bluuebunny/crossref_metadata_2025 huggingface dataset:
Deletion of a Recombined Ig Heavy Chain Transgene in B-Lineage Cells of Transgenic Mice
Authors: Heinzelmann Andy , Kumar Subbiah , Noggle Scott , Goedegebuur Ine , Sauer K. Morgan , Rath Satyajit , Durdik Jeannine M.
Published: December 2022

Fully recombined transgenes are stable in their transmission in the germline of transgenic mice, in common with the endogenous genetic complement of most mammalian somatic tissues, including the genes for lymphoid Ag receptors somatically generated from germline minigenes. There have, however, been isolated reports of unusual low frequency transgene losses in various transgenic mice. Here we show, using Southern blots and PCR-based assays, that plasmablast hybridomas and B cells from three independently derived founder lines of transgenic mice bearing a recombined heavy chain Ig transgene we have been studying show a significant net loss of transgene copies. This loss is more marked in the B cells expressing endogenous heavy chains than in those expressing transgenic heavy chains. We have also examined cells of the B lineage in the bone marrow, and a small degree of deletion is also evident in CD19+CD23−IgM− immature B-lineage cells. As greater deletion is observed in mature B cells, it is possible that the deletion process either continues into B cell maturity and/or provides a selective advantage. We have investigated the relationship between transgene expression and deletion, and we find that while thymocytes in these mice express the transgene well, T cell hybridomas derived from transgenic thymus do not show any loss of the transgene. Thus, a recombined Ig heavy chain transgene prominently undergoes somatic deletion in B-lineage cells independent of its insertion site or expression. This transgenic instability is significant to the analysis of genomic stability as well as to the design of gene therapy strategies.
Carrier-Induced, Hapten-Specific Suppression: A Problem of Antigen Presentation?
Authors: Renjifo Ximena , Wolf Stanley , Pastoret Paul-Pierre , Bazin Hervé , Urbain Jacques , Leo Oberdan , Moser Muriel
Published: December 2022

Prior immunity against a carrier protein has been shown to modulate the serologic response to injected haptens attached to the same carrier. In particular, a carrier/hapten-carrier immunization protocol induces marked suppression for IgG2a anti-hapten Ab production but does not interfere with anti-carrier Ab responses. Although the phenomenon of epitopic suppression has been amply demonstrated, the mechanism underlying the suppression remains unknown. The selective deficiency in IgG2a secretion suggests that IFN-γ-producing Th1 cells are not properly activated. We and others have shown that the nature of the APCs present during the first encounter with the Ag influences the development of selected Th populations in vivo; dendritic cells (DCs) seem to be required for the induction of primary, Th1-type responses. Since carrier priming induces the clonal expansion of specific B cells that appear to efficiently capture the Ag, we hypothesized that the hapten-carrier conjugate may be presented by B cells in preimmunized animals. Therefore, we immunized mice to the conjugate by injecting syngeneic DCs pulsed in vitro with the Ag. Our data show that an injection of DCs and IL-12 prevents epitopic suppression, suggesting that it may result from defective Ag presentation.
Following Antigen Challenge, T Cells Up-Regulate Cell Surface Expression of CD4 In Vitro and In Vivo
Authors: Ridgway William , Fassò Marcella , Fathman C. Garrison
Published: December 2022

The low precursor frequency of Ag-specific T cells has raised significant barriers to studying the T cell response in vivo. We demonstrate that T cells up-regulate the cell surface expression of CD4 following Ag recognition, which identifies Ag-specific T cells in vitro and in vivo and allows their characterization. The CD4high cell subpopulation contains the Ag-specific population as indicated by Ag-induced proliferation and limiting dilution analyses. The use of the CD4high marker will allow analysis of the dynamics of the T cell immune response in vivo, the study of the suboptimal T cell response to Ag, and the identification of T cells which are reactive to known and unknown autoantigens.
Role of Maternal Ig in the Induction of Cκ-Specific CD8+ T Cell Tolerance
Authors: Rueff-Juy Dominique , Faure Mathias , Drapier Anne-Marie , Cazenave Pierre-André
Published: December 2022

Although the influence of maternal Ig on the B cell repertoire and subsequent Ab response has been extensively studied, much less attention has been devoted to their effects on T cell responses of the offspring. To address this question, we have studied the influence of maternal κ-positive Ig (Igκ) on the Cκ-specific CD8+ T cell response of κ knock-out (κ−/−) pups resulting from various crosses and foster nursings. These systems allowed control of physiologic transmission of Igκ at defined periods of ontogeny. Our data show that conventional transfer of maternal Ig via the placenta plus colostrum/milk or adoptive transfer via only the colostrum/milk were the most efficient at tolerizing Cκ-specific CD8+ responses. Surprisingly, tolerance was not detected in κ−/− pups born to κ+/− females obtained by cesarean delivery and suckled by κ−/− mothers (transplacental supply only). Tolerance, which was strong until 5 wk of age, was reversible and waned with the decrease of Igκ serum concentration. Depletion of CD4+ T cells at the time of Cκ peptide immunization abolished the tolerance of Cκ-specific CD8+ T cells. These data suggest that an oral supply of Ig is very efficient at inducing and maintaining tolerance of Cκ-specific CD8+ T cells, at least for several weeks after birth, and that suppression rather than deletion is responsible for this tolerance. In addition, they strengthen the view that tolerance of CD8+ T cells to a soluble Ag is never permanently acquired even if it is present in large quantities during ontogeny.
Engagement of CD99 Induces Up-Regulation of TCR and MHC Class I and II Molecules on the Surface of Human Thymocytes
Authors: Choi Eun Young , Park Weon Seo , Jung Kyeong Cheon , Kim Soon Ha , Kim You Young , Lee Wang Jae , Park Seong Hoe
Published: December 2022

