bluuebunny/crossref_metadata_2025 huggingface dataset:
Complete Freund's adjuvant-induced T cells prevent the development and adoptive transfer of diabetes in nonobese diabetic mice.
Authors: Qin H Y , Sadelain M W , Hitchon C , Lauzon J , Singh B
Published: December 2022

Insulin-dependent diabetes mellitus is an autoimmune disease that is characterized by the destruction of insulin-producing beta cells in the islet of Langerhans. We have recently reported that the induction of the disease in nonobese diabetic (NOD) mice can be prevented by a single injection of CFA. In this study, we have explored the cellular basis and the time course of the disease protection. Since CFA contains a mycobacterial cell wall that has adjuvant property, we investigated the protective role of mycobacteria in young NOD mice. Mice injected with Mycobacterium tuberculosis or Mycobacterium bovis (BCG vaccine) at 4 wk of age were also found to be protected from diabetes. We have found that complete protection from diabetes is only achieved by administration of CFA between 4 and 10 wk of age. Draining lymph node cells or spleen cells from CFA-treated NOD mice transfer the protection. Adoptive transfer of spleen cells from CFA-treated mice with spleen cells from acutely diabetic mice delayed the induction of disease into irradiated recipient mice. CFA-treated old NOD mice were also resistant to passive transfer of disease by spleen cells from acutely diabetic mice. Depletion of the Thy 1.2+ cells or CD4(+)-bearing T cells abrogated the protection. However, disease can be induced in the protected mice by cyclophosphamide treatment. We also found that thymocytes from NOD mice responded only weakly to mitogen Con A. CFA treatment, however, restored the ability of these cells to respond to Con A. Finally, our results suggest that T cells induced after CFA treatment of NOD mice prevent both the induction and effector phases of the disease.
Induction of differentiation in a B lymphoma X B lymphocyte hybrid line. II. Intraclonal heterogeneity in growth, secretion of IgM, and cytokine production in response to lipopolysaccharide.
Authors: Little J , Alling D W , Asofsky R
Published: December 2022

Murine B lymphocyte clones have proven to be useful models to study aspects of B lymphocyte growth and development. It had been previously shown that induction of TH2.2 (B lymphoma X B lymphocyte) with LPS resulted in differentiation into IgM-secreting cells (a feature similar to other B cells lines such as BCL1), and secretion of granulocyte-macrophage-CSF. Many transformed and nontransformed lines (including TH2.2) have been shown to generate progeny heterogenous in size and secretion of product when exposed to mitogen. We extend this study to demonstrate heterogeneity in secretion of IgM, granulocyte-macrophage-CSF, IL-3, and IL-6 in LPS-induced TH2.2. Clones generated by limiting dilution were heterogenous with respect to size, type, and quantity of product secreted. Microscopic clones ranged from 10 to about 1000 cells in size and could not be grown further; the majority secreted one product (mainly IgM). These clones were very efficient secretors of IgM and probably consist mainly of terminal-secreting cells. Microscopic clones secreting cytokine were also efficient producers. Visible clones from LPS-induced cultures grew to the same size as uninduced clones and were often restricted in secretion of products. Although the majority secreted three of four products (52%), many secreted IgM only or IgM and one cytokine. Although there was a strong tendency for clones to secrete multiple products, almost every secretory phenotype could be found. Amounts of different products secreted were not correlated, suggesting an additional level of independent control of this variable. Restriction of secretion was not due to genetic variation because visible clones originally restricted in secretion almost always produced all products when expanded and retested. These findings indicate that cells of TH2.2 were heterogeneous with respect to growth and secretion in the presence of LPS and were individually programmed for differentiative responses. The programming of cells was not permanent, but subject to dynamic change.
Immunosuppressive activity of [MeBm2t]1-, D-diaminobutyryl-8-, and D-diaminopropyl-8-cyclosporin analogues correlates with inhibition of calcineurin phosphatase activity.
Authors: Nelson P A , Akselband Y , Kawamura A , Su M , Tung R D , Rich D H , Kishore V , Rosborough S L , DeCenzo M T , Livingston D J
Published: December 2022

Calcineurin, a Ca2+/calmodulin-dependent phosphatase, has recently been identified as a common target for cyclophilin A-cyclosporin A and FK506 binding protein 12-FK506 complexes. This study has examined the structure activity relationships of cyclosporin A (CsA) and three functionally distinct analogues, [MeBm2t]1-CsA, D-diaminobutyryl-8-CsA (Dab8-CsA), and D-diaminopropyl-8-CsA (Dap8-CsA). Immunosuppressive potency in T cell activation models, NF kappa B activation, and IL-2 mRNA transcription has been compared with analogue affinity for cyclophilin A and inhibition of calcineurin phosphatase activity. CsA, Dap8-CsA, and Dab8-CsA bind to cyclophilin A with a similar affinity (Ki 4 to 5 nM as measured by inhibition of prolyl cis-trans isomerase activity), however, Dap8-CsA and Dab8-CsA inhibit T cell activation less than CsA. Although [MeBm2t]-CsA has weak affinity for cyclophilin A (Ki 540 nM), its immunosuppressive potency is similar to that of CsA. Both cyclophilin A-CsA and cyclophilin A-[MeBm2t]1-CsA complexes inhibit calcineurin phosphatase activity in vitro (Ki 114 and 67 nM, respectively). In Jurkat cells exposed to CsA or the analogues for 2 h, endogenous calcineurin phosphatase activity in cell lysates was inhibited by CsA and [MeBm2t]1-CsA (drug concentrations causing 50% reduction in 32PO4 release of 8 and 55 nM, respectively) in proportion to inhibition of T cell activation, IL-2 mRNA transcription, and NF kappa B activation. Dap8-CsA and Dab8-CsA had a minimal effect on endogenous calcineurin phosphatase activity in Jurkat cell lysates. These findings correlate the functional activity of CsA and structural analogues with calcineurin phosphatase activity and support calcineurin as a target for drug action. The Dap8 and Dab8 modifications of CsA, occurring in residue 8, which is exposed to solvent in the cyclophilin A-CsA complex, appears to significantly alter complex affinity for calcineurin.
Accessory cell function of keratinocytes for superantigens. Dependence on lymphocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction.
Authors: Nickoloff B J , Mitra R S , Green J , Zheng X G , Shimizu Y , Thompson C , Turka L A
Published: December 2022

