bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipopolysaccharide-induced protein tyrosine phosphorylation in human macrophages is mediated by CD14.
Authors: Weinstein S L , June C H , DeFranco A L
Published: December 2022

Induced protein tyrosine phosphorylation is an early intracellular event in LPS-stimulated murine macrophages that appears to play a role in signal transduction. We have now demonstrated that LPS also increases protein tyrosine phosphorylation in human monocyte-derived macrophages and in the human monocytic cell line, THP-1. This response was rapidly elicited by biologically active forms of LPS or lipid A, at concentrations of these bacterial components that stimulate anti-bacterial responses by human macrophages. Inhibition of the LPS-induced tyrosine phosphorylation response with the tyrosine kinase inhibitor, herbimycin A, was accompanied by the inhibition of the secretion of TNF-alpha by human macrophages. These results extend previous work with murine macrophages and provide further support for the hypothesis that induced protein tyrosine phosphorylation is an important signaling reaction in macrophages after LPS exposure. In addition, CD14, which is thought to be a receptor for LPS, appeared to mediate the induced phosphorylation response in human macrophages and THP-1 cells at low LPS concentrations. Two antibodies against CD14, 3C10 and 60b, which have been shown to prevent LPS binding to CD14, specifically inhibited the protein tyrosine phosphorylation induced by nanogram per milliliter concentrations of LPS in these cells. The antibody-mediated inhibition did not appear to involve engagement of surface FcR because a preparation of F(ab')2/Fab fragments of the 60b antibody also prevented LPS-induced tyrosine phosphorylation. At higher concentrations of LPS (> or = 10 ng/ml), however, anti-CD14 antibodies did not prevent the induction of protein tyrosine phosphorylation, suggesting that a lower affinity CD14-independent pathway also mediates the tyrosine phosphorylation response. Because CD14-dependent and CD14-independent recognition of LPS appear to lead to the same functional responses by macrophages, induced protein tyrosine phosphorylation may be part of a shared intracellular signaling pathway.
Co-stimulatory signal delivered by CD73 molecule to human CD45RAhiCD45ROlo (naive) CD8+ T lymphocytes.
Authors: Dianzani U , Redoglia V , Bragardo M , Attisano C , Bianchi A , Di Franco D , Ramenghi U , Wolff H , Thompson L F , Pileri A
Published: December 2022

CD73 is a molecule expressed by a subset of CD8+ human T lymphocytes and is involved in T cell activation. CD73 expression and function were analyzed in peripheral blood CD45RAhiCD45ROlo (naive) and CD45RAloCD45ROhi (memory) CD8+ cells. We found that CD73 was expressed by a majority of naive cells (74 +/- 12%), whereas fewer memory cells were CD73+ (29 +/- 10%). Moreover, CD73 was selectively expressed by the CD11b- subset of naive CD8+ cells, which were almost all CD73+. The same result was found on CD8+ cord blood lymphocytes, which prevalently display the naive phenotype. Naive CD8+ CD11b- cells were almost unresponsive to CD3 engagement, but this apparent anergy was completely overcome when CD3 and CD73 were simultaneously cross-linked by plastic-immobilized CD73 and CD3 mAb, showing that CD73 delivers an accessory signal that allows their activation via the CD3/TCR. This costimulatory signal was tenfold more potent than that induced by CD28 ligation. A phosphotyrosine analysis by Western blotting showed that cross-linking of CD73 induced the phosphorylation of two proteins with a molecular mass of approximately 28 and 100 kDa respectively, whereas ligation of CD3 induced phosphorylation of many substrates. When CD3 and CD73 were simultaneously triggered these substrates were hypophosphorylated. Because CD73 is linked to the cell surface by a GPI anchor, the transduction of this signal is probably mediated by a lateral interaction with transmembrane molecules. This hypothesis was assessed by cocapping, which showed that CD73 associates strongly with CD45RC, moderately with CD8, and weakly with CD3. These data suggest that CD73 signaling is coupled to both tyrosine kinase and phosphatase activities.
Germline and productive C epsilon gene expression during in vivo IgE responses.
Authors: Thyphronitis G , Katona I M , Gause W C , Finkelman F D
Published: December 2022

In vitro studies have established that Ig isotype switching typically involves deletion of CH genes that are located between VDJ and the CH gene that will be expressed, and is preceded by transcription of a germline (g) form of that CH gene. Increases in g epsilon transcript levels are induced by the cytokine IL-4, and always precede switching to IgE. To evaluate whether a similar relationship occurs in vivo, we examined IL-4 mRNA, g epsilon RNA, productive (p) epsilon mRNA, and serum IgE levels in two in vivo systems: one in which the injection of anti-IgD antibody induces mIgD+ B cells to switch to the expression of IgE and to secrete this isotype, and a second in which the injection of anti-IgE antibody stimulates IgE secretion by B cells that had been induced to express membrane IgE by earlier treatment with anti-IgD antibody. Increases in IL-4 transcript levels in anti-IgD-injected mice were followed within 24 h by increases in g epsilon RNA, and, one to two days later, by increased p epsilon mRNA and serum IgE levels. IL-4 antagonists blocked the g epsilon and p epsilon RNA and serum IgE responses in these mice, whereas the injection of otherwise untreated mice with IL-4 stimulated, within 24 h, a large increase in g epsilon RNA levels, followed 1-2 days later by a small increase in p epsilon mRNA. Injection of anti-IgD-primed mice with anti-IgE antibody also stimulated increases in IL-4, g epsilon and p epsilon RNA levels; however, the increases in IL-4 and g epsilon RNA were considerably smaller, and the increases in p epsilon mRNA and serum IgE considerably larger, than those observed in anti-IgD antibody-injected mice. IL-4 antagonists blocked the anti-IgE antibody-induced g epsilon RNA response, but not the p epsilon mRNA or serum IgE responses. Thus, IL-4 is required for the induction of g epsilon RNA in at least two in vivo systems, increased g epsilon RNA levels precede increases in p epsilon RNA levels in vivo as in vitro, and neither IL-4 nor g epsilon RNA is required to induce B cells that have already switched to IgE expression to differentiate into IgE-secreting cells.
Transforming growth factor beta 1 repression of the HLA-DR alpha gene is mediated by conserved proximal promoter elements.
Authors: Reimold A M , Kara C J , Rooney J W , Glimcher L H
Published: December 2022

