bluuebunny/crossref_metadata_2025 huggingface dataset:
Cutting Edge: Presence of Progenitors Restricted to T, B, or Myeloid Lineage, but Absence of Multipotent Stem Cells, in the Murine Fetal Thymus
Authors: Kawamoto Hiroshi , Ohmura Koichiro , Katsura Yoshimoto
Published: December 2022

The most immature population of fetal thymus (FT) cells has been shown to generate not only T but also B and myeloid cells. The present study was undertaken to clarify whether such a multipotent activity of the earliest population of FT cells is attributed to multipotent hemopoietic progenitors or to a mixture of lineage-restricted progenitors. Examination of individual FT progenitors by a recently established clonal assay system, which is able to determine the developmental potential of each progenitor toward T, B, and myeloid lineages, elucidated that a large majority of progenitors in FT were restricted to the T cell lineage. Presence of a small number of B or myeloid lineage-restricted progenitors was also disclosed. No multipotent progenitors, however, were detected in FT. These results are consistent with our recent finding that restriction of hemopoietic stem cells to T, B, and myeloid lineages takes place in the fetal liver.
Cutting Edge: Negative Regulation of Human T Cell Activation by the Receptor-Type Protein Tyrosine Phosphatase CD148
Authors: Tangye Stuart G. , Wu Jun , Aversa Gregorio , de Vries Jan E. , Lanier Lewis L. , Phillips Joseph H.
Published: December 2022

T cell activation represents a balance between positive and negative signals delivered via distinct cell surface molecules. Many cytoplasmic protein tyrosine phosphatases are involved in regulating cellular responses by antagonizing the action of protein tyrosine kinases. CD148 is a receptor-type protein tyrosine phosphatase expressed by all human mononuclear cells. We have investigated the effect of CD148 on TCR-mediated activation of human T cells. Overexpression of wild-type, but not a phosphatase-deficient, CD148 in Jurkat T cells inhibited TCR-mediated activation, evidenced by reduced expression of the early activation Ag CD69, inhibition of tyrosine phosphorylation of many intracellular proteins including the critical protein tyrosine kinase ZAP-70, and impairment of mitogen-activated protein kinase activation. Taken together, these results suggest that CD148 is an important phosphatase involved in negatively regulating the proximal signaling events during activation of Ag-specific T cells.
Cutting Edge: IL-4-Independent Induction of Airway Hyperresponsiveness by Th2, But Not Th1, Cells
Authors: Cohn Lauren , Tepper Jeffrey S. , Bottomly Kim
Published: December 2022

We investigated the role of Th1 or Th2 cells in airway hyperresponsiveness (AHR), because both IFN-γ and IL-4 and IL-5-producing CD4 T cells have been identified in the airways of asthmatics. After transfer of in vitro-generated TCR transgenic Th1 or Th2 cells and exposure to inhaled Ag, Th2 cells induced AHR and airway eosinophilia, whereas Th1 cells induced neutrophilic inflammation without AHR. Next, to determine the precise effector function of IL-4 in Th2 cell-induced AHR, we transferred IL-4−/− Th2 cells into wild-type and IL-4−/− recipient mice. After exposure to inhaled Ag, both groups of mice exhibited AHR with markedly reduced airway eosinophilia. Thus, IL-4 production by Th2 cells is not essential for the induction of AHR, but is critical for the migration of eosinophils from lung tissue into the airways.
B Lymphocytes Are Critical Antigen-Presenting Cells for the Initiation of T Cell-Mediated Autoimmune Diabetes in Nonobese Diabetic Mice
Authors: Serreze David V. , Fleming Sara A. , Chapman Harold D. , Richard Scott D. , Leiter Edward H. , Tisch Roland M.
Published: December 2022

Nonobese diabetic (NOD) mice genetically deficient in B lymphocytes (NOD.Igμnull) are resistant to T cell-mediated autoimmune insulin-dependent diabetes mellitus (IDDM). Ig infusions from diabetic NOD donors did not abrogate IDDM resistance in NOD.Igμnull mice. However, T cell responses to the candidate pancreatic β cell autoantigen glutamic acid decarboxylase (GAD), but not the control Ag keyhole limpet hemocyanin, were eliminated in NOD.Igμnull mice. To initially test whether they contribute to IDDM as APC, NOD B lymphocytes were transferred into NOD.Igμnull recipients. B lymphocytes transferred into unmanipulated NOD.Igμnull recipients were rejected by MHC class I-restricted T cells. Stable T and B lymphocyte repopulation was achieved in irradiated NOD.Igμnull mice reconstituted with syngeneic bone marrow admixed with NOD B lymphocytes. IDDM susceptibility was restored in NOD.Igμnull mice reconstituted with syngeneic marrow plus B lymphocytes, but not with syngeneic marrow only. T cell responses to GAD were restored only in NOD.Igμnull mice reconstituted with syngeneic marrow plus B lymphocytes. Hence, B lymphocytes appear to contribute to IDDM in NOD mice as APC with a preferential ability to present certain β cell Ags such as GAD to autoreactive T cells.
Molecular Mechanisms of Immune-Mediated Lysis of Murine Renal Cancer: Differential Contributions of Perforin-Dependent Versus Fas-Mediated Pathways in Lysis by NK and T Cells
Authors: Sayers Thomas J. , Brooks Alan D. , Lee Jong-Keuk , Fenton Robert G. , Komschlies Kristin L. , Wigginton Jon M. , Winkler-Pickett Robin , Wiltrout Robert H.
Published: December 2022