A growing body of evidence points to a role for epidermal keratinocytes as active participants in immunologic reactions. Inasmuch as certain T cell-mediated skin diseases, such as psoriasis and atopic dermatitis, are triggered by microbial infection, we asked whether multipassaged human keratinocytes could provide the costimulatory signals necessary to induce autologous T cell proliferation in response to bacterial-derived super-antigens. On exposure to IFN-gamma, keratinocytes are induced to express HLA-DR and HLA-DQ class II MHC Ag, and the lymphocyte function-associated Ag-1 counter-receptor intercellular adhesion molecule-1 (ICAM-1). This change in keratinocyte phenotype is accompanied by the ability of these cells to support T cell proliferation induced by two different bacterial-derived superantigens, staphylococcal enterotoxins A and B. Superantigen-driven proliferation in the presence of IFN-gamma-treated keratinocytes was significantly inhibited (70-90% reduction) by mAb against the LFA-1 alpha- or beta-chain or ICAM-1. Proliferation was not inhibited by mAb against the CD28 ligands BB-1 or B7, even though these keratinocytes express BB-1. In addition to previous defined roles for class II MHC Ag, stimulation of LFA-1 on the T cells by ICAM-1 on the keratinocytes also plays an important costimulatory role in this superantigen-mediated response. The accessory cell capability of keratinocytes was not unique to superantigen driven responses as PHA, as well as anti-CD3 mAb also induced vigorous T cell proliferation when IFN-gamma-treated keratinocytes were added. However, IFN-gamma-treated keratinocytes consistently failed to provoke an allogeneic response. These data demonstrate that 1) keratinocytes can serve as accessory cells for T cell proliferation using a variety of different stimuli, 2) the LFA-1/ICAM-1 interaction plays a major role in keratinocyte-mediated costimulation, and 3) previous reports in which IFN-gamma-treated keratinocytes failed to support T cell proliferation to nominal or alloantigens, may reflect impaired Ag presentation via class II MHC molecules, rather than lack of necessary costimulatory signals. These findings highlighting the accessory cell function of keratinocytes may have implications for our understanding of the pathogenesis of immunologic disorders of the skin.
Peptide epitope induced apoptosis of human cytotoxic T lymphocytes. Implications for peripheral T cell deletion and peptide vaccination.
Authors: Suhrbier A , Burrows S R , Fernan A , Lavin M F , Baxter G D , Moss D J
Published: December 2022

Cloned CTL can be induced to undergo apoptosis with cognate peptide epitopes. This phenomenon has been attributed to self recognition and self destruction of individual, isolated CTL and/or Ag-induced cell death. It has been claimed that these mechanism are responsible for extrathymic elimination of primed CTL. In our study the contribution of all known effector mechanisms (single cell self killing, CTL-CTL killing, backward killing, bystander killing, Ag-induced cell death, and veto) to peptide epitope-induced destruction of CTL was evaluated. CTL-CTL killing was found to be the dominant mechanism, with a significant contribution from bystander killing. CTL were as susceptible to peptide-mediated lysis as conventional target cells. CTL, which were actively killing target cells, were not refractory to the lytic mechanisms of other CTL but paradoxically, avoid destruction by their own lytic mediators when delivering the lethal hit. These data imply that mature primed CTL are not deleted in the periphery as a direct result of the lysis of target cells, although, under certain circumstances peripheral CTL-CTL killing may be envisaged when cognate peptide is used for vaccination.
Intestinal intraepithelial lymphocytes are activated and cytolytic but do not proliferate as well as other T cells in response to mitogenic signals.
Authors: Sydora B C , Mixter P F , Holcombe H R , Eghtesady P , Williams K , Amaral M C , Nel A , Kronenberg M
Published: December 2022

Compared with T lymphocytes from other organs, intestinal intraepithelial lymphocytes (IEL) proliferate weakly in response to CD3/TCR ligation, and they do not respond at all to treatment with other mitogenic stimuli. These signals also failed to induce expression of the IL-2R alpha-chain on the surface of most IEL. IEL from germ-free mice, from V gamma 1.1-transgenic mice, and from beta 2-microglobulin-deficient mice also gave a weak proliferative response. Therefore, the low proliferative response is not linked to the level of exposure to gut bacterial flora, the V gamma region expressed by the TCR-gamma delta + IEL, or the presence of class I molecules that may be recognized by CD8+ IEL. The relatively small amount of proliferation in response to TCR signaling, therefore, is not likely to be the result of induction of anergy caused by previous contact with Ag. In contrast, ligation of the CD3/TCR complex could elicit a rapid cytotoxic response and serine esterase release by IEL. The unusual functional capabilities and the activation state of IEL are independent of the TCR isotype expressed by these cells. Freshly isolated IEL have a high intracellular microtubule-associated protein kinase-2 (MAP-2K) activity level, further suggesting that these cells are activated despite their weak proliferative response. Consistent with this, MAP-2K is tyrosine-phosphorylated in both untreated and PMA-treated IEL. In contrast, MAP-2K activation and tyrosine phosphorylation occur in other T cells only when they are activated by PMA or other treatments. MAP-2K activity also is elevated in IEL from germ-free mice, demonstrating that activation does not depend on normal levels of exposure to bacterial flora. The activation of protein kinases such as MAP-2K could reflect the differentiation state of IEL or Ag receptor stimulation of some of these cells by epithelial cells in the preparation.
V(D)J recombination generates a high frequency of nonstandard TCR D delta-associated rearrangements in thymocytes.
Authors: Carroll A M , Slack J K , Mu X
Published: December 2022

