (MAI) by elaboration of the inflammatory monokines TNF-alpha, IL-1 beta, and IL-6, which are of central importance for the protection against bacterial and parasitic infections. Peak TNF-alpha mRNA levels were of brief duration, being maximal at 1.5 h, and were only slightly higher than background levels at 4 h. Increases of IL-1 beta and IL-6 mRNA levels, on the other hand, persisted for 48 to 72 h. In contrast to LPS, MAI induced the production of only small amounts of TNF-alpha protein in the first 12 h and of large amounts of IL-1 beta and IL-6 protein between 3 and 72 h. MAI-induced TNF-alpha transcripts, in contrast to LPS induced TNF-alpha transcripts, were highly unstable. Their accumulation was blocked and their t 1/2 significantly decreased by the protein kinase C inhibitor staurosporine. In contrast, LPS-induced increases of TNF-alpha mRNA levels and MAI-induced increases of IL-1 beta and IL-6 mRNA levels were PKC independent. The cAMP- and cGMP-dependent protein kinase inhibitors, KT5720 and KT5823, respectively, and the tyrosine kinase inhibitors herbimycin and erbstatin had no effect on the MAI-dependent mRNA accumulation of TNF-alpha, IL-1 beta, and IL-6. W7, a calmodulin-dependent protein kinase inhibitor, was inhibitory in all cases. Thus, MAI-induced TNF-alpha mRNA accumulation is of short duration and PKC dependent. MAI-induced TNF-alpha protein production is low, possibly resulting in a mitigated antimicrobial effect.
Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is mediated by mannose receptors in addition to complement receptors.
Authors: Schlesinger L S
Published: December 2022

We have examined macrophage receptors that mediate phagocytosis of virulent strains (Erdman and H37Rv) and an attenuated strain (H37Ra) of the intracellular pathogen, Mycobacterium tuberculosis. Adherence of the three strains to monocyte-derived macrophages (MDM) is markedly enhanced (>threefold) in the presence of low levels of fresh serum and requires heat-labile serum components because heat inactivation of serum reduces adherence by 65 +/- 5 to 71 +/- 2%. In the presence and absence of serum, adherence of the three strains to MDM is comparable. By electron microscopy, all bacteria are ingested and reside in phagosomes. C receptors (CR) play an important role in adherence of the three strains to MDM in the presence and absence of serum. mAb against CR1, CR3, and CR4 inhibit adherence of Erdman M. tuberculosis in fresh serum by 75 +/- 3% and inhibit the low level of adherence of Erdman (71 +/- 13%), H37Rv (72 +/- 1%), and H37Ra (64 +/- 14%) M. tuberculosis in the absence of serum. Mannose receptors (MR) play an important role in mediating macrophage adherence of the virulent strains but not the attenuated strain of M. tuberculosis. Preincubation of MDM with soluble mannan or mannose-BSA consistently and significantly inhibits adherence of Erdman and H37Rv (up to 60 +/- 7%) but not H37Ra (0 +/- 1 to 5 +/- 5% enhancement of adherence) in the absence of serum. Down-modulation of macrophage MR on mannan substrates inhibits adherence of Erdman (52 +/- 8%) and H37Rv (55 +/- 6%) but not H37Ra (2 +/- 2% enhancement of adherence). Preincubation of MDM with soluble N-acetylglucosamine-BSA also significantly inhibits adherence of the virulent strains (42 +/- 3%). Preincubation of MDM with glucose-BSA minimally inhibits adherence of the three strains (2 +/- 4 to 12 +/- 5%). Anti-MR antibody inhibits adherence of Erdman (57 +/- 2%) and H37Rv (44 +/- 4%) but not H37Ra (4 +/- 5% enhancement of adherence). Inhibition of adherence of zymosan was comparable with that seen with virulent strains of M. tuberculosis in these studies. Down-modulation of macrophage MR also inhibits adherence of Erdman (48 +/- 9%) and H37Rv (20 +/- 2%) in the presence of serum. Simultaneous blockade of MR and CR does not further inhibit adherence of the virulent M. tuberculosis strains over that seen with blocking CR alone.(ABSTRACT TRUNCATED AT 400 WORDS)
Recognition of human melanoma cells by HLA-A2.1-restricted cytotoxic T lymphocytes is mediated by at least six shared peptide epitopes.
Authors: Slingluff C L , Cox A L , Henderson R A , Hunt D F , Engelhard V H
Published: December 2022

HLA-A2.1-associated peptides were extracted from human melanoma cell lines and used to study epitopes for melanoma-specific HLA-A2.1-restricted CTL. CTL were generated from tumor-involved nodes by in vitro stimulation, initially with autologous melanoma cells and subsequently with allogeneic A2.1+ melanoma cells. These CTL lysed autologous melanoma plus four allogeneic HLA-A2.1+ melanomas, including an HLA-A2.1-transfected melanoma. K562, HLA-A2- melanomas and HLA-A2+ nonmelanomas were not lysed. HLA-A2.1 molecules were purified from human melanoma cell lines by immunoaffinity column chromatography of detergent-solubilized cell pellets. Peptides bound to the MHC molecules were acid eluted and fractionated by reversed phase HPLC. Individual fractions were assessed for their ability to reconstitute melanoma-specific epitopes by addition to the HLA-A2.1+ Ag-processing mutant, 721.174XCEM.T2 (T2). Five peaks of reconstitution were observed. Second dimension HPLC separations of reconstituting fractions revealed evidence for two distinct reconstituting peptides within one of these peaks. Based on these data, a minimum of six distinct peptides associated with HLA-A2.1 and recognized by melanoma-specific CTL are present on these different melanoma lines. These data document the presence of multiple peptide-defined CTL epitopes that are shared by at least three unrelated human melanoma cell lines.
Identification of a chemotactic domain of the pro-inflammatory S100 protein CP-10.
Authors: Lackmann M , Rajasekariah P , Iismaa S E , Jones G , Cornish C J , Hu S , Simpson R J , Moritz R L , Geczy C L
Published: December 2022