CD99 is a cell surface molecule involved in the aggregation of lymphocytes and apoptosis of immature cortical thymocytes. Despite its high level expression on immature cortical thymocytes, the functional roles of this molecule during thymic selection are only now being elucidated. Examination of the effects of CD99 engagement on the expression kinetics of the TCR and MHC class I and II molecules, which are involved primarily in thymic positive selection, revealed a marked up-regulation of these proteins on the surface of immature thymocytes. This increase was the result of accelerated mobilization of molecules stored in cytosolic compartments to the plasma membrane, rather than increased RNA and protein synthesis. Confocal microscopic analysis revealed the changes in subcellular distribution of these molecules. When CD99 was engaged, TCR and MHC class I and II molecules were concentrated at the plasma membrane, particularly at cell-cell contact sites. The TCRlow subpopulation of immature double positive thymocytes was much more responsive to CD99-mediated up-regulation than was the TCRhigh population. These findings suggest that CD99-dependent up-regulation may have possible implication in positive selection during thymocyte ontogeny.
Somatic Hypermutation of an Artificial Test Substrate Within an Igκ Transgene
Authors: Klotz Emily L. , Hackett John , Storb Ursula
Published: December 2022

We have characterized a novel substrate for somatic hypermutation, confirming that non-Ig sequences can be targeted for mutation and demonstrating that this substrate allows for the rapid assay for mutations. An artificial sequence containing alternating EcoRV and PvuII sites (EPS) was inserted into the Vκ167 transgene, which is known to be a target for mutation. To assay for somatic hypermutation, the EPS is amplified using flanking transgene primers, and the PCR product is subsequently digested with either EcoRV or PvuII. A mutation is seen as the appearance of a larger fragment, indicating a base change in a restriction enzyme site. The original transgene, Vκ167/EPS, showed evidence of a low level of mutation in both splenic hybridomas and Peyer’s patch-derived or immunized splenic B220+ cells with high peanut agglutinin levels. Two derivative lines of Vκ167/EPS were made, Vκ167/POX and Vκ167/PEPS. While none of the Vκ167/POX transgenic lines demonstrated mutation, the Vκ167/PEPS transgene was highly mutated in B220+ splenic B cells with high peanut agglutinin levels at a frequency similar to that of endogenous Ig genes. An analysis of splenic RNA from the unimmunized transgenic mice indicated that the levels of stable message in splenic B cells could not be correlated with the mutation seen in GC B cells. The mutable Vκ167/PEPS transgenic line is a unique tool to study somatic hypermutation in vivo.
Expression Screening of a Yeast Artificial Chromosome Contig Refines the Location of the MouseH3aMinor Histocompatibility Antigen Gene
Authors: Zuberi Aamir R. , Christianson Gregory J. , Dave Sonal B. , Bradley Julie A. , Roopenian Derry C.
Published: December 2022

The H3 complex, on mouse Chromosome 2, is an important model locus for understanding mechanisms underlying non-self Ag recognition during tissue transplantation rejection between MHC-matched mouse strains. H3a is a minor histocompatibility Ag gene, located within H3, that encodes a polymorphic peptide alloantigen recognized by cytolytic T cells. Other genes within the complex include β2-microglobulin and H3b. A yeast artificial chromosome (YAC) contig is described that spans the interval between D2Mit444 and D2Mit17, a region known to contain H3a. This contig refines the position of many genes and anonymous loci. In addition, 23 new sequence-tagged sites are described that further increase the genetic resolution surrounding H3a. A novel assay was developed to determine the location of H3a within the contig. Representative YACs were modified by retrofitting with a mammalian selectable marker, and then introduced by spheroplast fusion into mouse L cells. YAC-containing L cells were screened for the expression of the YAC-encoded H3aa Ag by using them as targets in a cell-mediated lympholysis assay with H3aa-specific CTLs. A single YAC carrying H3a was identified. Based on the location of this YAC within the contig, many candidate genes can be eliminated. The data position H3a between Tyro3 and Epb4.2, in close proximity to Capn3. These studies illustrate how genetic and genomic information can be exploited toward identifying genes encoding not only histocompatibility Ags, but also any autoantigen recognized by T cells.
Stringent Allele/Epitope Requirements for MART-1/Melan A Immunodominance: Implications for Peptide-Based Immunotherapy
Authors: Bettinotti Maria P. , Kim Christina J. , Lee Kang-Hun , Roden Matthew , Cormier Janice N. , Panelli Monica , Parker Kenneth K. , Marincola Francesco M.
Published: December 2022

The exclusiveness of the relationship between peptide and HLA alleles, combined with their extensive polymorphism, emphasizes the need for immunization strategies based on endogenous processing of full length proteins (containing multiple epitopic determinants) for presentation to T cells. This could allow vaccination regardless of the patient’s HLA phenotype, assuming that individual molecules can be efficient T cell Ags in association with various HLA alleles. An endogenous system of Ag presentation was developed using dendritic cells infected with recombinant viral vectors expressing the melanoma-associated Ag MART-1/Melan A. CD8+ T cells from melanoma patients were activated in vitro by coincubation with infected dendritic cells and tested for recognition of HLA-A-matched melanoma targets. This allowed the analysis of T cell induction in association with any HLA-A allele of a given patient’s phenotype. In this system, MART-1/Melan A could not efficiently immunize in association with HLA-A alleles other than A*0201, including the one residue variant from A*0201: HLA-A*0226. Clonal analysis of MART-1/Melan A-specific CTL confirmed that MART-1/Melan A immunodominance is strongly restricted to the AAGIGILTV/HLA-A*0201 combination. The stringent epitope/allele requirements for MART-1/Melan A/TCR interactions were not associated with limitations in the TCR repertoire. In conclusion, autologous induction of MART-1/Melan A CTL by whole Ag processing and presentation is restricted to a unique allele/ligand combination and is excluded by minimal changes in HLA structure. Thus, whole protein vaccination for small m.w. Ags may provide no further advantage over a peptide-based approach.
T Cells Specific for a Polymorphic Segment of CD45 Induce Graft-Versus-Host Disease with Predominant Pulmonary Vasculitis
Authors: Chen Wei , Chatta Gurkamal S. , Rubin William D. , Clark Joan G. , Hackman Robert C. , Madtes David K. , Ligitt Denny H. , Kusunoki Yoichiro , Martin Paul J. , Cheever Martin A.
Published: December 2022