The standard products of V(D)J recombination are coding junctions, which encode Ag receptor polypeptides, and their commonly excised reciprocal products, signal junctions. Additional nonstandard products also have been detected, mostly in artificial recombination substrate studies. The occurrence of nonstandard products, including pseudonormal, hybrid, and open/shut junctions, indicates significant indeterminacy of the V(D)J recombinase. However, the incidence of nonstandard products for endogenous Ag receptor genes in vivo has not been specifically addressed. The data presented here show that for the TCR-delta locus, D element-associated recombination in mouse thymocytes results in a high incidence of nonstandard recombination products. D delta 1-D delta 2 rearrangements, both chromosome retained and excised episomal products, were studied by polymerase chain reaction amplification, cloning, and sequence analysis. The proximity of D delta 1 and D delta 2 elements, and the fact that both are flanked by 5' and 3' recombination signal sequences with 12-bp and 23-bp spacers, respectively, results in frequent pseudonormal joining. The resulting products are signal junctions retained on the chromosome. Excised episomal products include coding junctions, hybrid junctions formed in apparent violation of the 12/23 spacer rule, and standard signal junctions; some signal junctions show evidence of imprecise cleavage. Evidence for open/shut and/or oligonucleotide capture events was also seen. Similar rearrangements were detectable in thymocytes of mutant scid mice. These findings indicate a high degree of indeterminacy of V(D)J recombinase-mediated D delta 1-D delta 2 rearrangement in both wild-type and scid thymocytes. This indeterminacy affects the productive potential of TCR-delta loci.
Multiple B29 containing complexes on murine B lymphocytes. Common and stage-restricted Ig-associated polypeptide chains.
Authors: Ishihara K , Wood W J , Wall R , Sakaguchi N , Michnoff C , Tucker P W , Kincade P W
Published: December 2022

The B29 gene is specifically expressed in all cells of the B lymphocyte lineage, and the B29 protein is disulfide-linked to the protein product of at least one other gene, known as mb-1. The noncovalent association of these heterodimers with Ig H chains is thought to be required for surface expression and signal transmission by B cell Ag receptors. We now demonstrate by two-color immunofluorescence a direct correlation between B29 density and surface Ig expression on normal spleen and bone marrow cells. The proportion of B29 in Ag receptor complexes appears to be relatively constant across major B lymphocyte subpopulations. Multiple B29-containing heterodimers were resolved on normal spleen cells by surface labeling, immunoprecipitation, two-dimensional gel analysis, and immunoblotting. As with lymphoma cells in our earlier study, the conditions of detergent extraction were critical to detection of certain species. Many laboratories have observed a family of 69- to 85-kDa heterodimers that are extracted with digitonin. These species are clearly Ig-associated, and are coprecipitated with anti-Ig antibodies. We found that extraction with Triton X-100 revealed an additional pair of 52- to 58-kDa heterodimers, where B29 was disulfide-bonded to a protein of approximately 23 kDa. The latter was detectable by immunoblotting with antibodies to extracellular, but not cytoplasmic, portions of mb-1. We found that, with mature cells, both conventional and low molecular mass heterodimers were solubilized with digitonin, but only detectable if Triton was present during immunoprecipitation. Thus, a protein having partial serologic identity with mb-1 forms heterodimers that are cryptic on splenic B cells, and possibly not directly associated with surface Ig molecules. In contrast, both types of heterodimers were readily detectable on late stage pre-B cells, regardless of detergent used for extraction or antibody used for immunoprecipitation. In that situation, both low- and high molecular mass heterodimers were associated with surface Ig. These findings increase our understanding of the B lymphocyte Ag receptor complex and indicate that its components may change as a function of differentiation.
IFN-gamma modulation of epithelial barrier function. Time course, reversibility, and site of cytokine binding.
Authors: Adams R B , Planchon S M , Roche J K
Published: December 2022

The single cell-thick intestinal epithelium forms a crucial barrier between the host and environment, and is modeled in vitro by a monolayer of polarized, highly differentiated T84 epithelial cells impermeable to most macromolecules because of functional intercellular tight junctions. Absence of a permeability defect across the monolayer, either transcellular or paracellular, is indicated by development of a transepithelial electrical resistance of > or = 1000 ohm-cm2, reported to be markedly diminished by exposure to a T lymphocyte cytokine, IFN-gamma. We sought to define this phenomenon in four ways by determining its duration and reversibility; the uniqueness of type II (gamma) IFN as opposed to type I (alpha) IFN; the surface of the polarized columnar epithelium likely involved in responding to IFN-gamma; and whether a specific surface membrane receptor on the epithelial cell participates in the response. Using a special apparatus that allows differential cytokine exposure of monolayer surfaces, our data demonstrate 1) only the monolayer's basolateral surface is IFN-gamma responsive, whereas the apical (microvillous) surface is no; 2) the alteration in electrical resistance of epithelium is prolonged (5 days), even after a single (24 h) exposure to IFN-gamma, but nevertheless is reversible; 3) the effect is likely receptor-ligand mediated, because it can be partially blocked by IFN-gamma receptor-specific monoclonal Ig; 4) an alteration in tight junction function (a paracellular pathway) rather than cell necrosis or a transcellular pathway is responsible for IFN-gamma-induced monolayer dysfunction because permeability to a 44,000-Da macromolecule (horseradish peroxidase) did not increase, and intracytoplasmic T84 cell enzymes were not released into the media; and 5) the biologic phenomenon could not be induced by a species (alpha) of class I IFN, making IFN-gamma reasonably unique in this regard. Given the proximity; activation status, and capacity of T lymphocytes for cytokine production in mucosa, we suggest that IFN-gamma-induced changes in epithelial permeability may be a major cause of altered intestinal barrier function in vivo.
Culture of human monocytes with granulocyte-macrophage colony-stimulating factor results in enhancement of IFN-gamma receptors but suppression of IFN-gamma-induced expression of the gene IP-10.
Authors: Finbloom D S , Larner A C , Nakagawa Y , Hoover D L
Published: December 2022

