bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevention of acute graft-versus-host disease by treatment with a novel immunosuppressant. Cholera toxin B subunit.
Authors: Yankelevich B , Brown E , Mazumder A
Published: December 2022

Transplantation of allogeneic bone marrow (BM) is often complicated by the development of acute graft-vs-host disease (GVHD) caused by contaminating T cells in BM inocula because of activation of the host reactive T effector cells. Using a murine model for acute GVHD caused by injection of parental C57Bl/6 splenocytes into unirradiated (C57Bl/6 x DBA/2) F1 hybrids, we demonstrated that pretreatment of the inocula with a novel immunosuppressant, B subunit of cholera toxin (CT-B) impaired the ability of C57Bl/6 T cells to induce acute GVHD in F1 recipients. F1 mice injected with CT-B-treated C57Bl/6 splenocytes did not develop significant splenomegaly, and no antihost CTLs were found in their spleens. Moreover, these mice did not suffer from aplastic anemia, nor from general immunosuppression. Immunofluorescence studies suggest that treatment of the inducing inocula with CT-B selectively prevents accumulation of the host-reactive CD8+ T cells in F1 mice. Furthermore, our experiments demonstrated that CT-B treatment does not impair the ability of BM progenitors to form colonies in semisolid culture or in lethally irradiated hosts. Thus, taken together, our data suggest that ex vivo CT-B treatment can be used in allogeneic BM transplantation to prevent acute GVHD.
Cytotoxic NK1.1 Ag+ alpha beta T cells with intermediate TCR induced in the liver of mice by IL-12.
Authors: Hashimoto W , Takeda K , Anzai R , Ogasawara K , Sakihara H , Sugiura K , Seki S , Kumagai K
Published: December 2022

Systemic administration of IL-12 greatly reduced the hepatic metastases of i.v.-injected liver metastatic EL4 tumor cells in C57BL/6 +/+ and nu/nu mice. Cytotoxic assay in vitro revealed that administration of IL-12 greatly enhanced cytotoxicity of hepatic mononuclear cells (MNC) against various NK- sensitive and -resistant tumor targets, including EL4 cells, whereas only slight or moderate augmentation of the cytotoxicity was observed in splenocytes in normal and nude mice. After IL-12 administration, hepatic MNC increased in number and showed vigorous proliferation in vitro. Hepatic MNC of control C57BL/6 +/+ mice contain alpha beta T cells with intermediate TCR (TCRint) as well as alpha beta T cells with bright TCR, whereas hepatic MNC of nu/nu mice have only TCRint cells. These TCRint cells are found to be NK1.1 Ag+ (NK1+ TCRint). Systemic administration of IL-12 into normal and nude mice markedly augments the NK1 expression of NK1+ TCRint cells (NK1high TCRint), which is comparable to or brighter than that of NK cells in the liver, whereas alpha beta T cells with bright TCR or gamma delta T cells in the liver are NK1-. Depletion of either NK1.1+ or CD3+ cells, but not CD8+ cells, of hepatic MNC from IL-12-treated normal mice by respective Abs and C in vitro abrogate their cytotoxicity. These results revealed that TCRint cells are potent cytotoxic effector cells and suggest that NK1high TCRint cells are the main antimetastatic population in the liver, and that TCRint cells are functionally different from regular T cells with bright TCR.
Differential requirement for protein tyrosine kinase Fyn in the functional activation of antigen-specific T lymphocyte clones through the TCR or Thy-1.
Authors: Lancki D W , Qian D , Fields P , Gajewski T , Fitch F W
Published: December 2022

The protein tyrosine kinase Fyn has been shown to be involved in signal transduction through the TCR and the glycosyl-phosphatidylinositol-linked surface molecule Thy-1 expressed on T cells. In this study, we examine the requirement for Fyn expression in signaling through the TCR or Thy-1 using a panel of Ag-specific T cell clones derived from fyn-/- mutant mice. These clones do not express normal Fyn protein, as measured by immune-complex kinase reaction using anti-Fyn Ab. Stimulation through the TCR, either by APC bearing relevant Ag or by immobilized anti-CD3 mAb, resulted in comparable levels of proliferation, lymphokine production, and cytolysis by clones from both wild-type and fyn-/- mice. In contrast, stimulation through Thy-1, using soluble (or cross-linked) anti-Thy-1 mAb, was deficient, as measured by these responses. Thus, Fyn expression is selectively required for functional activation through Thy-1 in these T cell clones.
Differential expression of endoglin on fetal and adult hematopoietic cells in human bone marrow.
Authors: Rokhlin O W , Cohen M B , Kubagawa H , Letarte M , Cooper M D
Published: December 2022

Endoglin, a glycoprotein that is expressed by human endothelial cells, binds TGF-beta 1 and -beta 3 with high affinity. It was originally identified with the 44G4 mAb that was produced against a human pre-B cell line. We now report that another anti-pre-B cell mAb, 29-G8, reacts with pro-B and pre-B leukemic cells, but not with mature B and T cells, and recognizes a different epitope of endoglin. The 29-G8 mAb bound specifically to recombinant endoglin and immunoprecipitated a phosphorylated homodimeric glycoprotein with subunits of M(r) 95,000 from the 697 pre-B cell line. This new Ab removed all of the molecules identified by the prototypic 44G4 anti-endoglin Ab, but the reverse was not true. A subpopulation of 29-G8+ endoglin molecules on this pre-B cell line was unreactive with the 44G4 mAb, thus suggesting that these anti-endoglin Abs see different epitopes that may discriminate different species of endoglin molecules. Flow cytometric analysis with the 29-G8 mAb revealed two endoglin-positive subpopulations in fetal bone marrow: early B-lineage precursor cells (CD19+ and CD34+), and proerythroblasts (CD71+ and glycophorin A+). In adult bone marrow, only the proerythroblast subpopulation was observed. Stromal cells derived from fetal bone marrow also reacted strongly with the 29-G8 and 44G4 Abs, and these cells responded with enhanced proliferation after stimulation with either TGF-beta 1 or the anti-endoglin Abs. Thus, endoglin, a specialized component of the TGF-beta receptor system, may play a physiologic role in the stromal-hemopoietic cell interactions occurring during development.
Constitutive endocytosis and degradation of CD22 by human B cells.
Authors: Shan D , Press O W
Published: December 2022

