bluuebunny/crossref_metadata_2025 huggingface dataset:
Staphylococcal Enterotoxin A Induces Survival of VH3-Expressing Human B Cells by Binding to the VH Region with Low Affinity
Authors: Domiati-Saad Rana , Lipsky Peter E.
Published: December 2022

Staphylococcal enterotoxins (SE) are bacterial superantigens that bind to MHC class II molecules and to the Vβ-chain of the TCR, and subsequently activate T cells expressing specific Vβ regions. In this study, we have studied the effects of SEA on human B cell activation, and specifically the capacity of SEA to function as a B cell superantigen in vitro. We show herein that SEA failed to induce B cell proliferation and differentiation in the absence of T cells. However, SEA induced survival of B cells uniquely expressing VH3-containing IgM, independently of light chain utilization. The sequences of VH3 IgM gene products were determined and found to include a number of members of the VH3 family with a variety of different D and JH gene segments. Analysis of the sequences of VH3 gene products revealed possible sites in framework region 1 and/or framework region 3 that could be involved in SEA-mediated activation of VH3-expressing B cells. Binding studies showed that SEA interacts with the VH3 domain of Ig with low, but detectable affinity. These results indicate that SEA functions as a B cell superantigen by interacting with VH3 gene segments of Ig.
Characterization and Differentiation of an Early Murine Yolk Sac-Derived IL-7-Independent Pre-Pro-B Cell Line
Authors: Lu Li-Sheng , Auerbach Robert
Published: December 2022

We describe a unique, stable pre-pro-B cell line (YS-PPB) derived from AA4.1+ yolk sac cells from day 10 mouse embryos. This cell line, discovered fortuitously during the course of studies of in vitro B cell differentiation, is independent of IL-7 supplementation for long term expansion in vitro. YS-PPB cells as well as clonal sublines expressed AA4.1, CD43, B220, Sca-1, CD19, heat stable antigen, MHC class I, IL-7R, and FcγR, but did not express cytoplasmic μ-chain, surface IgM (sIgM), or MHC class II molecules. PCR analysis showed that the cells expressed TdT, λ5, and RAG-1 genes, but that their Ig genes were still in germline configuration. The cell line was dependent on direct contact with S17 stromal cells for growth, but, in contrast to bone marrow stem cells, required no additional growth factors for maintenance and expansion. When stimulated with IL-7 and LPS, YS-PPB cells and cells from all tested clonal sublines differentiated into sIgM+ B cells in vitro. Irradiated mice reconstituted with YS-PPB cells yielded spleens containing 38% sIgM+ donor-derived B cells, demonstrating that YS-PPB cells, although stably arrested in development at the boundary between pre-pro-B and pro-B stages of B cell differentiation, still retain their competence to differentiate into mature, Ig-producing B cells when transferred to a normal host environment. Thus, this new cell line can provide a reproducible source of B cell precursors arrested at that critical time in B cell differentiation when the machinery for Ig gene rearrangement is in place but rearrangement has not yet occurred.
Cytokine-Like Factor-1, a Novel Soluble Protein, Shares Homology with Members of the Cytokine Type I Receptor Family
Authors: Elson Greg C. A. , Graber Pierre , Losberger Christophe , Herren Suzanne , Gretener Denise , Menoud Laurent N. , Wells Timothy N. C. , Kosco-Vilbois Marie H. , Gauchat Jean-François
Published: December 2022

In this report we describe the identification, cloning, and expression pattern of human cytokine-like factor 1 (hCLF-1) and the identification and cloning of its murine homologue. They were identified from expressed sequence tags using amino acid sequences from conserved regions of the cytokine type I receptor family. Human CLF-1 and murine CLF-1 shared 96% amino acid identity and significant homology with many cytokine type I receptors. CLF-1 is a secreted protein, suggesting that it is either a soluble subunit within a cytokine receptor complex, like the soluble form of the IL-6R α-chain, or a subunit of a multimeric cytokine, e.g., IL-12 p40. The highest levels of hCLF-1 mRNA were observed in lymph node, spleen, thymus, appendix, placenta, stomach, bone marrow, and fetal lung, with constitutive expression of CLF-1 mRNA detected in a human kidney fibroblastic cell line. In fibroblast primary cell cultures, CLF-1 mRNA was up-regulated by TNF-α, IL-6, and IFN-γ. Western blot analysis of recombinant forms of hCLF-1 showed that the protein has the tendency to form covalently linked di- and tetramers. These results suggest that CLF-1 is a novel soluble cytokine receptor subunit or part of a novel cytokine complex, possibly playing a regulatory role in the immune system and during fetal development.
Sequence-Based Typing Provides a New Look at HLA-C Diversity
Authors: Turner Seán , Ellexson Mary E. , Hickman Heather D. , Sidebottom David A. , Fernández-Viña Marcelo , Confer Dennis L. , Hildebrand William H.
Published: December 2022

Although extensive HLA-A and HLA-B polymorphism is evident, the true diversity of HLA-C has remained hidden due to poor resolution of HLA-C Ags. To better understand the polymorphic nature of HLA-C molecules, 1823 samples from the National Marrow Donor Program research repository in North America have been typed by DNA sequencing and interpreted in terms of HLA-C diversification. Results show that HLA-Cw*0701 was the most common allele with a frequency of 16%, whereas 28% of the alleles typed as Cw12-18 (serologic blanks). The frequency of homozygotes was 9.8% as compared with previous studies of 18% for sequence-specific primers and 50% for serology. Most startling was the frequency at which new alleles were detected; 19 new HLA-C alleles were detected, representing a rate of ∼1 in 100 samples typed. These new HLA-C alleles result from 29 nucleotide substitutions of which 4 are silent, such that coding substitutions concentrated about the Ag-binding groove predominate. Polymorphism at the HLA-C locus therefore resembles that at the HLA-A and HLA-B loci more than previously believed, indicating that antigenic stress is driving HLA-C evolution. However, sequence conservation in the α-helix of the first domain and a clustering of unique amino acids around the B pocket indicate that HLA-C alleles respond to antigenic pressures differently than HLA-A and HLA-B. Finally, because the samples characterized were predominantly from Caucasians, we hypothesize that HLA-C polymorphism will equal or exceed that of the HLA-A and -B loci as DNA sequence-based typing is extended to include more non-Caucasian individuals.
Secretory IgA Induces Degranulation of IL-3-Primed Basophils
Authors: Iikura Motoyasu , Yamaguchi Masao , Fujisawa Takao , Miyamasu Misato , Takaishi Toshiaki , Morita Yutaka , Iwase Takashi , Moro Itaru , Yamamoto Kazuhiko , Hirai Koichi
Published: December 2022