We previously reported the purification and partial amino acid sequence of a novel murine cytokine designated CP-10, which has chemotactic activity for murine polymorphonuclear cells (PMN) and macrophages. The complete cDNA encoding an 88-amino acid polypeptide has been isolated and the sequence is presented here. Transient transfection of CP-10 cDNA into CV-1 cells confirmed the chemotactic activity of rCP-10 for murine PMN. CP-10 has sequence homology with members of the S100 family of Ca(2+)-binding proteins with pronounced amino acid sequence similarities within the putative N- and C-terminal Ca(2+)-binding sites, but differences within their connecting hinge and C-terminal regions. We have confirmed the hypothesis of Kligman and Hilt that functional specificity of individual members of the S100 protein family may reside in the hinge region. A synthetic peptide corresponding to the hinge region of CP-10 (CP-10(42-55) was compared with native CP-10 in chemotaxis and skin test assays. Native CP-10 had potent activity for phagocytic cells, but not lymphocytes, in vitro (optimal activity, 10(-11) to 10(-13) M) and elicited a sustained recruitment of neutrophils and mononuclear cells over 24 h in vivo. The hinge-region peptide had strong chemotactic activity for murine phagocytic cells (optimal activity, 10(-10) - 10(-11) M) but elicited only a transient infiltration of neutrophils over 4 to 8 h after intradermal injection. Results indicate that although the hinge region contributes significantly to the functional specificity of the S100 protein CP-10, sustained cellular recruitment typical of a delayed type hypersensitivity response is apparently dependent on the structural integrity of the protein.
Differential regulation of formation of multinucleated giant cells from concanavalin A-stimulated human blood monocytes by IFN-gamma and IL-4.
Authors: Takashima T , Ohnishi K , Tsuyuguchi I , Kishimoto S
Published: December 2022

We found that in vitro multinucleated giant cells (MGC) could be produced by incubation of highly purified human blood monocytes with Con A alone, and the effect of Con A was dose- and time-dependent. Any of the cytokines considered as macrophage-activating factor, such as IFN-gamma, IL-2, IL-4, GM-CSF, or TNF-alpha, did not induce MGC by itself. When added to monocyte cultures, however, IFN-gamma enhanced Con A-induced MGC formation in a dose-dependent manner. In contrast, IL-4 suppressed this response in a dose- and time-dependent manner. IL-4 antagonized the enhancing effect of IFN-gamma on Con A-induced MGC formation. This ability to suppress the formation of MGC was completely abrogated after treatment with anti-IL-4 antibody. In addition, the involvement of monokines (IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha) in Con A-induced monocyte fusion was investigated by adding various antimonokine polyclonal antibodies in cultures. Normal rabbit IgG, anti-IL-1 alpha rabbit antibody, and anti-IL-1 beta rabbit antibody had no effect on Con A-induced MGC formation. However, anti-TNF-alpha rabbit antibody had suppressed the monocyte fusion induced by Con A in a dose-dependent manner, and a high dose of anti-IL-6 rabbit antibody had a partially suppressive effect. Anti-TNF-alpha mAb also had an inhibitory effect on monocyte fusion. Furthermore, the enhancing effect of IFN-gamma on this response was entirely abrogated by anti-TNF-alpha rabbit antibody. There was a highly significant positive correlation between the fusion rates of monocytes and the levels of TNF-alpha produced by monocytes (r = 0.68, p < 0.0005). Our results indicate that T cell-derived lymphokines, such as IFN-gamma and IL-4, have mutually antagonistic effects on Con A-induced human MGC formations in vitro, in which the expression of TNF-alpha in human monocytes plays a key role in the fusion process of monocytes.
Circulating HIV-specific CD8+ cytotoxic T cells express CD38 and HLA-DR antigens.
Authors: Ho H N , Hultin L E , Mitsuyasu R T , Matud J L , Hausner M A , Bockstoce D , Chou C C , O'Rourke S , Taylor J M , Giorgi J V
Published: December 2022