To study the character of graft-vs-host disease (GVHD) induced by T cells specific for hemopoietic cells, T cells specific for a polymorphic segment of CD45 were transferred into CD45 congenic mice. C57BL/6 mice that express the CD45b allele were immunized with a 13 mer peptide representing the polymorphic segment (p257–268) of CD45a protein. Conversely, C57BL/6 mice congenic for CD45a were immunized with the CD45b peptide. CD4+ T cells specific for allelic CD45 peptides were elicited. Importantly, T cells specific for CD45 peptides proliferated specifically and vigorously in response to spleen cells expressing the appropriate polymorphic CD45 protein. T cells specific for CD45 induced a substantial graft-vs-host response (GVHR) with predominant early pulmonary vasculitis and later more widespread interstitial mononuclear cell infiltration and alveolitis. No GVHR was induced in bone marrow chimeras expressing only donor hemopoietic cells. Thus, donor T cell recognition of host hemopoietic cells is sufficient to elicit GVHR, but the classical skin, liver, and gut manifestations of GVHD were not observed. The CD45-specific T cells used secreted Th1 cytokines, but without detectable soluble IL-2. Studies using CD45-specific T cells with different effector functions might allow further dissection of donor cell requirements for GVHD syndromes.
Phosphorylation of the Cytoplasmic Domain of E-Selectin Is Regulated During Leukocyte-Endothelial Adhesion
Authors: Yoshida Masayuki , Szente Brian E. , Kiely Jeanne-Marie , Rosenzweig Anthony , Gimbrone Michael A.
Published: December 2022

E-selectin, a selectin expressed on activated vascular endothelium, supports rolling and stable adhesion of leukocytes at sites of inflammation. Previously, we have reported that leukocyte adhesion to cultured endothelial cells induces association of the cytoplasmic domain of E-selectin with cytoskeletal elements, suggesting that outside-in signaling may occur during E-selectin-mediated adhesion. To investigate this potential signaling function of E-selectin, HUVEC activated with recombinant human IL-1β (10 U/ml, 4 h) were labeled with [32P]orthophosphate, and E-selectin was immunoprecipitated using mAb H18/7. Autoradiography revealed constitutive phosphorylation of E-selectin in these cells and time-dependent dephosphorylation following adhesion of HL-60 cells. Cross-linking of cell surface E-selectin using H18/7 and a polyclonal secondary Ab induced E-selectin dephosphorylation, as did adhesion of beads coated with recombinant P-selectin glycoprotein ligand-1 (PSGL-1), an E-selectin ligand. Using adenoviral vector-mediated transfection in HUVEC of a tail-less E-selectin and phosphoamino acid analysis, we documented phosphorylation occurring exclusively within the cytoplasmic domain and involving serine residues. Additional experiments using a series of cytoplasmic domain mutants of E-selectin expressed in COS-7 cells localized the regions that were constitutively phosphorylated. Preincubation with okadaic acid and sodium vanadate abrogated adhesion-induced dephosphorylation of E-selectin. Thus, E-selectin, which is constitutively phosphorylated in cytokine-activated human endothelial cells, undergoes an enzymatically regulated dephosphorylation following leukocyte adhesion. This process appears to be triggered by multivalent ligand binding and/or cross-linking of cell surface E-selectin. Ligand-dependent regulation of the phosphorylation of E-selectin’s cytoplasmic domain provides additional evidence for a transmembrane signaling function of this molecule during leukocyte-endothelial interactions.
CD4+ T Cells Migrate into Inflamed Skin Only If They Express Ligands for E- and P-Selectin
Authors: Tietz Wolfgang , Allemand Yvonne , Borges Eric , von Laer Dorothea , Hallmann Ruppert , Vestweber Dietmar , Hamann Alf
Published: December 2022

Previous data suggested a role of endothelial selectins in skin homing of lymphocytes. In the current study, we have analyzed the expression and functional role of E-and P-selectin ligands on CD4+ T cells induced in vivo upon skin sensitization, using soluble selectin-Ig chimera and blocking Abs. Only low numbers of CD4+ cells expressing significant levels of E- or P-selectin ligands were present in s.c. lymph nodes of untreated mice (0.5–1.5% and 2–4%, respectively). Induction of a delayed-type hypersensitivity reaction increased the percentage of E-selectin-binding CD4+ cells in the draining lymph nodes up to 6 to 9% and that of P-selectin-binding cells up to 14%. The majority of E- and P-selectin-binding cells displayed an activated phenotype as judged by the increase in IL-2R, CD71, or cell size. The populations of E- and P-selectin-binding cells were largely overlapping; all E-selectin-binding cells also bound to P-selectin, whereas only a subfraction of P-selectin-binding cells reacted with E-selectin. Both E- and P-selectin-binding CD4+ cells, isolated by FACS, efficiently migrated into inflamed, but not normal skin, whereas P- or E-selectin ligand-negative CD4+ T cells did not. Abs against one of the two endothelial selectins partially inhibited the entry of isolated, ligand-positive cells, whereas a combination of Abs against both selectins almost completely abrogated skin homing. These data indicate that the expression of functional ligands for E- and for P-selectin is essential for homing of CD4+ T cells into the inflamed skin.
Differential Regulation of Eosinophil Adhesion and Transmigration by Pulmonary Microvascular Endothelial Cells
Authors: Yamamoto Hideaki , Sedgwick Julie B. , Busse William W.
Published: December 2022