The initiation and promulgation of chronic inflammation are controlled in part by the various pro-inflammatory and anti-inflammatory cytokines present at the site of injury. IFN-gamma and granulocyte-macrophage CSF (GM-CSF) are two cytokines that can contribute to the inflammatory state and possess both pro- and anti-inflammatory properties. However, the characterization of the interaction between GM-CSF-cultured monocytes and IFN-gamma is poorly documented. In this report we show that culture of human peripheral blood monocytes for up to 6 days in the presence of GM-CSF results in an eightfold increase in the level of IFN-gamma R expression, as determined by radioligand binding. The IFN-gamma R on these cells maintains a specificity typical of that observed in fresh monocytes. Only IFN-gamma, not IFN-alpha or -beta, blocks the binding of IFN-gamma to its receptor, and anti-IFN-gamma R antibodies block at least 80% of binding of IFN-gamma to these cultured cells. However, in spite of increased receptor expression, GM-CSF-cultured monocytes have a diminished response to IFN-gamma, as measured by the induction of the gene for IP-10 (a member of the platelet factor-4/IL-8 family). On the other hand, IFN-gamma-induced activation of the DNA-binding protein FcRF gamma is maintained in GM-CSF-cultured monocytes. Therefore, suppression of IFN-gamma-mediated IP-10 induction is not the result of a global abrogation of signal transduction across the IFN-gamma R but a more selective inhibition that appears to occur downstream of the receptor.
Lung surfactant suppresses oxygen-dependent bactericidal functions of human blood monocytes by inhibiting the assembly of the NADPH oxidase.
Authors: Geertsma M F , Broos H R , van den Barselaar M T , Nibbering P H , van Furth R
Published: December 2022

Surfactant is known to lower the surface tension in alveoli and affects the antibacterial functions of alveolar and peritoneal macrophages. We investigated the effects of surfactant on the bactericidal functions and oxidative metabolism of human blood monocytes and granulocytes. Monocytes incubated with surfactant ingest this material and subsequently exhibit an impaired ability to kill ingested bacteria. Granulocytes incubated with surfactant do not ingest this material, and their bactericidal functions are not affected. However, granulocytes that have ingested surfactant-coated Staphylococcus aureus display an impaired ability to kill these bacteria. Moreover, in monocytes and granulocytes that contain surfactant--the latter by ingestion of surfactant-coated S. aureus--the intracellular production of H2O2 is impaired due to inhibition of the assembly of the NADPH oxidase. Together these results demonstrate that surfactant inside monocytes and granulocytes inhibits the capacity of these cells to kill bacteria intracellularly by impairing oxygen-dependent killing mechanisms.
Role of beta 1, beta 2 integrins and ICAM-1 in lung injury after deposition of IgG and IgA immune complexes.
Authors: Mulligan M S , Wilson G P , Todd R F , Smith C W , Anderson D C , Varani J , Issekutz T B , Miyasaka M , Tamatani T , Myasaka M
Published: December 2022

After intrapulmonary deposition of IgG or IgA immune complexes, injury has been recently shown to be CD18-dependent in both cases and E-selection-dependent only in the former case. In our studies further evaluation of the requirements for beta 1 and beta 2 integrins and intercellular adhesion molecule-1 (ICAM-1) has been undertaken. In the IgG immune complex model, which is neutrophil dependent, anti-CD11a reduced injury (as measured by changes in permeability and hemorrhage) by 61 and 43%, respectively, whereas a newly developed anti-CD11b produced minimal protection (16 and 19%, respectively). Treatment of rats with increasing doses (1.5- and 3.0-fold) of antibody to rat CD11b failed to demonstrate additional protective effects in this model of injury. Anti-ICAM-1 reduced the parameters of injury by 61 and 78%, respectively, while anti-VLA-4 reduced the injury parameters by 40 and 35%, respectively. There were reductions in lung content of myeloperoxidase, roughly corresponding to the protective effects of the interventions. In the IgA immune complex model of injury, in which lung macrophages appear to be the effector cells, anti-CD11a reduced the injury parameters (permeability and hemorrhage) by 36 and 33%, respectively, whereas anti-CD11b reduced the parameters of injury by 63 and 67%, respectively. In this model, anti-ICAM-1 reduced the parameters of injury by 61 and 56%, respectively, while anti-VLA-4 reduced the parameters by 77 and 62%, respectively. The cell content of bronchoalveolar lavage fluids revealed changes that have been shown to reflect protective interventions in both models of immune complex-induced injury. These findings suggest that, in IgG and IgA immune complex models of lung injury, both VLA-4 and ICAM-1 are required, although lymphocyte function-associated Ag-1 is the predominant beta 2 integrin requirement in the IgG immune complex-induced model of injury and Mac-1 is the predominant requirement for IgA immune complex-induced lung injury. Thus, engagement in the lung of adhesion molecules in a manner leading to injury depends on the nature of the inflammatory stimulus and the type of phagocytic cells involved in the development of injury.
Platelet-derived growth factor (PDGF) activates primitive hematopoietic precursors (pre-CFCmulti) by up-regulating IL-1 in PDGF receptor-expressing macrophages.
Authors: Yan X Q , Brady G , Iscove N N
Published: December 2022