Mice bearing the experimental murine renal cancer Renca can be successfully treated with some forms of immunotherapy. In the present study, we have investigated the molecular pathways used by NK and T cells to lyse Renca cells. Renca cells normally express low levels of Fas that can be substantially enhanced by either IFN-γ or TNF-α, and the combination of IFN-γ + TNF-α synergistically enhances cell-surface Fas expression. In addition, cells pretreated with IFN-γ and TNF-α are sensitive to lysis mediated by Fas ligand (FasL)-expressing hybridomas (dllS), cross-linking of anti-Fas Abs or soluble Fas (FasL). Lysis via Fas occurs by apoptosis, since Renca shows all the typical characteristics of apoptosis. No changes in levels of bcl-2 were observed after cytokine treatments. We also examined cell-mediated cytotoxic effects using activated NK cells and T cells from gld FasL-deficient mice, and perforin-deficient mice, as well as wild-type C57BL/6 and BALB/c mice. Interestingly, the granule-mediated pathway predominated in killing of Renca by activated NK cells, while the Fas/FasL pathway contributed significantly to cell-mediated killing of Renca by activated T cells. These results suggest that killing of Renca tumor cells by immune effector cells can occur by both granule and Fas-mediated cytotoxicity. However, for the Fas-mediated pathway to function, cell surface levels of Fas need to be increased beyond a critical threshold level by proinflammatory cytokines such as IFN-γ and TNF-α.
Critical Involvement of Tcf-1 in Expansion of Thymocytes
Authors: Schilham Marco W. , Wilson Anne , Moerer Petra , Benaissa-Trouw Barry J. , Cumano Ana , Clevers Hans C.
Published: December 2022

T cell maturation in Tcf-1−/− mice deteriorates progressively and halts completely around 6 mo of age. During fetal development thymocyte subpopulations seem normal, although total cell numbers are lower. By 4 to 6 wk of age, obvious blockades in the differentiation of CD4−8− thymocytes are observed at two distinct stages (CD44+25+ and CD44−25−), both of which are normally characterized by extensive proliferation. This lack of thymocyte expansion and/or differentiation was also observed when Tcf-1−/− progenitor cells from the aorta-gonad-mesonephros region (embryonic day 11.5), fetal liver (embryonic day 12.5/14.5), and fetal bone marrow (embryonic day 18.5) were allowed to differentiate in normal thymic lobes (fetal thymic organ cultures) or were injected intrathymically into normal recipients. Despite these apparent defects in thymocyte differentiation and expansion, adult Tcf-1−/− mice are immunocompetent, as they generate virus neutralizing Abs at normal titers. Furthermore, their peripheral T cells have an activated phenotype (increased CD44 and decreased CD62L expression) and proliferate normally in response to Ag or mitogen, suggesting that these cells may have arisen from the early wave of development during embryogenesis and are either long lived or have subsequently been maintained by peripheral expansion. As Tcf-1 is a critical component in the Wnt/β-catenin signaling pathway, these data suggest that Wnt-like factors play a role in the expansion of double-negative thymocytes.
Affinity and Kinetic Analysis of the Molecular Interaction of ICAM-1 and Leukocyte Function-Associated Antigen-1
Authors: Tominaga Yuichi , Kita Yasuo , Satoh Atsushi , Asai Satoshi , Kato Kimitoshi , Ishikawa Koichi , Horiuchi Tadashi , Takashi Tohru
Published: December 2022

LFA-1 is a member of the β2 integrin family, and interacts with ICAM-1, a member of the Ig superfamily containing five Ig-like domains. Interaction of LFA-1 with ICAM-1 is important in a number of cellular events, including Ag-specific T cell activation and leukocyte transendothelial migration, which are known to be typically transient and highly regulated. In this study, we have used surface plasmon resonance technology to study the ICAM-1/LFA-1 interaction at the molecular level. A soluble form of LFA-1 (sLFA-1), normally expressed as two noncovalently associated membrane-bound subunits, has been produced, and its interaction with ICAM-1 has been examined. The kinetic analysis of a monomeric sLFA-1 binding to the first two domains of ICAM-1 expressed as a chimeric IgG fusion protein (D1D2-IgG) revealed that sLFA-1 was bound to the D1D2-IgG chimera with a Kd of 500 nM and dissociated with a kdiss of 0.1 s−1. Monomeric membrane-bound LFA-1 purified from plasma membranes showed a similar kinetic to sLFA-1. These results suggest that the monovalent interaction between ICAM-1 and LFA-1 has a primarily high affinity and a slow dissociation rate constant as compared with other adhesion molecules, suggesting a potential mechanism for firm adhesion.
Large Protein Fragments as Substrates for Endocytic Antigen Capture by MHC Class II Molecules
Authors: Castellino Flora , Zappacosta Francesca , Coligan John E. , Germain Ronald N.
Published: December 2022

Although the binding sites of MHC class II molecules can accommodate longer ligands, peptides of 15 to 20 residues are the primary form of processed Ag recovered from class II dimers isolated from living cells. These peptides are derived from intact Ags by proteolysis in endocytic organelles, where binding to class II dimers also occurs. Whether generation of these short peptides typically precedes association with class II molecules, or whether class II molecules initially bind to unfolded proteins or large protein fragments, followed by degradation of the unprotected regions, remains unknown. Here we report the identification of an SDS-stable, long-lived, 120-kDa complex composed of two class II dimers bound to a common large Ag fragment. This complex is produced within the endocytic pathway from newly synthesized MHC class II molecules following exposure of the cells to exogenous hen egg lysozyme. These data suggest that a major pathway of Ag processing involves the initial binding of class II heterodimers to large protein substrates upon exposure of regions with suitable motifs, followed by cleavage and/or trimming of the exposed protein around this bound region. This sequence of events during Ag processing may provide a partial molecular explanation for the immunodominance of certain determinants in protein Ags.
CD3/TCR Complex-Associated Lymphocyte Activation Gene-3 Molecules Inhibit CD3/TCR Signaling
Authors: Hannier Sigrid , Tournier Muriel , Bismuth Georges , Triebel Frédéric
Published: December 2022

