bluuebunny/crossref_metadata_2025 huggingface dataset:
Surface expression of functional IgE binding protein, an endogenous lectin, on mast cells and macrophages.
Authors: Frigeri L G , Liu F T
Published: December 2022

IgE-binding protein (epsilon BP) is a galactoside-specific lectin containing an S-type carbohydrate-recognition domain. It was originally identified in rat basophilic leukemia cells and is now known to be identical to a macrophage surface Ag, Mac-2, and lectins designated as CBP 35/L-34/RL-29. It has also been related to a nonintegrin laminin-binding protein isolated from mouse macrophages. In this report we have shown the following: epsilon BP is present in variable amounts in several mast cell lines, and the surface expression of epsilon BP in these cell lines is quite variable and does not correlate with the total amount of epsilon BP in the cell. epsilon BP is displayed on the cell surface in a manner that is reversible by lactose, most likely through attachment to yet unidentified glycoconjugates. The putative epsilon BP binding sites on the cell surface can be readily demonstrated by using radiolabeled epsilon BP, and the sites are present in comparable amounts in various cell lines. Expression of epsilon BP on the cell surface can be regulated; the most notable example is the upregulation of surface epsilon BP on RBL cells activated through the high-affinity IgE receptor by IgE immune complexes. Cell-surface epsilon BP is functional as measured by its ability to promote adhesion of trypsinized rabbit erythrocytes to mast cells and macrophages. On the basis of these results and reported properties of related lectins, we propose that the lectin represented by epsilon BP is a new class of cell-adhesion protein.
Release of newly synthesized platelet-activating factor (PAF) from human polymorphonuclear leukocytes under in vivo conditions. Contribution of PAF-releasing factor in serum.
Authors: Miwa M , Sugatani J , Ikemura T , Okamoto Y , Ino M , Saito K , Suzuki Y , Matsumoto M
Published: December 2022

The behavior of platelet-activating factor (PAF) produced in stimulated human polymorphonuclear leukocytes (PMN) was investigated in the presence of serum under conditions close to those existing in vivo. When the cells were stimulated in the presence of the serum obtained from a PAF acetylhydrolase (PAF-AH)-deficient Japanese subject, over 60% of synthesized PAF was detected in the extracellular medium by bioassay, scintillation proximity RIA and selected ion monitoring/gas chromatography/mass spectrography analysis. The release of PAF from PMN after stimulation with FMLP and A23187 was also observed in the presence of normal serum treated with acid to inactivate PAF-AH. The heterogeneity of the molecular species of extracellular PAF was similar to that of intracellular PAF produced in stimulated PMN in the presence of PAF-AH-deficient serum, ruling out the possibility that a specific molecular species of PAF was preferentially released from the cells in the presence of the serum. As these data suggested the occurrence of PAF-releasing factor(s) in the serum, an attempt was made to partially purify this factor from PAF-AH-deficient serum and acid-treated normal serum by ammonium sulfate fractionation and column chromatography with DEAE-Cellulofine and Sepharose CL-6B. The molecular mass of PAF-releasing factor revealed on a TSK gel G3000 SW HPLC column was 240 kDa, which was different from that of albumin. The binding assay, newly developed for this study, revealed that the PAF-binding activity of PAF-releasing factor is stronger than that of albumin, and that the PAF-releasing factor forms a complex with PAF at low concentration (10(-9) M). PAF bound to this factor was difficult to be hydrolyzed by serum PAF-AH. On the other hand, the PAF/PAF-releasing factor complex had aggregatory activity toward washed rabbit platelets. These observations suggest that certain protein(s) releases and carries the PAF newly synthesized by PMN in blood plasma/serum. Thus it appears that PAF functions as an autacoid in vivo, along with other mediators.
Induction of C3 expression in astrocytes is regulated by cytokines and Newcastle disease virus.
Authors: Rus H G , Kim L M , Niculescu F I , Shin M L
Published: December 2022

Synthesis of complement proteins and their regulation in resident cells of the central nervous system are important pathophysiologic factors that can affect the outcome of inflammatory central nervous system diseases. Primary cultures of rat astrocytes constitutively express C3 mRNA and produce C3 protein; both of them were enhanced by LPS or by a live as well as inactivated Newcastle disease virus, a neurotropic paramixovirus. TNF, IL-1 beta, and IL-8 also increased the levels of C3 mRNA and protein whereas IL-1 alpha and IL-6 had no effect, although all of these cytokines are inducible by LPS. LPS stimulation in the presence of cycloheximide decreased the LPS-mediated C3 mRNA induction by 60%. These data suggest that LPS effect on C3 regulation is mediated directly by LPS as well as by LPS-induced cytokines. Interestingly, C3 mRNA induced by Newcastle disease virus or inactivated Newcastle disease virus was inhibited by protein kinase inhibitors, H-7 and staurosporine, whereas these inhibitors had no effect on C3 induction mediated by LPS or cytokines, indicating the existence of different signal transduction pathways.
Exon encoding the antigen-binding site of MHC class II beta-chains is divided into two subregions with different evolutionary histories.
Authors: Sigurdardóttir S , Borsch C , Gustafsson K , Andersson L
Published: December 2022