In bronchial asthma, eosinophils (EOS) adhere to, and migrate across, the lung microvasculature to exert their effector functions in the airways. This study was conducted to determine the effect of cytokines on adhesion molecule expression on human pulmonary microvascular endothelial cells (HPMEC) and the influence of these molecules on EOS adhesion and transmigration in vitro. Unlike ICAM-1 expression (>80% positive cytokine-treated HPMEC by flow cytometry), VCAM-1 expression varied with the cytokine(s) pretreatment; the order of potency was: TNF-α + IL-4 (82.2 ± 4.2% positive cells) > TNF-α (41.8 ± 5.1%) > IL-1β (20.8 ± 4.7%). IL-4 alone had no effect on either ICAM-1 or VCAM-1 expression. EOS adhesion to cytokine-treated HPMEC followed the same order as that observed for VCAM-1 expression. Interestingly, EOS migration across cytokine-treated HPMEC varied inversely with VCAM-1 expression on, and EOS adhesion to, HPMEC; IL-1β (21.2 ± 1.4% migration) > TNF-α (12.6 ± 2.6%) > TNF-α + IL-4 (9.1 ± 2.0%). EOS adhesion was greatest with TNF-α + IL-4-treated HPMEC, was dependent on VCAM-1, and inhibited with anti-α4 integrin mAb (67.7 ± 7.5% inhibition, p < 0.0005). In contrast, the highest EOS migration occurred across IL-1β-treated HPMEC and was inhibited by anti-β2 integrin mAb (40.4 ± 2.5% inhibition, p < 0.005). Viable HPMEC were required for EOS migration but not adhesion. Our results suggest that EOS adhesion and transmigration are differentially regulated by VCAM-1 and ICAM-1 expression and the interaction of these adhesion proteins with their respective counterligands, i.e., α4 and β2 integrins on EOS.
Inhibition of Inducible Nitric Oxide Synthase by Peroxisome Proliferator-Activated Receptor Agonists: Correlation with Induction of Heme Oxygenase 1
Authors: Colville-Nash Paul R. , Qureshi Saima S. , Willis Dean , Willoughby Derek A.
Published: December 2022

Genetic knock-out in mice of peroxisome proliferator-activated receptor-α (PPARα) can prolong inflammation in response to leukotriene B4. Although cyclooxygenase 2 has been shown to be induced by PPAR activation, the effect of PPAR agonists on the key inflammatory enzyme systems of nitric oxide synthase (NOS) and stress proteins has not been investigated. The effect on these of naturally occurring eicosanoid PPAR agonists (leukotriene B4 and 8(S)-hydroxyeicosatetraenoic acid, which are PPARα selective; PGA2, PGD2, PGJ2, and Δ12PGJ2, which are PPARγ selective) and the synthetic PPARα agonist Wy14,643 was examined in activated RAW264.7 murine macrophages. Leukotriene B4 and 8(S)-hydroxyeicosatetraenoic acid stimulated nitrite accumulation, indicative of enhanced NOS activity. PGA2, PGD2, PGJ2, Δ12PGJ2, and Wy14,643 reduced nitrite accumulation, with Δ12PGJ2 being the most effective. The mechanism behind this reduction was examined using Western blotting. Inhibition of nitrite accumulation was associated with a fall in inducible NOS protein and an induction of heme oxygenase 1, correlating both dose dependently and temporally. Other proteins examined (cyclooxygenase 2, heme oxygenase 2, heat shock protein 70, and glucose-regulated protein 78) were unaffected. The data suggest that naturally occurring PPAR agonists can inhibit the inducible NOS enzyme pathway. This inhibition may be mediated by modulation of the stress protein, heme oxygenase 1. Thus, the generation of eicosanoid breakdown products during inflammation may contribute to its eventual resolution by activation of the PPAR system. This system may thus represent a novel target for therapeutic intervention in inflammatory disease.
Tolerance Induction by Anti-CD2 Plus Anti-CD3 Monoclonal Antibodies: Evidence for an IL-4 Requirement
Authors: Punch Jeffrey D. , Tono Takeshi , Qin Lihui , Bishop D. Keith , Bromberg Jonathan S.
Published: December 2022

Anti-CD2 mAb plus anti-CD3 mAb induce alloantigen specific tolerance. We sought to determine whether Th2 cytokines are involved in the induction of tolerance in this model. Addition of anti-IL-4 mAb or anti-IL-10 mAb to anti-CD2 plus anti-CD3 treatment abrogated tolerance and resulted in graft survivals of 26 ± 4 and 25 ± 5 days, respectively. Splenocytes from the anti-IL-4 mAb and anti-IL-10 groups had greater proliferation in response to alloantigen than either tolerant or naive groups. Cytokine analysis of MLR supernatants showed increased IL-10 in the tolerant group and increased IFN-γ in the anti-IL-4 mAb treated group. Donor-specific alloantibody responses in untreated immune animals had a predominantly Th1 (IgG2a) alloantibody response, while the tolerogenic regimen reduced the ratio of IgG2a:IgG1 titers. The addition of anti-IL-4 mAb to the tolerogenic regimen partly restored the Th1-related IgG2a response. Tolerance did not develop in IL-4 knockout animals treated with anti-CD2 plus anti-CD3 (mean graft survival, 27 ± 5 days). Restoration of IL-4 to IL-4 knockout animals by gene transfer with plasmid DNA resulted in prolongation of survival to 46 ± 7 days, while adoptive transfer of wild-type splenocytes into IL-4 knockout recipients resulted in indefinite graft survival (>60 days) and indefinite survival of second donor-type grafts. IL-10 gene transfer to IL-4 knockout recipients did not prolong graft survival (28 ± 4). These results demonstrate that tolerance in this model is mediated at least in part by Th2-type cells that secrete IL-4, promote IL-10 and IgG1 production, and inhibit alloantigen reactivity.
Characterization of a Novel Bax-Associated Protein Expressed in Hemopoietic Tissues and Regulated During Thymocyte Apoptosis
Authors: He Huiling , Hershberger Pamela A. , McCarthy Susan A.
Published: December 2022