We examined whether secretory IgA (sIgA), known to mediate eosinophil stimulation, has an effect on basophil functions. An immobilized preparation of sIgA, but not of monomeric IgA, induced histamine release (approximately 15% of total histamine contents) from human basophils in vitro. sIgA-induced basophil histamine release was totally dependent on pretreatment with IL-3. IL-5 and granulocyte-macrophage CSF also primed basophils for sIgA-mediated release. Exogenous divalent ions, i.e., Ca2+ and Mg2+, were essential for sIgA-mediated basophil degranulation, and the degranulation was completed within 45 min. A newly synthesized lipid mediator, leukotriene C4, was also liberated from IL-3-primed, sIgA-stimulated basophils. Enzyme digestion experiments revealed that the (Fc)2·secretory component portion of sIgA is important for sIgA-mediated basophil activation, but the functional binding sites of sIgA on basophils were surmised to be different from FcαR. These observations reveal the novel finding that sIgA is able to stimulate basophils as well as eosinophils. Since sIgA is the most abundant Ig isotype in the secretions from mucosal tissues, and basophils are active participants in allergic late-phase reactions, sIgA-mediated basophil mediator release is potentially involved in exacerbation of the inflammation associated with allergic disorders.
Sustained Phosphorylation of Cytosolic Phospholipase A2 Accompanies Cycloheximide- and Adenovirus-Induced Susceptibility to TNF
Authors: O’Brien Jennifer B. , Piddington Debra L. , Voelkel-Johnson Christina , Richards Debra J. , Hadley Leslie A. , Laster Scott M.
Published: December 2022

In this report we examine the phosphorylation state of cytosolic phospholipase A2 (cPLA2) in C3HA fibroblasts that have been treated with TNF, cycloheximide (CHI), or a combination of both compounds. Our experiments show that TNF and CHI, when used independently, caused the rapid phosphorylation of cPLA2 (within 10 min). In both cases, cPLA2 was subsequently dephosphorylated to pretreatment levels by 40 min. In addition, under these conditions [3H]arachidonic acid was not released, and we could not detect a change in the activity of cPLA2 in vitro. In contrast, in cells treated with a combination of TNF and CHI, we found that the dephosphorylation of cPLA2 was inhibited, and cPLA2 remained phosphorylated for up to 2 h. In vitro we found that sustained phosphorylation of cPLA2 was accompanied by a 60 to 80% increase in the activity of cPLA2. The sustained phosphorylation of cPLA2 also occurred in cells infected with the adenovirus mutant dl309, suggesting that sustained phosphorylation may be a general requirement for the activation of cPLA2 in apoptotic cells. We also found that sustained phosphorylation of phosphoproteins is not a general consequence of apoptotic death, since the phosphorylation of p42 mitogen-activated protein kinase was not sustained. Finally, we show that the phosphatase inhibitor orthovanadate acts as does CHI to render cells susceptible to TNF, suggesting that resistance to TNF may depend on TNF’s ability to induce the expression of tyrosine or dual specificity phosphatase(s).
Pre-B Cell Receptor-Mediated Selection of Pre-B Cells Synthesizing Functional μ Heavy Chains
Authors: Kline Gregory H. , Hartwell Laura , Beck-Engeser Gabrielle B. , Keyna Ulrike , Zaharevitz Samantha , Klinman Norman R. , Jäck Hans-Martin
Published: December 2022

Ig gene rearrangements could generate VH-D-JH joining sequences that interfere with the correct folding of a μ-chain, and thus, its capability to pair with IgL chains. Surrogate light (SL) chain might be the ideal molecule to test the capacity of a μ-chain to pair with a L chain early in development, in that only pre-B cells that assemble a membrane μ-SL complex would be permitted to expand and further differentiate. We have previously identified two SL chain nonpairing VH81X-μ-chains with distinct VH-D-JH joining regions. Here, we show that one of these VH81X-μ-chains does not rescue B cell development in JH knock-out mice, because flow cytometric analysis of bone marrow cells from VH81X-μ transgenic JH knock-out mice revealed normal numbers of pro-B cells, but essentially no pre-B and surface IgM+ B cells. Immunoprecipitation analysis of transfected pre-B and hybridoma lines revealed that the same μ-chain fails to pair not only with SL chain but also with four distinct κ L chains. These findings demonstrate that early pre-B cells are selected for maturation on the basis of the structure of a μ-chain, in particular its VH-D-JH joining or CDR3 sequence, and that one mechanism for this selection is the capacity of a μ-chain to assemble with SL chain. Therefore, we propose a new function of SL chain in early B cell development: SL chain is part of a quality control mechanism that tests a μ-chain for its ability to pair with conventional L chains.
Endogenous Basic Fibroblast Growth Factor Is Essential for Cyclin E-CDK2 Activity in Multiple External Cytokine-Induced Proliferation of AIDS-Associated Kaposi’s Sarcoma Cells: Dual Control of AIDS-Associated Kaposi’s Sarcoma Cell Growth and Cyclin E-CDK2 Activity by Endogenous and External Signals
Authors: Murakami-Mori Kaoru , Mori Shunsuke , Nakamura Shuji
Published: December 2022