The evolution of the Ag-binding site of polymorphic class II molecules was investigated by comparing the pattern of silent and replacement substitutions in the first domain exon of DQB and DRB alleles in two distantly related mammalian species, man and cattle. We show that the first domain can be subdivided into two regions, corresponding to the beta-strand and alpha-helical regions, with distinct evolutionary histories. The data for the alpha-helical region are in conflict with the standard phylogeny of mammalian class II genes. In this region, there is only weak locus divergence at the protein level and the frequency of silent substitutions is extremely low between nonorthologous genes (i.e., DQB-DRB) within species. We propose that the major underlying cause for the observed sequence similarity in the alpha-helical region is due to a selective constraint restricting the sequence divergence at the protein level. This selective constraint may be related to the interaction between different isotypic forms of polymorphic class II beta-chains and a common ligand. The extremely low frequencies of silent substitutions between nonorthologous genes within species are most likely due to the transfer of sequence information between loci by the occurrence of gene conversion-like events in the particular gene segment.
Immune response to Moloney murine leukemia virus nonviral, tumor-associated antigens fails to provide in vivo tumor protection.
Authors: Jiang D , Flyer D C
Published: December 2022

Two distinct populations of CTL have previously been shown to be generated in lymphocyte cultures derived from the spleens of C57BL/6 mice that have rejected Moloney murine leukemia virus:Moloney sarcoma virus (MoMuLV:MSV)-induced tumors. One population is specific for MoMuLV viral Ag whereas the other appears to be directed against a nonviral, tumor-associated Ag (TAA). Using a virus-negative variant of the MoMuLV-induced lymphoma MBL-2 that has retained the expression of the MuLV:TAA, we attempted to further characterize the MuLV:TAA-specific CTL population. First, this same pattern of CTL reactivity was observed using a variety of immunization protocols indicating that the TAA-specific CTL population was not an artifact of the original immunization protocol but was a reproducible component of the MoMuLV CTL response. Moreover, CTL precursor frequency analysis indicates that the MuLV:TAA-specific CTL represent approximately 60% of the CTL detected in in vitro cytotoxicity assays. However, when the role of MuLV:TAA CTL in the in vivo rejection of MoMuLV-induced tumors was examined, no role for the MuLV:TAA-specific CTL response could be determined. Immunization protocols that had been shown to give rise to both CTL populations were capable of protecting mice from tumor development after a challenge with the parental MBL-2 tumor cell line but not the virus-negative variant MBLv cell line. In addition, immunization with the variant, shown to give rise to only MuLV:TAA-specific CTL capable of lysing both MBL-2 and MBLv in vitro, failed to protect mice from a tumor challenge of either cell type.
Human postnatal thymocytes generate phenotypically immature CD3dim, CD5dim, CD1abright progeny in organ culture.
Authors: Merkenschlager M , Fisher A G
Published: December 2022

We previously described an organ culture system that supports the in vitro development of human fetal thymocytes in murine alymphoid thymic rudiments without addition of exogenous factors. This approach to study human T cell precursors is limited by the requirement for fetal tissue. We show that human thymocytes from pediatric sources can be expanded under similar conditions. Organ cultures generated predominantly CD4 CD8 double positive cells, most of which maintained the phenotype CD3dim, CD5dim, and CD1abright, characteristic for double positive thymocytes ex vivo. This culture system should facilitate in vitro studies on human thymocyte development and repertoire selection.
A toxic shock syndrome toxin-1 peptide that shows homology to mycobacterial heat shock protein 18 is presented as conventional antigen to T cells by multiple HLA-DR alleles.
Authors: Ramesh N , Spertini F , Scholl P , Geha R
Published: December 2022

We observe that PBMC from most adults (16 of 18 subjects tested) show a small but significant in vitro proliferative response to a 30-amino acid-long peptide (peptide 2, amino acids 34-63) derived from toxic shock syndrome toxin. By contrast, PBMC from newborn blood and thymocytes do not proliferate to this peptide, and furthermore, peptide 2 did not displace the binding of radiolabeled TSST-1 to MHC class II positive cells, nor did it induce IL-1 beta mRNA in monocytes, indicating that this peptide does not behave as a superantigen. Proliferation of PBMC to peptide 2 could be blocked by anti-HLA-DR, but not by anti-HLA-DP or DQ mAb, suggesting that HLA-DR molecules are the restriction elements for the recognition of this peptide by T cells. This premise was further confirmed by demonstrating that mouse L cells transfected with human HLA-DR, but not HLA-DP or DQ molecules, supported the proliferation of purified T cells to peptide 2. Studies with subjects of known HLA-DR types showed that all types tested are capable of responding to this peptide, PBMC from adults exposed to mycobacterial Ag showed significantly better proliferative response to peptide 2 than unexposed adults. Studies with truncations of this peptide suggest that a "core" region of eight amino acids that is conserved between low m.w. heat shock proteins and peptide 2 may be critical to T cell recognition of this peptide. The universal presentation of peptide 2 by HLA-DR molecules may contribute to the widespread natural immunity observed against toxic shock syndrome toxin.
A common precursor for CD4+ T cells producing IL-2 or IL-4.
Authors: Röcken M , Saurat J H , Hauser C
Published: December 2022