Platelet-derived growth factor (PDGF-BB) is known to stimulate growth of fibroblasts and smooth muscle cells via interaction with specific PDGF-B receptors on their surfaces. Hematopoietic precursor cells are not known to respond directly to PDGF, and PDGF receptors have never been reported on normal hematopoietic cells. We report a consistent stimulatory effect of PDGF-B on primitive pluripotential hematopoietic cells (pre-CFCmulti) in suspension cultures of mouse bone marrow that resembles effects obtainable previously only with IL-1. Further, we show that the effect depends on plastic-adherent accessory cells and is abrogated by neutralizing antibodies to IL-1 beta, that PDGF strongly up-regulates IL-1 mRNA levels in marrow macrophages but not in marrow fibroblast lines, and finally provide a first report of expression of RNA transcripts for PDGF-B receptors in normal marrow macrophages. Together, the results suggest the existence of a novel cytokine loop implicating monocyte/macrophages as cells reactive to PDGF and highlighting IL-1 induction as a significant feature of their PDGF response.
Growth inhibition of a B cell clone mediated by ligation of IL-4 receptors or membrane IgM.
Authors: Bishop G A , Ramirez L M , Koretzky G A
Published: December 2022

The development of B cell tolerance is believed to involve negative signaling to the B cell derived from the binding of Ag to the B cell surface Ig (sIg). B cell clones that receive negative signals via sIg may provide useful models for studying the mechanisms of negative signaling. We have recently identified a previously undescribed mouse B cell clone, CHB3, which receives growth-inhibitory signals through the binding of IL-4 to its IL-4R or through ligation of its sIgM, but not its sIgD, molecules. Data presented here demonstrate that the negative signal delivered by sIgM, but not that delivered by IL-4R, requires protein kinase C activation and elevated intracellular Ca2+, and is associated with the tyrosine phosphorylation of a number of proteins. Thus, the IL-4R signaling pathway appears to be divergent from the sIgM-mediated pathway. However, growth inhibition mediated via both sIgM and IL-4R can be partially counteracted by a signal delivered through the MHC class II molecule.
IL-7 enhances the expression of IL-3 and granulocyte-macrophage-CSF mRNA in activated human T cells by post-transcriptional mechanisms.
Authors: Dokter W H , Sierdsema S J , Esselink M T , Halie M R , Vellenga E
Published: December 2022

The stromal derived growth factor IL-7 was studied for its ability to modulate cytokine expression in human T cells. IL-7 alone did not induce IL-3 or granulocyte-macrophage-CSF (GM-CSF) mRNA. However, IL-7 enhanced the Con A-induced IL-3 and GM-CSF mRNA accumulation in a dose-dependent way. mRNA stability studies revealed that the effect of IL-7 was caused by post-transcriptional stabilization of the IL-3 and GM-CSF transcripts. Upon Con A treatment, the IL-3 and GM-CSF mRNA decayed with a t1/2 of approximately 90 and 50 min, respectively. Costimulation with Con A plus IL-7 stabilized both transcripts to t1/2 of greater than 2 h for IL-3 mRNA and 90 min for GM-CSF mRNA. Using nuclear run-on assays, we showed that the transcription rate of both genes was not affected by IL-7. Furthermore, it appeared that the effect of IL-7 was independent on protein synthesis, because cycloheximide did not abolish the promotive effect of IL-7. Finally, it was shown that in accordance with the mRNA results IL-7 enhanced the secretion of GM-CSF protein in Con A-activated T cells. After 12 h of stimulation T cells cultured in the presence of Con A secreted 575 +/- 309 pg GM-CSF/ml (x +/- SD, n = 5), which increased to 1425 +/- 758 pg/ml in the presence of Con A plus IL-7 (p < 0.01). In summary, these data demonstrate that IL-7 augments the expression and secretion of CSF in activated human T cells.
Cholera toxin inhibits T cell receptor signaling by covalent modification of the CD3-zeta subunit.
Authors: Haack B M , Emmrich F , Resch K
Published: December 2022

In the Jurkat T cell line, triggering of the TCR leads to activation of phospholipase C, resulting in an increase in inositol trisphosphate (IP3) release followed by a rise in intracellular Ca2+. This signaling pathway is interrupted by cholera toxin (CTX) treatment. To possibly explain this inhibition, we demonstrate that CTX can affect the TCR/CD3 complex itself by causing a covalent modification of the CD3-zeta subunit. After exposure of Jurkat cells to CTX, CD3-zeta increases its apparent m.w. and becomes more acidic in isoelectric focusing. The time course of the modification correlates well with the reduction in IP3 generation and Ca2+ release after CTX treatment, suggesting that the modification of zeta might be the cause of the impaired TCR/CD3 signaling. As is true for the CTX-mediated decrease in TCR signaling, the change in CD3-zeta was cAMP-independent and cannot be evoked by the enzymatically inactive CTX-B subunit alone.
Cross-reactive T cell clones from unrelated individuals reveal similarities in peptide presentation between HLA-B27 and HLA-DR2.
Authors: López D , Barber D F , Villadangos J A , López de Castro J A
Published: December 2022

