bluuebunny/crossref_metadata_2025 huggingface dataset:
Lymphocyte Adhesion to Epithelia and Endothelia Mediated by the Lymphocyte Endothelial-Epithelial Cell Adhesion Molecule Glycoprotein
Authors: Shieh Chi-Chang , Sadasivan Bhanu K. , Russell Gary J. , Schön Michael P. , Parker Christina M. , Brenner Michael B.
Published: December 2022

Upon encountering the relevant vascular bed, lymphocytes attach to endothelial adhesion molecules, transmigrate out of circulation, and localize within tissues. Lymphocytes may then be retained at microanatomic sites, as in tissues, or they may continue to migrate to the lymphatics and recirculate in the blood. Lymphocytes also interact transiently, but with high avidity, with target cells or APC that are infected with microbes or have taken up exogenous foreign Ags. This array of adhesive capabilities is mediated by the selective expression of lymphocyte adhesion molecules. Here, we developed the 6F10 mAb, which recognizes a cell surface glycoprotein designated lymphocyte endothelial-epithelial cell adhesion molecule (LEEP-CAM), that is distinct in biochemical characteristics and distribution of expression from other molecules known to play a role in lymphocyte adhesion. LEEP-CAM is expressed on particular epithelia, including the suprabasal region of the epidermis, the basal layer of bronchial and breast epithelia, and throughout the tonsillar and vaginal epithelia. Yet, it is absent from intestinal and renal epithelia. Interestingly, it is expressed also on vascular endothelium, especially high endothelial venules (HEV) in lymphoid organs, such as tonsil and appendix. The anti-LEEP-CAM mAb specifically blocked T and B lymphocyte adhesion to monolayers of epithelial cells and to vascular endothelial cells in static cell-to-cell binding assays by ∼40–60% when compared with control mAbs. These data suggest a role for this newly identified molecule in lymphocyte binding to endothelium, as well as adhesive interactions within selected epithelia.
A Novel Human CC Chemokine, Eotaxin-3, Which Is Expressed in IL-4-Stimulated Vascular Endothelial Cells, Exhibits Potent Activity Toward Eosinophils
Authors: Shinkai Akeo , Yoshisue Hajime , Koike Masamichi , Shoji Emi , Nakagawa Satoshi , Saito Akiko , Takeda Tsuyoshi , Imabeppu Susumu , Kato Yuzuru , Hanai Nobuo , Anazawa Hideharu , Kuga Tetsuro , Nishi Tatsunari
Published: December 2022

IL-4 has been shown to be involved in the accumulation of leukocytes, especially eosinophils, at sites of inflammation by acting on vascular endothelial cells. To identify novel molecules involved in the IL-4-dependent eosinophil extravasation, cDNA prepared from HUVEC stimulated with IL-4 was subjected to differential display analysis, which revealed a novel CC chemokine designated as eotaxin-3. The human eotaxin-3 gene has been localized to chromosome 7q11.2, unlike most other CC chemokine genes. The predicted mature protein of 71 aa showed 27–42% identity to other human CC chemokines. The recombinant protein induced a transient increase in the cytosolic Ca2+ concentration and in vitro chemotaxis on eosinophils. Furthermore, in cynomolgus monkeys, the accumulation of eosinophils was observed at the sites where the protein was injected. Eotaxin-3 inhibited the binding of 125I-eotaxin, but not 125I-macrophage inflammatory protein-1α, to eosinophils and acted on cell lines transfected with CCR-3, suggesting that eotaxin-3 recognized CCR-3. IL-13 as well as IL-4 up-regulated eotaxin-3 mRNA in HUVEC, whereas neither TNF-α, IL-1β, IFN-γ, nor TNF-α plus IFN-γ did. The expression profile of eotaxin-3 is different from those of eotaxin, RANTES, and monocyte chemoattractant protein-4, which are potent eosinophil-selective chemoattractants and are induced by either TNF-α or TNF-α plus IFN-γ. These results suggest that eotaxin-3 may contribute to the eosinophil accumulation in atopic diseases.
CD28 Costimulation Accelerates IL-4 Receptor Sensitivity and IL-4-Mediated Th2 Differentiation
Authors: Kubo Masato , Yamashita Masakatsu , Abe Ryo , Tada Tomio , Okumura Ko , Ransom John T. , Nakayama Toshinori
Published: December 2022

The development of Th1 and Th2 cells is determined by the type of antigenic stimulation involved in the initial cell activation step. Evidence indicates that costimulatory signals, such as those delivered by CD28, play an important role in Th2 development, but little is known about how CD28 costimulation contributes to Th2 development. In this study, TCR cross-linking was insufficient for Th2 development, while the addition of CD28 costimulation drastically increased Th2 generation through the IL-4-mediated pathway. Th2 generation following CD28 costimulation was not simply explained by the enhancement of IL-4 production in naive T cells. To generate Th2 cells after TCR cross-linking only, it was necessary to add a 20- to 200-fold excess of IL-4 generated after TCR and CD28 stimulation. TCR cross-linking increased the expression level and binding property of the IL-4R, but enhanced the sensitivity to IL-4 only slightly. In contrast, as evidenced by the enhanced phosphorylation of Jak3, the IL-4Rα-chain, and STAT6 following IL-4 stimulation, CD28 costimulation increased IL-4R sensitivity without affecting its expression and binding property. This evidence of the enhancement of IL-4R sensitivity increases our understanding of how CD28 costimulation accelerates Th2 development.
Induction of Allograft Tolerance in the Absence of Fas-Mediated Apoptosis
Authors: Li Xian Chang , Li Yongsheng , Dodge Ingrid , Wells Andrew D. , Zheng Xin Xiao , Turka Laurence A. , Strom Terry B.
Published: December 2022