The lymphocyte activation gene-3 (LAG-3) molecule is a T cell activation Ag closely related to CD4 at the gene and protein levels. We investigated whether LAG-3 itself may down-regulate the immune response by interfering with TCR signaling. The binding of Ab to the LAG-3 molecule followed by cross-linking (XL) inhibits cell proliferation and cytokine secretion in response to CD3XL on activated T cells. LAG-3XL-induced down-regulation is associated with functional unresponsiveness, as well as with high CD25 expression levels and reversion by exogenous IL-2. It is also associated with a down-modulation of CD3/TCR complex expression. At the biochemical level, LAG-3XL inhibits calcium response to CD3 stimulation. This inhibition is observed with different LAG-3- and CD3-specific mAbs on condition that the two receptors are cross-linked together. Finally, the capping of CD3 was shown to induce cocapping of LAG-3 molecules. Together, these results show that CD3/TCR complex-associated LAG-3 molecules can play an active role in negatively regulating the CD3/TCR activation pathway. They ultimately suggest that LAG-3 is an inhibitory receptor in activated T lymphocytes.
Genomics, Isoforms, Expression, and Phylogeny of the MHC Class I-Related MR1 Gene
Authors: Riegert Patricia , Wanner Valérie , Bahram Seiamak
Published: December 2022

A growing number of non-MHC-encoded class I-related molecules have been shown to perform diverse, yet essential, functions. These include T cell presentation of bacterially derived glycolipidic Ags by CD1, transcytosis of maternal IgG by the neonatal Fc receptor, enriched presence and plausible function within exocrine fluids of the Zn-α2-glycoprotein, subversion of NK cytolytic activity by the CMV UL18 gene product, and, finally, crucial involvement in iron homeostasis of the HFE gene. A recently described member of this family is the MHC class-I related (MR1) gene. The most notable feature of MR1 is undoubtedly its relatively high degree of sequence similarity to the MHC-encoded classical class I genes. The human chromosome 1q25.3 MR1 locus gives rise not only to the originally reported 1,263-bp cDNA clone encoding a putative 341-amino acid polypeptide chain, but to many additional transcripts in various tissues as well. Here we define the molecular identity of all human and murine MR1 isoforms generated through a complex scenario of alternative splicing, some encoding secretory variants lacking the Ig-like α3 domain. Moreover, we show ubiquitous transcription of these MR1 variants in several major cell lineages. We additionally report the complete 18,769-bp genomic structure of the MR1 locus, localize the murine orthologue to a syntenic segment of chromosome 1, and provide evidence for conservation of a single-copy MR1 gene throughout mammalian evolution. The 90% sequence identity between the human and mouse MR1 putative ligand binding domains together with the ubiquitous expression of this gene favor broad immunobiologic relevance.
Production of IgM Hexamers by Normal and Autoimmune B Cells: Implications for the Physiologic Role of Hexameric IgM
Authors: Hughey C. Todd , Brewer Joseph W. , Colosia Ann D. , Rosse Wendell F. , Corley Ronald B.
Published: December 2022

Secreted IgM is predominantly found as pentameric molecules, but IgM can also be secreted as hexamers by B cell lines. Murine hexamers activate the complement cascade more efficiently than pentamers, but the physiologic significance of hexameric IgM remains unknown. Here, we report that IgM hexamers and pentamers are cleared from the circulation with similar kinetics, suggesting that the predominance of pentameric IgM in vivo reflects the regulation of polymer assembly and secretion in responding B cells. Normal IgM-secreting B cells, particularly those from the peritoneal cavity, are capable of secreting abundant hexameric IgM in vitro. The disparity between the ability of B cells to secrete IgM hexamers in vitro and the paucity of this polymer in vivo suggest that IgM hexamers might be deleterious. In support of this, we demonstrate that the autoantibodies from a number of patients with cold agglutinin (CA) disease include both IgM hexamers and pentamers. The CA IgM hexamers lyse human erythrocytes in the presence of human complement more efficiently than CA IgM pentamers, suggesting a potential role for hexameric IgM in the pathogenesis of this autoimmune syndrome.
Peripheral Blood-Derived CD34+ Progenitor Cells: CXC Chemokine Receptor 4 and CC Chemokine Receptor 5 Expression and Infection by HIV
Authors: Ruiz Margaret E. , Cicala Claudia , Arthos James , Kinter Audrey , Catanzaro Andrew T. , Adelsberger Joseph , Holmes Kevin L. , Cohen Oren J. , Fauci Anthony S.
Published: December 2022

The present study demonstrates cell surface expression of both CXC chemokine receptor 4 (CXCR4) and CC chemokine receptor 5 (CCR5), major coreceptors for T cell-tropic and macrophage-tropic strains of HIV, respectively, on CD34+ progenitor cells derived from the peripheral blood. CD34+ progenitor cells were susceptible to infection by diverse strains of HIV, and infection could be sustained for prolonged periods in vitro. HIV entry into CD34+ progenitor cells could be modulated by soluble CD4, HIV gp120 third variable loop neutralizing mAb and the cognate ligands for the CXCR4 and CCR5 HIV coreceptors. This study suggests that a significant proportion of the circulating progenitor cell pool may serve as a reservoir for HIV that is capable of trafficking the virus to diverse anatomic compartments. Furthermore, the infection and ultimate destruction of these progenitor cells may explain in part the defective lymphopoiesis in certain HIV-infected individuals despite effective control of virus replication during highly active antiretroviral therapy.
Haemophilus influenzaeType b Polysaccharides-Protein Conjugate Vaccine Elicits a More Diverse Antibody Repertoire in Infants Than in Adults
Authors: Adderson Elisabeth E. , Wilson Patricia M. , Cunningham Madeleine W. , Shackelford Penelope G.
Published: December 2022