CD38, a molecule with multilineage distribution but unknown function, and the MHC class II molecule HLA-DR (DR) have markedly elevated levels of expression on CD8+ cells of HIV-infected people. This study investigated the expression of CD38 and DR Ag on circulating HIV-specific CD8+ CTL in HIV-seropositive subjects. Purified CD8+ lymphocytes from 22 participants in the University of California at Los Angeles Multicenter AIDS Cohort Study were screened for CTL activity against autologous EBV-immortalized lymphoblast targets infected with vaccinia vectors that carried HIVIIIB gag, pol, and env genes. Sixty-seven percent (14 of 21), 64% (14 of 22), and 9% (2 of 22), respectively, of the subjects had HIV-specific CD8+ CTL activity against gag, pol, and env proteins. CD8+ cells from 11 of the subjects who had high CTL activity were then FACS-separated using three-color immunofluorescence sorting. Circulating DR-CD38- CD8+ cells had little activity. Highly purified DR+CD38+ CD8+ cells had higher HIV-specific CTL activity than other CD8+ cells. DR+CD38- or DR-CD38+ CD8+ cells also mediated significant activity, but only about half as much on a per cell basis as DR+CD38+ CD8+ cells. This is the first report that the CD38 molecule is expressed in vivo on Ag-specific CD8+ CTL, and confirms previous reports that DR is expressed on these cells. Both asymptomatic HIV-seropositive subjects (144 +/- 132/mm3) and AIDS patients (253 +/- 178/mm3) had markedly elevated levels of DR+CD38+ CD8+ cells compared with the levels in HIV-seronegative controls (7 +/- 3/mm3). However, the level of anti-HIV CTL activity was not correlated with the level of DR+CD38+ CD8+ cells, indicating that enumeration of this lymphocyte population by flow cytometry most likely will not be a useful surrogate for measuring functional CTL activity. Low levels of HIV-specific CTL activity, especially against gag, were correlated with lower CD4+ cells numbers, suggesting that the loss of CD8+ T cell cytotoxic activity against HIV that has been reported to occur with advancing HIV disease progression may reflect in part the extent of CD4+ cell immunodeficiency in HIV-infected subjects.
Monoclonal antibody to rubella virus capsid protein recognizes a beta-cell antigen.
Authors: Karounos D G , Wolinsky J S , Thomas J W
Published: December 2022

The high frequency of insulin-dependent diabetes (IDDM) in children with congenital rubella suggests that the infectious agent may trigger the autoimmune process. To explore the immunologic relationship between rubella virus and IDDM, we examined a panel of mAb that recognizes rubella virus capsid and envelope glycoproteins for reactivity with islet cell Ag. One mAb, C9, which recognizes a defined domain within the capsid protein of rubella virus, was found to react with extracts from a rat beta-cell tumor and normal rat and human islets. Using one and two dimensional immunoblot analysis of rat beta-cell tumor extracts, the C9-like epitope was found to reside on a 52 kDa protein that is also the target of autoantibodies from human IDDM and nonobese diabetic mice. To confirm this cross-reactivity, antibodies in diabetic sera were absorbed to the recombinant rubella virus capsid protein, eluted, and then shown to react with the 52 kDa insulinoma protein. These data show that an immunogenic epitope on the rubella virus capsid protein is mimicked by a similar structure on a beta-cell protein. These findings suggest that rubella virus has the potential to sensitize susceptible individuals for an autoimmune response to beta-cell Ag and identify one mechanism that may contribute to beta destruction in IDDM.
Characterization of the mouse autoantigen La (SS-B). Identification of conserved RNA-binding motifs, a putative ATP binding site and reactivity of recombinant protein with poly(U) and human autoantibodies.
Authors: Topfer F , Gordon T , McCluskey J
Published: December 2022

To facilitate the study of autoimmunity to the nuclear Ag La (SS-B) we have isolated and characterized cDNA encoding the mouse La (SS-B) protein. Mouse La (SS-B) protein has 76.7% identity to both human and bovine La (SS-B) proteins and the previously recognized RNP-binding motifs were noted to be highly conserved across the three species. Examination of the primary sequence of human and bovine La (SS-B) newly identifies the site of a potential ATP-binding motif G/AXXXXGK which is preserved in mouse La (SS-B) despite a unique 16 amino acid insertion present in this region of the mouse protein. Analysis of mouse genomic DNA suggests a single gene encodes mouse La (SS-B) and no restriction fragment polymorphisms were identified in the three mouse strains investigated. Two alternate 5' untranslated regions were identified in mouse La (SS-B) cDNA and La (SS-B) mRNA was identified in a wide range of murine tissues consistent with its ubiquitous expression. Recombinant mouse La (SS-B) protein purified from Escherichia coli was shown to bind to poly(U) agarose and to react with sera containing anti-La (SS-B) antibodies obtained from patients with Sjögren's syndrome. The ability to produce recombinant mouse La (SS-B) should provide a valuable reagent for the study of the cellular autoimmune response as well as immunologic tolerance to La (SS-B) Ag in mice.
Immunization with recombinant BCG-SIV elicits SIV-specific cytotoxic T lymphocytes in rhesus monkeys.
Authors: Yasutomi Y , Koenig S , Haun S S , Stover C K , Jackson R K , Conard P , Conley A J , Emini E A , Fuerst T R , Letvin N L
Published: December 2022