Using certain immunosuppressive regimens, IL-2 knockout (KO) mice, in contrast to wild-type (wt) controls, are resistant to the induction of allograft tolerance. The mechanism by which IL-2 regulates allograft tolerance is uncertain. As IL-2 KO mice have a profound defect in Fas-mediated apoptosis, we hypothesized that Fas-mediated apoptosis of alloreactive T cells may be critical in the acquisition of allograft tolerance. To definitively study the role of Fas in the induction of transplantation tolerance, we used Fas mutant B6.MRL-lpr mice as allograft recipients of islet and vascularized cardiac transplants. Alloantigen-stimulated proliferation and apoptosis of Fas-deficient cells were also studied in vivo. Fas mutant B6.MRL-lpr (H-2b) mice rapidly rejected fully MHC-mismatched DBA/2 (H-2d) islet allografts and vascularized cardiac allografts with a tempo that is comparable to wt control mice. Both wt and B6.MRL-lpr mice transplanted with fully MHC-mismatched islet allografts or cardiac allografts can be readily tolerized by either rapamycin or combined costimulation blockade (CTLA-4Ig plus anti-CD40L mAb). Despite the profound defect of Fas-mediated apoptosis, Fas-deficient T cells can still undergo apoptotic cell death in vivo in response to alloantigen stimulation. Our study suggests that: 1) Fas is not necessarily essential for allograft tolerance, and 2) Fas-mediated apoptosis is not central to the IL-2-dependent mechanism governing the acquisition of allograft tolerance.
Autocrine IL-12 Is Involved in Dendritic Cell Modulation Via CD40 Ligation
Authors: Bianchi Roberta , Grohmann Ursula , Vacca Carmine , Belladonna Maria L. , Fioretti Maria C. , Puccetti Paolo
Published: December 2022

Ligation of CD40 on dendritic cells (DC) triggers production of IL-12. Using an adoptive transfer model we have previously shown that rIL-12 acts directly on DC to enhance presentation of an otherwise poorly immunogenic tumor peptide. Using the same experimental model, we now describe a similar adjuvanticity of CD40 ligation on peptide presentation by DC. We also explore the possibility that the IL-12 resulting from CD40 ligation directly affects the APC function of DC, mediating or contributing to the adjuvant effect of CD40 ligation. CD40 engagement in vitro and rIL-12 at concentrations in the range induced by CD40 ligation were equally effective in priming DC for presentation of the tumor peptide in vivo. Remarkably, the copresence in vitro of neutralizing Ab to IL-12, but not to TNF-α, IL-1β, or IFN-γ, ablated the enhancing effect of CD40 engagement on the APC function of DC. These data suggest a major role for autocrine IL-12 in DC modulation via CD40 ligation.
Differential Responses of Invariant Vα24JαQ T Cells and MHC Class II-Restricted CD4+ T Cells to Dexamethasone
Authors: Milner Joshua D. , Kent Sally C. , Ashley Timothy A. , Wilson S. Brian , Strominger Jack L. , Hafler David A.
Published: December 2022

NK T cells are a T cell subset in the human that express an invariant α-chain (Vα24invt T cells). Because of the well-described immunomodulation by glucocorticoids on activation-induced cell death (AICD), the effects of dexamethasone and anti-CD3 stimulation on Vα24invt T cell clones and CD4+ T cell clones were investigated. Dexamethasone significantly enhanced anti-CD3-mediated proliferation of Vα24invt T cells, whereas CD4+ T cells were inhibited. Addition of neutralizing IL-2 Ab partially abrogated dexamethasone-induced potentiation of Vα24invt T cell proliferation, indicating a role for autocrine IL-2 production in corticosteroid-mediated proliferative augmentation. Dexamethasone treatment of anti-CD3-stimulated Vα24invt T cells did not synergize with anti-Fas blockade in enhancing proliferation or preventing AICD. The Vα24invt T cell response to dexamethasone was dependent on the TCR signal strength. In the presence of dexamethasone, lower doses of anti-CD3 inhibited proliferation of Vα24invt T cells and CD4+ T cells; at higher doses of anti-CD3, which caused inhibition of CD4+ T cells, the Vα24invt T cell clones proliferated and were rescued from AICD. These results demonstrate significant differences in TCR signal strength required between Vα24invt T cells and CD4+ cells, and suggest important immunomodulatory consequences for endogenous and exogenous corticosteroids in immune responses.
Inhibition of Kit Expression by IL-4 and IL-10 in Murine Mast Cells: Role of STAT6 and Phosphatidylinositol 3′-Kinase
Authors: Mirmonsef Paria , Shelburne Christopher P. , Fitzhugh Yeatman C. , Chong Hey Jin , Ryan John J.
Published: December 2022