HLA-B27- responder cells were stimulated in vitro with B*2705+ lymphoblastoid cell lines and alloreactive CTL clones were obtained by limiting dilution. Of the CD3+ CD4-CD8+ HLA-B27-specific CTL clones obtained, two of them, possessing the same TCR, cross-reacted with HLA-DR2. The fine specificity of these CTL was established with HLA-B27 and HLA-DR2 subtypes. They recognized the B*2701 to B*2706 subtypes, but only DR2Dw2. Lysis of DR2+ target cells was specifically inhibited by anti-CD3, anti-class II, and anti-DR mAb, but not with an anti-CD8 antibody. The monoclonal nature of the cross-reaction was established by the mutual inhibition of HLA-B27 and DR2Dw2 cells in cold target competition experiments. The DR2 protein involved in the cross-reaction was the heterodimer carrying the B5*0101 product, as shown by using L cell transfectants expressing each of the two molecules encoded in the DR2Dw2 haplotype. A correlation between the fine specificity of these CTL clones and the amino acid sequences of HLA-B27 and HLA-DR2 subtypes revealed a shared structural motif between HLA-B27 and the DR2 B5*0101 chain, which could be related to the observed cross-reaction. This motif was contributed for by several residues located in adjacent beta strands, at the floor of the peptide-binding site. The contribution of two of these residues, as well as other beta-pleated sheet residues to HLA-B27 allorecognition by the cross-reactive CTL clones was directly demonstrated with site-directed mutants. These results suggest that the dual reactivity pattern reflects presentation of identical or structurally related peptide by HLA-B27 and HLA-DR2Dw2. As T cell cross-reactivity between HLA-B27 and HLA-DR2 was previously found in cells from an unrelated individual the results reported here are likely to reflect an intrinsic property of HLA-B27, rather than the fortuitous finding of a rare clonal reaction pattern. We speculate on the potential implications of these results for the pathogeny of HLA-B27-associated spondyloarthropathies.
Mouse soluble IFN gamma receptor as IFN gamma inhibitor. Distribution, antigenicity, and activity after injection in mice.
Authors: Ozmen L , Gribaudo G , Fountoulakis M , Gentz R , Landolfo S , Garotta G
Published: December 2022

With the purpose to use a soluble receptor as an IFN gamma antagonist in vivo, we assessed the immunogenicity, the half-life, the bioavailability, and the activity of the soluble receptor after injection into mice. No significant immunogenicity was detected after multiple chronic injections of mouse (Mo)IFN gamma-R or injections of MoIFN gamma-R in emulsion with CFA. Pharmacokinetic experiments using radiolabeled MoIFN gamma-R revealed that the half-life of the soluble MoIFN gamma-R is 3 h in the blood and 6 h in the lymphoid organs. When the MoIFN gamma-R protein was traced by the capacity of the animal sera to displace the binding of radiolabeled MoIFN gamma-R from affinity purified rabbit anti-MoIFN gamma-R antibodies, a blood half-life of 1 h was determined. Finally, the capacity of the injected receptor to bind and neutralize the IFN gamma-mediated antiviral activity was assessed in vivo. Treatment with IFN-gamma or IFN-alpha A/D protected the mice from the lethal infection with encephalomyocarditis virus. Mice treated with MoIFN-gamma-R neutralized the protective effect of IFN-gamma.
Exogenous IL-7 promotes the growth of CD3-CD4-CD8-CD44+CD25+/- precursor cells and blocks the differentiation pathway of TCR-alpha beta cells in fetal thymus organ culture.
Authors: Plum J , De Smedt M , Leclercq G
Published: December 2022

Addition of human rIL-7 to fetal thymic organ culture started at day 13, 14, or 15 did not influence the number of cells generated during a 12-day culture period. However, the IL-7 treatment resulted in a preferential expansion of cells with a phenotype characteristic for cells at an early step of differentiation. The cells were CD4-CD8-CD3-CD2- and SCA-1+. Analysis of the coordinate expression of CD44 and CD25 on these cells showed that the majority of the cells were either CD44+CD25- or CD44+CD25intermediate. TCR-alpha beta cells were present but in a significantly lower number as compared to the control cultures. The cell number of TCR-gamma delta cells was increased. All these effects were moderate after 6 days, but unequivocal after 12 days of culture. Treatment of the fetal organ culture with mAb-neutralizing murine IL-7 resulted in an inhibition of the proliferation of the fetal thymocytes. No particular subset studied was preferentially inhibited. By using a model of reconstitution of 14-day embryonic thymuses depleted of thymocytes by deoxyguanosine and reconstituted with fetal day 13 liver cells and set up in organ culture with or without IL-7, it was shown in a clear cut way that IL-7 indeed promotes expansion of the early precursor cells and TCR-gamma delta cells, but prevents the generation of TCR-alpha beta cells. In addition, reconstitution experiments were set up in the presence of mAb-neutralizing murine IL-7. This treatment resulted in the inhibition of the growth of the fetal thymocytes without inhibiting preferentially a particular subset. These data indicate that IL-7 acts at an early step of T cell differentiation and plays a role to expand precursor cells, but prevents this population from additional differentiation towards the TCR-alpha beta pathways, whereas the differentiation towards TCR-gamma delta cells is not influenced or even enhanced.
Endogenous generation and presentation of the ovalbumin peptide/Kb complex to T cells.
Authors: Shastri N , Gonzalez F
Published: December 2022