Because the transmission of HIV is likely to occur through cell-associated virus, an effective HIV vaccine should be capable of eliciting HIV-specific CTL. We have employed the simian immunodeficiency virus (SIV)/rhesus monkey model to explore the use of the attenuated tuberculosis bacillus, Calmette Guérin bacillus (BCG), as a vaccine vehicle to elicit AIDS virus-specific CTL. BCG was engineered to express SIVmac gag under the control of hsp70 regulatory sequences. Immunization with this rBCG-SIVmac gag elicited MHC class I-restricted, CD8+ SIVmac gag-specific CTL in rhesus monkeys. In fact, SIVmac gag-specific CTL could be cloned readily from peripheral blood lymphocytes of these immunized monkeys. These findings provide further evidence for the power of BCG as a vaccine vector and its continued exploration as a vehicle for eliciting HIV-specific immunity.
Memory effectors: a potent, IL-4-secreting helper T cell population that develops in vivo after restimulation with antigen.
Authors: Bradley L M , Duncan D D , Yoshimoto K , Swain S L
Published: December 2022

Resting CD4+ memory T cells from the spleens of mice primed more than 6 wk earlier with KLH in adjuvant produce IL-2 and IL-3, but only low levels of IL-4 and IFN-gamma, and undetectable IL-5 after specific restimulation in vitro. In contrast, when previously primed mice are boosted by i.v. injection of soluble KLH, a CD4+ T cell population develops in the spleen that can produce greatly increased levels of IL-4, and variably increased IL-2, IFN-gamma, and/or IL-3. The ability of these memory effector Th cells to be restimulated to secrete cytokines is maximal by 3 days after in vivo boosting with Ag, and declines rapidly thereafter. Memory effector Th are induced in the spleens of KLH-primed mice that have been depleted of precursor T cells by thymectomy more than 25 wk previously, suggesting that they are derived from a memory T cell population. Cell separation on the basis of CD45RB or MEL-14, surface markers that are down-regulated on memory CD4+ cells, indicates that memory effector Th are in the CD45RBlo and MEL-14- subsets of CD4+ T cells. The frequency of KLH-specific splenic CD4+ T cells with the capacity to secrete cytokines in limiting dilution cultures is very low in unprimed mice (1/3 x 10(4) to 1/9 x 10(4)), but increases as much as 50-fold after priming. IL-2, but not IL-4, can be detected in the supernatants of stimulated unprimed CD4+ T cells. Both IL-2- and IL-4-producing cells are readily detected after priming, and frequencies of IL-2-secreting cells (1/250 to 1/4000) are up to ninefold greater than those for IL-4 secreting cells. Boosting elicits variable (two to eightfold) increases in the frequencies of both IL-2- and IL-4-producing CD4+ T cells. However, high IL-4 titers associated with memory effector Th function can be obtained when significant frequency changes are not detected after boosting. The data suggest that reactivation of resting memory Th in vivo induces their differentiation into effector Th with the capacity to secrete much higher levels of IL-4. Although the capacity to produce high levels of IL-4 is associated with both primary and memory CD4+ effector populations, memory effector Th are the higher producers.(ABSTRACT TRUNCATED AT 400 WORDS)
Differential recruitment of viral and allo-epitopes into the MHC class I antigen processing pathway of a novel mutant of Ltk- cells. HSV/MHC class I restriction/immune recognition/antigen processing/antigen presentation/influenza virus.
Authors: Lippé R , Kolaitis G , Michaelis C , Tufaro F , Jefferies W A
Published: December 2022

It is generally thought that the Ag processing pathways for endogenously synthesized proteins are the same for allo and viral Ag processing. However, this view does not take into consideration the diversity of specialized maturation and assembly pathways for viruses. In particular, viral assembly that takes place within intracellular membranes may require unique Ag processing steps. In this study we sought to assess this possibility. Hence, we describe the CTL response against a murine Ltk- cell derivative, gro29, which was previously shown to be defective in the propagation of herpes simplex virus type 1 (HSV-1). In HSV-1-infected gro29 cells, viral polypeptides are synthesized in normal amounts and viral assembly takes place. However, transport of the assembled particles is defective in these cells, resulting in the accumulation of noninfectious virus in cytoplasmic vesicles, and a reduction in the release of viral particles by at least 2000-fold. We show that the rate of transport of individual endogenous proteins through the organelles of the secretory pathway is also impaired, but only by roughly 50%, suggesting that the defect in this cell line affects the transport of particles to a greater extent than the transport of individual proteins. It is also shown that allogeneic and influenza A- specific CTL responses are indistinguishable between gro29 and Ltk- cells, as is the response against target cells pretreated with a influenza A derived synthetic peptide. By contrast, HSV-1-infected gro29 cells are approximately eightfold less sensitive than infected Ltk- cells to lysis by HSV-1-specific CTL. This illustrates that in contrast to the allogeneic and influenza specific responses, the recruitment of herpes virus-specific Ag into the Ag-processing pathway is dependent on a cellular function that is also required for viral maturation and egress. We believe that this is the first demonstration of this phenomenon.
IL-10 inhibits T cell-independent but not T cell-dependent responses in vitro.
Authors: Peçanha L M , Snapper C M , Lees A , Yamaguchi H , Mond J J
Published: December 2022