The c-kit protooncogene encodes a receptor tyrosine kinase that is known to play a critical role in hemopoiesis and is essential for mast cell growth, differentiation, and cytokine production. Studies have shown that the Th2 cytokine IL-4 can down-regulate Kit expression on human and murine mast cells, but the mechanism of this down-regulation has remained unresolved. Using mouse bone marrow-derived mast cells, we demonstrate that IL-4-mediated Kit down-regulation requires STAT6 expression and phosphotidylinositide-3′-kinase activation. We also find that the Th2 cytokine IL-10 potently down-regulates Kit expression. IL-4 enhances IL-10-mediated inhibition in a manner that is STAT6 independent and phosphotidylinositide-3′-kinase dependent. Both IL-4- and IL-10-mediated Kit down-regulation were coupled with little or no change in c-kit mRNA levels, no significant change in Kit protein stability, but decreased total Kit protein expression. Inhibition of Kit expression by IL-4 and IL-10 resulted in a loss of Kit-mediated signaling, as evidenced by reduced IL-13 and TNF-α mRNA induction after stem cell factor stimulation. These data offer a role for STAT6 and phosphotidylinositide-3′-kinase in IL-4-mediated Kit down-regulation, coupled with the novel observation that IL-10 is a potent inhibitor of Kit expression and function. Regulating Kit expression and signaling may be essential to controlling mast cell-mediated inflammatory responses.
Requirement for Shc in TCR-Mediated Activation of a T Cell Hybridoma
Authors: Pratt Joanne C. , van den Brink Marcel R. M. , Igras Vivien E. , Walk Scott F. , Ravichandran Kodimangalam S. , Burakoff Steven J.
Published: December 2022

Engagement of the TCR determines the fate of T cells to activate their functional programs, proliferate, or undergo apoptosis. The intracellular signal transduction pathways that dictate the specific outcome of receptor engagement have only been partially elucidated. The adapter protein, Shc, is involved in cytokine production, mitogenesis, transformation, and apoptosis in different cell systems. We found that Shc becomes phosphorylated on tyrosine residues upon stimulation of the TCR in DO11.10 hybridoma T cells; therefore, we investigated the role of Shc in activation-induced cell death in these cells by creating a series of stably transfected cell lines. Expression of Shc-SH2 (the SH2 domain of Shc) or Shc-Y239/240F (full-length Shc in which tyrosines 239 and 240 have been mutated to phenylalanine) resulted in the inhibition of activation-induced cell death and Fas ligand up-regulation after TCR cross-linking. Expression of wild-type Shc or Shc-Y317F had no significant effect. In addition, we found that Shc-SH2 and Shc-Y239/240F, but not Shc-Y317F, inhibited phosphorylation of extracellular signal-regulated protein kinase and production of IL-2 after TCR cross-linking. These results indicate an important role for Shc in the early signaling events that lead to activation-induced cell death and IL-2 production after TCR activation.
Levels of Ly-49 Receptor Expression Are Determined by the Frequency of Interactions with MHC Ligands: Evidence Against Receptor Calibration to a “Useful” Level
Authors: Manilay Jennifer O. , Waneck Gerald L. , Sykes Megan
Published: December 2022

Ly-49 receptor expression was studied in NK cells that developed in fully MHC-mismatched mixed bone marrow chimeras, in which host and donor MHC ligands were expressed solely on various proportions of hemopoietic cells or on both hemopoietic and nonhemopoietic cells. When hemopoietic cells were the only source of MHC ligand, a strong correlation between the level of down-regulation of Ly-49A, Ly-49C, and Ly-49G2 and the number of hemopoietic cells expressing their MHC ligands was observed on both donor and host NK cells. In some animals with low levels of donor hemopoietic chimerism, NK cells of donor origin expressed Ly-49 receptors at higher levels than was observed in normal mice of the same strain. This unexpected observation is inconsistent with the receptor calibration theory, which states that expression of Ly-49 inhibitory receptors is calibrated to an optimal level to maintain an NK cell repertoire that is sensitive to perturbations in normal class I ligand expression. Our data suggest a model in which Ly-49 receptors down-modulate in accordance with the frequency of their interactions with ligand-bearing cells, rather than a model in which these receptors calibrate to a specific “useful” level in response to ligands present in their environment.
Lipopolysaccharide Induces in Macrophages the Synthesis of the Suppressor of Cytokine Signaling 3 and Suppresses Signal Transduction in Response to the Activating Factor IFN-γ
Authors: Stoiber Dagmar , Kovarik Pavel , Cohney Solomon , Johnston James A. , Steinlein Peter , Decker Thomas
Published: December 2022