Transforming growth factor beta 1 (TGF-beta 1) is a pleiotropic cytokine that decreases the expression of class II MHC Ag in the melanoma cell line Hs294T(c). To investigate the mechanism of this repression, we have examined the effect of TGF-beta 1 on expression of the HLA-DR alpha gene. Both the constitutive level of HLA-DR protein and DR alpha mRNA were repressed by treatment with TGF-beta 1. The proximal 176 bp of the DR alpha promoter were sufficient to confer TGF-beta 1 repression on a reporter gene. Deletional and mutational analysis of the DR alpha promoter revealed that the conserved S and X1 promoter elements were important for basal expression of DR alpha and also mediated the down-regulation by TGF-beta 1. Mobility shift assays and in vivo footprinting showed no change in occupancy of the proximal DR alpha promoter after TGF-beta 1 treatment. These results identify the DNA elements that mediate repression of the HLA-DR alpha gene by TGF-beta 1 and suggest that TGF-beta 1 acts at these sites without causing a change in promoter occupancy.
Hepatitis C virus-specific CTL responses in the liver of chimpanzees with acute and chronic hepatitis C.
Authors: Erickson A L , Houghton M , Choo Q L , Weiner A J , Ralston R , Muchmore E , Walker C M
Published: December 2022

Hepatitis C virus (HCV)-specific CTL responses were evaluated in two chimpanzees (Pan troglodytes) during the acute and chronic phases of HCV infection. CD8+ T lymphocytes were isolated from liver tissue homogenates using anti-CD8 antibody-coated magnetic beads and then stimulated with anti-CD3 antibodies, IL-2, and irradiated human PBMC using limiting dilution culture conditions. HCV-specific cytotoxic activity of expanding CD8+ cell lines was assessed against autologous lymphoblastoid cell lines infected with recombinant vaccinia virus vectors encoding HCV Ag. CD8+ T cell lines specific for structural and nonstructural proteins of HCV were established from both animals. Cytolytic activity was blocked with anti-CD8 or anti-class I MHC antibodies, indicating that class I MHC molecules were involved in presentation of viral Ag to the CTL. Overlapping synthetic peptides were used to define a 12 amino acid segment of the nonstructural 3 (NS3) protein recognized by CTL lines from both chimpanzees. Studies with truncated peptides revealed that these CD8+ cell lines were directed against overlapping epitopes presented by distinct class I restriction elements of the chimpanzee MHC complex. CD8+ cell lines with identical specificities for an NS3 epitope were generated from one chronically infected animal at 16 and 28 wk postinfection. These results indicate that virus-specific CTL populations persist in the liver for months, but are unable to resolve chronic HCV infection.
IL-10 inhibits the synthesis of migration inhibitory factor and migration inhibitory factor-mediated macrophage activation.
Authors: Wu J , Cunha F Q , Liew F Y , Weiser W Y
Published: December 2022

IL-10, in addition to being a cytokine synthesis-inhibitory factor, is a cytokine that exerts multiple effects on various cell types. Recombinant human migration inhibitory factor (MIF) inhibits the migration of human monocytes as well as that of guinea pig and murine macrophages. In addition, it has recently been shown to activate human monocyte-derived macrophages to suppress the growth of and/or kill both intracellular parasites and extracellular tumor targets in vitro and to have adjuvant activity in vivo. In this study, we examined the interactions between IL-10 and rMIF. We demonstrate that IL-10 reduces the production of MIF from T cells and abolishes rMIF-mediated migration inhibition of human monocytes. Incubation of IL-10 together with rMIF diminishes rMIF-induced intracellular killing of Leishmania donovani by human monocyte-derived macrophages and inhibits nitric oxide production and nitric oxide synthase activity by murine macrophages.
Macrophage inflammatory protein-1 alpha rapidly modulates its receptors and inhibits the anti-CD3 mAb-mediated proliferation of T lymphocytes.
Authors: Zhou Z , Kim Y J , Pollok K , Hurtado J , Lee J K , Broxmeyer H E , Kwon B S
Published: December 2022

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is a member of the intercrine/chemokine family which consists of basic, heparin-binding, small molecular weight proteins. We have previously shown that a T cell line, CTLL-R8, carried high-affinity receptors for MIP-1 alpha and the proliferation of CTLL-R8 cells was inhibited by murine recombinant (mr) MIP-1 alpha. We extended our previous studies to murine resting splenic T lymphocytes to determine whether the inhibition of T cell proliferation is a general property of MIP-1 alpha. The resting splenic T cells carried approximately 680 high-affinity binding sites for mrMIP-1 alpha; more than 90% of the primary T cells carried MIP-1 alpha receptors. When the T cells were stimulated with immobilized anti-CD3 mAb in the presence of accessory cells, the MIP-1 alpha binding was reduced. The lowest binding was obtained 2 h after anti-CD3 mAb stimulation due to the internalization of MIP-1 alpha receptors. mrMIP-1 alpha inhibited the anti-CD3 mAb-mediated proliferation of murine splenic T lymphocytes. The maximum inhibition was obtained when mrMIP-1 alpha was added 30 min before anti-CD3 mAb stimulation. Slight inhibition of T cell proliferation was observed when mrMIP-1 alpha was added at the same time as anti-CD3 mAb stimulation. These results indicate that T lymphocytes are regulated negatively by MIP-1 alpha, which occurs when the T cells are exposed to MIP-1 alpha before activation. The negative effect of MIP-1 alpha seems to be mediated in part by the inhibition of IL-2 production, for there was a reduction in both the IL-2 mRNA levels and the IL-2 activity in supernatants from T cells preincubated with MIP-1 alpha before anti-CD3 mAb stimulation.
T cells of patients with the Wiskott-Aldrich syndrome have a restricted defect in proliferative responses.
Authors: Molina I J , Sancho J , Terhorst C , Rosen F S , Remold-O'Donnell E
Published: December 2022