Conjugation of bacterial polysaccharides (PS) to protein carriers confers the ability to elicit protective serum Ab in infants, who respond poorly to plain PS. The serum Ab of young children immunized with Haemophilus influenzae type b (Hib) PS conjugate vaccine varies with age and Ag formulation. To understand these age-related changes in human anti-Hib PS immune responses we determined the variable region gene sequences encoding anti-Hib PS mAbs of infants immunized with Hib oligosaccharide-diphtheria toxin vaccine. The anti-Hib PS repertoire of children differs from that of adults. A smaller proportion of mAbs from children have high affinity for Hib PS, and the overall variable region gene repertoire of infants is more diverse than that in adults. Variable region genes encoding high affinity mAbs of infants are similar to the restricted repertoire described in adults. Low affinity anti-Hib PS mAbs of infants are encoded by a heterogeneous group of genes that are uncommonly observed in the adult repertoire. Abs with high affinity for Hib PS from infants, like most mAbs from adults, react only with Hib PS and the structurally similar PS of Escherichia coli K100, whereas low affinity mAbs of infants are polyreactive. The low affinity anti-Hib PS mAbs of infants immunized with Hib oligosaccharide-diphtheria toxin vaccine vaccine are not reflected in serum Ab. However, the differences between the variable region gene repertoires of adults and infants may account for the distinct immunologic characteristics of the anti-Hib PS responses in young children immunized with other vaccine formulations.
Comparison of Humoral Immune Responses Elicited by DNA and Protein Vaccines Based on Merozoite Surface Protein-1 fromPlasmodium yoelii, a Rodent Malaria Parasite
Authors: Kang Yang , Calvo Paul A. , Daly Thomas M. , Long Carole A.
Published: December 2022

Immunization with DNA vaccines encoding relevant Ags can induce not only cell-mediated immune response but also humoral immune responses against pathogenic microorganisms in several animal models. Our previous results demonstrated that, when the C terminus (PyC2) of Plasmodium yoelii merozoite surface protein-1 (MSP-1), a leading vaccine candidate against erythrocytic stages of malaria, was expressed as a fusion protein (GST-PyC2) with glutathione S-transferase (GST), it elicited Ab-mediated protective immune responses in BALB/c mice. In our present study, we wished to examine the humoral responses to a DNA vaccine (V3) encoding GST-PyC2. The GST-PyC2 expressed in V3-transfected Cos 7 cells was recognized by a protective monoclonal Ab to PyC2 (mAb302), although the secreted product had undergone N-linked glycosylation. When BALB/c mice were immunized with V3 plasmid, anti-PyC2 Abs were successfully induced. These Abs immunoprecipitated native PyMSP-1 protein and competed with mAb302 for binding to its epitope at a level similar to those elicited by GST-PyC2 protein immunization. However, these Abs had significantly lower titers and avidities, and different isotype profiles and protective capacities against a lethal erythrocytic stage challenge, than those resulting from immunization with GST-PyC2 protein. Most surprising was the finding that, in contrast to protein immunization, there was no significant increase in the avidity of either GST-specific or PyC2-specific IgG Abs during the course of DNA immunization. This suggests that there may be little or no affinity maturation of specific Abs during DNA immunization in this system.
Apoptosis of Epithelial Cells and Macrophages Due to Infection with the Obligate Intracellular PathogenChlamydia psittaci
Authors: Ojcius David M. , Souque Philippe , Perfettini Jean-Luc , Dautry-Varsat Alice
Published: December 2022

We have characterized the cytotoxic activity of the obligate intracellular bacterium Chlamydia psittaci, which resides within a membrane-bound vacuole during the 2-day infection cycle. We have established that infected epithelial cells and macrophages die through apoptosis, which is measurable within 1 day of infection and requires productive infection by the bacteria. Inhibition of host cell protein synthesis has no effect on cell death, but blocking bacterial entry or bacterial protein synthesis prevents apoptosis, implying that bacterial growth is required for death of the host cell. Apoptosis was confirmed through the use of electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, gel agarose electrophoresis of fragmented DNA, and propidium-iodide labeling of host cell nuclei. Although infected cells died preferentially, both infected and uninfected cells became apoptotic, suggesting that the infected cells may secrete proapoptotic factors. Inhibition of either of two proapoptotic enzymes, caspase-1 or caspase-3, did not significantly affect Chlamydia-induced apoptosis. These results suggest that, as in the case of apoptosis due to Bax expression or oncogene dysregulation, which initiate the apoptotic program within the cell interior, the Chlamydia infection may trigger an apoptotic pathway that is independent of known caspases. As apoptotic cells secrete proinflammatory cytokines, Chlamydia-induced apoptosis may contribute to the inflammatory response of the host.
Chronically HIV-1-Infected Monocytic Cells Induce Apoptosis in Cocultured T Cells
Authors: Chen Houchu , Yip Y. K. , George Italas , Tyorkin Max , Salik Erez , Sperber Kirk
Published: December 2022