The goal of this study was to investigate how bacterial LPS affects macrophage responsiveness to the activating factor IFN-γ. Pretreatment of macrophages with LPS for <2 h increased the transcriptional response to IFN-γ. In contrast, simultaneous stimulation with IFN-γ and LPS, or pretreatment with LPS for >4 h, suppressed Stat1 tyrosine 701 phosphorylation, dimerization, and transcriptional activity in response to IFN-γ. Consistently, the induction of MHCII protein by IFN-γ was antagonized by LPS pretreatment. Neutralizing Abs to IL-10 were without effect on LPS-mediated suppression of Stat1 activation. Decreased IFN-γ signal transduction after LPS treatment corresponded to a direct induction of suppressor of cytokine signaling3 (SOCS3) mRNA and protein. Under the same conditions socs1, socs2, and cis genes were not transcribed. In transfection assays, SOCS3 was found to suppress the transcriptional response of macrophages to IFN-γ. A causal link of decreased IFN-γ signaling to SOCS3 induction was also suggested by the LPS-dependent reduction of IFN-γ-mediated Janus kinase 1 (JAK1) activation. Further consistent with inhibitory activity of SOCS3, LPS also inhibited the JAK2-dependent activation of Stat5 by GM-CSF. Our results thus link the deactivating effect of chronic LPS exposure on macrophages with its ability to induce SOCS3.
Differentiation of NK1.1+, Ly49+ NK Cells from flt3+ Multipotent Marrow Progenitor Cells
Authors: Williams Noelle Sevilir , Klem Jennifer , Puzanov Igor J. , Sivakumar P. V. , Bennett Michael , Kumar Vinay
Published: December 2022

To delineate factors involved in NK cell development, we established an in vitro system in which lineage marker (Lin)−, c-kit+, Sca2+ bone marrow cells differentiate into lytic NK1.1+ but Ly49− cells upon culture in IL-7, stem cell factor (SCF), and flt3 ligand (flt3L), followed by IL-15 alone. A comparison of the ability of IL-7, SCF, and flt3L to generate IL-15-responsive precursors suggested that NK progenitors express the receptor for flt3L. In support of this, when Lin−, c-kit+, flt3+ or Lin−, c-kit+, flt3− progenitors were utilized, 3-fold more NK cells arose from the flt3+ than from the flt3− progenitors. Furthermore, NK cells that arose from flt3− progenitors showed an immature NK1.1dim, CD2−, c-kit+ phenotype as compared with the more mature NK1.1bright, CD2+/−, c-kit− phenotype displayed by NK cells derived from flt3+ progenitors. Both progenitors, however, gave rise to NK cells that were Ly49 negative. To test the hypothesis that additional marrow-derived signals are necessary for Ly49 expression on developing NK cells, flt3+ progenitors were grown in IL-7, SCF, and flt3L followed by culture with IL-15 and a marrow-derived stromal cell line. Expression of Ly49 molecules, including those of which the MHC class I ligands were expressed on the stromal or progenitor cells, as well as others of which the known ligands were absent, was induced within 6–13 days. Thus, we have established an in vitro system in which Ly49 expression on developing NK cells can be analyzed and possibly experimentally manipulated.
Human Endothelial Cells Augment Early CD40 Ligand Expression in Activated CD4+ T Cells Through LFA-3-Mediated Stabilization of mRNA
Authors: Murakami Kenji , Ma Weilie , Fuleihan Ramsay , Pober Jordan S.
Published: December 2022

Human endothelial cells (EC) augment CD40 ligand (CD40L) expression on PHA-activated CD4+ T cells at early times (e.g., 4–6 h). Fixed EC, devoid of mRNA, are comparable to living EC in their capacity to augment early CD40L expression on CD4+ T cells. Fixed EC increase T cell mRNA expression of both IL-2 and CD40L compared with PHA alone at 6 h. EC are unable to increase the rate of transcription of CD40L compared with PHA alone as measured with a promoter-reporter gene, although they do increase transcription of an IL-2 promoter-reporter gene. Fixed EC prolong the half-life of CD40L mRNA >2-fold. Inclusion of anti-human LFA-3 (CD58) mAb or pretreatment of EC with an LFA-3 antisense oligonucleotide blocks EC-induced increases in CD40L expression, whereas mAb to ICAM-1 or pretreatment with ICAM-1 antisense oligonucleotide does not. Moreover, mAb to LFA-3 reverses the capacity of EC to prolong the half-life of CD40L mRNA, whereas mAb to ICAM-1, even in combination with mAb to ICAM-2, does not. We conclude that EC use LFA-3 to increase early CD40L protein expression on newly activated CD4+ T cells by stabilizing CD40L mRNA.
Attenuation of MHC Class II Expression in Macrophages Infected withMycobacterium bovisBacillus Calmette-Guerin Involves Class II Transactivator and Depends on theNramp1Gene
Authors: Wojciechowski Wojciech , DeSanctis Juan , Skamene Emil , Radzioch Danuta
Published: December 2022