AIDS-associated Kaposi’s sarcoma (KS) cell, a key element for development of KS lesions, proliferates in response to external cytokines, such as oncostatin M, the soluble IL-6R-IL-6 complex, TNF-α, and IL-1β. In addition, the KS cell-produced basic fibroblast growth factor (bFGF) was reported to function as an autocrine growth factor. However, little is known of the exact roles of these external growth factors and endogenous bFGF on proliferation of KS cells, and underlying intracellular events have remained to be defined. We obtained evidence that anti-bFGF Ab abolished growth of KS cells by preventing S phase entry of the cell cycle, even in the presence of the external growth factors. Blockade of the FGF action profoundly inhibited cyclin E expression and cyclin-dependent kinase-2 (CDK2) activity, but not D-type cyclin expression and CDK4 activity. Exogenously added acidic FGF (aFGF), which generated a rapid tyrosine phosphorylation of FGFR1 and FGFR2 on KS cells, reversed the inhibitory effects of anti-bFGF Ab. Thus, FGF actions are essential for cyclin E-CDK2 activity and S phase entry. We also observed that the presence of external growth factors markedly induced cyclin E-CDK2 activity and S phase entrance, while the addition of aFGF or bFGF alone was insufficient to induce these responses. All this evidence shows that integration of the activities of external growth factors and endogenous bFGF is required for full activation of cyclin E-CDK2 activity and KS cell proliferation.
Receptor-Specific Allelic Exclusion of TCRVα-Chains During Development
Authors: Boyd Richard , Kozieradzki Ivona , Chidgey Ann , Mittrücker Hans-Willi , Bouchard Dennis , Timms Emma , Kishihara Kenji , Ong Christopher J. , Chui Daniel , Marth Jamey D. , Mak Tak W. , Penninger Josef M.
Published: December 2022

Expression of a single Ag receptor on lymphocytes is maintained via allelic exclusion that generates cells with a clonal receptor repertoire. We show in normal mice and mice expressing functionally rearranged TCRαβ transgenes that allelic exclusion at the TCRα locus is not operational in immature thymocytes, whereas most mature T cells express a single TCRVα-chain. TCRVα allelic exclusion in mature thymocytes is regulated through a CD45 tyrosine phosphatase-mediated signal during positive selection. Using functional and genetic systems for selection of immature double TCRVα+ thymocytes, we show that peptide-specific ligand recognition provides the signal for allelic exclusion, i.e., mature T cells maintain expression of the ligand-specific TCRVα-chain, but lose the nonfunctional receptor. Whereas activation of TCRVβ-chains or CD3ε leads to receptor internalization, TCRVα ligation promotes retention of the TCR on the cell surface. Although both TCRVα- and TCRVβ-chains trigger phosphotyrosine signaling, only the TCRVβ-chain mediates membrane recruitment of the GTPase dynamin. These data indicate that TCRVα-directed signals for positive selection control allelic exclusion in T cells, and that developmental signals can select for single receptor usage.
IL-2 Receptor α-Chain Expression Is Independently Regulated in Primary and Secondary Lymphoid Organs
Authors: Demaison Christophe , Fiette Laurence , Blanchetière Valérie , Schimpl Anneliese , Thèze Jacques , Froussard P.
Published: December 2022

The IL-2R is composed of three chains: IL-2Rα, IL-2Rβ, and IL-2Rγ. In mice, IL-2Rα is critical and determines IL-2 binding to the tripartite IL-2R complex. To extend our previous studies, which demonstrated that IL-2 regulates IL-2Rα expression in vitro, we have analyzed expression in IL-2-deficient mice in vivo. As in control animals, CD4−CD8− thymocytes and bone marrow-derived B220+ pre-B cells were Il-2Rα positive. In contrast, activated lymph node and splenic CD4 T cells (CD4+CD69+) were found to be IL-2Rα negative, whereas ∼20% of the same cell populations from the MLR/lpr strain, which also accumulate large numbers of CD4-activated T cells in the presence of intact IL-2, retained expression. A similar pattern of IL-2Rα expression was found among splenic CD8 cells from IL-2−/− and IL-2+/− animals. These findings demonstrate that in primary lymphoid organs, IL-2 is not directly involved in IL-2Rα expression. However, at the level of mature lymphocytes, and more specifically CD4 T cells, IL-2 remains in vivo, as in vitro, the most critical cytokine controlling both IL-2Rα expression and sensitivity to IL-2.
Initial Cytokine Exposure Determines Function of Macrophages and Renders Them Unresponsive to Other Cytokines
Authors: Erwig Lars-Peter , Kluth David C. , Walsh Garry M. , Rees Andrew J.
Published: December 2022