The Wiskott-Aldrich syndrome (WAS) is a disease of profound thrombocytopenia and severe immune defects caused by an unidentified defective X chromosome gene. In this study, T lymphocyte function is examined using a panel of allospecific WAS patient T cell lines, previously found to express the abnormal disease gene and the cytoarchitectural defect characteristic of the disease. Although T cell lines from normal individuals proliferate vigorously in response to immobilized anti-CD3 mAb OKT3 and SPV-T3b, five of seven WAS patient T cell lines failed to proliferate and two lines showed significantly decreased proliferation when challenged with the immobilized anti-CD3 mAb. The deficient responsiveness of the WAS T cell lines to immobilized anti-CD3 mAb is a restricted defect, because the cells proliferate normally when challenged with allospecific Ag, PHA, or PMA plus ionomycin. Addition of anti-CD28 mAb did not correct the deficient proliferation of the WAS cells challenged with immobilized anti-CD3. Deficient response of the WAS T cell lines to immobilized anti-CD3 was detected also when earlier events of the proliferation process, IL-2 production and up-regulation of activation Ag CD69 and CD28, were measured. On the other hand, WAS cell lines did not differ from normal cell lines in binding of anti-CD3 mAb, mobilization of Ca2+ in response to soluble OKT3, and tyrosine phosphorylation and GTP binding of the CD3 zeta-chain in response to OKT3. Cumulatively, these findings demonstrate a striking restricted defect in the proliferative response of WAS T cells, which because it is found in cell lines free of secondary changes that occur in the patient circulation must be a reflection of the inherited defective disease gene product.
Induction of multiple heart autoantibodies in mice with coxsackievirus B3- and cardiac myosin-induced autoimmune myocarditis.
Authors: Neumann D A , Rose N R , Ansari A A , Herskowitz A
Published: December 2022

When mice of certain strains are infected with the cardiotropic virus coxsackievirus B3 or are immunized with mouse cardiac myosin, myocarditis, accompanied by immune recognition of the myocardium, ensues. That both target organ injury and peripheral immunization lead to similar disease manifestations raise questions about the role of tissue damage in eliciting autoantibodies. A/J mice were infected with coxsackievirus B3 or immunized with mouse cardiac myosin and killed at weekly intervals for 4 wk. A portion of each heart was examined histologically for evidence of myocarditis and antibodies were eluted from the remaining heart tissue. Heart eluates and serum were tested for IgG antibodies to myosin and to adenine nucleotide translocator and branched chain ketoacid dehydrogenase molecules by ELISA. At each sampling time, coxsackievirus B3-infected mice exhibited high titers of circulating antibodies to myosin, adenine nucleotide translocator, and branched chain ketoacid dehydrogenase with successively increasing myocardial deposition of antibodies of each specificity. Among myosin-immunized mice, antibodies of all three specificities were eluted from the myocardium and were found in the serum, but only antibodies to myosin were present in appreciable amounts in the circulation. Antibodies to myosin, adenine nucleotide translocator, and branched chain ketoacid dehydrogenase were rarely observed in serum and heart eluates from control animals. This study indicates that myocardial injury may be a prerequisite for the induction of cardiac autoimmunity and suggests that as tissue damage accrues, recognition of additional cardiac Ag may occur.
Epithelial transcytosis of monomeric IgA and IgG cross-linked through antigen to polymeric IgA. A role for monomeric antibodies in the mucosal immune system.
Authors: Kaetzel C S , Robinson J K , Lamm M E
Published: December 2022

We recently demonstrated that the polymeric IgR (pIgR) mediates epithelial transcytosis of immune complexes (IC) containing dimeric IgA (dIgA). In vivo, this "excretory" pathway could allow direct elimination of IgA IC at the mucosal sites where they are likely to form. In contrast, IC containing only monomeric IgA (mIgA) were not transported, consistent with the specificity of pIgR for polymeric IgA. However, the potential exists in vivo that monomeric Ig like mIgA or IgG could become associated through binding to multivalent Ag with IC containing dIgA, and that such mixed IC could act as ligands for pIgR. In the present work, using Madin-Darby canine kidney epithelial cells that express pIgR, we showed that 125I-labeled anti-DNP mIgA or IgG in the same IC with unlabeled dIgA antibody and DNP-BSA Ag was vectorially transported from the basolateral to the apical surface and then released. However, 125I-mIgA IC or 125I-IgG IC (without dIgA) and 125I-mIgA or 125I-IgG in the presence of dIgA antibody (but without Ag) were not transported, demonstrating the necessity for monomeric Ig to be in an IC with dIgA to be transported. Transcytosis of mixed mIgA/dIgA or IgG/dIgA IC was mediated by pIgR because no transport was observed in untransfected, wild-type Madin-Darby canine kidney cells lacking pIgR. The data demonstrate that mIgA and IgG can participate along with dIgA in the "excretory" pathway for local elimination of IgA IC, thus providing a means by which monomeric antibodies have the potential to participate in the mucosal immune system.
Mitogenic CD2 monoclonal antibody pairs predispose peripheral T cells to undergo apoptosis on interaction with a third CD2 monoclonal antibody.
Authors: Rouleau M , Mollereau B , Bernard A , Metivier D , Rosenthal-Allieri M A , Charpentier B , Senik A
Published: December 2022