The natural resistance associated macrophage protein 1 (Nramp1) gene determines the ability of murine macrophages to control infection with a group of intracellular pathogens, including Salmonella typhimurium, Leishmania donovani, and Mycobacterium bovis bacillus Calmette-Guérin (BCG). The expression of the resistant allele of the Nramp1 gene in murine macrophages is associated with a more efficient expression of several macrophage activation-associated genes, including class II MHC loci. In this study, we investigated the molecular mechanisms involved in IFN-γ-induced MHC class II expression in three types of macrophages: those expressing a wild-type allele of the Nramp1 gene (B10R and 129/Mφ), those carrying a susceptible form of the Nramp1 gene (B10S), and those derived from 129-Nramp1-knockout mice (129/Nramp1-KO). Previously, we published results showing that Ia protein expression is significantly higher in the IFN-γ-induced B10R macrophages, compared with its susceptible counterpart. In this paper, we also show that the higher expression of Ia protein in B10R cells is associated with higher I-Aβ mRNA expression, which correlates with a higher level of IFN-γ-induced phosphorylation of the STAT1-α protein and subsequently with elevated expression of class II transactivator (CIITA) mRNA, compared with B10S. Furthermore, we demonstrate that the infection of macrophages with M. bovis BCG results in a down-regulation of CIITA mRNA expression and, consequently, in the inhibition of Ia induction. Therefore, our data explain, at least in part, the molecular mechanism involved in the inhibition of I-Aβ gene expression in M. bovis BCG-infected macrophages activated with IFN-γ.
Role of Complement Component C1q in the IgG-Independent Opsonophagocytosis of Group BStreptococcus
Authors: Butko Peter , Nicholson-Weller Anne , Wessels Michael R.
Published: December 2022

We investigated the role of complement component C1q in the IgG-independent opsonophagocytosis of type III group B Streptococcus (GBS) by peripheral blood leukocytes. We report that C1q binds to type III GBS both in normal human serum deficient in IgG specific for type III capsular polysaccharide and in a low-ionic strength buffer. The dissociation constant Kd ranged from 2.0 to 5.5 nM, and the number of binding sites Bmax ranged from 630 to 1360 molecules of C1q per bacterium (CFU). An acapsular mutant strain of GBS bound C1q even better than the wild type, indicating that the polysaccharide capsule is not the receptor for C1q. In serum, binding of C1q to GBS was associated with activation of the classical complement pathway. However, normal human serum retained significant opsonic activity after complete depletion of C1q, suggesting that the serum contains a molecule that is able to replace C1q in opsonization and/or complement activation. Mannan-binding lectin, known to share some functions with C1q, appeared not to be involved, since its depletion from serum had little effect on opsonic activity. Excess soluble C1q or its collagen-like fragment inhibited phagocytosis mediated by normal human serum, suggesting that C1q may compete with other opsonins for binding to receptor(s) on phagocytes. We conclude that, although C1q binds directly to GBS, C1q binding is neither necessary nor sufficient for IgG-independent opsonophagocytosis. The results raise the possibility that additional unknown serum factor(s) may contribute to opsonization of GBS directly or via a novel mechanism of complement activation.
Mice with a Targeted Mutation in Lymphotoxin-α Exhibit Enhanced Tumor Growth and Metastasis: Impaired NK Cell Development and Recruitment
Authors: Ito Daisuke , Back Timothy C. , Shakhov Alexander N. , Wiltrout Robert H. , Nedospasov Sergei A.
Published: December 2022

Mice deficient in lymphotoxin (LT)-α lack peripheral lymph nodes and Peyer’s patches and have profound defects in development of follicular dendritic cell networks, germinal center formation, and T/B cell segregation in the spleen. Although LTα is known to be expressed by NK cells as well as T and B lymphocytes, the requirement of LTα for NK cell functions is largely unknown. To address this issue, we have assessed NK cell functions in LTα-deficient mice by evaluating tumor models with known requirements for NK cells to control their growth and metastasis. Syngeneic B16F10 melanoma cells inoculated s.c. grew more rapidly in LTα−/− mice than in the wild-type littermates, and the formation of experimental pulmonary metastases was significantly enhanced in LTα−/− mice. Although LTα−/− mice exhibited almost a normal total number of NK cells in spleen, they showed an impaired recruitment of NK cells to lung and liver. Additionally, lytic NK cells were not efficiently produced from LTα−/− bone marrow cells in vitro in the presence of IL-2 and IL-15. These data suggest that LTα signaling may be involved in the maturation and recruitment of NK cells and may play an important role in antitumor surveillance.
The Human B Cell Response to IL-13 Is Dependent on Cellular Phenotype as Well as Mode of Activation
Authors: Ford Dwayne , Sheehan Catherine , Girasole Christopher , Priester Rory , Kouttab Nicola , Tigges John , King Thomas C. , Luciani Andrea , Morgan John W. , Maizel Abby L.
Published: December 2022