The CD22 B lymphocyte-surface Ag is an important component of the B cell-surface IgM (sIgM)/B cell receptor complex and has been shown to regulate B cell activation. In addition, this molecule has been shown to be an effective target for immunotherapy of B cell malignancies using immunotoxins and radioimmunoconjugates. In this report we describe the internalization and metabolic degradation of this molecule under constitutive conditions and after stimulation of B cells with phorbol dibutyrate or mAbs binding to sIgM, CD19, and CD22. Flow cytometry, "neuraminidase protection," and "neuraminidase shift" assays demonstrated that CD22 is internalized constitutively by unstimulated B cell lines and subsequently degraded in an acidic intracellular compartment (presumably lysosomes) without detectable recycling of the molecule back to the cell surface. Ligation of CD22 with anti-CD22 mAbs markedly increased CD22 internalization but did not affect the rate of intracellular degradation of CD22, suggesting that anti-CD22 mAbs perturb the intracellular trafficking of CD22. In contrast, CD22 internalization and degradation was unaffected by stimulation of B cell lines with phorbol dibutyrate or ligation of other components of the B cell receptor complex (e.g. CD19, sIgM) with mAbs. These patterns of internalization and degradation under constitutive and stimulated conditions contrast with those reported for other lymphoid differentiation Ags (e.g., the TCR, CD3, CD4, and the transferrin receptor), and may help explain the utility of this molecule as a target for immunoconjugate therapy.
Heavy chain variable region, light chain variable region, and heavy chain CDR3 influences on the mono- and polyreactivity and on the affinity of human monoclonal rheumatoid factors.
Authors: Crouzier R , Martin T , Pasquali J L
Published: December 2022

Monoreactive high affinity pathologic autoantibodies were supposed previously to derive through somatic mutation from polyreactive low affinity autoantibodies that are encoded by a small set of unmutated V region genes in fetal and neonatal B cells. However, recent data exploring the physiologically expressed Ab repertoire and the importance of the stochastically generated heavy chain CDR3 (H-CDR3) in autoreactivity suggest that this scheme is incomplete. Here we analyzed via gene-swapping experiments and site-directed mutagenesis the relative contributions of the mutations in the light chain variable region (VL) and the heavy chain variable region (VH) domains and of the H-CDR3 in the autoreactivity of two IgM rheumatoid factors (RF), one a polyreactive low affinity Ab, the other a monoreactive high affinity Ab. These two RFs derived from the same V kappa III (humkv325) and VH1 (51p1) genes, but differed from each other by a few mutations and by the structure of the H-CDR3. The analysis of the reactivity patterns of different combinations of wild-type and in vitro engineered hybrid gene products clearly demonstrates the main influence of the H-CDR3 in the autoAb activity profiles. The results directly demonstrate the previously proposed hypothesis, namely, that the H-CDR3 plays a critical role in distinguishing poly- from monospecific RF. However, the data also indicate that self polyreactivity is a very fragile property and is dependent upon the primary structure of the VH segment.
Peptides control the gain and loss of allele specificity by mutated MHC class I molecules.
Authors: Hengel H , Burke K , Kyburz D , Zinkernagel R M , Koszinowski U H
Published: December 2022

To analyze the molecular basis of MHC class I allele-restricted peptide recognition, a set of eight Ld/Lq mutants was constructed and tested for peptide recognition by allele-restricted and peptide-specific CTL. The MHC molecules H-2Ld and H-2Lq differ at six amino acid positions (95, 97, 107, 116, 155, 157) located within the alpha 2 domain of the molecule. Both molecules present the lymphocytic choriomeningitis virus (LCMV) nucleoprotein-derived peptide RPQASGVYM and the murine cytomegalovirus (MCMV) pp89-derived peptide YPHFMPTNL to the respective virus-specific CD8+ CTL is a strictly allele-restricted fashion. All mutated MHC class I molecules did still bind the LCMV peptide and seven of eight mutants retained MCMV peptide binding. The exchange Arg-->Trp at position 97 of Lq in pocket C of the peptide binding groove prevented binding of the MCMV ligand and this loss was compensated by the additional exchange of Ile-->Leu in position 95 (pocket F). Within the Lq molecule, single mutations at either position 97 on the floor of the groove or position 155 of the wall sufficed for a gain of LCMV peptide recognition by Ld-restricted CTL. Altogether, six of eight mutants resulted in a gain of recognition by CTL specific for the other allele. Thus, six of the eight mutants lost MHC-restricted recognition and were accepted by both Ld- as well as Lq-restricted CTL when presenting the LCMV peptide. Only one case of simultaneous recognition of the MCMV peptide by both Ld- as well as Lq-restricted CTL was noted. In other mutations, a gain of recognition by Ld-restricted CTL was associated with a loss of recognition of Lq-restricted CTL. Analysis of extracted MCMV peptide from mutant molecules excluded quantitative differences in presented MCMV peptide as a reason for the lack of CTL recognition. Altogether, the results show that, rather than aminoacids at certain residue positions, individual peptides govern MHC allele specificity of CTL recognition.
Rearrangement and expression of the human psi C lambda 6 gene segment results in a surface Ig receptor with a truncated light chain constant region.
Authors: Stiernholm N B , Verkoczy L K , Berinstein N L
Published: December 2022