Peptide fragments are displayed on the APC surface by MHC class I molecules as ligands for appropriate TCR. Sequence analysis of MHC-bound peptide mixtures has suggested that naturally processed peptides are defined by their length and by the presence of MHC allele-defined consensus motifs. To define the minimal OVA peptide presented by Kb MHC, and the requirements for generation of endogenous OVA/Kb complex, APC were transfected with OVA cDNA constructs. We show that optimal stimulation of OVA/Kb-specific B3Z T cell hybrid by Kb-APC requires the OVA257-264 peptide (SIINFEKL, SL8) whether added exogenously, or when synthesized endogenously as precursor polypeptides. Thus, all information necessary for expression of the OVA/Kb complex is contained within the Kb octapeptide motif shared by SL8. Unexpectedly, the SL8/Kb or the influenza NP/Db complexes were also generated in APC even when the peptide coding sequences were placed in incorrect translational reading frames. By contrast, identical manipulations of the translational reading frame of the lacZ reporter gene reduced protein synthesis to undetectable levels demonstrating the remarkable efficiency with which endogenous peptide/MHC are generated in and presented by APC to T cells. These characteristics of the endogenous presentation may explain how large numbers of distinct peptide/MHC complexes are displayed on the cell surface and surveyed by TCR.
Influence of HLA genes on T cell receptor V segment frequencies and expression levels in peripheral blood lymphocytes.
Authors: Akolkar P N , Gulwani-Akolkar B , Pergolizzi R , Bigler R D , Silver J
Published: December 2022

The effect of the HLA complex on the TCR repertoire in human peripheral blood was assessed by using nine V beta- and V alpha-specific mAb and the quantitative polymerase chain reaction specific for 22 V beta segments. Studies in randomly selected and unrelated individuals failed to show any influence of the HLA complex on the TCR repertoire. In contrast, studies in large families with multiple siblings showed a strong influence on the TCR repertoire by the HLA complex. In pairwise comparisons, HLA-identical sibs had more similar patterns of V segment frequencies, as measured with the nine V segment-specific mAb, as well as more similar expression levels of V beta-specific RNA, as measured by quantitative polymerase chain reaction, than totally mismatched or haplo-identical sibs. When the amount of V beta-specific RNA expressed in CD4+ and CD8+ T cells was compared, it was found that V beta 2, 5.1, 9, and 20 were skewed toward CD4+ T cells; on the other hand, V beta 7 and 14 showed a bias in expression for CD8+ T cells, suggesting that the former were positively selected predominantly by HLA class II gene products whereas the latter V beta segments were positively selected predominantly by HLA class I gene products. These studies unequivocally document the effects of HLA genes on TCR V segment frequencies and expression levels in peripheral blood T lymphocytes.
Reactive nitrogen intermediates suppress the primary immunologic response to Listeria.
Authors: Gregory S H , Wing E J , Hoffman R A , Simmons R L
Published: December 2022

Reactive nitrogen intermediates (RNI), e.g., nitric oxide derived from a terminal guanido nitrogen atom of L-arginine, exhibit potent antimicrobial activity in vitro. The function of these intermediates in host defenses in vivo, however, is presently unclear. Experiments were undertaken to determine the role of RNI in the resolution of primary listerial infections of the liver. Serum RNI levels were elevated significantly in mice infected with Listeria monocytogenes. Moreover, a marked increase in RNI production was found in cultures of the parenchymal, as well as the nonparenchymal, liver cells obtained from Listeria-infected mice. RNI did not kill Listeria treated directly, however, nor were they a factor in the listericidal activity exhibited by hepatic cells. Rather, the elevated production of RNI during primary infection appeared to promote the replication of Listeria in vivo. Mice administered NG-monomethyl-L-arginine, a competitive inhibitor of RNI production, exhibited a 10- and a 100-fold reduction in the number of Listeria in their lives on days 3 and 7 postinfection, respectively. In vitro, NG-monomethyl-L-arginine stimulated the Ag-specific proliferation of T lymphocytes derived from Listeria-infected mice at concentrations that inhibited RNI production. These latter findings suggest that the elevated production of RNI during primary listerial infections suppresses host defenses by diminishing the proliferation and, consequently, the biologic response of immune cell populations.
Exogenous nitric oxide (NO) generation or IL-1 beta-induced intracellular NO production stimulates inhibitory auto-ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase in RINm5F cells.
Authors: Dimmeler S , Ankarcrona M , Nicotera P , Brüne B
Published: December 2022

Nitric oxide (NO) stimulates the auto-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which results in the inhibition of enzyme activity. In the present work we show that addition of exogenous NO or IL-1 beta-induced intracellular NO generation cause GAPDH ADP-ribosylation and inhibition of enzyme activity. Incubation of RINm5F cells with sodium nitroprusside (SNP) for 18 h caused a time- and dose-dependent inhibition of GAPDH activity. Half-maximal inhibition of GAPDH activity was observed with 80 microM of the NO donor, with maximal inhibition after roughly 6 h of incubation. In parallel, SNP induced endogenous ADP-ribosylation of GAPDH measured by a decreased incorporation of [32P]ADP-ribose from [32P]NAD+ in the cytosol of the SNP-treated cells. Stimulation of endogenous NO production by inducing the NO synthase by exposure to the cytokine IL-1 beta results in decreased GAPDH activity. IL-1 beta (10(-9) M) inhibited GAPDH activity about 55%, compared with control values. Production of nitrite and inhibition of GAPDH was reversed by the NO synthase inhibitor NG-monomethyl-L-arginine, indicating that endogenous generated NO was the effective molecule. Again, GAPDH inhibition was associated with NO-stimulated endogenous ADP-ribosylation of the enzyme. Western blot analysis of GAPDH excluded degradation of GAPDH by NO. NO-stimulated auto-ADP-ribosylation resulted in inhibition of the glycolytic enzyme GAPDH and may be relevant as a cytotoxic effect of NO. In concert with its inhibitory actions on iron-sulfur enzymes like aconitase and electron transport proteins of the respiratory chain NO may mediate autocytotoxic effect in beta-cells.
Cocapping of the leukoadhesin molecules complement receptor type 3 and lymphocyte function-associated antigen-1 with Fc gamma receptor III on human neutrophils. Possible role of lectin-like interactions.
Authors: Zhou M , Todd R F , van de Winkel J G , Petty H R
Published: December 2022