To investigate whether CD4+ T cells are predetermined to produce a given pattern of lymphokines, we have used a culture system that allows the controlled induction of either IL-2- or IL-4-producing CD4+ T cells. Single, freshly isolated murine CD4+ T cells were activated with Con A, rIL-2, and APC; the developing clones were split and then cultured for an additional 14 days with either rIL-2 alone or with rIL-2 and anti-CD3 stimulation. Subclones expanded in the presence of rIL-2 alone produced predominantly IL-2, although subclones derived from the same precursor and expanded in the presence of rIL-2 and a mitogenic antibody to CD3 released predominantly IL-4. Subclones expanded for 2 wk in the presence of rIL-2 plus a mitogenic mAb to CD3 released up to 60 times more IL-4 but only 1/90 the amount of IL-2 released by subclones derived from the same precursor cell and expanded with rIL-2. Both phenotypes can be derived from IL-2-producing precursor cells. These results demonstrate that IL-2-producing clones can be derived from the same cells as IL-4-producing clones and are most consistent with the view that the IL-2-producing Th1 or the IL-4-producing Th2 phenotype of a T cell clone is acquired during T cell differentiation and is not secondary to the expansion of distinct subpopulations that are predetermined to produce a specific cytokine pattern.
Purified HLA class II peptide complexes can induce adherence and activation of peptide-specific human T cell clones.
Authors: van Schooten W C , Maiore T , Jones K R , Paborsky L
Published: December 2022

An initial event in T cell activation is the specific adherence of T cells via their T cell receptor to the MHC peptide complex. We have studied this adherence by incubating T cells with preformed HLA DR4Dw4 peptide complexes attached to a solid support. Adherence of sodium 51Cr-labeled T cell clones specific for the influenza hemagglutinin peptide, HA 307-319, was maximal after 15 min and was specific for the HLA DR4Dw4-HA 307-319 complex. The binding was temperature dependent and could be blocked with azide or protein kinase C inhibitors, indicating that for adherence the T cells need to be metabolically active and have a functioning protein kinase C pathway. The adherence could be blocked with CD4- or CD3-reactive murine mAb, suggesting that the TCR and CD4 molecules work in concert to induce strong adherence to the HLA DR4Dw4-HA 307-319 complex. A subsequent event in T cell activation is proliferation, which is thought to need additional proteins such as IL-1 or other adhesion molecules. MHC peptide complexes coated on microtiter plates also induced proliferation in the human T cell clones. Removal of any monocytes by treatment of human T cell clones with anti-CD14 in conjunction with C, followed by purification over a nylon wool column, did not abrogate proliferation. After prolonged culture of the T cell clones in plates coated with peptide-pulsed HLA DR4Dw4 in the presence of IL-2, the T cell clones continued to proliferate in response to peptide. These results suggest that human T cell clones do not require a second signal from a monocyte or other APC to proliferate.
Cyclosporin A and FK506 mediate differential effects on T cell activation in vivo.
Authors: Bishop D K , Li W
Published: December 2022

Modified limiting dilution analysis techniques were used to evaluate the effects of the immunosuppressants cyclosporin A (CsA) and FK506 on alloantigen-induced T cell activation in vivo. Treatment of sponge matrix allograft recipients with either CsA or FK506 inhibited lymphocytic infiltration of the allograft, a process thought to be dependent on local lymphokine production. In addition, both immunosuppressants markedly reduced the absolute number of lymphocytes recovered from the draining lymph nodes (LN) and prevented CTL activation in the LN. However, Ag-primed helper T lymphocytes (HTL) were present in the draining LN of sponge allograft recipients treated with CsA, but not in recipients treated with FK506. T cell depletion experiments were performed to determine the phenotype of primed HTL in the LN of untreated and CsA-treated sponge allograft recipients. In untreated sponge allograft recipients, CD4+ and CD8+ Ag-primed HTL were present in the draining LN in equivalent numbers. In contrast, the majority of primed HTL in the LN of CsA-treated sponge allograft recipients were CD8+, rather than CD4+ T cells. These observations indicate that CsA and FK506 exert distinct in vivo effects at the level of HTL priming, and CD4+ and CD8+ HTL exhibit differential sensitivity to CsA in vivo.
Allogeneic chimerism in scid mice after neonatal transfer of bone marrow.
Authors: Cowing C , Gilmore G L
Published: December 2022

Allogeneic chimeras are valuable tools for studies of complex immune cell interactions in vivo. Mice with severe combined immune deficiency (scid) should be ideal hosts for chimerism with allogeneic bone marrow cells as these animals lack mature T and B lymphocytes capable of reacting against donor alloantigens. However, it has been difficult to achieve full reconstitution of adult scid mice even using coisogenic bone marrow grafts without prior irradiation of the recipient. We explored ways to generate complete reconstitution of scid mice with allogeneic bone marrow. Unirradiated adult scid recipients of allogeneic bone marrow were only marginally reconstituted. Adult scid mice pretreated with 250 R were reconstituted with allogeneic bone marrow as measured by serum IgM concentration, peripheral lymphoid cellularity, and mitogen responses, but a potentially important immunologic deficit was found in these mice: 250 R caused a 70% loss of scid macrophages and dendritic cells which persisted at least 5 months. By contrast, when scid mice were injected i.p. with allogeneic bone marrow within the first 24 h after birth, rapid and complete reconstitution of both T and B cell lineages was achieved, and the animals had APC that were both donor and host in origin. Considering the extent and duration of engraftment (43 wk) by allogeneic cells in neonatally transplanted scid mice, it was anticipated that their bone marrow would be chimeric. However, the bone marrow contained very few donor-derived cells, suggesting that lymphopoiesis may be taking place in other organs in these chimeras.
Stimulation of human monocytes via CD45, CD44, and LFA-3 triggers macrophage-colony-stimulating factor production. Synergism with lipopolysaccharide and IL-1 beta.
Authors: Gruber M F , Webb D S , Gerrard T L
Published: December 2022