When stimulated for a few days with the mitogenic GT2 + T11(1) CD2 mAb pair and IL-2, resting T cells were induced to proliferate but the introduction into the cultures of a third CD2 mAb resulted in apoptotic cell death of 40 to 60% of the cells. The death signal was active on T cells entered the cell cycle without causing an apparent cell cycle block and without discriminating between the G1 and S phases. Apoptosis was not prevented by cycloheximide or actinomycin D, indicating that the death program was already expressed in preactivated cells awaiting an appropriate signal. A series of Abs, directed at various cell surface molecules, were unable to trigger apoptosis (except CD3 mAb), whereas most of the CD2 mAb tested were active, provided the third CD2 mAb was recognizing an epitope different from GT2 and T11(1). Mere aggregation of CD2 molecules did not seem to be the triggering signal of apoptosis, because cross-linking cell-bound GT2 + T11(1) with an Ab to mouse IgG had no effect, suggesting that a conformational change was imposed on CD2 molecules by the third CD2 mAb. Stimulation performed in the presence of IL-2 predisposed both CD45R0+ and CD45RA+ T cells to apoptosis, whereas stimulation in the presence of IL-4 primed only CD45RA+ T cells to undergo this process. Monocytes and potent co-signals of the CD2 pathway were unable to prevent CD2-induced apoptosis. Thus, successive engagements of the CD2 molecule of mature T cells by two and three CD2 mAbs recognizing distinct epitopes can provide in short term cultures signals for proliferation and apoptosis, depending on the activation state of the cells.
Molecular cloning and expression of early T cell costimulatory molecule-1 and its characterization as B7-2 molecule.
Authors: Chen C , Gault A , Shen L , Nabavi N
Published: December 2022

The interaction of T cell CD28/CTLA-4 receptors with B7-1 activation Ag on APC represents an important costimulatory pathway in T cell activation. However, it is now evident that this costimulatory pathway is neither unique nor universal for the activation of T cells. Our previous study indicated that a 60-kDa membrane protein, recognized by mAb 2D10, was expressed before B7 by activated murine B cells. This molecule was critically involved in activation of T cells in response to auto- and alloantigens. In the present study, we report on the isolation of a cDNA for this early T cell costimulatory molecule (ETC-1). ETC-1, like B7-1, is a member of the Ig supergene family and is composed of 303 amino acids. Nucleic acid sequence comparison indicated that ETC-1 is identical to the B7-2 molecule. When expressed in Chinese hamster ovary cells, ETC-1 showed profound T cell costimulatory activity as demonstrated by its ability to enhance CD4 T cell proliferation in response to Con A or anti-CD3 stimulation. Furthermore, ETC-1 also bound to both CD28-Ig and CTLA4-Ig fusion proteins. These results strongly support the notion that the interaction of ETC-1/B7-2 with CD28 or CTLA-4 receptors represents an alternative T cell costimulatory pathway.
Primary activation of V gamma 9-expressing gamma delta T cells by Mycobacterium tuberculosis. Requirement for Th1-type CD4 T cell help and inhibition by IL-10.
Authors: Pechhold K , Wesch D , Schondelmaier S , Kabelitz D
Published: December 2022

Purified peripheral blood gamma delta T cells proliferated vigorously in response to killed Mycobacterium tuberculosis (M. tb.) in the presence of PBMC but not in the presence of T cell-depleted (E-) feeder cells. Addition of graded numbers of autologous CD4 T cells to E- feeder cells reconstituted in a dose-dependent fashion the response of V gamma 9-expressing gamma delta T cells to M. tb. IL-2 was identified as the major CD4 T cell-derived helper factor required for gamma delta T cell proliferation after stimulation with M. tb. In addition, neutralizing anti-IFN-gamma but not anti-IFN-alpha Ab inhibited the responsiveness of V gamma 9 T cells, suggesting that endogenously produced IFN-gamma was involved in the activation of gamma delta T cells by M. tb. Although gamma delta T cells could not proliferate on their own in the absence of CD4 T cells (or exogenous IL-2), the appearance of IL-2 receptors (CD25) was triggered in the absence of CD4 T cells. Furthermore, IL-10 strongly inhibited the activation of V gamma 9 T cells among unfractionated PBMC responder cells. Similarly, the responsiveness of purified gamma delta T cells to M. tb. occurring in the presence of CD4 T cells was strongly inhibited by IL-10, whereas the activation occurring in the presence of exogenous IL-2 was not impaired. These results show that interactions with Th1-type CD4 T cells are required for efficient activation of peripheral blood gamma delta T cells by M. tb. In addition, our results have practical implications for creating experimental conditions aimed at identifying V gamma 9-selective (myco)bacterial ligands.
Induction of a protective human polysaccharide-specific antibody response in hu-PBL SCID mice by idiotypic vaccination.
Authors: Reason D C , Kitamura M Y , Lucas A H
Published: December 2022