We have tested the possible physical interactions between the iC3b receptor (CR3), lymphocyte function-associated Ag-1, and class III Fc gamma receptor (Fc gamma RIII) at neutrophil surfaces. Cells were labeled using fluorochrome-conjugated Fab or F(ab')2 fragments of antireceptor mAb. Labeled receptors were capped using second-step F(ab')2 fragments of goat anti-mouse Fab antiserum. After 20 min at 37 degrees C, 68% of the cells capped the anti-CR3 plus second-step complex. Capping was time, temperature, and cytochalasin B sensitive. When capped cells were probed with Fab' or F(ab')2 fragments of anti-Fc gamma RIII labeled with a distinct fluorochrome, 41% of the cells cocapped Fc gamma RIII. Indistinguishable results were obtained when potential antibody combining sites within caps were blocked with a large excess of Fab or F(ab')2 fragments. When Fc gamma RIII was capped, 49% of the cells cocapped CR3. Similarly, LFA-1 cocapped with both CR3 and Fc gamma RIII. Importantly, other membrane components including HLA class I, Mo5, CD13, CR type 1, and IL-8 receptors and N-4-nitrobenzo-2-oxa-1, 3-diszole L-alpha-dimyristoyl phosphatidylethanolamine did not cocap with CR3. However, the positive control Con A did cocap with CR3 and Fc gamma RIII. We next evaluated the effect of saccharides on CR3-Fc gamma RIII cocapping and found that 0.15 M N-acetyl-D-glucosamine (NADG), alpha-methyl-D-mannoside, and D-mannose significantly inhibited cocapping by 70, 58, and 48%, respectively. No inhibition was obtained using glucose, galactose, N-acetyl-neuraminic acid, fucose, sorbitol, fructose, or sucrose. Similarly, Fc gamma RIII-lymphocyte function-associated-1 cocapping was inhibited by NADG. However, the cocapping of CR3 with lymphocyte function-associated-1 or Con A were not affected by 0.15 M NADG, which suggests that NADG inhibition of leukoadhesin-Fc gamma RIII cocapping is not due to a general effect of NADG on capping. Inasmuch as Fc gamma RIII is a glycophospholipid-linked membrane protein, we speculate that it interacts with CR3 and/or lymphocyte function-associated-1 via lectin-like interactions.
Antitumor activity of the single-chain immunotoxin BR96 sFv-PE40 against established breast and lung tumor xenografts.
Authors: Friedman P N , Chace D F , Trail P A , Siegall C B
Published: December 2022

We have constructed a single-chain immunotoxin consisting of the variable H and L chains of the carcinoma-reactive mAb BR96, fused to the binding defective protein toxin, PE40. This molecule, BR96 sFv-PE40, has been shown to be extremely cytotoxic toward a variety of BR96 Ag-expressing tumor cell lines. When administered i.v. into athymic mice carrying L2987 tumor xenografts, BR96 sFv-PE40 was cleared rapidly from the blood with a half-life of approximately 30 min. This is in comparison to a chemical conjugate, chiBR96-LysPE40, that remained in the blood for almost 2 h. In addition, the smaller single-chain immunotoxin (67 kDa) penetrates the tumor faster than the larger chemical conjugate (190 kDa). Using a variety of administration schedules and doses, we treated established human tumor xenografts in athymic mice with both the single-chain immunotoxin BR96 sFv-PE40 and the chemical conjugate chiBR96-LysPE40. In both L2987 lung carcinoma and MCF-7 breast carcinoma models, we found that BR96 sFv-PE40 completely regressed the tumor xenografts. With an administration schedule of q4dx5, the tumors were totally regressed and did not reappear. The chiBR96-LysPE40 conjugate produced partial tumor regressions, although at near maximum tolerated dose. These results show that the single-chain immunotoxin, BR96 sFv-PE40, is a potent antitumor agent.
CD28 engagement by B7/BB-1 induces transient down-regulation of CD28 synthesis and prolonged unresponsiveness to CD28 signaling.
Authors: Linsley P S , Bradshaw J , Urnes M , Grosmaire L , Ledbetter J A
Published: December 2022

Costimulatory molecules on the APC regulate T cell growth by providing signals that regulate responses to TCR occupancy. One such molecule is B7/BB-1, which triggers a T cell activation pathway by binding the CD28 and/or CTLA-4 cell-surface molecules. Expression and signaling activity of CD28 have been shown to increase after T cell activation by various polyclonal activators. Here we show that CD28 expression and signaling activity in activated T cells decrease after ligand binding to CD28. Stimulation of CD28 on PHA- or PMA-activated T cells by cross-linked mAb 9.3 or by co-culture with B7+ Chinese hamster ovary (CHO) cells caused a marked reduction of CD28 mRNA levels within 4 h. The decrease in CD28 mRNA was transient, and by 24 h of CD28 stimulation, CD28 mRNA was found at approximately initial levels. In contrast, CTLA-4 mRNA levels were usually up-regulated by CD28 triggering. Cell-surface expression of CD28, but not CD2 or CD3, decreased by 12 to 24 h after addition of B7+ CHO cells, but returned to initial levels or higher by 48 h. The ability of CD28 cross-linking on PMA-activated CD4+ cells to trigger calcium mobilization was also reduced by treatment with B7+ CHO cells, and remained reduced even after cell-surface expression of CD28 returned to normal levels. Thus, engagement of the CD28 receptor by its natural ligand B7/BB-1 leads to a transient down-regulation of CD28 synthesis and a prolonged unresponsiveness to CD28 signaling. This represents a novel mechanism for regulation of costimulatory signals delivered by interactions of CD28 with the B7/BB-1 counter receptor.