Stimulation of human monocytes with immobilized mAb directed against the CD45, CD44, or LFA-3 Ag induced the production of macrophage-CSF (M-CSF). M-CSF-specific transcripts appeared at 3 h poststimulation, were further increased by 12 h, and were still detectable at 24 h. M-CSF gene expression was accompanied by the induction of small but detectable amounts of M-CSF protein. LPS and IL-1 beta, but not IL-6 or TNF-alpha, dramatically augmented the ability of anti-CD45, anti-CD44, and anti-LFA-3 antibodies to induce M-CSF secretion but failed to stimulate M-CSF secretion in the absence of antibody. M-CSF activity in the culture supernatants was first detectable at 8 h of culture, peaked at day 2, and declined thereafter. Purified F(ab)2 fragments of anti-CD45 antibody were also effective in inducing M-CSF message and secretion, indicating that the Fc gamma RII is not involved in this response. Stimulation of cells with antibodies to the monocyte surface Ag MAC-1, LFA-1, and ICAM-1 did not result in M-CSF secretion. Moreover, LPS and IL-1 beta failed to synergize with these Ag in inducing M-CSF production. Together, these results indicate that stimulation of monocytes via the cell surface Ag CD45, CD44, and LFA-3 can trigger M-CSF production. However, second signals that can be provided by IL-1 beta or LPS are required to regulate optimal M-CSF protein secretion.
Immunization with antigen bound to bispecific antibody induces antibody that is restricted in epitope specificity and contains antiidiotype.
Authors: Snider D P
Published: December 2022

Mice can be efficiently immunized in the absence of adjuvant using chemically cross-linked bispecific antibody (biAb) that bind to both class II MHC molecules and a protein Ag of interest. In our experiments, mice were immunized with the protein Ag hen egg lysozyme (HEL) bound to several different biAb, each of which contained a different mAb specific for a distinct (nonoverlapping) epitope of HEL. Primary and secondary serum antibody responses of the immunized mice were analyzed for their specificity for different epitopes of HEL. The results show that immunization with each HEL-biAb complex produced a bias in the epitope specificity of the primary antibody response. This bias was determined by the individual specificity of the anti-HEL mAb used in each biAb. The primary response was dominated by antibody reacting with epitopes distinct from that bound by the mAb in the immunizing complex, and was deficient in antibody that recognized the epitope bound by the biAb during immunization. This bias in antibody specificity was maintained during the secondary antibody response that followed a single challenge with soluble HEL alone. However, an additional challenge with HEL induced a switch in the specificity pattern, with increased amounts of antibody against the epitope that was previously ignored. In addition, immunization with Ag bound to biAb resulted in a substantial primary anti-Id response, detected by serum antibody specific only for the Fab'2 fragment of the mAb used in the biAb. These studies illustrate two unique features of immunization using biAb that allow for fine manipulation of the epitope specificity and anti-Id repertoires of the antibody response to whole protein Ag.
Comparative study of the T cell response to two allelic forms of a malarial vaccine candidate protein.
Authors: Rzepczyk C M , Csurhes P A , Saul A J , Jones G L , Dyer S , Chee D , Goss N , Irving D O
Published: December 2022

T cell responses to two allelic forms of the merozoite surface Ag 2 (MSA2) of Plasmodium falciparum were mapped in mice using the rMSA2 proteins, Ag 1609 which has the sequence of the FCQ27/PNG strain and Ag 1615 which has the sequence of the Indochina 1 strain. Lymph node cells of BL/10 and B10.BR mice immunized with either Ag 1609 or Ag 1615 responded to both Ag in in vitro proliferation assays. Lymph node cells of BALB/c mice did not respond. The T cell determinants recognized by the responder strains were mapped to conserved and variant regions of these Ag using overlapping synthetic peptides. The determinants recognized by each mouse strain were distinct. Marked difference in sequence between the central regions of the two rMSA2 proteins did not affect antigenic processing of the conserved N and C terminal regions. Hence lymph node cells of BL/10 mice immunized with either Ag 1615 or Ag 1609 recognized an immunodominant T cell determinant at the highly conserved N terminal end within the sequence YSNTFINNAYNMSIR (peptide 3b) and B10.BR mice similarly immunized recognized an immunodominant determinant at the highly conserved C terminal within the sequence CTDGNKENCGAATSL (peptide 23). Several peptides identified as containing immunodominant T cell determinants specific to BL/10 mice induced peptide-specific T cells in both BL/10 and B10.BR mouse strains when used as immunogens. However, the ability of the peptide-primed T cells to proliferate in response to the rMSA2 proteins was confined to BL/10 mice. An example of this was observed with peptides 3b and N (KNESKYSNTFINNAYNMSIRRSMAN). Peptide N was able to prime B10.BR and BL/10 mice for an enhanced antibody response when these mice were subsequently immunized with Ag 1615 even though Ag 1615-specific T cell proliferation was not detected in B10.BR mice primed with N. The study concluded that 1) conserved sequences such as peptide N when used in vaccines may give rise to MSA2-specific memory Th cells amenable to boosting by subsequent exposure to all parasite strains and 2) peptide priming may be a useful pathway for inducing defined memory Th cells in a wider population and for preferentially inducing T dependent over T independent responses to some malarial Ag.
Two regions of the H-2 Dd promoter are responsive to dimethylsulfoxide in line 1 cells by a mechanism distinct from IFN-gamma.
Authors: Cerosaletti K M , Woodward J G , Lord E M , Frelinger J G
Published: December 2022