The constant region of the human Ig lambda locus consists of seven tandemly organized J-C gene segments. Although it has been established that the J-C lambda 1, J-C lambda 2, J-C lambda 3, and J-C lambda 7 gene segments are functional, and code for the four distinct Ig lambda isotypes found in human serum, the J-C lambda 4, J-C lambda 5, and J-C lambda 6 gene segments are generally considered to be pseudogenes. Although one example of a functional J-C lambda 6 gene segment has been documented, in the majority of cases, J-C lambda 6 is rendered nonfunctional by virtue of a single duplication of four nucleotides, creating a premature translational arrest. We show here that rearrangements to the J-C lambda 6 gene segment do occur, and that such a rearrangement encodes an Ig lambda protein that lacks the terminal end of the constant region. We also show that this truncated protein is expressed on the surface with the IgH chain, creating an unusual surface Ig (sIg) receptor (sIg delta CL). Cells that express this receptor on the surface do so at significantly reduced levels compared with clonally related variants, which express sIg receptors with conventional Ig lambda L chains. However, the effects of sIg cross-linking on tyrosine phosphorylation and surface expression of the CD25 and CD71 Ags are similar in cells that express conventional sIg receptors and in those that express sIg delta CL receptors, suggesting that the latter could possibly function as an Ag receptor.
Active immunotherapy of cancer with a nonreplicating recombinant fowlpox virus encoding a model tumor-associated antigen.
Authors: Wang M , Bronte V , Chen P W , Gritz L , Panicali D , Rosenberg S A , Restifo N P
Published: December 2022

Some tumor cells express Ags that are potentially recognizable by T lymphocytes and yet do not elicit significant immune responses. To explore new immunotherapeutic strategies aimed at enhancing the recognition of these tumor-associated Ags (TAA), we developed an experimental mouse model consisting of a lethal clone of the BALB/c tumor line CT26 designated CT26.WT, which was transduced with the lacZ gene encoding beta-galactosidase, to create CT26.CL25. The growth rate and lethality of CT26.CL25 and CT26.WT were virtually identical despite the expression by CT26.CL25 of the model tumor Ag in vivo. A recombinant fowlpox virus (rFPV), which is replication incompetent in mammalian cells, was constructed that expressed the model TAA, beta-galactosidase, under the influence of the 40-kDa vaccinia virus early/late promoter. This recombinant, FPV.bg40k, functioned effectively in vivo as an immunogen, eliciting CD8+ T cells that could effectively lyse CT26.CL25 in vitro. FPV.bg40k protected mice from both subcutaneous and intravenous tumor challenge by CT26.CL25, and most surprisingly, mice bearing established 3-day pulmonary metastasis were found to have significant, Ag-specific decreases in tumor burden and prolonged survival after treatment with the rFPV. These observations constitute the first reported use of rFPV in the prevention and treatment of an experimental cancer and suggest that changing the context in which the immune system encounters a TAA can significantly and therapeutically alter the host immune response against cancer.
Chloromethyl ketones block induction of nitric oxide synthase in murine macrophages by preventing activation of nuclear factor-kappa B.
Authors: Kim H , Lee H S , Chang K T , Ko T H , Baek K J , Kwon N S
Published: December 2022

N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), serine protease inhibitors, block many cytotoxic functions of immune cells including superoxide anion production, cytokine release, cell-mediated cytolysis, and nitric oxide (NO)-related macrophage functions. IFN-gamma/LPS-induced NO production from murine peritoneal macrophages was inhibited by TPCK and TLCK in a dose-dependent manner (EC50s: approximately 20 microM for TPCK and approximately 30 microM for TLCK). Viability exceeded 91% with 25 microM TPCK and with 80 microM TLCK. When TPCK treatment was delayed until 1 h of activation, the inhibitory effect was markedly reduced. After 2 h of the activation, TPCK was not effective anymore. Addition of either TNF-alpha or conditioned media from IFN-gamma/LPS-activated macrophage culture did not prevent the inhibitory effect of TPCK. Neither TPCK nor TLCK reduced enzymatic NO production from macrophage lysates. Lysates from TPCK-treated cells did not generate NO even after supplementing necessary cofactors for NO synthase. Immunoblotting analysis showed that simultaneous treatment of TPCK with IFN-gamma/LPS abolished the NO synthase expression, whereas delayed addition of TPCK was either partially effective or not effective at all. Furthermore, TPCK treatment reduced the concentration of mRNA for NO synthase without decreasing mRNA stability. Thus, the serine protease inhibitors directly blocked an early event in expression of NO synthase. Electrophoretic mobility shift assay indicated that TPCK blocked the activation of nuclear factor-kappa B, a transcription factor necessary for NO synthase induction. TPCK also blocked disappearance of I kappa B from cytosolic fraction, and nuclear translocation of NF-kappa B subunits p50 and p65. Delaying the addition of TPCK by 10 min partially prevented the inhibition of the NF-kappa B activation process and allowed partial resuming of NO production. Thus, TPCK inhibited NO synthase induction by blocking NF-kappa B activation.
Eosinophil major basic protein induces degranulation and IL-8 production by human eosinophils.
Authors: Kita H , Abu-Ghazaleh R I , Sur S , Gleich G J
Published: December 2022