The human Ab repertoire to the Haemophilus influenzae type b (Hib) polysaccharide (PS) is dominated by Abs that use the kappa II-A2 VL region and that express an idiotype (Id) designated Hibld-1. In this study we determined whether a human Hib PS-specific Ab response could be induced by idiotypic manipulation. We prepared a bispecific vaccine consisting of the F(ab')2 fragment of a mAb specific for Hibld-1, coupled to the F(ab')2 fragment of a mAb specific for CD3, a component of the TCR complex. This bispecific idiotypic vaccine stimulated production of human Abs to Hib PS in severe combined immunodeficient mice engrafted with normal human adult PBLs. The induced Abs uniformly expressed Hibld-1 and protected neonatal rats from Hib bacteremia. Experiments using additional conjugates demonstrated that covalent coupling of the CD3-specific moiety to the anti-Id was required for immunogenicity in this model, a result suggesting that engagement of B cell Id and proximate delivery of T cell signals are both necessary for B cell activation and differentiation. These findings demonstrate that human Ids can serve as targets for induction of a protective anti-PS Ab response.
Divergence of human alpha-chain constant region gene sequences. A novel recombinant alpha 2 gene.
Authors: Chintalacharuvu K R , Raines M , Morrison S L
Published: December 2022

IgA is the major Ig synthesized in humans and provides the first line of defense at the mucosal surfaces. The constant region of IgA heavy chain is encoded by the alpha gene on chromosome 14. Previous studies have indicated the presence of two alpha genes, alpha 1 and alpha 2, with alpha 2 existing in two allotypic forms, alpha 2 m(1) and alpha 2 m(2). Here we report the cloning and complete nucleotide sequence determination of a novel human alpha gene. Nucleotide sequence comparison with the published alpha sequences suggests that the gene arose as a consequence of recombination or gene conversion between the two alpha 2 alleles. We have expressed the gene as a chimeric protein in myeloma cells indicating that it encodes a functional protein. The novel IgA resembles IgA2 m(2) in that disulfide bonds link H and L chains. This novel recombinant gene provides insights into the mechanisms of generation of different constant regions and suggests that within human populations, multiple alleles of alpha may be present providing IgAs of different structures.
Conserved amino acid residues in the complementarity-determining region 1 of the TCR beta-chain are involved in the recognition of conventional Ag and Mls-1 superantigen.
Authors: Kang J , Chambers C A , Pawling J , Scott C , Hozumi N
Published: December 2022

Superantigens activate T cells by interacting primarily with the V beta region of the TCR. This report describes a series of studies performed to elucidate the role of the conserved amino acid motif (Asp-His-Asn) in the complementarity-determining region 1 (CDR1) of the TCR V beta chains that recognize murine endogenous superantigen Mls-1. By using site-directed mutagenesis of the Mls-1-reactive TCR V beta 6 gene, it is shown that the alterations of the conserved CDR1 motif disrupt Mls-1 superantigen and conventional Ag recognition in vitro. The loss of V beta 6 (mutant)+ TCR reactivity to Mls-1 superantigen is apparently dependent on the partner alpha-chain in the V beta 6/V alpha 3 TCR pairing shows some reactivity to Mls-1, whereas other TCR pairings do not. The examination of the developmental fate of the mutated form of the V beta 6 chain in Mls-1+ mice by using retroviral vector-mediated gene transfer confirms the critical role played by the CDR1 residues in Mls-1 recognition in vivo. Collectively, the results indicate that the CDR1 of the TCR V beta 6 chain is involved in interacting with peptide/MHC as well as in Mls-1 recognition. The observation that the conserved residues in selective TCR V beta chains are imparting a significant contribution to Ag recognition suggests a molecular basis for the intrinsic bias of some V beta chains for MHC molecules.
Role of IL-6 in promoting growth of human EBV-induced B-cell tumors in severe combined immunodeficient mice.
Authors: Durandy A , Emilie D , Peuchmaur M , Forveille M , Clement C , Wijdenes J , Fischer A
Published: December 2022

To investigate the role of IL-6 as a growth factor in EBV-induced B lymphoproliferative disorders in immune-suppressed patients, we analyzed two B cell lines derived from two different patients for IL-6 production, expression of the p80 chain of the IL-6 receptor, and the effects on cell growth in vitro and in vivo of neutralizing mAbs specific for IL-6. One of the cell lines (LCL41) was shown to produce large amounts of IL-6 and to express IL-6 receptors. Its in vitro growth was weakly inhibited by the anti-IL-6 MAb. After inoculation into SCID mice, this cell line provoked human B cell tumors, the growth of which was controlled by the anti-IL-6 mAb. Indeed, four i.v. infusions of 0.1 mg at days 30 to 42 after i.p. inoculation of cells led to complete remission in most mice and long term survival in 40% of cases. In contrast, the other B cell line (LCL48) produced smaller amounts of IL-6; its growth was not inhibited in vitro by the anti-IL-6 Ab and was poorly blocked in vivo in SCID mice (40% of remissions and 20% of long-term survival). In addition, a clone derived in vivo in SCID mice from LCL48 was completely IL-6-independent. These results demonstrate that B cell tumors transformed in vivo by EBV in immune-suppressed patients are heterogeneous with respect to IL-6 requirements for proliferation. An antitumoral effect in some of them can be achieved by neutralizing the IL-6-dependent proliferative loop.
Role of IL-6 in activation of T cells for acquired cellular resistance to Listeria monocytogenes.
Authors: Liu Z , Simpson R J , Cheers C
Published: December 2022

We have previously shown that IL-6 is a major cytokine in the serum of mice infected with the intracellular bacterium Listeria monocytogenes, and that injection of rIL-6 before, but not after, infection promotes the recovery of mice from listeriosis. Here we demonstrate that IL-6 is required in the early stages of infection, in that injection of anti-IL-6 Ab 24 or 4 h before infection, but not 24 h after, led to a massive increase in bacterial numbers 4 or more days after infection. On the other hand, injection of rIL-6 before infection significantly suppressed bacterial numbers in the liver and spleen 3 to 4 days after infection and increased the production of IFN-gamma by in vitro cultured CD4+ and CD8+ T cells in response to specific Ag. rIL-6 did not protect SCID mice, which lack both T and B lymphocytes, against Listeria infection. The protective effect of rIL-6 was neutralized by the injection of Ab to IFN-gamma. We conclude that IL-6 plays an essential role in the activation of T cells to produce IFN-gamma, the cytokine that is central to acquired cellular resistance to intracellular bacteria.
Crystal-induced neutrophil activation. V. Differential production of biologically active IL-1 and IL-1 receptor antagonist.
Authors: Roberge C J , de Médicis R , Dayer J M , Rola-Pleszczynski M , Naccache P H , Poubelle P E
Published: December 2022