Normal mature quiescent human B lymphocytes, isolated as a function of buoyant density, require activation for up-regulation of IL-13R constituents. Cell activation through a combination of surface Ig and CD40 receptor ligation leads to the most substantial message production for IL-13Rα1. Functional consequences of this receptor variation, in initially quiescent cells, includes demonstrable effects on cellular proliferation in response to ligand exposure. Variations in the method of surface activation, with particular emphasis on the CD40 receptor, reveals that immobilized CD40 ligand may be sufficient, in and of itself, to up-regulate IL-13Rα1, which may bear significance for B-lymphocyte bystander proliferation. Regulation of the IL-13Rα1 protein and message also differs as a function of cellular phenotype. Although values are greater in memory than naive B cells, as they are initially isolated from extirpated tonsils, variations in the magnitude of message and protein, as a function of surface stimulation, are more substantial in the naive subset. The magnitude of variation in message production in naive cells is associated with a more vigorous proliferative response to IL-13 than seen in memory lymphocytes. The cellular response to IL-13, as a function of activation and phenotype, is the converse of that demonstrated for IL-2. Evaluation of proliferation, receptor message, ligand binding protein production, and the response to putatively synergistic cytokines reveals that IL-2 is the predominant lymphokine utilized by memory cells. This is in contradistinction to IL-13, which along with IL-4, are the predominant moieties for naive lymphocytes.
The Importance of the Light Chain for the Epitope Specificity of Human Anti-U1 Small Nuclear RNA Autoantibodies Present in Systemic Lupus Erythematosus Patients
Authors: Hoet René M. A. , Pieffers Martijn , Stassen Maurice H. W. , Raats Jos , de Wildt Ruud , Pruijn Ger J. M. , van den Hoogen Frank , van Venrooij Walther J.
Published: December 2022

Abs to U1 RNA are frequently found in patients suffering from systemic lupus erythematosus overlap syndromes and Ab titers correlate with disease activity. We describe the isolation of the first human anti-U1 RNA autoantibodies from a combinatorial IgG library made from the bone marrow of a systemic lupus erythematosus patient. With the use of phage display technology, two anti-U1 RNA single-chain variable fragment (scFv) Abs were selected. Both high affinity anti-U1 RNA Ab fragments (Kd ∼ 1 nM) recognize stem II of U1 RNA and were derived from the same heavy chain gene (VH3–11) and the same λ (3r) light chain gene although somatic mutations, predominantly present in the complementarity-determining regions, are different. Experiments, in which the heavy chain genes of both anti-U1 RNA scFvs were reshuffled with the original light chain repertoire of the patient resulted, after selection on stem loop II, in a large number of RNA-binding Ab fragments. All these stem loop II-specific RNA binding clones used a similar, but not identical, 3r λ light chain. When scFvs were selected from the reshuffled libraries by stem loop IV, representing the other autoantigenic site of U1 RNA, most selected Ab clones did react with stem loop IV, but no longer with stem loop II. The stem loop IV-reactive Ab clones contained different, not 3r-related, light chains. These results point to a major role for the light chain in determining the sequence specificity of these disease-related anti-U1 RNA Abs. The possibility that secondary light chain rearrangements are involved in this autoimmune response is discussed.
A Monoclonal Antibody Directed Against the Murine Macrophage Surface Molecule F4/80 Modulates Natural Immune Response toListeria monocytogenes
Authors: Warschkau Holger , Kiderlen Albrecht F.
Published: December 2022

Whole spleen cell cultures from SCID mice release high levels of IFN-γ when exposed to heat-killed Listeria monocytogenes (HKL). This microbe-induced and T cell-independent response depends on both macrophages (MΦ) and NK cells: HKL-stimulated MΦ release TNF-α and IL-12, which together activate NK cells for IFN-γ release. We show here that this cytokine-mediated activation cascade can be modulated by a mAb against the MΦ surface glycoprotein F4/80. HKL-induced IL-12, TNF-α, and IFN-γ in SCID whole spleen cell cultures was inhibited by coincubation with anti-F4/80 mAb whereas IL-1 and IL-10 were enhanced. Both effects were apparent at mRNA and protein release levels. Whereas inhibitory activities were F4/80 Ag specific, stimulatory effects were Fc dependent and nonspecific. Furthermore, cytokine inhibition by anti-F4/80 was only apparent when MΦ and NK cells were present simultaneously and in close vicinity, indicating that direct cell-to-cell contact is a prerequisite. These data suggest a novel pathway for microbe-induced MΦ/NK cell interaction involving direct cell-to-cell signaling and give the first evidence for a functional role of the MΦ surface glycoprotein F4/80.
Oral Administration of Lipopolysaccharide Exacerbates Collagen-Induced Arthritis in Mice
Authors: Yoshino Shin , Sasatomi Eizaburo , Mori Yoki , Sagai Masaru
Published: December 2022

We investigated whether oral administration of LPS exacerbated collagen-induced arthritis (CIA) in mice, which was an experimental model of autoimmune disease. CIA was induced by s.c. injection of type II collagen emulsified with CFA into the base of the tail (day 0) followed by a booster injection on day 21. To examine the ability of LPS to exacerbate CIA, varying doses of LPS were orally administered on day 50. The results showed that administration of LPS was followed by reactivation of CIA in a dose-related fashion. Histologically, on day 55 there were marked edema of synovium proliferated by day 50, new formation of fibrin, and intense infiltration of neutrophils accompanied with a large number of mononuclear cells. Severe destruction of cartilage and subchondral bone was also observed on day 70. The reactivation of CIA by oral administration of LPS was associated with increase in anti-type II collagen IgG and IgG2a Abs as well as varying kinds of cytokines including IL-12, IFN-γ, IL-1β, and TNF-α. Polymyxin B sulfate given either orally or i.v. suppressed the recurrence of CIA. Increased amounts of LPS were found in sera of mice given the endotoxin orally. LPS from Salmonella enteritidis, Salmonella typhimurium, and Klebsiella pneumoniae and its component, lipid A from Escherichia coli, also reactivated the disease. These findings suggest that LPS from intestinal bacteria may play a role in the exacerbation of autoimmune joint inflammation.
A Common TCR V-D-J Sequence in Vβ13.1 T Cells Recognizing an Immunodominant Peptide of Myelin Basic Protein in Multiple Sclerosis
Authors: Hong Jian , Zang Ying C. Q. , Tejada-Simon Maria V. , Kozovska Milena , Li Sufang , Singh Rana A. K. , Yang Deye , Rivera Victor M. , Killian James K. , Zhang Jingwu Z.
Published: December 2022