The functional properties of infiltrating macrophages (Mφ) must be tightly regulated to facilitate appropriate responses to complex conditions in an inflammatory focus. This study was designed to ascertain whether uncommitted Mφ that have been exposed to combinations of cytokines with opposing functions develop properties dictated by one cytokine or by cytokine mixtures. Uncommitted rat bone marrow-derived Mφ (BMDMs) were incubated with IFN-γ, TNF-α, TGF-β, IL-4, IL-6, and IL-10 alone or sequentially in combinations. After 48 h, function was assessed by nitric oxide (NO) generation, uptake of apoptotic neutrophils, and β-glucuronidase expression. IFN-γ followed 4 h later by TNF-induced NO generation. The pretreatment of BMDMs before IFN-γ priming with TNF, TGF-β, and IL-4 suppressed NO generation by 87%, 92%, and 85%, respectively; IL-10 had no effect. The same cytokines administered at 4 h after IFN priming had no effect on NO generation. The uptake of apoptotic polymorphonuclear leukocytes was augmented by TNF (40% vs 29% controls; p < 0.05) and decreased by IFN-γ, IL-10, and IL-4. The TNF response was unaffected by subsequent treatment with IFN-γ, IL-4, or IL-10. Similarly, the decreased polymorphonuclear leukocyte uptake induced by IFN-γ, IL-4, or IL-10 was unaffected by the subsequent addition of TNF. β-glucuronidase expression was increased by TGF-β and decreased by IFN-γ. These responses were not modified by cytokines with the opposing function. Thus, the functional response of BMDMs to complex mixtures of cytokines was determined by the first cytokine to which they were exposed. Once activated, BMDMs become unresponsive to alternative activating signals, a finding which has obvious implications for Mφ function in vivo.
Cutting Edge: FcγRII-B1 Regulates the Presentation of B Cell Receptor-Bound Antigens
Authors: Minskoff Stacey A. , Matter Karl , Mellman Ira
Published: December 2022

Fcγ receptors (FcγRII) on B lymphocytes negatively regulate B cell receptor (BCR)-dependent activation upon cross-linking of the two receptors. The mechanism reflects the ability of the FcγRII cytoplasmic tail to recruit specific phosphatases that inactivate elements of the BCR-signaling cascade. We now show that cross-linking also blocks the processing and presentation of BCR-bound Ag. This occurs because the FcγRII isoform typically expressed by B cells (FcγRII-B1) is incompetent for endocytosis. When cross-linked, FcγRII-B1 acts as a dominant negative inhibitor of BCR endocytosis. In contrast, cross-linking of endocytosis-competent FcγRII isoforms did not inhibit endocytosis or processing of BCR-bound Ag. Thus, FcγRII-B1 acts not only to prevent B cell activation under conditions of Ab excess, but also to prevent clonotypic T cell activation by inhibiting the ability of B cells to generate specific MHC class II-bound TCR ligands.
Quiescence-Inducing and Antiapoptotic Activities of IL-15 Enhance Secondary CD4+ T Cell Responsiveness to Antigen
Authors: Dooms Hans , Desmedt Marjory , Vancaeneghem Sabine , Rottiers Pieter , Goossens Vera , Fiers Walter , Grooten Johan
Published: December 2022

IL-15 shows functional redundancy with IL-2 due to its usage of the β and γc subunit of the IL-2R. Yet, the requirement of IL-15 for an IL-15Rα chain for high affinity interaction and the separate cellular sources of IL-2 and IL-15 suggest divergent activities for both cytokines. We compared the growth-inducing and proapoptotic or antiapoptotic activities of IL-15 and IL-2 on mature CD4+ T lymphocytes in the presence or absence of TCR occupancy. We found that the nature of IL-15 activity was critically dependent on the activation status of the T cells. In the absence of TCR triggering, IL-15 did not exert the growth factor activity of IL-2, but induced a quiescent phenotype, characterized by maintenance of the cells in the G0/G1 phase of the cell cycle and down-regulation of CD25, CD71, and CD95 expression. In the presence of appropriate TCR engagement, the IL-15-induced quiescent T cells were resistant against TCR-induced cell death and proliferated strongly. IL-2-treated cells, on the contrary, were sensitized to cell death, resulting in a negative feedback on cellular expansion and weak proliferative responsiveness. Consecutive action of IL-15 during the distinct phases of an in vitro immune response markedly increased the cell output of a second antigenic stimulation, as compared with IL-2. These results imply that during immune reactivity in vivo, IL-15 may take over from the transiently available IL-2 the role of survival factor but not of growth factor, hence promoting the long term maintenance of resting, Ag-experienced CD4+ T cells.
TNF-Related Apoptosis-Inducing Ligand (TRAIL) Induces Apoptosis in Fas Ligand-Resistant Melanoma Cells and Mediates CD4 T Cell Killing of Target Cells
Authors: Thomas Wayne D. , Hersey Peter
Published: December 2022

We have previously shown that melanoma cells were resistant to apoptosis induced by TNF family members Fas ligand (FasL), TNF-α, and CD40L. FasL also was not involved in CD4 T cell-mediated killing of melanoma cells. In the present study, we have tested melanoma cells for their susceptibility to apoptosis induced by human TNF-related apoptosis-inducing ligand (TRAIL) and the ability of a mAb against TRAIL to inhibit apoptosis and CD4 CTL-mediated killing of melanoma and Jurkat target cells. The results show that TRAIL-induced apoptosis in cells from 7 of 10 melanoma cell lines tested as well as in Jurkat T cells. Susceptibility to apoptosis was increased in some of the cell lines by treatment with cyclohexamide or actinomycin D. The melanoma cells were resistant to apoptosis induced by FasL, TNF-α, and CD40L. mAb M180 against TRAIL inhibited apoptosis induced by TRAIL. It was also found to inhibit CD4 CTL-mediated killing of Jurkat T cells as well as autologous and allogeneic melanoma cells. The degree of inhibition produced by the mAb varied between different clones of CTL and according to the susceptibility of the target cells to TRAIL-induced apoptosis. These results suggest that TRAIL is an important mediator of cell death induced by CTL and may have an important therapeutic role against human melanoma.
Protein Kinase C Regulates Fas (CD95/APO-1) Expression
Authors: Wang Ruoxiang , Zhang Liying , Yin Deling , Mufson R. Allan , Shi Yufang
Published: December 2022