We have previously developed a human macrophage hybridoma model system to study the effect of HIV-1 infection on monocytic function. Upon coculture of one chronically (35 days postinfection) HIV-1-infected human macrophage hybridoma cell line, 43HIV, there was a dose-dependent decrease in the viability of cocultured Ag-stimulated T cells associated with an increase in DNA strand breaks. Enhanced apoptosis was determined by labeling with biotinylated dUTP and propidium iodide, increased staining with annexin V, increased side light scatter and expression of CD95, and decreased forward light scatter and expression of Bcl-2. There was also increased DNA strand breaks as determined by propidium iodide staining in unstimulated T cells cocultured with 43HIV and in T cells stimulated with anti-CD3 mAb and PHA. Pretreatment with 5145, a human polyclonal anti-gp120 Ab that recognizes the CD4 binding region, as well as with an anti-Fas ligand mAb blocked apoptosis in CD4+ T cells but not in CD8+ T cells. A soluble factor with a Mr below 10,000 Da was defined that induced apoptosis in CD4+ and CD8+ T cells and B cells. SDS-PAGE analysis of the active fractions revealed a band of 6000 Da that, after electroelution, had proapoptotic activity. The pI of the activity was estimated to be between 6.5 and 7.0. In conclusion, chronically HIV-1-infected monocytic cells induce apoptosis in bystander-, Ag-, anti-CD3-, and mitogen-stimulated T cells by multiple factors, which may contribute to the depletion of lymphocytes induced by HIV-1.
Expression and Participation of Eotaxin During Mycobacterial (Type 1) and Schistosomal (Type 2) Antigen-Elicited Granuloma Formation
Authors: Ruth Jeffrey H. , Lukacs Nicholas W. , Warmington Kelly S. , Polak Tim J. , Burdick Marie , Kunkel Steven L. , Strieter Robert M. , Chensue Stephen W.
Published: December 2022

Eotaxin participation was analyzed during types 1 and 2 lung granuloma formation induced by embolizing Sepharose beads coupled to purified protein derivative (PPD) of Mycobacterium bovis or soluble Ags derived from Schistosoma mansoni eggs. Eotaxin was monitored by protein ELISA and semiquantitative reverse-transcriptase PCR mRNA analysis. Both types 1 and 2 granulomas released eotaxin, but levels were sixfold greater (on day 4) in the type 2 than for the type 1 or foreign body granulomas. Transcripts for eotaxin, IL-4, and CCR3 (eotaxin receptor) were also enhanced during type 2 granuloma formation. Anti-IL-4 treatment impaired eotaxin mRNA in lungs with type 2 granulomas, indicating that IL-4 promoted local eotaxin expression. In vivo, anti-eotaxin treatment caused modest reductions in the size of both types 1 and 2 lesions, with negligible effect on eosinophil recruitment. Surprisingly, anti-eotaxin treatment abrogated IFN-γ-producing cells in regional lymph nodes during the type 1 PPD response. Lymph nodes draining both types 1 and 2 lesions showed enhanced CCR3 mRNA, but this followed the time of maximum eotaxin protein and mRNA expression. Correlative, in vitro studies revealed that graded doses of eotaxin increased IFN-γ production from PPD-sensitive regional lymph node cultures, while monocyte-chemotactic protein-1, an important macrophage chemoattractant, had the opposite effect. These findings indicate that eotaxin expression is not limited to type 2 hypersensitivity granulomas, but also promotes IFN-γ production during mycobacterial responses.
Immunoregulatory Roles of IL-10 in Innate Immunity: IL-10 Inhibits Macrophage Production of IFN-γ-Inducing Factors but Enhances NK Cell Production of IFN-γ
Authors: Shibata Yoshimi , Foster L. Ann , Kurimoto Masashi , Okamura Haruki , Nakamura Reiko M. , Kawajiri Katsuhide , Justice J. Paul , Van Scott Michael R. , Myrvik Quentin N. , Metzger W. James
Published: December 2022

In our study of the immunoregulatory roles of IL-10 in innate immunity, nonantigenic phagocytosable chitin particles were administered i.v. to IL-10-deficient (knockout (KO)) mice or KO mice pretreated with anti-NK1.1 or anti-IFN-γ Abs. The results established that chitin treatment of KO mice increased superoxide anion release from alveolar macrophages (Mφ) to a level much higher than that in wild-type (WT) mice. The results also suggested that the NK cell is the source of IFN-γ that is primarily responsible for this alveolar Mφ priming. To further study the roles of IL-10-inhibiting chitin-induced IFN-γ production, we used spleen cell cultures. The experiments showed that IL-12, IL-18, and TNF-α, which were produced by chitin-stimulated Mφ, contributed to the IFN-γ-inducing activity of chitin. Our results established that exogenous IL-10 inhibited chitin-induced IFN-γ production in spleen cell cultures from both KO and WT mice. Exogenous IL-10 also inhibited IL-12 and TNF-α production by chitin-stimulated Mφ. Exogenous IL-10 decreased IL-12- or IL-18-induced IFN-γ levels in KO but not in WT NK cell cultures. However, exogenous IL-10 enhanced IFN-γ levels when NK cells were stimulated simultaneously with both IL-12 and IL-18 in KO and WT cultures. Our in vitro data indicate that IL-10 has differential effects on chitin-induced IFN-γ production. However, the inhibitory effects of endogenous IL-10 appear to be dominant in the chitin-induced alveolar Mφ priming response in vivo.
Effect of Cytotoxic Necrotizing Factor-1 on Actin Cytoskeleton in Human Monocytes: Role in the Regulation of Integrin-Dependent Phagocytosis
Authors: Capo Christian , Meconi Sonia , Sanguedolce Muriel-Véronique , Bardin Nathalie , Flatau Gilles , Boquet Patrice , Mege Jean-Louis
Published: December 2022