Eosinophil granule proteins, such as major basic protein (MBP), eosinophil peroxidase (EPO), and eosinophil cationic protein (ECP), possess a wide range of biologic activities including the ability to activate other cells, such as basophils, neutrophils, and platelets. Here we have analyzed the effects of these proteins on eosinophils themselves. MBP and EPO, at concentrations as low as 0.1 micrograms/ml, induced eosinophil degranulation as measured by release of eosinophil-derived neurotoxin (EDN); in contrast, ECP, at 1 micrograms/ml, was inactive. MBP (10 micrograms/ml) and EPO (0.1 micrograms/ml) induced EDN release comparable with one of the strongest agonists for eosinophils, secretory IgA. Pretreatment of cells with dibutyryl cAMP or cytochalasin B completely abolished the EDN release induced by MBP and EPO, suggesting that the effects of MBP and EPO are not due to cytotoxic lysis of the cells. Degranulation induced by MBP was only partially dependent on calcium, and no elevation of intracellular Ca2+ concentration ([Ca2+]i) was observed in eosinophils stimulated with MBP. MBP stimulated the production, up to eightfold, of IL-8 by eosinophils in a dose-dependent manner. The MBP-stimulated expression of IL-8 mRNA by eosinophils was confirmed by reverse transcription-PCR. The MBP-stimulated production of IL-8 was inhibited by actinomycin D, but not by cyclosporin A. Furthermore, MBP and calcium ionophore ionomycin synergistically induced production of leukotriene C4 from eosinophils. Thus, MBP and EPO may act as autocrine mediators in the pathogenesis of eosinophil-associated diseases, such as bronchial asthma.
TNF-alpha induces tyrosine phosphorylation of mitogen-activated protein kinase in adherent human neutrophils.
Authors: Rafiee P , Lee J K , Leung C C , Raffin T A
Published: December 2022

Recombinant human TNF-alpha induces increased tyrosine phosphorylation of several proteins in human neutrophils (PMN) adhered to serum-coated plastic. When PMN are kept in suspension, TNF does not induce significant tyrosine phosphorylation. In adherent PMN, a 42-kDa protein (p42) displayed the most striking increase in tyrosine phosphorylation after TNF stimulation. Cell lysates of TNF-stimulated PMN were separated by two-dimensional gel electrophoresis and were immunoblotted with either anti-phosphotyrosine (alpha-PY) mAb or anti-mitogen-activated protein kinase (alpha-MAPK) mAb. Both Abs detected p42, and the spots were superimposable. Cell lysates were immunoprecipitated with agarose-conjugated alpha-PY mAb, electrophoresed, and then immunoblotted with alpha-MAPK Ab; alternatively, cell lysates were immunoprecipitated with agarose-conjugated alpha-MAPK Ab, electrophoresed, and then immunoblotted with alpha-PY mAb. In both cases, p42 was detected. These results demonstrate that p42 is a member of the MAPK family. TNF induces a time-and dose-dependent increase in tyrosine phosphorylation of p42 and MAPK activity. The degree of p42 tyrosine phosphorylation parallels the level of MAPK activity. MAPK activity was determined by measuring 32P phosphorylation of a synthetic peptide containing the recognition site on myelin basic protein for MAPK. PMN pretreatment with genistein, a tyrosine kinase inhibitor, inhibited the TNF-induced increase in tyrosine phosphorylation and MAPK activity. These results indicate that TNF signaling involves activation of MAPK.
Molecular basis of the complement C7 M/N polymorphism. A neutral amino acid substitution outside the epitope of the allospecific monoclonal antibody WU 4-15.
Authors: Würzner R , Fernie B A , Jones A M , Lachmann P J , Hobart M J
Published: December 2022

The allotypes of the C7 M/N polymorphism are determined by comparing the ELISA reaction pattern of the allospecific mAb WU 4-15 with polyclonal anti-C7 IgG. To characterize the molecular basis of this polymorphism, the WU 4-15 epitope was mapped by expression of cDNA fragments. It was found to be located within the boundary region of the two short consensus repeats of C7 (amino acid residues 595-612). Coincidentally an A or C substitution was found in the course of investigation of the gene structure at nucleotide 1759. This leads to an electrically neutral Pro/Thr substitution at residue 565 and alternative Sau961 or RsaI restriction sites, respectively. Enzymatic digestion or sequencing of PCR-amplified genomic DNA from C7 M/N-typed individuals showed a perfect correspondence of the genotype with the phenotype of the C7 M/N polymorphism. In contrast, no association was found with the genotypes of a second polymorphic site at residue 367. Thus, a threonine at amino acid residue 565 allows access of WU 4-15 to its epitope but its substitution with proline almost completely inhibits access in the native molecule, although the epitope is about 40 amino acid residues distant from the polymorphic site in the primary structure. Furthermore, the different conformational structure is probably responsible for the hypomorphic appearance of C7*N.
Cleavage of lymphocyte surface antigens CD2, CD4, and CD8 by polymorphonuclear leukocyte elastase and cathepsin G in patients with cystic fibrosis.
Authors: Döring G , Frank F , Boudier C , Herbert S , Fleischer B , Bellon G
Published: December 2022