Members of the Bcl-2 protein family have been implicated as critical intracellular regulators of apoptosis. Most studies of this protein family have utilized transformed and/or transfected cell lines expressing high levels of these proteins. In the current study, we have analyzed normal murine lymphoid cells and tissues and have detected a previously unreported protein of approximately 16 kDa recognized by an anti-Bax Ab. This 16-kDa protein is abundant in hemopoietic tissues of both wild-type and Bax knock-out mice, it can heterodimerize with Bax in normal lymphocytes, and it is dramatically down-modulated in thymocytes in response to apoptotic stimuli. These results suggest that this protein may have antiapoptotic activity and may participate in the regulation of apoptosis in normal lymphocytes.
Human and Nonhuman Primate Lymphocytes Engrafted into SCID Mice Reside in Unique Mesenteric Lymphoid Structures
Authors: Donze Hollie Hale , Cummins James E. , Schwiebert Rebecca S. , Fultz Patricia N. , Jackson Susan , Mestecky Jiri
Published: December 2022

The present study compares the location and phenotype of B lineage lymphocytes in tissues from SCID mice engrafted with PBMC of human, chimpanzee, and pig-tailed macaque origin. In mice repopulated with both human and nonhuman primate lymphocytes, plasma cells were found in the peritoneal cavity in vascularized structures located in the mesentery near the pancreas, intestines, and spleen. The predominant isotype of the plasma cells was IgG; IgM and IgA cells were also present. κ and λ light chains were expressed by 62% and 38% of the Ig-containing cells, respectively. J chain expression occurred in most cells irrespective of the Ig isotype. In the SCID mice engrafted with human lymphocytes, a few IgM-containing cells were found in the spleen; plasma cells were not found in other tissues, including the intestine. The aggregation of plasma cells did not appear to be a result of infection with EBV. T cells were rarely found in the lymphoid aggregates but were recovered from the spleen and peritoneal lavage. Human Ig levels in the serum of engrafted mice reflected the isotype distribution of the cells with IgG > IgM ≥ IgA.
Generation of Switch Hybrid DNA Between Ig Heavy Chain-μ and Downstream Switch Regions in B Lymphocytes
Authors: Müller Jürgen R. , Giese Thomas , Henry Diane L. , Mushinski J. Frederic , Marcu Kenneth B.
Published: December 2022

Ig heavy chain isotype switching in B lymphocytes is known to be preceded by transcription of a portion of the particular heavy chain gene segment that is targeted for recombination. Here, we describe an active role for these transcripts in the switch recombination process. Using an in vitro assay that exposes an artificial switch-μ (Sμ) minisubstrate to switch region transcripts in the presence of nuclear extracts from switching cells, we demonstrate that free 3′ ends of the Sμ sequence are extended onto switch region transcripts by reverse transcription. The activity was induced in splenic B lymphocytes upon activation with LPS or CD40 ligand. This in vitro process is thought to be relevant to in vivo class switching for two reasons: 1) although only one-third of the Sμ minisubstrate actually contains Sμ sequence, all crossovers between switch regions occurred in the Sμ portion; and 2) treatment of B lymphocytes with IL-4, which enriches for switching to Sγ1, increases the ratio of Sμ-Sγ1 to Sμ-Sγ3 hybrids by 16% after LPS treatment and by 37% after CD40 ligand activation, implicating this Sμ-primed reverse transcription of switch region transcripts as a novel mechanism of regulating the specificity of isotype switching. Further evidence for an active role of switch region transcripts was obtained by expressing Sα RNA in trans in the Bcl1B1 B lymphoma line. Endogenous Sμ-Sα switch circles were detected in Bcl1B1 cells expressing exogenous Sα RNA but not in mock-transfected cells.
A Developmental Stage-Specific Promoter Directs Germline Transcription of DβJβ Gene Segments in Precursor T Lymphocytes
Authors: Sikes Michael L. , Gomez Rey J. , Song Jing , Oltz Eugene M.
Published: December 2022

The tissue- and stage-specific assembly of Ag receptor genes is regulated by transcriptional control elements positioned within Ig and TCR loci. To further understand the role of cis-acting elements in these regulatory mechanisms, we have characterized a transcriptional promoter that drives germline expression of TCRβ gene segments in vivo. The activity of this promoter, termed PDβ, is restricted to a highly conserved 400-bp region located directly upstream from Dβ1-coding sequences. Maximal PDβ activity requires a TATA element situated within the Dβ1 recombination signal sequences and consensus binding sites for the ubiquitous SP1 and the T cell-specific GATA-3 transcription factors. When linked to active enhancer elements, PDβ directs transcription in most cell types; however, the TCRβ enhancer (Eβ) stimulates PDβ function specifically in precursor T lymphocytes. These findings suggest that PDβ/Eβ interactions may contribute to differential regulation of regions within the TCRβ locus during thymocyte development.
Host NK Cells Are Required for the Growth of the Human Filarial ParasiteBrugia malayiin Mice
Authors: Babu Subash , Porte Patricia , Klei Thomas R. , Shultz Leonard D. , Rajan T. V.
Published: December 2022