Fas (CD95/APO-1) is a transmembrane protein of the TNF/neuron growth factor receptor family. Ligation of Fas by specific Abs or Fas ligand (FasL/CD95 ligand) induces rapid apoptotic cell death in a variety of cell types. Despite progress in understanding the death signals transduced from Fas, very little is known with regard to the mechanisms by which Fas expression is regulated. Using our previously established murine T cell hybridoma model A1.1, we show that specific protein kinase C (PKC) inhibitors could block activation-induced Fas expression and apoptosis. The activation of PKC with PMA or 1-oleoyl-2-acetyl-sn-glycerol could mimic the TCR signal by inducing the expression of Fas but not FasL. PKC-dependent Fas expression was also observed in several murine and human tumor cell lines. Since the inhibition of Ca2+ redistribution by an inhibitor of intracellular Ca2+ mobilization, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride, inhibited TCR-induced FasL but not Fas, the expression of Fas appears to be independent of Ca2+ mobilization. Significantly, expression of the newly identified Fas-regulatory gene, TDAG51, was found to be dependent upon the activity of PKC. PKC activation only induced Fas expression in cells expressing wild-type TDAG51. Thus, Fas expression is likely mediated by PKC through TDAG51.
Maturation of B Cell Precursors Is Impaired in Thymic-Deprived Nude and Old Mice
Authors: Szabo Paul , Zhao Kesheng , Kirman Irena , Le Maoult Joel , Dyall Rubendra , Cruikshank William , Weksler Marc E.
Published: December 2022

We have previously reported that bone marrow B cell precursors from thymic-deprived nude and old mice express less recombination-activating gene-1 (RAG-1) mRNA than they do in young euthymic mice. We now report that both nude and old mice have decreased bone marrow pre-B cells and that fewer pre-B cells express RAG protein. This combination of events appears to be the basis for the lower level of bone marrow RAG mRNA in thymic-deprived mice. A link between thymic function and B cell development was suggested by the similar kinetics of thymic involution and of declining bone marrow RAG-1 gene expression during aging. Support for this hypothesis was obtained by demonstrating that injection of supernatant medium from activated CD8+ but not CD4+ young T cells from mice increases RAG mRNA, RAG protein, and the number of bone marrow pre-B cells in nude and old mice. Furthermore, in vivo CD8+ T cells also regulate bone marrow RAG gene expression. Thus, mice deficient in CD8+ T cells expressed levels of RAG-1 mRNA in their bone marrow that were only 10% of those observed in normal or CD4+ T cell-deficient mice. IL-16 was detected in the supernatant medium from activated T cell cultures, and injection of nanogram quantities of recombinant IL-16 (rIL-16) into nude or old mice increased the levels of RAG mRNA in bone marrow B cell precursors and the number of bone marrow pre-B cells. We conclude that the impaired development of B cells within the bone marrow of thymic-deprived nude and old mice can be reversed, at least in part, by the administration of rIL-16.
Distinct Stage-Specific cis-Active Transcriptional Mechanisms Control Expression of T Cell Coreceptor CD8α at Double- and Single-Positive Stages of Thymic Development
Authors: Zhang Xiao-Li , Seong Rho , Piracha Rafia , Larijani Mani , Heeney Matthew , Parnes Jane R. , Chamberlain John W.
Published: December 2022

Developing thymocytes that give rise to CD8+ (cytotoxic) and CD4+ (helper) αβ-TCR T lymphocytes go through progressive stages of expression of coreceptors CD8 and CD4 from being negative for both (the double-negative stage), to coexpressing both (the double-positive (DP) stage), to a mutually exclusive sublineage-specific expression of one or the other (the single-positive (SP) stage). To delineate the mechanisms underlying regulation of CD8 during these developmental transitions, we have examined expression of a series of mouse CD8α gene constructs in developing T cells of conventional and CD8α “knock-out” transgenic mice. Our results indicate that cis-active transcriptional control sequences essential for stage- and sublineage-specific expression lie within a 5′ 40-kb segment of the CD8 locus, ∼12 kb upstream of the CD8α gene. Studies to characterize and sublocalize these cis sequences showed that a 17-kb 5′ subfragment is able to direct expression of the CD8α gene up to the CD3intermediate DP stage but not in more mature DP or SP cells. These results indicate that stage-specific expression of CD8α in developing T cells is mediated by the differential activity of multiple functionally distinct cis-active transcriptional control mechanisms. It will be important to determine the relationship of “switching” between these cis mechanisms and selection.
Bacterial Surface Proteins Recognized by CD4+ T Cells During Murine Infection with Listeria monocytogenes
Authors: Campbell Daniel J. , Shastri Nilabh
Published: December 2022

Optimal immunity to the Gram-positive pathogen Listeria monocytogenes (LM) requires both CD8+ and CD4+ antigen-specific T cell responses. Understanding how CD4+ T cells function in an immune response to LM and how bacterial proteins are processed to peptide/MHC class II complexes in infected cells requires identification of these proteins. Using LacZ-inducible, LM-specific CD4+ T cells as probes, we identified two immunogenic LM proteins by a novel expression cloning strategy. The antigenic peptides contained within these proteins were defined by deletion analysis of the genes, and their antigenicity was confirmed with synthetic peptides. The nucleotide sequences of the genes showed that they encode previously unknown LM proteins that are homologous to surface proteins in other bacterial species. Consistent with their surface topology, mild trypsin treatment of LM protoplasts ablated T cell recognition of these Ags. These findings establish a general strategy for identifying unknown CD4+ T cell Ags and demonstrate that LM surface proteins can provide the peptides for presentation by MHC class II molecules that are specific targets for CD4+ T cells during murine LM infection.
Molecular Cloning and Immunologic Reactivity of a Novel Low Molecular Mass Antigen ofMycobacterium tuberculosis
Authors: Coler Rhea N. , Skeiky Yasir A. W. , Vedvick Thomas , Bement Teresa , Ovendale Pamela , Campos-Neto Antonio , Alderson Mark R. , Reed Steven G.
Published: December 2022