The line 1 lung carcinoma is a spontaneous BALB/c tumor deficient in class I Ag expression at the protein and mRNA levels. Exposure of line 1 cells to 3% DMSO or IFN-gamma increases class I Ag protein and mRNA dramatically. We have examined the regulation of class I Ag induction by DMSO in line 1 cells. We found DMSO induces class I Ag expression in line 1 cells by a mechanism distinct from IFN, because the kinetics of class I Ag induction by these agents were dramatically different, 7 days vs 3 days, and DMSO did not act through an IFN second messenger. At the molecular level, class I H chain transcription in line 1 cells was low. Treatment with 3% DMSO or IFN-gamma increased H chain transcription four-fold and sevenfold, respectively, indicating that class I H chain expression is regulated at the level of transcription in line 1 cells. Using reporter gene constructs, we mapped the regions in the Dd H chain promoter that increase H chain expression after DMSO treatment of line 1 cells. Two regions of the Dd promoter, D1, from -210 to -133 bp, and D2, from -125 to -61 bp, were found to be independently responsive to DMSO. These regions were also responsive to IFN-gamma in line 1 cells. However, consistent with our cellular results, DMSO and IFN induction of class I H chain expression differed at the molecular level as determined by D1 point mutations that diminished IFN-gamma responsiveness but did not alter induction by DMSO. Thus, DMSO appears to regulate class I transcription through multiple regions of the class I H chain promoter in line 1 cells by a mechanism distinct from IFN-gamma.
Analysis of the AP-1 sites in the IL-2 promoter.
Authors: Jain J , Valge-Archer V E , Rao A
Published: December 2022

We have investigated the role of the two AP-1 sites, located at approximately -150 and -180 bp relative to the transcription start site, in induction of the IL-2 promoter through the TCR/CD3 complex. We show that only the proximal (-150 bp) AP-1 site is functional in vitro, as judged by its ability to bind nuclear proteins from T cells stimulated with Ag or anti-CD3 epsilon. The inducible nuclear proteins binding to this site have the characteristics of AP-1, as judged by their kinetics of induction, the ability to compete and be competed efficiently by a metallothionein AP-1 site oligonucleotide, and their reaction with antibodies to Fos and Jun proteins. Mutations in the proximal AP-1 site greatly diminish or abrogate induction of the IL-2 promoter, indicating that the site is also functional in vivo. Although the distal (-180 bp) AP-1 site is incapable of direct binding to nuclear proteins from activated T cells, a mutation in this site diminishes IL-2 promoter induction, suggesting that this site may also be functional in vivo. Cotransfection of a 5' IL-2-chloramphenicol acetyltransferase plasmid with c-Fos and/or c-Jun enhances the induction of IL-2-chloramphenicol acetyltransferase activity, confirming that the IL-2 promoter contains a functional AP-1 site. Both AP-1 sites may be targets for c-Fos action, as inferred from the results of experiments in which c-Fos was cotransfected with internal deletion mutants of the IL-2 promoter lacking either AP-1 site. Northern analysis indicates that mRNAs for at least six members of the Fos/Jun family (c-fos, fosB, fra-1, c-jun, junB, and junD) are expressed in activated Ar-5 cells; thus the AP-1 sites of the IL-2 promoter may bind different dimeric Fos/Jun complexes at different times after T cell activation, perhaps mediating both positive and negative regulation of the IL-2 promoter.
Gene transfer demonstrates that the V gamma 1.1C gamma 4V delta 6C delta T cell receptor is essential for autoreactivity.
Authors: Kikuchi G E , Roberts K , Shevach E M , Coligan J E
Published: December 2022

Murine T cell lines and hybridomas derived from the epidermis that express the V gamma 1.1C gamma 4V delta 6C delta TCR and may, therefore, recognize an autoantigen, secrete cytokines spontaneously in culture. In addition, activation of these cells requires engagement of the vitronectin receptor (VNR) by extracellular matrix proteins. To further evaluate the role of the TCR, the VNR, and the putative autoantigen in the activation of this T cell subset, we cloned complete cDNA encoding the V gamma 1.1C gamma 4 and V delta 6C delta TCR and transfected the cDNA constructs into a TCR- murine hybridoma and into a TCR- variant of the human Jurkat line. The murine transfectant spontaneously produced IL-2 in culture and IL-2 production could be inhibited by anti-CD3, anticlonotypic mAb to the transfected TCR, and anti-VNR mAb, as well as by RGDS. These results demonstrate that transfection of the gamma delta TCR confers to recipient T cells the phenotype of constitutive activation, as well as dependence on engagement of the VNR as an accessory molecule. In contrast, the Jurkat gamma delta transfectant failed to produce cytokines spontaneously, although the transfected TCR was capable of signal transduction after stimulation by anti-TCR mAb. Surprisingly, neither the murine transfectant nor the human transfectant could be induced to respond to autoantigen bearing cells in coculture assays. One interpretation of these results is that coexpression on the surface of the same cell of the V gamma 1.1 V delta 6 TCR, the VNR, and a putative autoantigen are necessary for T cell activation in this system.
Monoclonal antibodies directed to different epitopes in the CD3-TCR complex induce different states of competence in resting human T cells.
Authors: Schwinzer R , Franklin R A , Domenico J , Renz H , Gelfand E W
Published: December 2022