Cytotoxic necrotizing factor-1 (CNF1) is isolated from pathogenic strains of Escherichia coli and catalyzes the activation of Rho GTPases by the deamidation of a glutamine residue. This toxin induces stress fiber formation, cell spreading, and membrane folding and promotes phagocytosis competence in epithelial cells. We show that CNF1 induces morphologic changes in monocytic cells: polarized-like shape in THP-1 cells, lamellipodia, and cell spreading in adherent monocytes. CNF1 also increased filamentous actin (F-actin) content in a time- and dose-dependent manner. In addition, the toxin profoundly reorganized the actin cytoskeleton: redistribution of F-actin in polarized deformations of THP-1 cells and disorganization of microfilament network in monocytes. We also studied the effects of CNF1 on phagocytosis. It markedly impaired the ingestion of unopsonized zymosan involving CR type 3. However, CNF1 had no effect on the uptake of iC3b-coated zymosan or IgG-mediated phagocytosis of SRBC. In addition, CNF1 induced clustering of CR3 and FcγRII (CD32) but selectively impaired the colocalization of CR3 with F-actin. It is likely that CNF1-induced reorganization of actin cytoskeleton down-modulates integrin activation-dependent phagocytosis by preventing the codistribution of CR3 with F-actin. CNF1 may control some features of integrin-dependent phagocytosis in myeloid cells through its action on Rho GTP binding proteins and cytoskeletal organization.
Differential Binding of IgG and of a HIV gp41 Peptide by the B Chain and A Chain Globular Head Sequences of C1q, Respectively
Authors: Kojouharova Mihaela S. , Panchev Ivelin D. , Tchorbadjieva Magdalena I. , Reid Kenneth B. M. , Hoppe Hans-Jürgen
Published: December 2022

Two individual globular head regions (ghA and ghB) of the heterotrimeric C1q molecule (containing A, B, and C chains) were expressed in a bacterial expression system using a coproduction with the bacterial chaperone GroESL. The purified proteins were soluble and monomeric, as shown by gel-filtration analysis. No association into homotrimers was seen, which indicates that the ability to form heterotrimers is coupled with the discrimination against homotrimeric self-association. The individual globular heads retained their binding activities toward two ligands bound by the whole C1q molecule, i.e., IgG and the peptide P(601–613) derived from the HIV envelope glycoprotein gp41. The differential binding activities displayed for these ligands indicated a degree of structural independence of the binding sites from the regions responsible for heterotrimerization. It was found, using single chain recombinant anti-C1q Abs, that the binding sites on C1q for IgG and gp41 do not overlap, and this observation is also consistent with the view that specialization between the C1q polypeptide chains takes place within the C1q molecule regarding their ligand-binding activities.
Mutations in the Activation Loop Tyrosines of Protein Tyrosine Kinase Syk Abrogate Intracellular Signaling But Not Kinase Activity
Authors: Zhang Juan , Kimura Teruaki , Siraganian Reuben P.
Published: December 2022

The protein tyrosine kinase Syk plays a pivotal role in mediating the high-affinity IgE receptor (FcεRI)-induced degranulation of mast cells. To examine the mechanism of Syk regulation, the two tyrosine residues at 519 and 520 in the putative activation loop of rat Syk were mutated to phenylalanine either singly or in combination. The various mutants were expressed in a Syk-negative variant of the RBL-2H3 (rat basophilic leukemia 2H3) mast cell line. In these transfected cell lines, mutant Syk did show increased tyrosine phosphorylation in vivo and increased enzymatic activity in vitro after FcεRI aggregation. There were conformational changes detected by an Ab when the wild-type and mutant Syk were either tyrosine phosphorylated or bound to tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif peptides. However, these mutant Syk were incapable of transducing FcεRI signaling. In cells in which the expression level of mutant Syk was similar to that of the wild-type Syk, FcεRI cross-linking induced no increase in cellular protein tyrosine phosphorylation, no increase in tyrosine phosphorylation of phospholipase C-γ2 and mitogen-activated protein kinase, and no histamine release. Overexpression of Y519F or Y520F Syk mutants partially reconstituted the signaling pathways. These results indicate that these tyrosines in the putative activation loop are not essential for the enzymatic activity of Syk or for the conformational changes induced by binding of tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif peptides. However, these tyrosines are necessary for Syk-mediated propagation of FcεRI signaling.
Endothelial Selectins and α4 Integrins Regulate Independent Pathways of T Lymphocyte Recruitment in the Pulmonary Immune Response
Authors: Wolber Frances M. , Curtis Jeffrey L. , Mály Petr , Kelly Robert J. , Smith Peter , Yednock T. A. , Lowe John B. , Stoolman Lloyd M.
Published: December 2022

The cell adhesion molecules (CAMs) required for T lymphocyte recruitment during pulmonary immune responses have not been defined. Our laboratories recently reported that intratracheal (IT) challenge of sensitized mice with SRBC induced prolonged expression of vascular P-selectin, E-selectin, and VCAM-1, particularly in areas of mononuclear leukocyte infiltration. A surge in the number of circulating T lymphocytes expressing selectin ligands preceded the peak accumulation of T cells in the lung. In addition, a significant percentage of the T cells recovered from the lung expressed selectin ligands as well. The current study demonstrates that cultured T lymphoblasts use both selectin ligands and α4 integrins to enter the airspace and interstitium during the response to SRBC. Fluorescently labeled T lymphoblasts, derived via activation on CD3 and growth in low dose IL-2, showed inflammation-specific recruitment into lungs harvested 24 h after cell infusion. Their flux paralleled the accumulation of host lymphocytes in the lung, with both peaking 2 to 4 days after SRBC challenge. Trafficking studies conducted over a 24-h period during peak lymphocyte accumulation in the lungs revealed preferential recruitment of labeled T lymphoblasts expressing P- and E-selectin ligands. In addition, mAb blockade of the α4 integrins and targeted deletion of an α(1,3)fucosyltransferase essential for selectin ligand synthesis each reduced labeled T lymphoblast trafficking to a significant degree. Furthermore, α4 integrin blockade reduced the trafficking of the selectin ligand-deficient cells into the airspace, confirming that its contribution is in part independent from the vascular selectins. These findings imply that both selectin ligands and α4 integrins participate in T lymphoblast recruitment during the pulmonary immune response to IT SRBC.
Localization of Quantitative Trait Loci Regulating Adjuvant-Induced Arthritis in Rats: Evidence for Genetic Factors Common to Multiple Autoimmune Diseases
Authors: Kawahito Yutaka , Cannon Grant W. , Gulko Pércío S. , Remmers Elaine F. , Longman Ryan E. , Reese Van R. , Wang Jianping , Griffiths Marie M. , Wilder Ronald L.
Published: December 2022