T cell responses to the immunodominant peptide (residues 83–99) of myelin basic protein are potentially associated with multiple sclerosis (MS). This study was undertaken to examine whether a common sequence motif(s) exists within the TCR complementarity-determining region (CDR)-3 of T cells recognizing the MBP83–99 peptide. Twenty MBP83–99-reactive T cell clones derived from patients with MS were analyzed for CDR3 sequences, which revealed several shared motifs. Some Vβ13.1 T cell clones derived from different patients with MS were found to contain an identical CDR3 motif, Vβ13.1-LGRAGLTY. Oligonucleotides complementary to the shared CDR3 motifs were used as specific probes to detect identical target CDR3 sequences in a large panel of T cell lines reactive to MBP83–99 and unprimed PBMC. The results revealed that, in contrast to other CDR3 motifs examined, the LGRAGLTY motif was common to T cells recognizing the MBP83–99 peptide, as evident by its expression in the majority of MBP83–99-reactive T cell lines (36/44) and PBMC specimens (15/48) obtained from randomly selected MS patients. The motif was also detected in lower expression in some PBMC specimens from healthy individuals, suggesting the presence of low precursor frequency of T cells expressing this motif in healthy individuals. This study provides new evidence indicating that the identified LGRAGLTY motif is preferentially expressed in MBP83–99-reactive T cells. The findings have important implications in monitoring and targeting MBP83–99-reactive T cells in MS.
IgG and Complement-Mediated Tissue Damage in the Absence of C2: Evidence of a Functionally Active C2-Bypass Pathway in a Guinea Pig Model
Authors: Wagner Eric , Platt Jeffrey L. , Howell David N. , Marsh Henry C. , Frank Michael M.
Published: December 2022

In vitro complement-mediated lysis of heavily sensitized sheep erythrocytes by C4-deficient (C4D) guinea pig and C2-deficient (C2D) human sera was demonstrated some years ago. It was postulated that these “complement-bypass” pathways resulted from activation of C1 and components of the alternative pathway. We used normal, C2D, and C4D guinea pigs in a Forssman shock model to test the in vivo relevance of the C2- and C4-bypass pathways of complement activation. High concentrations of both anti-Forssman Ab and C2D or C4D guinea pig serum induced efficient lysis of sheep erythrocytes in vitro. The most efficient lysis was observed when IgG Ab and C2D guinea pig serum were used. Blocking either the classical pathway (treatments with EGTA-Mg2+ or soluble recombinant complement receptor type 1 (sCR1)) or the alternative pathway (treatment with heating at 50°C, sCR1, or soluble recombinant CR1 lacking the first of the four long homologous repeat sequences (sCR1[desLHR-A])) inhibited lysis; both pathways were required for lysis of sheep erythrocytes by C2D and C4D guinea pig sera. i.v. injection of anti-Forssman Ab in normal guinea pigs resulted in rapid death from pulmonary shock, whereas C4D guinea pigs had no adverse effect. Surprisingly, C2D guinea pigs either died in a delayed fashion or had a sublethal reaction. sCR1 treatment prevented Forssman shock in both normal and C2D guinea pigs, whereas sCR1[desLHR-A] prevented Forssman shock only in C2D animals. Our results suggest that the C2-bypass pathway occurs in vivo to produce tissue damage. Activation of complement in the absence of C2 appears to be far more efficient than in the absence of C4.
Cutting Edge: TCRαβ+ CD8αα+ T Cells Are Found in Intestinal Intraepithelial Lymphocytes of Mice That Lack Classical MHC Class I Molecules
Authors: Gapin Laurent , Cheroutre Hilde , Kronenberg Mitchell
Published: December 2022

TCRαβ+ intestinal intraepithelial lymphocytes (IEL) can express either the typical CD8αβ heterodimer or an unusual CD8αα homodimer. Both types of CD8+ IEL require class I molecules for their differentiation, since they are absent in β2m−/− mice. To gain insight into the role of class I molecules in forming TCRαβ+ CD8+ IEL populations, we have analyzed the IEL in mice deficient for either TAP, β2m, CD1, or K and D. We find that K−/−D−/− mice have TCRαβ+ CD8αα+ IEL, although they are deficient for TCRαβ+ CD8αβ+ cells. This indicates that at least some TCRαβ+ CD8αα+ IEL require only nonclassical class I molecules for their development. Surprisingly, the TCRαβ+ CD8αα+ IEL are significantly increased in K−/−D−/− mice, suggesting a complex interaction between CD8+ IEL and class I molecules that might include direct or indirect negative regulation by K and D, as well as positive effects mediated by nonclassical class I molecules.
Cutting Edge: Differentiation of Antitumor CTL In Vivo Requires Host Expression of Stat1
Authors: Fallarino Francesca , Gajewski Thomas F.
Published: December 2022