Polypeptide Ags present in the culture filtrate of Mycobacterium tuberculosis were purified and evaluated for their ability to stimulate PBMC from purified protein derivative (PPD)-positive healthy donors. One such Ag, which elicited strong proliferation and IFN-γ production, was further characterized. The N-terminal amino acid sequence of this polypeptide was determined and used to design oligonucleotides for screening a recombinant M. tuberculosis genomic DNA library. The gene (Mtb 8.4) corresponding to the identified polypeptide was cloned, sequenced, and expressed in Escherichia coli. The predicted m.w. of the recombinant protein without its signal peptide was 8.4 kDa. By Southern analysis, the DNA encoding this mycobacterial protein was found in the M. tuberculosis substrains H37Rv, H37Ra, Erdman, and “C” strain, as well as in certain other mycobacterial species, including Mycobacterium avium and Mycobacterium bovis BCG (bacillus Calmette-Guérin, Pasteur). The Mtb 8.4 gene appears to be absent from the environmental mycobacterial species examined thus far, including Mycobacterium smegmatis, Mycobacterium gordonae, Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium scrofulaceum. Recombinant Mtb 8.4 Ag induced significant proliferation as well as production of IFN-γ, IL-10, and TNF-α, but not IL-5, from human PBMC isolated from PPD-positive healthy donors. Mtb 8.4 did not stimulate PBMC from PPD-negative donors. Furthermore, immunogenicity studies in mice indicate that Mtb 8.4 elicits a Th1 cytokine profile, which is considered important for protective immunity to tuberculosis. Collectively, these results demonstrate that Mtb 8.4 is an immunodominant T cell Ag of M. tuberculosis.
The Role of the Bacterial Membrane Protein ActA in Immunity and Protection Against Listeria monocytogenes
Authors: Darji Ayub , Bruder Dunja , zur Lage Susanne , Gerstel Birgit , Chakraborty Trinad , Wehland Jürgen , Weiss Siegfried
Published: December 2022

ActA, an essential virulence factor of Listeria monocytogenes, is an integral membrane protein that is required for intracellular motility, cell-to-cell spread, and rapid dissemination of the bacteria in the infected host. To reveal cytotoxic T cell responses against ActA we introduced a recombinant soluble form of ActA into the MHC class I-processing compartment of APC using a variant of listeriolysin mutated within its immunodominant MHC class I epitope. With this experimental system we demonstrate that T cells are induced against ActA during a sublethal infection with L. monocytogenes. However, adoptively transferred cytotoxic CD8+ T cells specific for ActA did not protect mice against a subsequent challenge with this pathogen. This was due to an inability of APC to present ActA by either MHC class I or class II molecules as long as ActA remained tethered to the surface of intracellular viable bacteria. ActA was only presented when L. monocytogenes were engineered to secrete ActA or when the bacteria were killed by antibiotics during the assay. These findings raise questions on the general use of membrane proteins of pathogens as candidates for subunit vaccines.
Effect of Human C-Reactive Protein on Chemokine and Chemotactic Factor-Induced Neutrophil Chemotaxis and Signaling
Authors: Zhong Wangjian , Zen Qin , Tebo Julie , Schlottmann Klaus , Coggeshall Mark , Mortensen Richard F.
Published: December 2022

C-reactive protein (CRP) is a unique serum pentraxin and the prototype acute phase reactant. CRP is a ligand for specific receptors on phagocytic leukocytes, and mediates activation reactions of monocytes/macrophages, but inhibits the respiratory burst of neutrophils (PMN). Since CRP selectively accumulates at inflammatory sites in which IL-8 is also produced, we tested the effects of CRP on the responsiveness of PMN to IL-8 and the bacterial chemotactic peptide, FMLP-phenylalanine (FMLPP). Purified human CRP inhibited the chemotactic response of PMN to IL-8 and FMLPP. A mouse IgM mAb that was generated against the leukocyte CRP receptor (CRP-R) also inhibited the chemotactic response. Incubation of purified CRP with activated PMN generated CRP-derived peptides that also inhibited chemotaxis. A synthetic CRP peptide (residues 27–38) that binds to the CRP-R had weak chemotactic activity, whereas two other CRP synthetic peptides (residues 174–185 and 191–205) inhibited chemotaxis of PMNs to both IL-8 and FMLPP. CRP did not alter receptor-specific binding of IL-8, but exerted its effect at the level of signaling. CRP augmented both IL-8- and FMLPP-induced mitogen-activated protein kinase (extracellular signal-regulated kinase-2) activity. CRP at acute phase levels increased both agonist-induced and noninduced phosphatidylinositol-3 kinase activity. The results suggest a role for CRP as a regulator of leukocyte infiltration at inflammatory sites.
Mature Mainstream TCRαβ+CD4+ Thymocytes Expressing L-Selectin Mediate “Active Tolerance” in the Nonobese Diabetic Mouse
Authors: Herbelin André , Gombert Jean-Marc , Lepault Françoise , Bach Jean-François , Chatenoud Lucienne
Published: December 2022