Neutrophils produce IL-1 when stimulated by monosodium urate (MSU) or calcium pyrophosphate dihydrate (CPPD) crystals. Neutrophils also generate the IL-1R antagonist (IL-1Ra), especially when incubated with granulocyte-macrophage CSF (GM-CSF) or TNF-alpha. We studied the simultaneous expression of IL-1 and IL-1Ra by GM-CSF- or TNF-alpha-treated neutrophils activated by MSU or CPPD. Neutrophils incubated with GM-CSF or TNF-alpha produced approximately 300 or 200 times more IL-1Ra than IL-1, respectively. Suboptimal concentrations of MSU or CPPD induced low amounts of IL-1 without affecting IL-1Ra. Interaction of GM-CSF- and TNF-alpha-treated neutrophils with MSU or CPPD up-regulated IL-1 while simultaneously down-regulating IL-1Ra. As a result, the bioactivity of IL-1 secreted was enhanced. Synergistic increases of IL-1 (but not IL-1Ra) mRNA levels were noted in GM-CSF- or TNF-alpha-treated neutrophils exposed to CPPD. Treatment of neutrophils with colchicine before incubation with GM-CSF or TNF alpha, inhibited crystal-induced IL-1 by 50 to 55%, but failed to significantly affect IL-1Ra. The IL-1Ra to IL-1 ratio was significantly increased by 185 to 220%. These results demonstrate that IL-1 and IL-1Ra production by human neutrophils are differentially regulated, that the combined presence of GM-CSF or TNF-alpha and microcrystals favor the production of biologically active IL-1 over that of IL-1Ra, and that colchicine selectively inhibits IL-1 without affecting IL-1Ra production.
Cytokine mRNA expression in salivary gland biopsies of Sjögren's syndrome.
Authors: Fox R I , Kang H I , Ando D , Abrams J , Pisa E
Published: December 2022

Sjögren's syndrome is a human autoimmune disease characterized by lymphocytic infiltration of salivary and lacrimal glands, hypergammaglobulinemia, and specific autoantibodies. The accessibility of the salivary gland to biopsy provides an opportunity to study cytokine mRNA expression at the site of organ-specific immune damage. Using reverse transcriptase and a quantitative PCR to measure cytokine mRNA, we found Sjögren's syndrome: 1) salivary gland CD4+ T cells produce over 40-fold more IL-2, IFN-gamma, and IL-10 mRNA than peripheral blood CD4+ T cells from the same patient or from normal controls; 2) salivary gland CD4+ T cells produced little IL-4 and IL-5 mRNA immediately after elution from the salivary gland, although these mRNAs could be induced by mitogen stimulation of Sjögren's syndrome salivary gland lymphocytes in vitro; 3) salivary gland epithelial cells produced over 40-fold more IL-1 alpha, IL-6, and TNF-alpha mRNA than epithelial cells from individuals with histologically normal salivary glands. Increased levels of IL-1 alpha, IL-6, IL-10, TNF-alpha, and IFN-gamma cytokines were found by ELISA assay in the saliva of Sjögren's syndrome patients, indicating that the elevated mRNA levels detected in their glandular tissue by PCR correlate with local protein synthesis. Our results demonstrate that CD4+ cells in the salivary glands of Sjögren's syndrome patients exhibit mRNA expression that is distinct from previously described Th1 and Th2 lymphocytes in mice or cytokines reported in other human autoimmune or allergic diseases. Also, we found that salivary gland epithelial cells may participate in the pathogenesis of Sjögren's syndrome biopsy by producing cytokines (IL-1 alpha, IL-6, and TNF-alpha).
Cytotoxic T cell repertoire selection. A single amino acid determines alternative class I restriction.
Authors: Bergmann C C , Tong L , Cua R V , Sensintaffar J L , Stohlman S A
Published: December 2022

CTL responses are governed by intracellular Ag processing, affinity of peptides for MHC class I molecules, and the T cell repertoire. In this report we demonstrate that a class I Dd-restricted 10-mer CTL epitope within the gp160 envelope glycoprotein of HIV-1 strain IIIB (residues 318-327) contains a 9-amino acid peptide (residues 319-327), which efficiently binds to both the Dd and Ld class I molecules in vitro. The potential for broadening the naturally limited CTL response to include presentation on the Ld class I molecules in vivo was examined using a minigene-based vaccine strategy to insure cytosolic expression of "preprocessed" forms of the gp160 epitope. Immunization with recombinant vaccinia viruses (vac) expressing either the gp160 10 mer or 9 mer, both including an initiation methionine (M318-327 and M319-327, respectively), induced predominantly Dd-restricted CTL specific for native gp160. By contrast, recombinant vac expressing eight gp160 amino acids (M320-327) generated predominantly Ld-restricted CTL which are specific for synthetic gp160 peptides but not native gp160. The ability to induce Ld-restricted CTL suggests that the absence of an Ld-restricted response to native gp160 cannot be attributed to a limited T cell repertoire, but to inefficient processing of gp160 for presentation on Ld. The switch in class I restriction, controlled by a single amino acid within one epitope, demonstrates that nonanchor residues have a profound effect on differential MHC restriction and CTL induction. Thus, minigene-based vaccines expressing minimal epitopes may be useful in inducing a more heterogeneous CTL response than previously appreciated.
Expression of the homotypic adhesion molecule E-cadherin by immature murine thymocytes and thymic epithelial cells.
Authors: Lee M G , Sharrow S O , Farr A G , Singer A , Udey M C
Published: December 2022