After the initial stages of activation, T cells are not able to proliferate on their own but become competent to proliferate in response to exogenously added lymphokines. In the present study we compared the capacity of mAb directed to CD3 (OKT3, Leu4, UCHT1) or to common epitopes on the alpha/beta T-cell receptor (BMA 031, BMA 032) to induce competence in purified resting T cells. Stimulation with either soluble anti-CD3 or anti-alpha/beta TCR mAb rendered cells competent to progress to DNA synthesis in response to exogenous IL-2. In contrast, only soluble BMA 031 and BMA 032 were able to induce responsiveness to IL-4; anti-CD3 mAb had either to be immobilized or used in combination with anti-CD28 mAb to induce responsiveness to IL-4. Further, BMA 031-induced IL-4 responsiveness was selectively found in the CD45RA+ T cell subset. Analysis of early activation events revealed that the capacity of soluble BMA 031 and BMA 032 to induce responsiveness to IL-4 did not correlate with the ability of these mAb to increase the level of cytosolic Ca2+ or to induce detectable tyrosine phosphorylation. On the other hand, soluble Leu4 (anti-CD3) triggered an increase in both intracellular Ca2+ and tyrosine phosphorylation but was unable to induce IL-4 responsiveness. These data indicate that the induction of IL-2 and IL-4 responsiveness requires different sets of activation signals which can be induced by stimulating different epitopes in the CD3-TCR complex. This supports the concept that distinct activation pathways are coupled to the CD3-TCR complex.
Regulation of ICAM-1 expression and function in human dermal fibroblasts by IL-4.
Authors: Piela-Smith T H , Broketa G , Hand A , Korn J H
Published: December 2022

ICAM-1 is found on the surface of many hematopoietic and nonhematopoietic cells and can function as an adhesive ligand for the integrin, leukocyte function-associated molecule-1 (LFA-1, CD11a/CD18). ICAM-1/leukocyte function-associated molecule-1 interaction has been shown to be of importance in many immune-mediated cell-cell adhesion reactions. In vitro, unstimulated human fibroblast cell cultures express low levels of ICAM-1. Using ELISA, cytofluorography, electron microscopy, Northern analysis, and an in vitro cell adherence assay, we demonstrate that treatment of human dermal fibroblasts with the cytokine IL-4 leads to an increase in cell surface ICAM-1 expression that is under transcriptional control as well as increased fibroblast adhesion to LFA-1-bearing T lymphocytes. The kinetics of increased ICAM-1 expression induced by IL-4 paralleled the increase in ICAM-1-dependent T lymphocyte adhesion. The increase in T cell adhesion was determined to be due to the effects of IL-4 on the fibroblasts and not the adhering T cells. Treatment of fibroblasts with IL-4 also resulted in enhanced binding of human rhinovirus, a recently reported additional ligand for ICAM-1. Virus binding was IL-4 dose dependent and could be inhibited with mAb to ICAM-1. Both the expression of ICAM-1 and the ICAM-1-dependent increase in T lymphocyte adhesion that was induced by IL-4 could be inhibited by preexposure of the fibroblasts to either IL-1 or IL-6, suggesting that multiple cytokines can have both positive and negative effects on human fibroblast ICAM-1 expression and function.
Comparison of autoantibodies to the collagen-like region of C1q in hypocomplementemic urticarial vasculitis syndrome and systemic lupus erythematosus.
Authors: Wisnieski J J , Jones S M
Published: December 2022

Hypocomplementemic urticarial vasculitis syndrome (HUVS) is an apparent autoimmune disorder that resembles SLE. We previously showed that C1q precipitins in HUVS sera are IgG autoantibody to human C1q. We have compared HUVS anti-C1q autoantibody to a similar autoantibody in the serum of some patients with SLE. As with anti-C1q autoantibody in SLE sera, the HUVS autoantibody binds only to the collagen-like region (CLR) of C1q. In both HUVS and SLE, IgG2 is the predominant subclass of IgG autoantibody and IgM autoantibody to C1q is uncommon. In both diseases, anti-C1q autoantibodies bind preferentially to surface-adsorbed C1q or CLR fragments compared to these antigens in solution. Finally, when HUVS or SLE autoantibodies were added to CLR-coated wells already bound, respectively, by SLE or HUVS autoantibodies, no increases in CLR binding were observed, suggesting that HUVS and SLE autoantibodies to C1q bind to the same CLR epitope(s).
Inhibition of apoptosis by calcium ionophores in IL-3-dependent bone marrow cells is dependent upon production of IL-4+.
Authors: Rodriguez-Tarduchy G , Malde P , Lopez-Rivas A , Collins M K
Published: December 2022

Calcium ionophores inhibit apoptosis in the IL-3-dependent cell line BAF3 and maintain the cells in a viable noncycling state. In this report, an identical effect of ionophore was also demonstrated on the multipotent IL-3-dependent progenitor cell line FDCP-MIX and on the primary IL-3-dependent cell population that could be cultured from murine bone marrow. Inhibition of apoptosis required extracellular calcium and could be blocked by cyclosporin A. Nuclei from IL-3-dependent cells were found to lack a calcium-activatable nuclease that degrades chromatin in the linker region between nucleosomes, unlike the nuclei of lymphoid cells. The mechanism of action of calcium ionophore could be divided into two distinct steps. First, ionophore induced the production of a survival factor that stimulated DNA synthesis and was identified as IL-4. Second, ionophore inhibited the cell cycle of the various IL-3-dependent cells. IL-4 production could be inhibited by cyclosporin A and required extracellular calcium, whereas cell cycle arrest did not. This implied that factor production was the step that was necessary for inhibition of apoptosis and maintenance of cell viability. This was confirmed by the use of an anti-IL-4R antibody, which blocked the inhibition of apoptosis induced by calcium ionophores.
Synthetic peptides anchor T cell-specific TNP epitopes to MHC antigens.
Authors: Ortmann B , Martin S , von Bonin A , Schiltz E , Hoschützky H , Weltzien H U
Published: December 2022