Pathogenic autoreactive T lymphocytes are mediators of spontaneous insulin-dependent diabetes in nonobese diabetic (NOD) mice. This is demonstrated by their capacity to transfer diabetes into syngeneic immunoincompetent recipients. In addition, especially in prediabetic NOD mice, peripheral CD4+ T lymphocytes were identified that are highly effective, in conventional mixing cotransfer experiments, at preventing disease transfer. The present data demonstrate that mature heat-stable Ag−TCRαβ+CD8− thymocytes from prediabetic NOD mice also express this inhibitory capacity. Selection using an L-selectin (CD62L)-specific Ab showed that TCRαβ+CD4+CD62L+ thymocytes, emerging from the mainstream differentiation pathway, concentrate this ability to regulate autoreactive effectors. Compared with mature TCRαβ+CD8− thymocytes, significantly lower numbers of TCRαβ+CD4+CD62L+ were sufficient to achieve an efficient inhibition of disease transfer into NOD-scid recipients. This protective ability was potentiated following in vitro culture in the presence of IL-7. In contrast, TCRαβ+CD62L− thymocytes, highly enriched in class I-restricted NK T cells, were unable to influence diabetes transfer. Identical results were obtained using thymocytes that have been cultured in vitro for 4 days in the presence of IL-7. These results support the active role in NOD mice of a thymus-derived CD4+ subset that controls peripheral pathogenic autoimmune effectors.
Expansion of Functional NK Cells in Multiple Tissue Compartments of Mice Treated with Flt3-Ligand: Implications for Anti-Cancer and Anti-Viral Therapy
Authors: Shaw Samuel G. , Maung Adrian A. , Steptoe Raymond J. , Thomson Angus W. , Vujanovic3 Nikola L.
Published: December 2022

The generation and activity of NK cells appear to be regulated by a particular set of cytokines. We examined the in vivo effects of recombinant human Flt3 ligand (Flt3-L), a recently cloned potent hemopoietic cytokine, on NK cell development in mice. Daily i.p. administration of Flt3-L consistently induced striking increases in both the absolute number and the total cytotoxic activity of mature nonactivated NK cells within various tissues. Dose- and time-dependent increases were observed in the bone marrow (∼2- and ∼11-fold, respectively), thymus (∼2.8- and ∼2.0-fold), blood (∼11- and ∼15-fold), spleen (∼10- and ∼9-fold), and liver (∼15- and ∼39-fold). In addition, IL-2 induced a rapid increase in NK activity, NK cell proliferative responses, generation of lymphokine-activated killer activity, and development of activated adherent NK cells, which were all significantly increased by Flt3-L treatment. Thus, in addition to its recently reported capacity to stimulate dendritic cell production, Flt3-L has a prominent biologic role in NK cell generation in vivo. This is probably a result of selectively induced expansion of NK cell progenitors (pro-NK cells), because Flt3-L stimulates in vitro proliferation of pro-NK cells without affecting the cytotoxicity of mature NK cells. The results also indicate that either alone or in combination with a potent activator of NK cells, such as IL-2, Flt3-L could be used to markedly augment the number and activity of NK cells, especially in the liver. Flt3-L appears to have considerable potential for therapy of both cancer and viral infection.
Intracellular Regulation of TRAIL-Induced Apoptosis in Human Melanoma Cells
Authors: Griffith Thomas S. , Chin Wilson A. , Jackson Glenn C. , Lynch David H. , Kubin Marek Z.
Published: December 2022

The observation that TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF cytokine family, induces apoptosis in a number of different tumor cell types led us to compare the tumoricidal effects of TRAIL to those of other TNF family molecules on human melanoma cells. We found that a high proportion of the melanoma cell lines tested were killed by TRAIL, whereas all the melanoma lines were resistant to the other TNF family cytokines tested. TRAIL-induced death was characterized by caspase activation and cellular protein cleavage within minutes of TRAIL addition, and death could be completely inhibited by the caspase inhibitors Ile-Glu-Thr-Asp (IETD) and Val-Ala-Asp (VAD), indicating the presence of a TRAIL receptor signaling pathway similar to that identified for Fas and TNF receptors. Specific TRAIL receptor expression was determined by RT-PCR, and the presence of mRNA encoding the “protective” TRAIL receptors did not correspond to resistance or sensitivity to TRAIL-induced apoptosis. Addition of protein synthesis inhibitors to TRAIL-resistant melanomas rendered them sensitive to TRAIL, indicating that the presence or the absence of intracellular apoptosis inhibitors may mediate resistance or sensitivity to TRAIL-mediated apoptosis. Expression of one such inhibitor, FLICE-inhibitory protein (FLIP), was highest in the TRAIL-resistant melanomas, while being low or undetectable in the TRAIL-sensitive melanomas. Furthermore, addition of actinomycin D to TRAIL-resistant melanomas resulted in decreased intracellular concentrations of FLIP, which correlated with their acquisition of TRAIL sensitivity. Collectively, our results indicate that TRAIL-induced apoptosis occurs through a caspase signaling cascade and that resistance is controlled by intracellular regulators of apoptosis.
Nuclear Translocation of Upstream Stimulating Factor 2 (USF2) in Activated Mast Cells: A Possible Role in Their Survival
Authors: Frenkel Shahar , Kay Gillian , Nechushtan Hovav , Razin Ehud
Published: December 2022

Multiple transcription factors are activated in the cytoplasm and translocated to the nucleus where they exert positive or negative control over cellular genes. Such subcellular traffic of transcription factors usually requires the presence of a positively charged nuclear localization sequence (NLS). Upstream stimulating factor 2 (USF2) is one of the few transcription factors that contain two potential domains for nuclear localization. In addition to the conventional basic NLS, USF2 contains a highly conserved USF-specific region that is involved in its nuclear translocation. In the present work, the induction of translocation of USF2 into the mast cell nucleus was observed and found to be dependent on activation of the cells either by IL-3 or IgE-Ag. It was also observed that the prevention of the translocation of USF2 to the nucleus, using a peptide derived from the specific USF-NLS region, significantly inhibited their IL-3-mediated survival. Thus, our findings show a direct connection between mast cell surface receptor-mediated USF2 nuclear translocation and cell viability.
TCRαβ Chains Associate with the Plasma Membrane Independently of CD3 and TCRζ Chains in Murine Primary T Cells
Authors: Zhang Jian , Salojin Konstantin , Gao Jian-Xin , Cameron Mark , Geisler Carsten , Delovitch Terry L.
Published: December 2022