Human lymphatic filariasis, which afflicts an estimated 120 million people worldwide, is caused by the large nematode parasites Wuchereria bancrofti and Brugia malayi. Filarial nematodes require both an arthropod vector and a mammalian host to complete their life cycle. Within the definitive (mammalian) host, the lymphatic filarial parasites reside in the lymph nodes and lymphatics, a seemingly hostile environment for infectious agents, since the location exposes them to the immune defenses of the host. We present data here that suggest that the growth of B. malayi in the mammalian host is dependent on host NK cell function. Comparisons of worm survival and development in different strains of mice with varying levels of NK cell activity reveal that NOD/LtSz-scid/scid and NOD/LtSz-scid/scid B2mnull mice (with diminished to absent NK cell activity respectively), are nonpermissive to worm growth, while C.B-17-scid/scid mice with normal NK cell activity are highly permissive. Depletion of NK cells in the permissive C57BL/6J-scid/scid mice renders them nonpermissive to B. malayi growth, whereas stimulation of NK cells in NOD/LtSz-scid/scid mice makes them permissive. Tgε26 mice, which lack NK and T cells, are nonpermissive, but, when reconstituted with NK cells by adoptive transfer of bone marrow cells from C57BL/6J-scid/scid mice, are rendered permissive. This requirement for NK cell activity may explain the site specificity of these parasites. Furthermore, these data suggest that the interaction of the host immune system with the filarial parasite is double edged, with both host protective and parasite growth-promoting activities emanating from the former.
LPS-Stimulated SJL Macrophages Produce IL-12 and IL-18 That Inhibit IgE Production In Vitro by Induction of IFN-γ Production from CD3intIL-2Rβ+ T Cells
Authors: Yoshimoto Tomohiro , Nagai Nobuhiko , Ohkusu Kazunobu , Ueda Haruyasu , Okamura Haruki , Nakanishi Kenji
Published: December 2022

SJL mice are known for their poor IgE production upon helminth infection. In this study, we have demonstrated that SJL standard B cells (85% IgM+ or B220+), prepared by complement-mediated T cell lysis, failed to proliferate and to produce IgE and IgG1 in response to LPS plus IL-4 in vitro. This diminished IgE production was restored by anti-IL-12 and enhanced by additional treatment with anti-IL-18, suggesting active suppression by the cells that produce IL-12 and IL-18. Indeed, SJL standard B cells were contaminated with Mac-1+ cells. Therefore, we removed macrophages by passing standard B cells through a Sephadex G-10 column (G10). Resultant cells (95% IgM+), designated as G10-B cells, responded to LPS and IL-4 by their proliferation and differentiation. G-10 treatment markedly diminished the proportion of B220− cells and Mac-1+ cells in SJL standard B cells. Furthermore, addition of SJL B220− cells dose dependently and MHC independently inhibited LPS plus IL-4-induced B cell growth and IgE production in SJL and BALB/c B cells. B220− cells in SJL standard B cells contained Mac-1+ cells (51%) and Fas ligand+ CD4−CD8− double-negative CD3intIL-2Rβ+ T cells (26%). Thus, IL-12 and IL-18 produced by LPS-stimulated Mac-1+ cells stimulate this unique subpopulation of T cells to produce IFN-γ, which in combination with Fas ligand, inhibits IgE production from the B cells. Our present results indicate that Mac-1+ cells and double-negative CD3intIL-2Rβ+ T cells, uniquely abundant in the spleens of SJL mice, inhibit IgE production, indicating their new role in IgE response.
Endothelial Myosin Light Chain Kinase Regulates Neutrophil Migration Across Human Umbilical Vein Endothelial Cell Monolayer
Authors: Saito Hajime , Minamiya Yoshihiro , Kitamura Michihiko , Saito Satoshi , Enomoto Katsuhiko , Terada Kunihiko , Ogawa Jun-ichi
Published: December 2022

Although extravasation of neutrophils is a critical step in acute inflammation, the role of the endothelial cytoskeleton in neutrophil transmigration has not been fully investigated. We used an in vitro model of neutrophil transmigration across a monolayer of HUVEC cultured on amniotic membrane. Human neutrophils were allowed to migrate across the HUVEC monolayer in response to a gradient leukotriene B4 and then the number of migrated neutrophils were counted microscopically. We also followed endothelial F-actin and myosin filament formation using rhodamine-phalloidin and anti-myosin Ab staining. Myosin light chain (MLC) phosphorylation in endothelial cells was determined by immunoprecipitation of 32P-labeled HUVEC with anti-myosin polyclonal Ab. Normally, neutrophil migration induced F-actin formation, myosin filament formation, and MLC phosphorylation in HUVEC. When HUVEC was pretreated with the myosin light chain kinase (MLCK) inhibitor, ML-9, neutrophil migration was diminished and F-actin formation, myosin filament formation, and MLC phosphorylation were inhibited. Pretreatments of HUVEC with the intracellular calcium ion chelator, bis-(O-aminophenoxyl)ethane-N, N, N′, N′-tetraacetic acid acetoxymethyl ester (BAPTA/AM), and the calmodulin antagonist, trifluoperazine, had similar effects. These results indicate that a calcium/calmodulin-dependent MLCK in endothelial cells regulates neutrophil transendothelial migration.
Circulating Form of Human Vascular Adhesion Protein-1 (VAP-1): Increased Serum Levels in Inflammatory Liver Diseases
Authors: Kurkijärvi Riikka , Adams David H. , Leino Rauli , Möttönen Timo , Jalkanen Sirpa , Salmi Marko
Published: December 2022