Polymorphonuclear leukocytes (PMN) accumulating in airways of patients with cystic fibrosis (CF) as a response to chronic endobronchial bacterial lung infection, release lysosomal serine proteinases such as PMN-elastase at concentrations of approximately 0.5 microM to 5 microM into the airway lumen. Immunohistology of CF lung material and fluorescence activated cell sorter analysis of sequential CF bronchoalveolar lavages demonstrated loss of the CD4 and CD8 Ag on CD3+ T lymphocytes in sputum-filled airways. In 10 CF sputum samples 1.0%, 19.1%, and 15.7% of all CD3+ T lymphocytes expressed CD4, CD8, and CD2, respectively. Incubation of CF sputum supernatant fluids with peripheral blood T lymphocytes resulted in total reduction of CD4 and CD8 but not CD2. Addition of alpha 1-proteinase inhibitor abolished surface Ag cleavage completely. Purified PMN-elastase and cathepsin G cleaved CD2, CD4, and CD8 on peripheral blood T lymphocytes at proteinase concentrations of 0.83 to 8.3 microM in a dose-dependent manner. Cleaved CD4 and CD8 were reexpressed on the surface of T lymphocytes after 24 h in the absence of PMN-elastase. Incubation of a CD4+ T cell clone with PMN-elastase lead to a significant reduction of cytotoxicity toward target cells and significantly reduced IL-2 and IL-4 production. The results suggest a temporary functional impairment of T lymphocytes in foci of high inflammation characterized by stimulated PMN, which may lower tissue destruction.
Analysis of the heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction.
Authors: Brezinschek H P , Brezinschek R I , Lipsky P E
Published: December 2022

The VH gene repertoire of human peripheral B cells was analyzed using PCR analysis of individual blood B cells. Because genomic DNA of single B cells was analyzed, data from both productive and nonproductive VDJ rearrangements were obtained. Nine out of 75 B cells contained both functional and nonfunctional rearrangement products, whereas 62/75 had a single productive VDJ rearrangement. The distribution of VH families was ordered in accordance with the germline complexity, although a bias toward VH3 and some of its members was found. This bias was noted in both the productively and nonproductively rearranged repertoires, indicating that it resulted from molecular and not selective processes. Evidence for negative selection of certain VH3 and VH4 family members was noted in that they were found less often as productive than nonproductive VDJ rearrangements. In addition, evidence for positive selection based on CDR3 was obtained, in that JH6 and DXP'1 were found at a higher frequency in the productively compared with the nonproductively rearranged repertoire. The CDR3 segments were markedly heterogenous, with a much greater degree of variability noted in the nonproductive VDJ rearrangements. Finally, a much greater frequency of mutations was noted in the nonproductively rearranged VH genes within individual B cells. These results have begun to delineate the human peripheral B cell repertoire and identify processes that shape it both positively and negatively.
Subverting lymph node trafficking by treatment with the Mel-14 monoclonal antibody to L-selectin does not prevent an effective host response to Sendai virus.
Authors: Hou S , Hyland L , Bradley L M , Watson S R , Doherty P C
Published: December 2022

A single 250-micrograms dose of the Mel-14 mAb to L-selectin greatly diminished the extent of L-selectin expression on lymphocytes and decreased (60 to 90%) the massive cellular recruitment to the cervical and mediastinal lymph nodes that follows intranasal infection of naive C57BL/6 mice with Sendai virus. The numbers of CD8+ CTL precursors in the mediastinal lymph nodes were considerably reduced on day 7, when compared with virus-infected mice given a control rat IgG2a, but potent CTL effectors were present in the lungs of both groups by day 10 after infection, and the overall magnitude of CTL precursor generation was not obviously compromised. The early dominance of Sendai virus-specific IgM Ab-forming cells was prolonged in the Mel-14-treated mice, whereas plasma cells producing virus-specific IgA were abnormally prominent in the lymph nodes but not in the spleen. The kinetics of virus-specific Ab-forming cells generation and the serum Ab response for the various IgG isotypes were also delayed. Thus, though L-selectin is clearly important for the localization of naive lymphocytes to regional lymph nodes, the Mel-14-treated mouse can still deal effectively with a virus that causes productive infection only in the respiratory tract. The spleen, where L-selectin does not determine lymphocyte trafficking, is a major site for the compensatory T cell and B cell responses.
Two waves of gamma delta T cells expressing different V delta genes are recruited into schistosome-induced liver granulomas.
Authors: Sandor M , Sperling A I , Cook G A , Weinstock J V , Lynch R G , Bluestone J A
Published: December 2022

Isolated granulomas provide a unique model to study T cells in the site of inflammation. Schistosoma mansoni-infected mice develop liver granulomas in response to schistosome egg deposition and the granulomas contain gamma delta T cells that appear to be activated (Pgp-1high and L-selectin low). Analysis of kinetics and TCR gene usage of granuloma gamma delta T cells revealed a limited TCR repertoire restricted to V gamma 1.1, V delta 4, and V delta 6 genes, suggesting that the occurrence of gamma delta T cells in the granuloma is influenced by TCR V gene usage. The V delta 4+, but not the V delta 6+, gamma delta T cells expressed CD69, a marker of recent activation. To determine if there was a preferred order of accumulation of the gamma delta T cells in granulomas, s.c. sponge grafts were implanted into schistosome-infected mice, schistosome eggs were injected into the grafts, and accumulated T cells were sequentially analyzed. The earliest gamma delta T cell immigrants expressed V delta 6 and later immigrants expressed V delta 4 V genes. Additional evidence for a role of TCR specificity in the accumulation of gamma delta T cells in granulomas is their absence from schistosome granulomas in TCR transgenic mice that express only a single MHC-specific gamma delta TCR. Finally, gamma delta T cell recruitment into the granulomas did not require beta 2-microglobulin, since gamma delta T cells were present in liver granulomas of beta 2-microglobulin gene-disrupted mice. The analysis of the influx of gamma delta T cells into schistosome-induced, liver granulomas and schistosome egg-containing sponges provides a model system to investigate the role, if any, of gamma delta T cells in schistosome infections.
Antigen-based heteropolymers facilitate, via primate erythrocyte complement receptor type 1, rapid erythrocyte binding of an autoantibody and its clearance from the circulation in rhesus monkeys.
Authors: Ferguson P J , Martin E N , Greene K L , Kuhn S , Cafiso D S , Addona G , Taylor R P
Published: December 2022