Lymphokines play a key role in T cell-independent (TI) Ag-induced responses. We recently showed that IL-10 potently inhibits Ig production stimulated by TI-2 Ag + IL-5. In view of our recent findings that the B cell activator can determine the effect lymphokines will have on Ig production and on isotype selection, we analyzed the effect of IL-10 on Ig production induced by other TI antigens and T cell-dependent Ag. These data demonstrate that IL-10 inhibited IL-5-induced Ig production stimulated by TI-1 and by TI-2 Ag, but had no effect on an in vitro T cell-dependent Ag-specific anti-SRBC-induced response or on a T cell-induced polyclonal response mediated by anti-CD3-activated T cell clones. IL-10 inhibited IgM, IgG1, IgG2a, and IgG3 secretion by B cells costimulated by LPS or anti-delta-dextran. IL-10 did not interfere with induction of class II MHC Ag expression or cell enlargement that was stimulated by LPS or anti-delta-dextran and had no detrimental effect on cell viability. IL-4 reversed the IL-10-mediated inhibition of IgG1 and IgM secretion stimulated by anti-delta-dextran or LPS-activated cells in the presence of IL-5. A 72 h, but not 24 h, exposure to IL-4 at initiation of culture with anti-delta-dextran + IL-5 + IL-10 was necessary to reverse the IL-10-mediated inhibition of IgM secretion. Our data suggest that IL-10 can selectively inhibit TI Ag-induced responses when other T cell-derived stimulatory lymphokines are not present and further emphasize the specific role of the B cell activator in influencing the responsiveness of B cells to lymphokines.
Coupling of GTP-binding to the T cell receptor (TCR) zeta-chain with TCR-mediated signal transduction.
Authors: Sancho J , Peter M E , Franco R , Danielian S , Kang J S , Fagard R , Woods J , Reed J C , Kamoun M , Terhorst C
Published: December 2022

The zeta-subunit of the TCR binds GTP and is a well characterized substrate for a TCR-activated tyrosine kinase. To examine the possible coupling of GTP-binding to zeta with TCR-mediated signal transduction, a mutant (termed J32-3.2) of the T cell line Jurkat (J32) was used. Anti-TCR/CD3 stimulation of the TCR/CD3+ J32-3.2 cells resulted in a weak stimulation of both the phosphatidyl inositol and tyrosine kinase signal transduction pathways, as measured by changes in the level of free intracellular calcium, tyrosine phosphorylation of TCR-zeta, CD3-epsilon and ZAP-70, p56lck, or p59fyn tyrosine kinase activity and IL-2 gene activation. The impaired responsiveness of J32-3.2 cells to anti-TCR/CD3 mAb correlated with a low basal level of GTP-binding to zeta. Furthermore, in J32-3.2 cells TCR activation by antibody ligation caused a weaker increase in GTP-binding to the zeta-chain, as compared with that of wild-type J32 cells, which indicates for the first time that GTP-binding to zeta can be modulated by extracellular signals and suggest that the role of GTP-binding to zeta is to couple the TCR to intracellular signal transduction mechanisms.
CD28 ligation by monoclonal antibodies or B7/BB1 provides an accessory signal for the cyclosporin A-resistant generation of cytotoxic T cell activity.
Authors: Van Gool S W , de Boer M , Ceuppens J L
Published: December 2022

Ligation of the T cell membrane Ag CD28 with mAb 9.3 or with its natural ligand B7/BB1 on accessory cells has been shown to provide a helper signal for stimulation through the TCR/CD3 complex. The present study was undertaken to investigate whether CD28 could function as an accessory signal receptor in the generation and effector phase of CTL activity. Purified resting human T cells were activated for 3 to 4 days with immobilized anti-CD3 mAb as the primary stimulus, and CTL activity was then measured by an anti-CD3-redirected 4-hr 51Cr release assay on Fc gamma R-bearing P815 target cells. When the concentration of immobilized anti-CD3 mAb as the primary signal for CTL generation was below threshold, CTL activity could be generated by addition of mAb 9.3 to the cultures. At optimal concentrations of immobilized anti-CD3, the addition of anti-CD28 did not further enhance the generation of CTL activity, but under these conditions generation of CTL activity was almost completely resistant to cyclosporin A (CsA) as a result of CsA-resistant IL-2 production. When 3T6 mouse fibroblasts, transfected with Fc gamma RII and B7, were used as accessory cells, anti-CD3 and B7 were also found to generate cytotoxic activity. Cytotoxic T cell generation under these conditions could be blocked by anti-B7 mAb, but was totally resistant to CsA. CTL activity could be generated by CD3 and CD28 ligation in both CD4(+) and CD8(+) subpopulations. Finally, we found that the activity of CTL lines (isolated from ascitic fluid of a patient with ovarian carcinoma and cultured in IL-2) was higher on B7-transfected targets than on the B7(-) targets. We conclude that CD28 ligation provides a major accessory signal for the CsA-resistant generation of CTL activity and that CD28-B7 interaction also enhances cytotoxic effector functions of CTL. These findings might have important implications for immunotherapeutic interventions.
Selective inhibition of T cell activation by an inhibitor of S-adenosyl-L-homocysteine hydrolase.
Authors: Wolos J A , Frondorf K A , Davis G F , Jarvi E T , McCarthy J R , Bowlin T L
Published: December 2022