Vascular adhesion protein-1 (VAP-1) is a dimeric 170-kDa endothelial transmembrane molecule that under normal conditions is most strongly expressed on the high endothelial venules of peripheral lymph nodes and on hepatic endothelia. It is a glycoprotein that mediates tissue-selective lymphocyte adhesion in a sialic acid-dependent manner. In this study, we report the detection of a soluble form of VAP-1 in circulation. We developed a quantitative sandwich ELISA using novel anti-VAP-1 mAbs and used it to determine the levels of soluble VAP-1 (sVAP-1) in the serum of healthy individuals and in patients with inflammatory diseases. In healthy persons, circulating sVAP-1 concentrations were 49 to 138 ng/ml. Immunoblotting studies revealed that the apparent molecular mass of dimeric sVAP-1 is slightly (∼10 kDa) higher than that of transmembrane VAP-1 under nonreducing conditions. In contrast, the electrophoretic mobilities of monomeric sVAP-1 and transmembrane VAP-1 were similar after reduction and boiling. Adhesion assays showed that the circulating sVAP-1 modulates lymphocyte binding to endothelial cells. Inflammation can cause an elevation of serum sVAP-1 levels, because sVAP-1 concentrations in patients with certain liver diseases were two- to fourfold higher than those in normal individuals. In contrast, rheumatoid arthritis and inflammatory bowel diseases were not associated with elevated levels of sVAP-1. These findings indicate that there is a functionally active, soluble form of VAP-1 in circulation and suggest that the serum level of sVAP-1 might be a useful marker of disease activity in inflammatory liver diseases.
Involvement of the Fas/Fas Ligand Pathway in Activation-Induced Cell Death of Mycobacteria-Reactive Human γδ T Cells: A Mechanism for the Loss of γδ T Cells in Patients with Pulmonary Tuberculosis
Authors: Li Baiqing , Bassiri Hamid , Rossman Milton D. , Kramer Peter , Eyuboglu A. Fusun-Oner , Torres Martha , Sada Eduardo , Imir Turgut , Carding Simon R.
Published: December 2022

Although the identity of T cells involved in the protection against Mycobacterium tuberculosis (Mtb) in humans remain unknown, patients with pulmonary tuberculosis (TB) have reduced numbers of Mtb-reactive, Vγ9+/Vδ2+ T cells in their blood and lungs. Here we have determined whether this γδ T loss is a consequence of Mtb Ag-mediated activation-induced cell death (AICD). Using a DNA polymerase-mediated dUTP nick translation labeling assay, 5% or less of freshly isolated CD4+ αβ or γδ T cells from normal healthy individuals and TB patients were apoptotic. However, during culture Mtb Ags induced apoptosis in a large proportion of Vγ9+/Vδ2+ peripheral blood T cells from healthy subjects (30–45%) and TB patients (55–68%); this was increased further in the presence of IL-2. By contrast, anti-CD3 did not induce any significant level of apoptosis in γδ T cells from healthy subjects or TB patients. Mtb Ag stimulation rapidly induced Fas and Fas ligand (FasL) expression by γδ T cells, and in the presence of metalloproteinase-inhibitors >70% of γδ T cells were FasL+. Blockade of Fas-FasL interactions reduced the level of Mtb-mediated γδ T cell apoptosis by 75 to 80%. Collectively, these findings demonstrate that Mtb-reactive γδ T cells are more susceptible to AICD and that the Fas-FasL pathways of apoptosis is involved. AICD of γδ T cells, therefore, provides an explanation for the loss of Mtb-reactive T cells during mycobacterial infection.
Cutting Edge: Selective Deamidation by Tissue Transglutaminase Strongly Enhances Gliadin-Specific T Cell Reactivity
Authors: van de Wal Yvonne , Kooy Yvonne , van Veelen Peter , Peña Salvador , Mearin Luisa , Papadopoulos George , Koning Frits
Published: December 2022

Celiac disease (CD) is caused by gluten ingestion in susceptible individuals. Tissue transglutaminase (tTG)-specific Abs are characteristic of CD, and increased tTG activity has been observed in the jejunal biopsies of patients. Here we demonstrate that tTG selectively deamidates gluten peptides, which results in strongly enhanced T cell-stimulatory activity. To our knowledge, this is the first example of an enzymatic modification of a food protein that affects T cell recognition. Moreover, these modifications may lead to the amplification of gluten-specific T cell responses in the gut and consequently may be important for the development of CD.
Human T Lymphocyte Proliferative Response to Resting Porcine Endothelial Cells Results from an HLA-Restricted, IL-10-Sensitive, Indirect Presentation Pathway But Also Depends on Endothelial-Specific Costimulatory Factors
Authors: Vallée Isabelle , Guillaumin Jean-Maurice , Thibault Gilles , Gruel Yves , Lebranchu Yvon , Bardos Pierre , Watier Hervé
Published: December 2022

To investigate the mechanisms of cellular rejection in pig-to-human xenotransplantation, the proliferation of different human purified lymphocyte subpopulations in response to swine leukocyte Ag class II-negative porcine aortic endothelial cells (PAEC) was measured in the presence or absence of human autologous adherent cells (huAPC). CD8+ lymphocytes proliferated moderately in the absence of huAPC, and the immune response was slightly increased when huAPC were added. CD56+ lymphocytes failed to proliferate in response to PAEC whether huAPC were present or not. CD4+ lymphocytes alone did not proliferate in response to PAEC, but a strong proliferative response was observed in the presence of metabolically active huAPC. This response was totally abolished by mAbs directed against HLA class II molecules or by pretreatment of huAPC by human IL-10. Even in the presence of huAPC, CD4+ lymphocytes failed to respond to fixed PAEC or to PAEC-lysates, suggesting that PAEC must be viable to support lymphocyte proliferation. Finally, none of the nonendothelial porcine adherent cells tested was able to induce human lymphocyte proliferation, despite the fact that they also provided a large set of xenogeneic peptides. Our results show that the indirect presentation pathway of xenoantigens by huAPC to CD4+ lymphocytes is crucial in the response to porcine endothelial cells, and that IL-10 could be of therapeutic interest to prevent human lymphocyte activation by this pathway. Furthermore, we demonstrated that stimulatory signals specifically provided by endothelial cells are also necessary for this huAPC-restricted proliferative response.
Protein Kinase C Is Involved in the Regulation of Both Signaling and Adhesion Mediated by DNAX Accessory Molecule-1 Receptor
Authors: Shibuya Akira , Lanier Lewis L. , Phillips Joseph H.
Published: December 2022