We investigated the feasibility of using the primate E complement receptor (CR1), in concert with Ag-based heteropolymers (AHP), as a potential therapy to remove autoantibodies from the circulation. AHP are prepared by cross-linking an anti-CR1 mAb with the acetylcholine receptor (AChR), the principal target Ag in myasthenia gravis. In vitro studies demonstrate that this methodology facilitates specific, rapid, and quantitative binding of an anti-AChR mAb to primate Es. In vivo experiments in rhesus monkeys indicate that AHP-mediated binding of an anti-AChR mAb to Es leads to the clearance of the mAb from the circulation. Once bound to the E via the AHP, the autoantibody is transported to the liver and spleen, where it is degraded without destruction of the E. It is therefore likely that the complexes of AHP and target mAb, when bound to Es, are recognized in vivo and processed by a mechanism quite similar to that which occurs when complement-opsonized immune complexes, bound to primate Es, are cleared from the circulation. It may be possible to extend and generalize this work to allow for the development of a simple, noninvasive therapy that can be made specific for the treatment of several different autoimmune diseases.
Age-dependent reduction of Bcl-2 expression in peripheral T cells of lpr and gld mutant mice.
Authors: Tamura A , Yui K
Published: December 2022

Autoimmune-prone lpr and gld mice carry defects in the apoptosis-mediating cell surface molecule Fas and its ligand, respectively. These mice develop lymphadenopathy because of an age-related accumulation of nonmalignant CD4- CD8- T cells in the peripheral lymphoid organs, suggesting a role for Fas-mediated apoptosis in peripheral T cell homeostasis. However, these accumulating cells are more susceptible to apoptosis ex vivo than peripheral T cells from control mice. To investigate the influence of additional regulatory elements on defects in the Fas-mediated apoptosis pathway, we analyzed the expression of Bcl-2 protein, a repressor of apoptosis, in T cells of lpr and gld mice. The expression levels of Bcl-2 in peripheral T cells of aged lpr and gld mice were significantly reduced when compared with their normal counterparts. Bcl-2 expression decreased with age in peripheral T cells, but not in thymocytes, suggesting that down-regulation of Bcl-2 protein occurs in the periphery. Analysis of T cell subsets indicated that CD4+ and CD4- CD8- T cells expressed significantly reduced levels of Bcl-2, whereas CD8+ cells maintain high levels of Bcl-2 expression. However, all peripheral T cell subsets including CD8+ cells were susceptible to glucocorticoid-induced apoptosis, indicating that there is no direct correlation between the levels of Bcl-2 expression and susceptibility to glucocorticoid-induced apoptosis. These studies suggest the presence of complex regulatory mechanisms for lymphocyte apoptosis and survival.
Role of environmental antigen in the development of IgG+ cells in the bursa of fabricius.
Authors: Ekino S , Riwar B , Kroese F G , Schwander E H , Koch G , Nieuwenhuis P
Published: December 2022

IgG+ cells were detected in the bursa of Fabricius after hatching by immunofluorescence staining of single cells and immunohistologic studies using mAb anti-Ig gamma-heavy chain. The frequency of IgG+ cells in the bursa increased rapidly immediately after hatching. In histologic studies, most of the IgG+ cells were found in the medullary areas of the bursal follicle. Isolation of the bursa from the gut by bursal duct ligation before hatching suppressed the development of IgG+ cells in the bursa after hatching. In addition, administration of Ags into the bursal lumen just before bursal duct ligation caused a significant increase of IgG+ cells in the bursa compared with the isolated bursa. However, parenteral administration of Ags had no effect on the frequency of IgG+ cells in the isolated and normal bursa. These results suggest that Ags in the bursa are a prerequisite for the development of IgG+ cells in the bursa. Although the majority of IgG+ bursal cells were IgM+ IgG+ double-positive cells, sorted Bu1+ bursal cells, which contained 99.9% of IgG+ cells but not plasma cells, synthesized de novo IgM but little or no IgG. Therefore, it is likely that surface IgG on the bursal cells is not produced locally, but is trapped by IgM+ bursal cells. We speculate that Ags derived from the bursal lumen form immune complexes within the bursa, which are subsequently trapped on the surface of IgM+ bursal cells mediated either by Fc-receptors, C3 receptors, or surface Ig.
Activation of the signal transducer and transcription (STAT) signaling pathway in a primary T cell response. Critical role for IL-6.
Authors: Henttinen T , Levy D E , Silvennoinen O , Hurme M
Published: December 2022

The T cell activation is initiated by interaction of specific Ags with TCR, followed by activation of intracellular biochemical events leading to activation of several genes. The activation of signal transducer and activator of transcription (STAT) proteins in a primary TCR-mediated activation of T cells have been explored. In purified human peripheral blood T cells, nuclear STAT proteins were activated approximately 3 h after activation by cross-linked anti-CD3 Abs. These STAT proteins were detected by using the IFN-gamma-activated sequence (GAS) and related oligonucleotides as probes in electrophoretic mobility shift assay. Analysis of the nuclear extracts with anti-STAT Abs indicated that they contained STAT-3 and additional proteins crossreactive with the STAT family. The induction of STAT activity was inhibited completely by pretreatment with either cycloheximide or cyclosporin A, thus indicating that the induction was due to a secondary factor produced by the activated T cells. As neutralizing anti-IL-6 Abs effectively down-regulated the early induction of STAT proteins and as exogenously added IL-6 rapidly activated DNA binding similar to TCR-mediated bindings, it can be concluded that IL-6 is the factor responsible for the activation of STAT proteins in a primary T cell response.
IL-12 as mediator and adjuvant for the induction of contact sensitivity in vivo.
Authors: Müller G , Saloga J , Germann T , Schuler G , Knop J , Enk A H
Published: December 2022