Cadherins mediate homotypic adhesion between lineage-related cells in epithelia and other tissues. One cadherin, E-cadherin, is also responsible for adhesion of murine epidermal Langerhans cells to keratinocytes in vitro, and may play a role in the localization of Langerhans cells in epidermis. The thymus is another tissue in which important adhesive interactions between bone marrow-derived cells and keratinizing epithelia occur. To determine whether cadherins might be involved in interactions between thymocytes and thymic epithelial cells, we examined thymocytes from C57BL/6 mice of various gestational ages for cadherin expression. Most day 14 (D14) and essentially all D16 isolated fetal thymocytes expressed cell surface E-cadherin. After D16, the proportion of fetal thymocytes expressing E-cadherin and the level of E-cadherin expressed by individual thymocytes decreased with increasing gestational age. A minority of neonatal thymocytes and very few adult thymocytes expressed E-cadherin. E-cadherin was maximally expressed by the least mature (CD4-CD8-, HSA (J11d)high, CD5 (Ly-1)low, CD25 (IL-2R alpha)+) thymocytes. P-cadherin, another epithelial cadherin, was not detected on thymocytes at any stage of development. Immunohistologic studies revealed that thymic epithelial cells also expressed E-cadherin. Similar levels of E-cadherin were expressed by neonatal and adult thymic epithelial cells in situ, and E-cadherin was easily demonstrable on the thymic epithelial cell line, TE-71. In contrast, P-cadherin was transiently expressed by thymic epithelial cells in situ, and only small amounts of P-cadherin were detected on TE-71 cells. These studies demonstrate that thymocytes and thymic epithelial cells each have the capacity to express the homotypic adhesion molecule E-cadherin. E-cadherin may play a role in developmentally regulated interactions between early thymocytes and thymic stromal cells.
Molecular context of a viral T cell determinant within a chimeric bacterial protein alters the diversity of its T cell recognition.
Authors: Lo-Man R , Martineau P , Betton J M , Hofnung M , Leclerc C
Published: December 2022

We genetically introduced two different viral CD4+ T cell epitopes within two internal sites of the Escherichia coli maltose-binding (MalE) protein. Affinity-purified hybrid MalE proteins were used to analyze the influence of the molecular environment on the presentation of inserted epitope to T cells. In the first model, the 120 to 132 PreS T cell epitope was inserted alone or with its C-terminal B cell epitope (132-145) at site 133 or 303 of MalE. The maltose-binding protein with PreS peptide inserts expressing the 120 to 132 sequence were able to induce in vivo and in vitro peptide-specific T cell response, whatever the length and the position of the insert. In the second model, the 103 to 115 T cell epitope from the C3 region of poliovirus type 1 (PV1) was inserted, with various flanking sequences, either at site 133 or 303 of MalE protein. The longer C3:86 to 115 insert induced poliovirus-specific T cell responses at both sites of MalE, whereas the C3:93 to 115 insert did it only at site 303 but not at site 133. Moreover, C3:103 to 115 specific T cell hybridomas discriminated between the processed peptides generated from the different chimeric proteins, as a result of differences in the length and the position of the inserted sequence. Therefore, in this experimental model the loss of in vivo immunogenicity of an antigenic determinant within a chimeric protein is related to the activation of a reduced T cell repertoire. These observations involve important consequences for the engineering of recombinant vaccines.
CD23 interacts with a new functional extracytoplasmic domain involving N-linked oligosaccharides on CD21.
Authors: Aubry J P , Pochon S , Gauchat J F , Nueda-Marin A , Holers V M , Graber P , Siegfried C , Bonnefoy J Y
Published: December 2022

Human CD21 has been described as a receptor for the C3d,g and iC3b proteins of complement, for the Epstein-Barr virus, and also for IFN-alpha. We reported recently that CD23, a low affinity receptor for IgE (Fc epsilon R2), is a new functional ligand for CD21. To determine the site of interaction of CD23 on CD21, we analyzed the ability of purified recombinant CD23 incorporated into fluorescent liposomes to bind CD21 mutants bearing various deletions of extracytoplasmic short consensus repeats (SCRs). We found that the site of interaction of CD23 on CD21 is on SCRs 5 to 8, with contribution of SCRs 1 and 2. Tunicamycin treatment of CD21-transfected K562 cells strongly inhibited the binding of CD23-liposomes, suggesting that an N-linked sugar, present on SCRs 5 to 8, is involved in the CD23/CD21 interaction. By mutating together or individually, the three asparagines present on SCRs 5 to 8, asparagines (Asn) 370 and 295, but not Asn 492, were shown to be involved critically in the binding of CD23. Furthermore, we mapped the binding sites of a panel of anti-CD21 mAbs and found that at least six epitopes can be detected on CD21. The mAbs that inhibit the most CD23 binding to CD21 map in SCRs 5 to 8. This study indicates that SCRs 5 to 8 represent a novel functional domain on the CD21 molecule, and is the first demonstration of an activity of an extracytoplasmic region of the CD21 outside of SCRs 1 to 4.
Recombinant soluble CD14 inhibits LPS-induced tumor necrosis factor-alpha production by cells in whole blood.
Authors: Haziot A , Rong G W , Bazil V , Silver J , Goyert S M
Published: December 2022