Several TNP-specific, H-2Kb-restricted mouse CTL clones were identified which specifically lysed target cells in the presence of tryptic digests of TNP-modified BSA. Glutaraldehyde fixation of cells revealed that the tryptic fragments did not require further cellular processing. Chromatographic fractionation of digested TNP-BSA identified the peptide TNP-BSA222-231, containing a TNP-modified lysine at BSA position 227, as the antigenic entity. The corresponding synthetic peptide was immunologically cross-reactive with the digest. All clones reactive with TNP-BSA222-231 cross-reacted with a similar peptide from mouse serum albumin (TNP-MSA126-135), favoring the assumption that TNP-BSA222-231 represents an artificial determinant, cross-reacting with some as yet unidentified, TNP-modified, Kb-associated self-peptides. Some of our clones also cross-reacted with tryptic digests of TNP-OVA or TNP-keyhole limpet hemocyanin. We interpret these findings to indicate that 1) a significant proportion of hapten (TNP) determinants for T cells are anchored to MHC via peptides; and 2) the amino acid sequence of these peptides may only partly define the specificity of the T cell-relevant hapten epitope, implying a particularly repetitive nature of these determinants. The production of T cell-antigenic hapten-peptide conjugates will hopefully open new roads to study immune responses to environmental allergens.
Antigen-specific (p30) mouse CD8+ T cells are cytotoxic against Toxoplasma gondii-infected peritoneal macrophages.
Authors: Kasper L H , Khan I A , Ely K H , Buelow R , Boothroyd J C
Published: December 2022

The importance of CD8+ T cells in immunity against Toxoplasma gondii is now well recognized. The mechanism by which these CD8+ T cells are able to confer this immunity is not yet understood. To examine the Ag specificity of this response, immune splenocytes from mice immunized with p30, a major surface parasite Ag, were evaluated for their ability to lyse peritoneal macrophages infected with three different strains of T. gondii. Macrophages infected with either the RH or P wild-type strain tachyzoites were lysed at varying E:T ratios by nylon wool nonadherent immune splenocytes whereas macrophages infected with a p30-deficient mutant (B mutant) of the P strain were not. The gene encoding p30 for the wild type and B mutant were amplified by the polymerase chain reaction. This revealed a nonsense mutation in the B mutant such that its primary translation product is predicted to be about two-thirds the size of the wild-type p30 molecule. mAb depletion studies indicate that the cytotoxic effect of the immune splenocytes is mediated by the CD8+ T cell population. Peritoneal macrophages infected with the three different strains (RH, P wild type, B mutant) from mice genetically restricted were not lysed by the immune CD8+ effector cell population. A cloned line (C3) of p30 Ag-specific CD8+ T cells exhibited significant cytotoxicity against syngeneic peritoneal macrophages infected with either the RH or P strain tachyzoites. There was no macrophage lysis observed by these CD8+ effector cells of either syngeneic macrophages infected with the B mutant or nonsyngeneic macrophages infected with the three different tachyzoite strains.
Pertussis toxin-induced lymphocytosis is associated with alterations in thymocyte subpopulations.
Authors: Person P L , Korngold R , Teuscher C
Published: December 2022

Pertussis toxin (Ptx), an important adjuvant for inducing certain organ-specific autoimmune diseases in mice, exerts multiple effects upon the immune system. In addition to its adjuvant effects, which include enhancement of delayed-type hypersensitivity and increased antibody production. Ptx elicits a marked lymphocytosis with a concomitant decrease in thymic weight. In vitro studies indicate that Ptx acts directly on thymocytes and that both susceptible and resistant populations exist. It is believed that these susceptible cells are released into the circulation and account, in part, for the T cell component of the lymphocytosis. We have used flow cytometry to analyze the CD4, CD8, and Thy-1 phenotypes of thymic and peripheral T cells from Ptx-treated mice. In the thymus, there is a dramatic decrease in the number of CD4+CD8+ (double positive) cells at all doses tested (0.25, 0.50, and 1.0 microgram) by day 4 after Ptx treatment. The double negative and single positive populations remain relatively constant. Analysis of Thy-1 expression reveals a significant reduction in Thy-1hi thymocytes, with little change in the Thy-1lo population. Thus Ptx primarily affects and depletes, in a dose-dependent fashion, thymic T cells with an immature phenotype. These results mimic those of corticosteroids, although neither prior adrenalectomy nor treatment with the antiglucocorticoid RU486 are able to prevent the effects of Ptx. In the periphery of Ptx-treated animals, the relative increase in the number of CD4+ T cells is more than that of CD8+ T cells. Double positive and Thy-1hi cells cannot be detected in appreciable numbers. These results are consistent with the concept that Ptx may drive immature thymocytes through accelerated maturation for release into the periphery as single positive, predominantly CD4+, Thy-1lo cells. Increased numbers of such cells may in part account for the immunopotentiating effects of Ptx, particularly as they relate to the induction of organ-specific autoimmune disease. Treatment with purified Ptx beta-oligomer fails to elicit any of the responses described above, indicating that the holotoxin is required for such activities.
Molecular cloning and expression pattern of a human gene homologous to the murine mb-1 gene.
Authors: Ha H J , Kubagawa H , Burrows P D
Published: December 2022