Defects in the enzymes involved in the pathway of S-adenosylmethionine (AdoMet) metabolism, or inhibition of those enzymes, results in profound immunodeficiency. We have examined MDL 28,842, a novel irreversible inhibitor of S-adenosyl-L-homocysteine hydrolase (AdoHcyase), an enzyme involved in AdoMet metabolism, to determine its effect on the immune system and to investigate its potential as an immunosuppressive agent. The stimulation of human mononuclear cell proliferation in vitro with Con A, a T cell mitogen, and PWM, a T-dependent B cell mitogen, were inhibited by MDL 28,842. The 50% inhibitory concentration for both were 0.33 microM. In murine spleen cells, MDL 28,842 was a potent, nontoxic, inhibitor of Con A-stimulated T cell proliferation (IC50 = 0.19 microM) but did not affect LPS-induced B cell proliferation. This selective suppression was also observed when enriched murine T and B cells were stimulated with mitogens, although S-adenosyl-L-homocysteine (AdoHcy), the substrate of AdoHcyase, was similarly elevated in both populations. In addition to proliferation in response to a number of stimuli, IL-2 production and the expression of IL-2R by mitogen-stimulated T cells were inhibited by MDL 28,842. These results suggest a direct effect of MDL 28,842 on T cells. In vivo, the antibody response to a T cell-dependent Ag, OVA, was inhibited by MDL 28,842. The response of splenic T cells from these animals to OVA in vitro were similarly depressed compared with controls. The results demonstrate that MDL 28,842 is a potent nontoxic immunosuppressive agent, which has selectivity for T cells and therefore may be useful in the treatment of T cell-mediated disorders, such as autoimmune disease and tissue transplantation.
Genomic composition and allelic polymorphisms influence V beta usage by the Mycoplasma arthritidis superantigen.
Authors: Cole B C , Balderas R A , Ahmed E A , Kono D , Theofilopoulos A N
Published: December 2022

The Mycoplasma arthritidis superantigen (MAM) is produced by an organism that causes systemic disease in rodents leading to chronic proliferative arthritis. MAM is a typical superantigen that requires presentation to T cells by MHC molecules without processing and T cell recognition of MAM occurs through the V beta chains of the TCR. Several major findings are presented here. First, different MAM-MHC class II isotype complexes may engage different sets of V beta TCR. Thus, activation of V beta 6- and V beta 8.3-bearing T cells is more dependent upon the I-E molecule of the murine H-2 MHC than is activation of cells bearing the V beta 5.1, 8.1, and 8.2 TCR. Secondly, both genomic composition and allelic polymorphisms at the V beta chain segment of the TCR exert profound effects upon the pattern of V beta that are used by MAM. Thus, in V beta b haplotype mice, MAM engages V beta 5.1, 6, and the V beta 8 family of TCR whereas in V beta a (C57BR) and V beta c (RIIIS) haplotype mice that lack various combinations of these V beta, activation of cells bearing V beta 1, 3.1, 7, and 16 can be demonstrated. These differences in V beta usage by MAM appear to be caused by both differences in the avidity of MAM for the various V beta s and to structural allelic polymorphisms in these V beta. Clonal expansion of specific V beta in vivo after injection of MAM is also dependent upon the genomic composition of the mice, because expansion of the V beta 8 TCR seen in V beta b haplotype mice (B10.RIII) whereas marked expansion of V beta 6 is seen in V beta a mice (C57BR). In as much as these TCR have been implicated in a number of experimental autoimmune diseases, MAM may represent an ideal model to evaluate the role of superantigens in the triggering of autoimmune disease.
Transcriptional analysis of inhibition of lipopolysaccharide response by anti-IgM.
Authors: Moore B B , Ariizumi K , Tucker P W , Yuan D
Published: December 2022

Murine splenic B cells, when stimulated with LPS, show a generalized enhancement of gene transcription. In addition to this general increase, there is a specific increase in microseconds mRNA production and differentiation to high rate IgM secretion. Anti-mu added concomitantly with LPS at the start of culture has been demonstrated to inhibit the LPS-induced increase in microseconds mRNA production without affecting the proliferative capacity of the cells. By "run-on" analysis of nascent transcription, we have shown that the effect of anti-mu is mediated by the abrogation of the up-regulation of transcription of the mu-gene induced by LPS. Furthermore, by assessing the site of transcription termination, it is possible to infer that alterations in 3'-end processing induced by LPS are also inhibited. We have also found that CAT3 gene activity driven by a number of promoter/enhancers with diverse regulatory motifs are inhibited by anti-mu. These results suggest that the effect of anti-mu cannot be restricted to interactions with a single regulatory element. Therefore, cross-linking of surface IgM may affect a number of genes involved in differentiation to Ig secretion.
Requirement for both H and L chain V regions, VH and VK joining amino acids, and the unique H chain D region for the high affinity binding of an anti-phosphotyrosine antibody.
Authors: Ruff-Jamison S , Glenney J R
Published: December 2022