CD14 functions as a cell surface receptor for LPS in the activation of monocytes/macrophages and neutrophils by endotoxin. To assess the utility of soluble forms of the CD14 receptor as a possible therapeutic for endotoxin shock, we have produced recombinant human soluble CD14 using a baculovirus expression system. We find that the recombinant protein is not only expressed on the surface of the insect cells as a glycosyl phosphatidylinositol (GPI)-anchored protein, but is also released into the culture medium as a soluble form that lacks the GPI anchor. Functional analyses of recombinant human soluble CD14 show that it binds specifically to LPS and can inhibit the LPS-induced release of TNF-alpha by macrophages and mononuclear cells as well as by cells in whole human blood when used at concentrations of approximately 70 micrograms/ml. Thus, soluble CD14 may be useful as an adjunct in the treatment of endotoxin shock.
Inflammatory granuloma formation is mediated by TNF-alpha-inducible intercellular adhesion molecule-1.
Authors: Lukacs N W , Chensue S W , Strieter R M , Warmington K , Kunkel S L
Published: December 2022

Recent studies have demonstrated a crucial role for TNF during inflammatory granuloma formation. In addition, TNF has been shown to up-regulate adhesion molecules that participate in cellular recruitment and lymphocyte activation. In the present study, we have examined the mechanism of TNF activation during Schistosoma mansoni egg granuloma formation and its relationship to the expression of ICAM-1. Our initial studies showed that high affinity human soluble TNFR coupled to the Fc portion of an Ig (sTNFR:Fc construct) could effectively diminish granuloma formation and lymphocyte activation in vivo. We have also assessed the increased expression of ICAM-1, its contribution to granuloma development, and its relationship with TNF during lesion formation. Increased steady state ICAM-1 mRNA expression was observed in primary egg granulomas when compared with normal lung and foreign body (Sephadex bead) granulomas, which suggests a role for ICAM-1 in Ag-induced lesion formation. Subsequent studies have demonstrated that sTNFR:Fc treatment down-regulated granuloma formation and ICAM-1 expression, thus suggesting one mechanism of TNF involvement in granuloma formation was through the induction of ICAM-1. Anti-ICAM-1 decreased the soluble egg Ag-specific T cell proliferation in vitro. In addition, passive immunization of mice with anti-ICAM-1 mAb during primary granuloma formation resulted in an attenuation of lesion development as compared with lesion development in a control Ab-treated group. The proliferative response to soluble egg Ag was also significantly reduced in ex vivo experiments that used spleen cells from the anti-ICAM-1 treated mice. These data demonstrate that both TNF and ICAM-1 participate in lymphocyte activation and granuloma formation and suggest that one mechanism of TNF in granuloma development is through TNF-induced ICAM-1 expression.
Restoration of IFN-gamma production and lymphocyte proliferation in visceral leishmaniasis.
Authors: Carvalho E M , Bacellar O , Brownell C , Regis T , Coffman R L , Reed S G
Published: December 2022

Visceral leishmaniasis is associated with a marked depression of T cell responses, which has been characterized by the absence of IL-2 and IFN-gamma production by lymphocytes on in vitro stimulation with Leishmania Ag. The aim of this study was to evaluate both the mechanism of these immunologic abnormalities and the restoration of in vitro T cell responses to Leishmania Ags. A total of 15 untreated visceral leishmaniasis patients were evaluated. Although IFN-gamma and IL-4 levels in the supernatants of lymphocyte cultures were very low or absent, mRNA for these cytokines and for IL-10 were observed in PBMCs. Addition of IFN-gamma plus IL-2 enhanced lymphocyte proliferation by 158%. Restoration of T cell proliferative responses and IFN-gamma production was also observed by the addition of a neutralizing mAb alpha-IL-10. Neutralizing mAb alpha-IL-4 did not restore T cell responses but alpha-IL-10 and alpha-IL-4 mAbs had a synergistic effect on lymphocyte proliferation. The IFN-gamma levels in supernatants of lymphocyte cultures stimulated with Leishmania chagasi Ag or L. chagasi Ag plus alpha-IL-4, alpha-IL-10, or alpha-IL-4 plus alpha-IL-10 mAbs were 26 +/- 30 pg/ml, 41 +/- 18 pg/ml, 146 +/- 73 pg/ml, and 174 +/- 106 pg/ml, respectively. These data indicate that Th2 cell activation occurs in visceral leishmaniasis and that in vitro production of IFN-gamma and lymphocyte proliferation can be restored by blocking the inhibitory effect of the Th2 cytokines on mononuclear cells.
B cell differentiation defects in common variable immunodeficiency are ameliorated after stimulation with anti-CD40 antibody and IL-10.
Authors: Eisenstein E M , Chua K , Strober W
Published: December 2022

In these studies we show that although purified B cells of patients with common variable immunodeficiency (CVI) have a normal capacity to proliferate, they manifest differentiation defects at multiple levels. Compared with controls, circulating CVI B cell populations contain reduced numbers of sIgG+ and sIgA+ cells with a commensurate increase in sIgM+ B cells, suggesting an in vivo defect in isotype switch. In addition, CVI B cells manifest Ig secretion defects on stimulation with either anti-CD40 and IL-10 or SAC and IL-2 and IL-10, which are of increasing severity for IgM, IgG, and IgA, respectively. These Ig secretion defects are not overcome by addition of a variety of cytokines, including TGF-beta, to anti-CD40-driven cultures. In further studies we show that despite the above abnormalities, CVI B cells are induced to express normal or near-normal levels of C mu, C gamma, and C alpha mRNA after 7 days of stimulation with anti-CD40 and IL-10. That this CH mRNA expression represents a recovery of CVI B cell differentiation is supported by studies of Ig secretion in which CVI B cells that are first stimulated for 7 days with anti-CD40 and IL-10 and then restimulated in coculture with activated normal allogeneic T cells and IL-10, secrete substantial levels of IgM and IgG and increased amounts of IgA. Overall, therefore, CVI B cell function can be significantly improved by maintenance in culture. These data suggest the abnormalities of B cell differentiation in CVI are reversible and that the defect is a form of B cell anergy.