The TCR is a multisubunit complex composed of the clonotypic α/β disulfide-linked heterodimer and noncovalently linked invariant CD3γε and CD3δε and TCRζ chains. Recent studies demonstrate that the surface expression of CD3 components can occur independently of the clonotypic TCR complexes in both thymocytes and splenic T cells. In this study, we report that free noncovalently associated TCRαβ heterodimers that exist independently of CD3 and TCRζ chains are expressed on the cell surface of immature thymocytes and peripheral T cells, but not of T cell lines and T cell hybridomas. This suggests that the regulation of surface expression of TCRαβ heterodimers differs between primary T cells and T cell lines or T cell hybridomas. The isolation and biochemical characterization of surface clonotype-independent CD3 complexes and free membrane-associated TCRαβ complexes may provide a structural basis for the quantitative difference in amount of T cell proliferation stimulated by anti-CD3ε and anti-TCRβ.
Molecular Cloning of Two Isoforms of the Guinea Pig C3a Anaphylatoxin Receptor: Alternative Splicing in the Large Extracellular Loop
Authors: Fukuoka Yoshihiro , Ember Julia A. , Hugli Tony E.
Published: December 2022

The anaphylatoxin C3a is released from C3 during complement activation. C3a is a potent spasmogen and has recently been described as an eosinophil and mast cell chemotactic factor that mediates a number of inflammatory reactions. Previously, we demonstrated the presence of a specific C3a receptor (C3aR) on guinea pig platelets. We report here the isolation of cDNA clones encoding for two isoforms of guinea pig C3aR (gpC3aR). Hydropathy analysis of the deduced amino acid sequence of both gpC3aR clones indicated seven transmembrane domains with a large extracellular (EC) loop between the fourth and fifth transmembrane domains, which is a known characteristic of the human C3aR. Northern blot analysis revealed that the gpC3aR was abundantly expressed on macrophages and in the spleen. A comparison of the deduced amino acid sequence of the larger gpC3aR (gpC3aR-L) with the recently cloned human C3aR indicated a 59.5% identity. The deduced amino acid sequence of the second, smaller cDNA clone was identical with gpC3aR-L, except that it lacked 35 amino acids in the large EC loop. Our evidence indicates that alternative splicing occurred in the large EC loop that accounts for these two isoforms. L cells separately expressing one of these two isoforms of the gpC3aR showed similar high-affinity C3a binding. An RT-PCR analysis documented that both forms of the C3aR were expressed in a variety of guinea pig tissues. The cloning and expression of these two natural forms of gpC3aR cDNA indicated that the deletion of the 35-residue portion of the large EC loop of gpC3aR-L did not alter C3a binding.
The MHC Class I-Restricted Immune Response to HIV-gag in BALB/c Mice Selects a Single Epitope That Does Not Have a Predictable MHC-Binding Motif and Binds to Kd Through Interactions Between a Glutamine at P3 and Pocket D
Authors: Mata Marielena , Travers Paul J. , Liu Qiang , Frankel Fred R. , Paterson Yvonne
Published: December 2022

Using a strain of Listeria monocytogenes that stably expresses and secretes HIV gag to deliver this Ag to the MHC class I pathway of Ag processing, we have identified the immunodominant CTL epitope to gag in the BALB/c mouse and shown that it is Kd restricted. The specific motif for the peptides that bind the MHC class I molecule H-2 Kd is believed to be a nonamer with residues tyrosine or phenylalanine in the second amino acid position and leucine or isoleucine in the carboxyl-terminal or ninth amino acid position as dominant anchoring positions. Surprisingly, the identified gag peptide, AMQMLKETI, does not contain an anchoring aromatic residue in position two although competition assays with other Kd-restricted epitopes indicated that it binds to Kd with comparable affinity. Using a theoretical molecular dynamics approach to probe the stability of peptide binding to MHC class I molecules, we show that the absence of an appropriate anchor residue at P2 in AMQMLKETI is compensated by favorable interactions of the glutamine at P3 with pocket D of Kd. These findings were verified experimentally, demonstrating the predictive power of this theoretical approach in analyzing MHC class I/peptide interactions. These studies also indicate that CTL epitope prediction that relies on dominant peptide motifs may not always identify the correct epitope.
SFA-1/PETA-3 (CD151), a Member of the Transmembrane 4 Superfamily, Associates Preferentially with α5β1 Integrin and Regulates Adhesion of Human T Cell Leukemia Virus Type 1-Infected T Cells to Fibronectin
Authors: Hasegawa Hitoshi , Nomura Tetsuhiko , Kishimoto Kyoko , Yanagisawa Kohsuke , Fujita Shigeru
Published: December 2022

In this study we have analyzed the adhesion molecules associated with and the biologic function of SFA-1/PETA-3 (CD151) in human T cell leukemia virus type 1 (HTLV-1)-infected T cells and in freshly isolated adult T cell leukemia (ATL) cells using an anti-CD151 mAb. The anti-CD151 mAb coprecipitated α5β1 integrin from HTLV-1-infected T cells. Conversely, an anti-α5 integrin mAb coprecipitated CD151. The anti-CD151 mAb inhibited the adhesion of HTLV-1-infected T cells to fibronectin but did not have any effect on their adhesion to laminin, collagen type I, or collagen type IV. Moreover, antisense CD151 oligonucleotide-treated HTLV-1-infected T cells showed significant inhibition of adhesion to fibronectin. These findings showed that the CD151 molecule was associated with the α5β1 integrin molecule and that it enhanced α5β1 integrin-mediated adhesion to fibronectin. In addition, the expression levels of CD151, α4β1 integrin, and α5β1 integrin on ATL cells from lymph nodes of lymphoma-type ATL patients were significantly higher than those on circulating ATL cells from leukemia-type ATL patients. This suggests that the increased expression of these integrins may contribute to lymphoma formation through the adhesion of ATL cells to the extracellular matrix and dendritic cells, rather than contributing to transmigration.