Adjuvant-induced arthritis (AIA) in rats is a widely used autoimmune experimental model with many features similar to rheumatoid arthritis (RA). To identify potential genetic regulatory mechanisms in RA, we conducted genome-wide linkage analysis in F2 progeny of arthritis-susceptible Dark Agouti (DA) and relatively resistant Fischer 344 (F344) inbred rats. We compared the data with our previously reported investigation of collagen-induced arthritis (CIA), which was expanded in the follow-up study reported in this work. We found two quantitative trait loci (QTLs) in common, i.e., Aia1/Cia1 on chromosome 20, which includes the MHC, and Aia3/Cia3 on chromosome 4. We also identified a second unique QTL in AIA, Aia2, on chromosome 4. Interestingly, the QTL region on chromosome 4 (Aia3/Cia3), like the MHC, appears to be involved in several other autoimmune diseases in rats, including insulin-dependent diabetes, thyroiditis, and experimental autoimmune uveitis. Moreover, an analysis of conserved synteny among rats, mice, and humans suggested that Aia2 and Aia3/Cia3, like Aia1/Cia1, contain candidate genes for several autoimmune/inflammatory diseases in mice and humans, including diabetes, systemic lupus erythematosus, inflammatory bowel disease, asthma/atopy, multiple sclerosis, and RA. The rat models appear to provide a powerful complementary approach to identify and characterize candidate genes that may contribute to autoimmune diseases in several species.
Cutting Edge: Antigen-Specific T Lymphocytes Regulate Lipopolysaccharide-Induced Apoptosis of Dendritic Cells In Vivo
Authors: De Smedt Thibaut , Pajak Bernard , Klaus Gerry G. B. , Noelle Randolph J. , Urbain Jacques , Leo Oberdan , Moser Muriel
Published: December 2022

The potent accessory properties of dendritic cells (DC) develop sequentially during a process termed “maturation.” Splenic DC undergo functional maturation in vivo in response to the bacterial product LPS and migrate from the marginal zone to the T cell areas. The redistribution of fully mature DC, which present Ags encountered in the periphery, in the T cell area is likely to result in T cell priming. Unexpectedly, we found that DC rapidly die by apoptosis once they have entered the T cell zone. Injection of OVA peptide in OVA-specific, TCR-transgenic mice strongly delays the LPS-induced apoptosis of DC in situ. We conclude that mature DC are programmed to die unless they receive a survival signal from T cells and that the regulation of DC survival may be a mechanism aimed at controlling the initiation and the termination of the immune response.
Cutting Edge: Chemotactic Activity of Soluble Fas Ligand Against Phagocytes
Authors: Seino Ken-ichiro , Iwabuchi Kazuhisa , Kayagaki Nobuhiko , Miyata Ryukou , Nagaoka Isao , Matsuzawa Akio , Fukao Katashi , Yagita Hideo , Okumura Ko
Published: December 2022

A recombinant soluble form of human Fas ligand (sFasL) was tested for its chemotactic activity against human and mouse polymorphonuclear neutrophils (PMN) by the Boyden chamber method. sFasL exhibited a potent chemotactic activity against both human and mouse PMN and HL-60 cells when differentiated into neutrophils or monocytes. A neutralizing anti-FasL mAb abolished the chemotactic activity, while control mAb did not. Ligation of Fas by either IgM- or IgG-type anti-Fas mAb also induced PMN migration. PMN derived from lpr mice that express few Fas molecules did not respond to sFasL. In contrast, those derived from lprcg mice that express Fas molecules with a mutated death domain normally responded to sFasL chemotaxis. These results directly indicated a chemotactic activity of sFasL against PMN and suggest a novel signaling function of Fas, which appears to be independent of the death domain-mediated apoptosis.
CD40 Ligand Is Pivotal to Efficient Control of Virus Replication in Mice Infected with Lymphocytic Choriomeningitis Virus
Authors: Thomsen A. Randrup , Nansen A. , Christensen J. Pravsgaard , Andreasen S. Ørding , Marker O.
Published: December 2022

CD40 ligand (CD40L) is an important molecule that is known to be involved in T-B collaboration and certain aspects of cell-mediated immunity. However, its role in antiviral immunity has not been clearly defined as of yet. Therefore, mice with a targeted defect in the gene encoding this molecule were infected with one of two strains of lymphocytic choriomeningitis virus differing markedly in their capacity to spread in the host. Infection with lymphocytic choriomeningitis virus is initially controlled primarily by CD8+ effector cells, whereas long-term immune surveillance also depends upon CD4+ cells and B cells. Our results reveal that the primary activation, clonal expansion, and differentiation of CD8+ T cells does not require expression of CD40L. However, lack of expression results in rapid impairment of CTL responsiveness and failure to permanently control virus replication. This happens not only in mice infected with the rapidly spreading virus strain but also at a late stage in mice infected with the strain of more limited potential for spreading. In the latter mice, virus replication is initially controlled very efficiently, but high levels of virus can be detected in the blood and internal organs ∼6 mo after virus inoculation. Since the impairment of immune function seems to be more pronounced in CD40L-deficient mice than in mice lacking either CD4+ cells or B cells, these results indicate that CD40L is pivotal to sustain efficient antiviral immune surveillance, including CD8+ T cells, and suggest that CD40L is critically involved in cellular interactions in addition to T-B cooperation.
Differential Requirements for ZAP-70 in TCR Signaling and T Cell Development
Authors: Kadlecek Theresa A. , van Oers Nicolai S. C. , Lefrancois Leo , Olson Sara , Finlay Deborah , Chu David H. , Connolly Kari , Killeen Nigel , Weiss Arthur
Published: December 2022