The mouse mb-1 gene was originally identified based on its restricted expression in B lineage cells. Predicted structural homology with the gamma chain of the CD3 complex on T cells led to the suggestion that the MB-1 protein might associate with surface Ig(sIg) on B cells and be involved in signal transduction. Other studies identified at least two proteins that are noncovalently associated with sIgM, one of which has recently been shown to be the product of the mb-1 gene. To identify genes specifically expressed in normal human B cells we constructed a B minus T lymphocyte subtraction library and isolated a cDNA clone highly homologous to murine mb-1 (m-mb-1). A full-length cDNA was subsequently isolated and found to encode a membrane glycoprotein of 226 amino acids. It included a leader sequence (32 amino acids), an extracytoplasmic domain (111 amino acids) containing six potential N-glycosylation sites and three cysteine residues for potential inter- or intrachain disulfide linkages, a transmembrane domain (22 amino acids), and an intracytoplasmic domain (61 amino acids). The amino acid sequence homology between human and mouse mb-1 was especially striking (approximately 92%) in the intracytoplasmic, transmembrane, and membrane-proximal extracellular domains but was less marked (approximately 42%) in the remaining extracytoplasmic portion. Interestingly, part of the 3'-untranslated region was also highly conserved between species, suggesting an important role for this region in the regulation of mb-1 expression. The human mb-1 (h-mb-1) cDNA hybridized with a mRNA species of approximately 1.2 kb on Northern blots. Similar to m-mb-1, the h-mb-1 transcripts could be detected in pre-B cell lines and fetal bone marrow, in normal, mitogen activated- and transformed B cells but not in myeloma plasma cells. h-mb-1 was not expressed in peripheral T cells nor by cells of other hemopoietic lineages or in brain, heart, muscle, lung, and kidney. Surprisingly, however, low levels of h-mb-1 transcripts were detectable in two early T lineage cell lines and in the fetal thymus. This suggests that mb-1 may have other functions in addition to its role in signal transduction in B lineage cells.
Formation of a bispecific antibody by the use of leucine zippers.
Authors: Kostelny S A , Cole M S , Tso J Y
Published: December 2022

A new method is described for the production of bispecific F(ab')2 heterodimers using leucine zippers. Two heterodimer-forming "zipper" peptides derived from the Fos and Jun proteins were respectively linked to the Fab' portions of two different mAb by gene fusion. The antibodies used were 145-2C11, which binds to murine CD3, and anti-Tac, which binds to the p55 chain of the human IL-2R. Anti-Tac Fab'-Jun and anti-CD3 Fab'-Fos were expressed individually as F(ab'-zipper)2 homodimers in the mouse myeloma cell line Sp2/0. When these homodimers were reduced at the hinge region to form monomers and then reoxidized together, the resulting end products were mostly F(ab'-zipper)2 heterodimers. Bispecific anti-CD3 x anti-Tac F(ab'-zipper)2 heterodimers produced by this method were shown to be highly effective in recruiting cytotoxic T cells to lyse IL-2R-bearing HuT-102 cells in vitro.
Regulation of the expression of ICAM-1 on human monocytes and monocytic tumor cell lines.
Authors: Möst J , Schwaeble W , Drach J , Sommerauer A , Dierich M P
Published: December 2022

In this study we investigated the mechanisms regulating expression of ICAM-1 that plays an important role for Ag presentation, on peripheral blood monocytes, and three myelomonocytic cell lines representing different stages of monocyte maturation. ICAM-1 expression on freshly isolated monocytes was low in all donors tested. After 16- to 20-h incubation, about a 20-fold increase was observed, whereas ICAM-1 expression on lymphocytes did not change. This marked increase in Ag expression was specific for ICAM-1, because expression of other monocyte Ag remained unchanged (alpha-chain of lymphocyte function-associated Ag-1) or decreased (ICAM-2, alpha-chains of CR3 and CR4, their common beta-chain and Fc gamma RI) during overnight incubation; only HLA-DR showed a slight increase. Enhanced expression of ICAM-1 was not caused by adherence; it was also not due to trace amounts of IFN-gamma possibly present under our culture conditions. Endotoxin contamination of the medium was excluded as a cause for the enhanced expression, and neither LPS nor its antagonist polymyxin B were able to alter ICAM-1 expression. Whole blood culture almost completely prevented up-regulation of ICAM-1 expression, thus apparently mimicking the in vivo situation. Culture of monocytes together with other PBMC had no significant influence on monocyte ICAM-1 levels. From 12 different cytokines tested for their ability to modulate "spontaneous" up-regulation of ICAM-1 in culture, only IFN-gamma was found to increase expression about twofold. This effect was also observed in whole blood culture. All other cytokines had no significant effect. In contrast, ICAM-1 expression on two of the three cell lines (KG1 and HL60) was inducible by TNF-alpha to a much higher degree than by IFN-gamma, whereas the results with U937 were comparable with those obtained for normal monocytes. The protein- and RNA-synthesis inhibitors cycloheximide and actinomycin D prevented expression of ICAM-1 in a dose-dependent fashion. In Northern blot experiments no difference in the amounts of mRNA was observed between freshly isolated and cultured monocytes, indicating that "spontaneous" induction of ICAM-1 is regulated at a posttranscriptional level. In contrast, stimulation with IFN-gamma caused a significant increase of detectable RNA comparable to that observed for Ag expression. These results disclose a very special regulation of ICAM-1 expression on monocytes which could be consistent with the view that ICAM-1 is actively down-regulated in vivo until the monocyte leaves the circulation.