To determine whether IL-12 serves as a regulator of contact sensitivity reactions, mice were painted with either 1.0% trinitrochlorobenzene or 0.5% dinitrofluorobenzene on abdominal skin. At various time points thereafter, regional lymph nodes or spleens were prepared for RNA extraction, and the signals for IL-12 p35 and p40 chain were sought by quantitative reverse transcriptase-PCR. Time course analysis showed a constitutive expression of p35 chain mRNA signals throughout the experiment (0 to 72 h), whereas the signal for the p40 chain was transiently induced in lymph node and spleen cells after 12 to 14 h. Cellular depletion experiments and double label in situ hybridization studies showed that dendritic cells were sources for a major part of the p40 chain message. The presence of functional IL-12 in culture supernatants was indirectly assessed by addition of anti-IL-12 antiserum and analysis of IFN-gamma production. Significant amounts of IFN-gamma could only be detected in supernatants of allergen-treated animals. Addition of anti-IL-12 antiserum inhibited IFN-gamma production by about 55%. In a further attempt to assess the role of IL-12 in contact sensitivity, anti-IL-12 antiserum was injected i.p. into mice, and ear swelling responses were assessed following challenge. Injection of anti-IL-12 antiserum significantly reduced ear swelling responses by 85%. Thus anti-IL-12 treatment almost completely prevented sensitization. To assess whether IL-12 would be able to overcome in vivo tolerance, UV-tolerized animals were treated with i.p. IL-12 in a contact allergy system. Treatment of mice with IL-12 not only prevented tolerance induction, but was able to reverse UV-induced tolerance. In aggregate, our data point to an important role for IL-12 as a mediator and adjuvant for the induction of contact sensitivity in vivo.
CD28 delivers costimulatory signals independently of its association with phosphatidylinositol 3-kinase.
Authors: Truitt K E , Shi J , Gibson S , Segal L G , Mills G B , Imboden J B
Published: December 2022

CD28, a cell-surface molecule expressed by T cells, delivers costimulatory signals during the activation of T cells by Ag. Stimulation of CD28 induces its association with phosphatidylinositol 3'-kinase (PI3-K), raising the possibility that PI3-K plays a critical role in CD28 signaling. We find, however, that wortmannin, a potent inhibitor of PI3-K, does not block CD28-mediated costimulation of Jurkat (a human T cell line) or of murine CD4+ T cells. To address further the role of PI3-K in CD28-mediated signaling, we expressed mutant murine CD28 molecules in Jurkat cells. Mutation of Tyr 170 of murine CD28 to Phe abrogates the association of murine CD28 with PI3-K but does not affect the ability of murine CD28 to augment IL-2 production by Jurkat cells in response to the combination of ionomycin and PMA. Conversely, a mutant of murine CD28 that has a Tyr at position 170 but has Phe substitutions at the remaining three cytoplasmic tyrosines retains the ability to associate with PI3-K and has an impaired ability to deliver a costimulus that augments IL-2 production. CD28, therefore, can deliver costimulatory signals independently of its interaction with PI3-K, and association with PI3-K is insufficient to mediate the full effector function of CD28. Optimal signaling by CD28 requires the integrity of one or more of the carboxyl-terminal three Tyr residues.
Differences in MHC class I self peptide repertoires among HLA-A2 subtypes.
Authors: Sudo T , Kamikawaji N , Kimura A , Date Y , Savoie C J , Nakashima H , Furuichi E , Kuhara S , Sasazuki T
Published: December 2022

To investigate how single amino acid substitutions in MHC class I molecules affect differences in peptide repertoires, we eluted and sequenced the naturally processed peptides from three HLA-A2 subtypes (HLA-A*0204, -A*0206, and -A*0207) that differ by a single amino acid residue substitution each with HLA-A*0201 at the floor of the binding groove. Allele-specific peptide motifs for each HLA-A2 subtype substantially differed from that of HLA-A*0201 in the dominant anchor residues. The relative signal intensities for 18 self peptides, determined by mass spectrometry, precisely reflected these peptide motifs. Some overlapping peptides were isolated from both HLA-A*0201 and a single HLA-A2 variant, but no peptide was ubiquitously found across all variants. To rationalize the differences in peptide motifs, possible conformations of each allele were computer modeled by energy minimization calculations based on the reported crystal structure of HLA-A*0201. According to our models, the differences in peptide motifs could be explained by substituted-residue-driven conformational changes for each MHC-peptide complex. These results demonstrate the fine differences between HLA-A2 subtype self peptide repertoires and contribute to the prediction of antigenic peptides.
Early IL-4 production by non-CD4+ cells at the site of antigen deposition predicts the development of a T helper 2 cell response to Schistosoma mansoni eggs.
Authors: Sabin E A , Pearce E J
Published: December 2022