Sequence analysis of a panel of antibodies to phosphotyrosine revealed predominant H and L chain V regions in the immune response and a unique D segment in the Py20 mAb, which exhibits a high affinity for phosphotyrosine. In order to determine the influence of somatic diversity on the high affinity binding of Py20, H and L chain V regions were expressed in Escherichia coli as an Fv dimer. Whereas the H or L chain V regions of Py20 alone were unable to bind phosphotyrosine, the Fv binds phosphotyrosine with an affinity comparable with the intact IgG as determined by fluorescence quenching experiments (1.55 x 10(-7) M vs 1.25 x 10(-7) M, respectively). Substitution of the Py20 V regions with other IgG V regions that differed greatly in sequence abolished binding. A high affinity Py20-combining site was dependent on the presence of the unique D-D segment. Replacement of the Py20 D-D region with a single homologous D region resulted in a decrease in affinity (5.9 x 10(-7) M). Substitution of this D-D region for the D region of another anti-phosphotyrosine antibody that is known to bind phosphotyrosine weakly (1 x 10(-3) M) conferred high affinity binding. Removal of three tyrosines from the first of the two D regions was accompanied by a fivefold reduction in affinity for phosphotyrosine. In addition, changing the VK and VH junctional amino acids resulted in a complete loss of binding. Therefore, the formation of the high affinity Py20 combining site requires both a H and L chain that are similar in sequence to those of Py20 including the unique D region and the junctional amino acids.
A primary intestinal helminthic infection rapidly induces a gut-associated elevation of Th2-associated cytokines and IL-3.
Authors: Svetić A , Madden K B , Zhou X D , Lu P , Katona I M , Finkelman F D , Urban J F , Gause W C
Published: December 2022

The immune response that is characteristic of parasitic helminth infections includes components associated with immediate-type hypersensitivity: elevated serum IgE, eosinophilia, and intestinal mast cell hyperplasia. In infection with the parasitic nematode, Heligmosomoides polygyrus, IL-4 mediates protective immunity, suggesting the presence of a host-protective Th2 response. In this investigation, we examined early stages of immune responsiveness to H. polygyrus infection to determine whether and at what stage a specific Th2-like pattern first appears. Using a quantitative reverse transcriptase-polymerase chain reaction assay, we analyzed changes in IL-2, IFN-gamma, IL-3, IL-4, IL-5, IL-6, IL-9, and IL-10 gene expression in the spleen, mesenteric lymph node, and Peyer's patch at various time points after infection. Our results demonstrate a highly specific and reproducible pattern of cytokine gene expression that remains localized to the enteric region. By 6 h after infection, IL-5 and IL-9 mRNA were elevated in the Peyer's patch and IL-3 was elevated by 12 to 24 h after infection. IL-4 RNA became elevated by 4 to 6 days after infection, but little change was observed in IFN-gamma, IL-2, or IL-10 mRNA levels. The early increases in IL-3, IL-5, and IL-9 gene expression after infection were probably T cell-independent, inasmuch as they were observed in Peyer's patches of congenitally athymic mice and anti-CD4, anti-CD8 mAb-treated conventional mice. However, treatment with these mAb considerably decreased cytokine gene expression 6 days after infection, and 8 days after infection, increased IL-4 gene expression in mesenteric lymph node cells was restricted to the CD4+ population. Thus, H. polygyrus infection induces cytokine gene expression that is restricted to some Th2-associated cytokines, is initiated by a T-independent response, and culminates in a T-dependent response.
Alpha 4 beta 1 recognition of the Hep II domain of fibronectin is constitutive on some hemopoietic cells but requires activation on others [corrected].
Authors: Sánchez-Aparicio P , Ferreira Júnior O C , Garcia-Pardo A
Published: December 2022

Leukocyte adhesion to the carboxyl-terminal region of fibronectin, a major component of extracellular matrices, involves recognition of the CS-1 site and the Hep II domain. We have previously shown that cultured T and B lymphoid cells constitutively attach via the alpha 4 beta 1 integrin to a 38-kDa fibronectin fragment that contains CS-1 and Hep II, and to a 58-kDa fragment that contains Hep II only. In this report we have studied the adhesion of other hemopoietic cells to the CS-1 and Hep II regions of fibronectin. Cultured monocytic cells and peripheral blood T lymphocytes constitutively bound to the 38-kDa fragment indicating that alpha 4 beta 1 was functional. These cells, however, were unable to bind to the 58-kDa fragment. On lymphoid cells both fragments were shown to bind to very close regions of alpha 4 beta 1 as indicated by the inhibition pattern of mAb to various alpha 4 epitopes, and by the good inhibitory capacity of soluble 38-kDa fragment on cell adhesion to 58-kDa fragment. These results suggested that alpha 4 beta 1 is present on certain cell populations as a partially active form able to recognize the "high affinity" ligand CS-1 but not the "low affinity" ligand Hep II. Binding of monocytic cells and peripheral blood T lymphocytes to the Hep II domain could be induced by several agents: first, long (48-h) and short (20-min) treatment with phorbol esters; second, cell incubation with the divalent cation Mn2+; third, and most effective, cell incubation with the mAb TS2/16, which is directed to the beta 1 integrin subunit. Binding to the 58-kDa fragment in all three cases was completely inhibited by mAb anti-alpha 4, thus confirming the involvement of alpha 4 beta 1 in the recognition of the Hep II domain. No major changes on alpha 4 beta 1 surface expression were observed after these treatments as determined by immunofluorescence analyses. Our results indicate that hemopoietic cells may differentially bind the CS-1 and Hep II ligands in fibronectin depending on the activation state of alpha 4 beta 1, a fact that may be relevant for the migration and function of leukocytes.
T cell epitope specificity in human allergic and nonallergic subjects to bee venom phospholipase A2.
Authors: Carballido J M , Carballido-Perrig N , Kägi M K , Meloen R H , Wüthrich B , Heusser C H , Blaser K
Published: December 2022