DNAX accessory molecule-1 (DNAM-1) is a signal-transducing adhesion molecule involved in the cytolytic function mediated by CTL and NK cells. In the present study, we have investigated various perimeters of DNAM-1-mediated signaling and adhesion. Although adhesion of DNAM-1 to its ligand does not require divalent cations, protein synthesis, or RNA transcription, activation of protein kinase C (PKC) is required for DNAM-1 functioning. Furthermore, mutation of the putative PKC-binding site in the cytoplasmic domain of DNAM-1 (Ser329 to Phe329) prevents both ligand binding and PMA-induced phosphorylation of the DNAM-1 receptor. These results indicate that PKC phosphorylates Ser329 of DNAM-1 and plays a critical role for both DNAM-1 adhesion and signaling.
Assembly of MHC Class I Molecules with Biosynthesized Endoplasmic Reticulum-Targeted Peptides Is Inefficient in Insect Cells and Can Be Enhanced by Protease Inhibitors
Authors: Deng Yuping , Gibbs James , Bačík Igor , Porgador Angel , Copeman James , Lehner Paul , Ortmann Bodo , Cresswell Peter , Bennink Jack R. , Yewdell Jonathan W.
Published: December 2022

To study the requirements for assembly of MHC class I molecules with antigenic peptides in the endoplasmic reticulum (ER), we studied Ag processing in insect cells. Insects lack a class I recognition system, and their cells therefore provide a “blank slate” for identifying the proteins that have evolved to facilitate assembly of class I molecules in vertebrate cells. H-2Kb heavy chain, mouse β2-microglobulin, and an ER-targeted version of a peptide corresponding to Ova257–264 were expressed in insect cells using recombinant vaccinia viruses. Cell surface expression of Kb-OVA257–264 complexes was quantitated using a recently described complex-specific mAb (25-D1.16). Relative to TAP-deficient human cells, insect cells expressed comparable levels of native, peptide-receptive cell surface Kb molecules, but generated cell surface Kb-OVA257–264 complexes at least 20-fold less efficiently from ER-targeted peptides. The inefficient assembly of Kb-OVA257–264 complexes in the ER of insect cells cannot be attributed solely to a requirement for human tapasin, since first, human cells lacking tapasin expressed endogenously synthesized Kb-OVA257–264 complexes at levels comparable to tapasin-expressing cells, and second, vaccinia virus-mediated expression of human tapasin in insect cells did not detectably enhance the expression of Kb-OVA257–264 complexes. The assembly of Kb-OVA257–264 complexes could be greatly enhanced in insect but not human cells by a nonproteasomal protease inhibitor. These findings indicate that insect cells lack one or more factors required for the efficient assembly of class I-peptide complexes in vertebrate cells and are consistent with the idea that the missing component acts to protect antigenic peptides or their immediate precursors from degradation.
Regulation of Tyrosine Phosphorylation in Isolated T Cell Membrane by Inhibition of Protein Tyrosine Phosphatases
Authors: Jin Yong-Jiu , Friedman Jeff , Burakoff Steven J.
Published: December 2022

Jurkat T cells activated by the phosphotyrosine phosphatase inhibitors H2O2 or vanadate were found to have a similar pattern of tyrosine phosphorylation when compared with T cells stimulated by anti-CD3 Ab cross-linking, suggesting that protein tyrosine phosphatase (PTP) inhibitors affect the early steps of TCR signaling. To study the role of PTPs in the most proximal membrane events of tyrosine phosphorylation, subcellular fractions of T cells were treated with the PTP inhibitors in the presence of ATP. In the membrane fraction, tyrosine phosphorylation of Lck, Fyn, and CD3ζ can be induced by PTP inhibitors, but not by anti-CD3. Detailed characterization of this cell-free system showed that the pattern and the order of induced tyrosine phosphorylation is similar to that induced in intact cells. Upon removal of the PTP inhibitor, the tyrosine-phosphorylated proteins, including Lck, Fyn, Syk, Zap70, and CD3ζ, are rapidly dephosphorylated. Preliminary characterizations indicate that a PTP distinct from CD45, SHP1, and SHP2 is present in T cell membranes and the inhibition of this yet unidentified PTP is most likely responsible for the Lck-dependent tyrosine phosphorylation triggered by PTP inhibitors.
Cyclosporin A-Resistant Transactivation of the IL-2 Promoter Requires Activity of Okadaic Acid-Sensitive Serine/Threonine Phosphatases
Authors: Nebl Gabriele , Meuer Stefan C. , Samstag Yvonne
Published: December 2022

Expression of the IL-2 gene requires activation of T cells through stimulation of the TCR and costimulation through accessory receptors. We have found recently that okadaic acid-sensitive Ser/Thr phosphatases are involved in a cyclosporin A-insensitive pathway that selectively transmits costimulatory signals. In this study, we analyzed whether activities of these phosphatases are necessary for the expression of the IL-2 gene. In both activated peripheral blood T lymphocytes and activated tumorigenic T cell lines, IL-2 gene expression was blocked at the transcriptional level by okadaic acid. The transcription factors active at the IL-2 promoter were differentially influenced: upon down-modulation of okadaic acid-sensitive phosphatases, transactivation by octamer, NF-κB, and NF of activated T cells proteins was abrogated, while transactivation by AP-1 proteins was even enhanced.