Cytokines play a major role in promoting naive Th cells to differentiate into Th1 or Th2 cells. While IL-4 is recognized as the primary pro-Th2 inducing cytokine, the identity of its cellular sources during the development of a Th2 response remains unclear. We have used Schistosoma mansoni eggs, potent stimulators of Th2 responses both during the natural progression of murine schistosomiasis and when experimentally isolated and injected into normal mice, to examine IL-4 production early in the evolution of an Ag-driven Th2 response. Analysis of peritoneal exudate cells by IL-4 specific reverse transcriptase-PCR and ELISPOT, at times following i.p. egg injection in naive C57BL/6 mice, revealed a marked, transient elevation in IL-4 production at 2 to 12 h after Ag exposure. This response was temporally accompanied by eosinophil and neutrophil infiltration and mast cell disappearance. The pattern of early IL-4 production and peritoneal cell infiltration was observed in egg-injected CD4+ cell-depleted and nude C57BL/6 mice, strongly suggesting that a non-T cell is the source of early IL-4 and that the stimulus leading to the egg-induced changes in cellular composition are T cell independent. In addition to IL-4 transcripts, peritoneal exudate cells from egg-injected T cell replete or deficient mice contained IFN-gamma and IL-12 transcripts. Control i.p. PBS injections led to no or minimal cytokine gene transcription. Early IL-4 was predictive of subsequent Th2 response development since, in contrast to C57BL/6 mice, egg-injected BALB/c mice demonstrated no detectable IL-4 production at 12 h and mounted a comparatively weak egg Ag-specific Th2 response.
Soluble CD23 directly activates monocytes to contribute to the antigen-independent stimulation of resting T cells.
Authors: Armant M , Rubio M , Delespesse G , Sarfati M
Published: December 2022

The majority of T cells at the site of an inflammatory lesion do not appear to be Ag-specific, but they still contribute to the inflammatory response. Herein, we report that sCD23 activates monocytes to participate in the stimulation of resting T cells in the absence of TCR engagement. First, sCD23 selectively triggers monokine release by purified monocytes in the absence of costimulus. It induces TNF-alpha, IL-1 beta, IL-8, granulocyte-macrophage-CSF, and prostaglandin E2 but no IL-10, IL-12, TGF-beta, or leukotriene B4. The sCD23-induced TNF-alpha production is significantly inhibited by IL-4 and IL-10 but not by TGF-beta. Also, monocytes activated by sCD23 express decreased levels of HLA-DR and increased levels of CD14, CD54, CD40, and B7 Ag. Next, we show that, in the presence of monocytes, sCD23 is a potent costimulator of IL-2 or IL-12-induced IFN-gamma production by resting T cells in the absence of exogenous Ag and that this effect is partially reduced by anti-TNF-alpha mAb. B cells cannot substitute for monocytes, and CD4+ and CD8+ T cells are equal responders. The data further indicate that monocyte-T cell contact, more particularly CD40-CD40L interactions, is required for IFN-gamma production in response to IL-2 plus sCD23, and the response to IL-12 plus sCD23 is CD40- and B7-independent but is still partially contact-dependent. It is proposed that sCD23, when produced locally at a site of immune response, may trigger an inflammatory process via monokine release and may further amplify it via the stimulation of bystander non-Ag-specific T cells.
Differential utilization of IFN-gamma-responsive elements in two maturationally distinct macrophage cell lines.
Authors: McDowell M A , Lucas D M , Nicolet C M , Paulnock D M
Published: December 2022

We have characterized the transcriptional response to IFN-gamma in two maturationally distinct macrophage populations: the mature RAW 264.7 cell line, phenotypically identical to thioglycollate-elicited peritoneal macrophages, and the less mature WEHI-3 cell line. We first investigated the use of two IFN-gamma-responsive regulatory elements, the interferon-stimulated response element (ISRE) and the gamma-activated sequence (GAS), in these cells. Transient transfection assays revealed that synthetic promoter constructs containing either the ISRE or GAS regulatory motif fused to a luciferase reporter gene were transcriptionally inactive in the WEHI-3 cell line. We then analyzed the expression in the two cell lines of a panel of known IFN-gamma-responsive genes that are transcriptionally controlled by different regulatory elements. RT-PCR analysis revealed that both cell lines responded to IFN-gamma treatment by up-regulating genes that are transcriptionally controlled by kappa B or W box DNA binding motifs. However, genes regulated by ISRE or GAS elements were induced by IFN-gamma only in the RAW 264.7 cell line. Kinetic analysis of the transcriptional activity of synthetic promoter constructs in the RAW 264.7 cell line showed rapid IFN-gamma induction through both the ISRE and GAS motifs, indicating that both elements are utilized early after IFN-gamma stimulation in mature macrophages. These results suggest that cis-acting DNA response element utilization, and the subsequent profiles of IFN-gamma-induced gene expression, differ in macrophages at different stages of maturation.
p21ras links Fc epsilon RI to NF-AT family member in mast cells. The AP3-like factor in this cell type is an NF-AT family member.
Authors: Prieschl E E , Pendl G G , Harrer N E , Baumruker T
Published: December 2022

Very recently, an AP3-like transcription factor regulating the chemokine gene MARC and an NF-AT family member regulating IL-5 were the first components of the transcription factor repertoire to be described as activated in mast cells after an allergic triggering. In this study, we show that with respect to cross-competition in a gel shift analysis using an NF-AT consensus oligonucleotide binding site, the antigenicity to a recently generated serum against T cell NF-AT, and the sensitivity to macrolide immunosuppressants, the AP3-like activity on the MARC promoter is indistinguishable from that described for NF-AT in T cells. Additionally, we show that this factor functions on the MARC chemokine promoter without the AP1 cofactor, a situation reminiscent of the function of NF-AT in Th2-type T cells. In all of these aspects, and strengthened further by a gel shift competition analysis, the AP3-like transcription factor is identical to the NF-AT family member recently described by an analogous set of experiments as regulating IL-5 in mast cells. Our finding that p21ras, but probably not protein kinase C, is necessary to activate this factor after Fc epsilon RI triggering indicates a situation in which a common transcription factor denominator in mast cells induces chemokine (MARC) and lymphokine (IL-5) gene expression in a manner closely similar to Th2-type T cells, which are induced along the ras/raf signal pathway.