Phospholipase A2 (PLA) is a biochemically fully defined glycoprotein, representing the main allergen of bee venom. We have established CD4+ T cell clones specific to PLA from subjects allergic and nonallergic to bee sting. By screening the epitope specificity of these clones with 62 synthetic overlapping dodecapeptides representing the PLA molecule, two immunogenic epitopes, PLA81-92 and PLA113-124, were identified. Additional screening, using longer peptides of up to 18 residues, revealed a third epitope at position PLA 45-62. These epitopes are recognized by specific T cell clones in association with HLA-DP and -DQ molecules, although HLA-DR-associated responses to PLA exist. Primary in vitro proliferation of unfractionated PBMC from bee sting allergic, hyposensitized, or hyperimmune subjects to PLA-derived peptides revealed the same immunogenic sites as found in the T cell clones. Among the different groups of individuals, proliferative responses to the PLA molecule and fragments thereof were generally higher in allergic patients than in nonallergic subjects. Thus, at least three linear epitopes are involved in T cell recognition of PLA, independently whether or not subjects are allergic.
Autoantigenic epitopes of the B and D polypeptides of the U1 snRNP. Analysis of domains recognized by the Y12 monoclonal anti-Sm antibody and by patient sera.
Authors: Hirakata M , Craft J , Hardin J A
Published: December 2022

Anti-Sm antibodies, a specific marker for SLE, are directed against the B'/B and D polypeptides of Sm small nuclear ribonucleoproteins. The Y12 monoclonal anti-Sm antibody (Y12 mAb), as well as many anti-Sm patient sera, recognize cross-reactive epitopes on the B'/B and D polypeptides. This immunoreactive site is of special interest since polypeptides B and D share little amino acid sequence homology. In the present study, we have sought to establish the autoantigenic domain of polypeptides B and D that accounts for this epitope. We tested the ability of the Y12 mAb and anti-Sm sera to immunoprecipitate truncated forms of polypeptides B and D translated in vitro from mRNA bearing 5' and 3' end deletions. Most anti-Sm sera bound epitopes at the carboxyl-terminus of polypeptide B, however, autoantigenic epitopes were also found at the amino-terminus (amino acids 1 to 83 and 104 to 115). Surprisingly, the Y12 mAb recognized nonoverlapping amino-terminal and carboxyl-terminal halves of polypeptide B. One putative Y12 mAb binding site (amino acids 104 to 115) indicated by carboxyl-terminal deletion studies was confirmed through recognition of a corresponding synthetic peptide. Deletion studies with polypeptide D demonstrated a major autoantigenic domain on the carboxyl-terminus (amino acids 85 to 119) that was necessary for recognition by the Y12 mAb and by 7/14 patient sera. These results indicate that a cross-reactive epitope on B'/B and D, as defined by the Y12 mAb, resides on at least two different domains of polypeptide B and localizes to the carboxyl-terminus of polypeptide D. From the shared homology of truncated forms of B and D polypeptides recognizable with the Y12 mAb, we suspect that some form of GRG motif is involved in developing the Y12 mAb epitope that may involve other residues and be largely conformational in nature.
Prostaglandin E2-induced changes in the phenotype, morphology, and lytic activity of IL-2-activated natural killer cells.
Authors: Linnemeyer P A , Pollack S B
Published: December 2022

During pregnancy, many large granular NK lineage cells that are recognized by mAb 4H12 accumulate at uterine implantation sites. Similar large, granular, 4H12+ NK cells accumulate during the 2nd wk of culture of IL-2-activated NK cells. Inasmuch as uterine NK cell lytic activity is suppressed during pregnancy, apparently due to the effects of PGE2, we have analyzed the effect of PGE2 on the phenotype, morphology, and lytic activity of IL-2-activated NK cells as a model for NK cell differentiation at uterine implantation sites. When cultures of IL-2-activated NK cells were supplemented with 1 microM PGE2, the cells increased in size and granularity, accompanied by an increase in the number of cells expressing the 4H12 Ag. This effect was specific to PGE2 because PGF2 alpha at the same concentration had no effect. In addition, changes in morphology and up-regulation of 4H12 Ag expression could be augmented by 100 mM dibutyryl cAMP, but not 100 mM dibutyryl cGMP, suggesting that the PGE2 effects are mediated by changes in cytoplasmic cAMP levels. When indomethacin was added to cultures of IL-2-activated NK cells at the beginning of the culture period, 4H12 expression was markedly reduced. Indomethacin also increased the cytolytic capacity of NK cells and reduced the number of cells that developed the large granular morphology characteristic of 4H12+ cells. When activated NK cells were sorted into 4H12+ and 4H12- populations and tested for their ability to kill YAC-1 cells, we found that 4H12+ NK cells have lower lytic activity as compared to the 4H12- subset. Furthermore, when activated NK cells were labeled with [3H]TdR, combined with 4H12 labeling, we found that the 4H12+ subset was not proliferative. These results are likely to have direct relevance to NK cell differentiation at uterine implantation sites where NK cell activity is suppressed by PGE2, 4H12 expression is up-regulated and NK cells differentiate into large, granular and relatively nonlytic cells termed granulated metrial gland cells.