The Syk/ZAP-70 family of protein tyrosine kinases is indispensable for normal lymphoid development. Syk is necessary for the development of B cells and epithelial γδ T cells, whereas ZAP-70 is essential for the normal development of T cells and TCR signaling. In this study, we show that although development of the αβ lineage was arrested in the thymus, CD3-positive T cells, primarily of the γδ lineage, were present in the lymph nodes of mice lacking ZAP-70. Moreover, in the absence of ZAP-70, dendritic epidermal T cells were fewer in number and of abnormal morphology, and intestinal intraepithelial lymphocytes, normally containing a large proportion of γδ T cells, were markedly reduced. These data suggest that γδ T cells show a variable dependence upon ZAP-70 for their development. Biochemical analyses of thymocytes revealed a lack of basal ζ-chain tyrosine phosphorylation. However, several other substrates were inducibly tyrosine phosphorylated following TCR stimulation. Thus, TCR-mediated signaling in ZAP-70-deficient thymocytes is only partially impaired. These studies suggest that Syk compensates only partially for the loss of ZAP-70, and that there is an absolute requirement of ZAP-70 for αβ T cells and epithelial γδ T cells, but not for some γδ T cells in peripheral lymphoid tissues.
p48/STAT-1α-Containing Complexes Play a Predominant Role in Induction of IFN-γ-Inducible Protein, 10 kDa (IP-10) by IFN-γ Alone or in Synergy with TNF-α
Authors: Majumder Sarmila , Zhou Lucy Z.-H. , Chaturvedi Priya , Babcock Gerald , Aras Sumer , Ransohoff Richard M.
Published: December 2022

Human IFN-γ-inducible protein, 10 kDa (hIP-10) and murine IP-10 (mIP-10) genes are induced by IFN-γ alone, and synergistically induced by TNF-α and IFN-γ. Upstream regions of the human and murine genes contain conserved regulatory motifs, including an IFN-stimulated response element (ISRE), which governs response of the mIP-10 gene to IFN-γ. Trans-acting factors mediating the IFN-γ response via ISRE remain incompletely defined. We examined ISRE-binding factors in the regulation of the hIP-10 gene. The requirement of p48 for hIP-10 induction by IFN-γ, with or without TNF-α, was demonstrated using p48-deficient U2A cells. An hIP-10 promoter-reporter mutant (mISRE3) that was relatively deficient for binding a related factor, IFN regulatory factor-1 (IRF-1) but competent for binding p48, was induced as well as the wild-type hIP-10 promoter, supporting the interpretation that p48 played a necessary and sufficient role in hIP-10 transcription. Genomic in vivo footprinting revealed IFN-γ/TNF-α-inducible binding at the ISRE consistent with the presence of p48 and associated factors, but not with IRF-1. Induction of hIP-10 by TNF-α/IFN-γ also required NFκB binding sites, which were protected in vivo and bound p65 homodimeric NFκB in vitro. These results documented the essential role of p48 (complexed with STAT-1α) for induction and sustained transcription of the IP-10 gene, strongly suggesting that IRF-1 is not required for IP-10 induction by these inflammatory cytokines.
Structural Aspects of the Interaction Between Heterogeneic Human Papillomavirus Type 1 E4-Specific T Cell Receptors and the Same Peptide/HLA-DQ8 Complex
Authors: Steele Jane C. , Young Stephen P. , Goodall Jane C. , Gallimore Phillip H.
Published: December 2022

TCR usage has been studied in a panel of Th cell clones specific for the same peptide epitope (P N S Q D R G R P R R S D), derived from the human papillomavirus type 1 (HPV1) E4 protein, and restricted through HLA-DQ8. After identifying the V, D, and J genes used by the TCRs and sequencing across the V(D)J junctions, five different α-chain sequences and five different β-chain sequences, comprising six independent clones, were identified. A structural model of our E4 peptide/HLA-DQ8 complex predicted that the guanidinyl side chain on the arginine residue at position 6 of the peptide could exist in different orientations. An intramolecular interaction between this arginine and the glutamine residue at position four appeared to control this orientation. Interacting HPV1 E4-specific TCRs would therefore have to recognize the complex in different conformations, and molecular modeling of the TCRs suggested that this could be achieved by changing the dimensions of the central pocket formed where the CDR3 loops of the TCR α- and β-chains converge. It is known that interactions between bound peptide and amino acid residues lining the peptide-binding cleft of HLA molecules are important for determining the conformation and orientation of the peptide/MHC complex. The suggestion here that intramolecular interactions between amino acids of close proximity on the bound peptide are also important adds a further level of complexity to the mechanism by which TCRs interact with Ag.
Invariant Chain Can Bind MHC Class II at a Site Other Than the Peptide Binding Groove
Authors: Wilson Nancy A. , Wolf Paula , Ploegh Hidde , Ignatowicz Leszek , Kappler John , Marrack Philippa
Published: December 2022

Invariant chain binds to class II molecules and guides them to the cell surface via the endosomes. Class II-associated invariant chain peptide (CLIP), a conserved sequence in an unstructured region of invariant chain, binds in the peptide binding groove of class II and is thought to be the major contributor to the interaction between invariant chain and class II molecules. However, other interaction sites between the two proteins may exist. The published data on this subject are conflicting. We have studied the ability of invariant chain to interact with a class II molecule in which the peptide binding groove of the protein is already occupied by a covalently attached peptide. Precipitation of these class II/peptide complexes with an Ab specific for this particular combination also precipitates invariant chain. This binding between class II/peptide and invariant chain is weak, and coprecipitation is only apparent in mild detergents. Thus, when the class II peptide binding groove is occluded by peptide and is not free to interact with CLIP, invariant chain can still bind the class II molecule at other lower affinity sites.