Several lines of evidence suggest that an IFN-γ-producing, Th1/Tc1 phenotype may be optimal for tumor rejection. Recent work has indicated that IFN signaling on tumor cells is important for protection against carcinogenesis. However, the potential involvement of IFN signaling among host immune cells has not been carefully examined. To this end, Stat1-deficient mice were employed as tumor recipients. In contrast to wild-type mice, Stat1−/− mice failed to reject immunogenic tumors and did not support regression of poorly immunogenic tumors when treated with an IL-12-based vaccine. T cells from immunized Stat1−/− mice produced 50% of the levels of IFN-γ and lacked cytolytic activity compared with wild-type mice, and NK lytic activity also was not observed. Lack of cytolytic function correlated with a failure to up-regulate serine esterase activity. Thus, IFN-mediated signaling on host cells is required for the development of antitumor lytic effector cells.
Deficiency of Transporter for Antigen Presentation (TAP) in Tumor Cells Allows Evasion of Immune Surveillance and Increases Tumorigenesis
Authors: Johnsen A. K. , Templeton D. J. , Sy M.-S. , Harding C. V.
Published: December 2022

Proteins involved in class I MHC (MHC-I) Ag processing, such as the TAP, are deficient in some human tumor cells. This suggests that antitumor responses by CD8 T cells provide selection pressure to favor outgrowth of cells with defective processing of tumor Ags. Nonetheless, this evidence is only correlative, and controlled in vivo experiments have been lacking to demonstrate that TAP deficiency promotes survival of tumor cells. To explore the role of Ag processing defects in tumor progression, matched panels of TAP1-positive and TAP1-negative tumor cell lines were generated from a parental transformed murine fibroblast line. Inoculation of C57BL/6 mice with TAP1-negative cells produced large and persistent tumors. In contrast, TAP1-positive cells did not generate lasting tumors, although small tumors were detected transiently and regressed spontaneously. Both TAP1-positive and TAP1-negative cells produced tumors in athymic mice, confirming that TAP-dependent differences in tumorigenicity were due to T cell-dependent immune responses. Inoculation of C57BL/6 mice with mixtures of TAP1-positive and TAP1-negative cells produced tumors composed exclusively of TAP1-negative cells, indicating in vivo selection for cells with TAP deficiency. Thus, loss of TAP function allows some tumor cells to avoid T cell-dependent elimination, resulting in selection for tumor cells with deficient Ag processing.
Naive and Memory T Lymphocytes Migrate in Comparable Numbers Through Normal Rat Liver: Activated T Cells Accumulate in the Periportal Field
Authors: Luettig Birgit , Pape Lars , Bode Ulrike , Bell Eric B. , Sparshott Sheila M. , Wagner Siegfried , Westermann Jürgen
Published: December 2022

Although the liver is known to contain a significant number of lymphocytes, migration of these through the compartments of the liver, parenchyma and periportal field, has not been studied. The periportal field, in particular, is affected in several immunological disorders of the liver. Populations of labeled naive, activated, and memory T cells were injected into congenic rats. The recipient livers and draining lymph nodes were removed at various time points, and cryostat sections were analyzed for the presence of donor cells using quantitative immunohistology. Donor cell proliferation and apoptosis were examined in vivo by BrdU (5 μM 5-bromo-2-deoxyuridine) incorporation and the TUNEL technique, respectively. Early after injection (0.5–1 h), naive, activated, and memory T cells were localized to the parenchyma and periportal field in comparable numbers. With time, all T cell subsets left the parenchyma but remained or, in the case of activated T cells, significantly accumulated in the periportal field. Furthermore, 12% of activated donor T cells proliferated in vivo within the periportal field, and 0.5% showed evidence of apoptosis. Taken together, not only activated and memory, but also naive T cells continuously migrate through the liver, showing a preference for the periportal field, and activated T cells mainly proliferate there. This may explain why many immunological liver diseases predominantly affect the periportal field.
Transcriptional Regulation of the Igκ Gene by Promoter-Proximal Pausing of RNA Polymerase II
Authors: Raschke Eva E. , Albert Thomas , Eick Dirk
Published: December 2022

Transcriptional regulation can occur at the level of initiation and RNA elongation. We report that the rearranged, nontranscribed Igκ gene in the pre-B cell line 70Z/3 harbors a paused RNA polymerase II (pol II) at a position between 45 and 89 bp downstream of the transcription initiation site. LPS, an inducer of NF-κB, activated Igκ gene transcription by increasing the processivity of pol II. TGF-β inhibited the LPS-induced transcription of the Igκ gene, but not initiation and pausing of pol II. A rearranged copy of the Igκ gene was introduced into 70Z/3 cells using an episomal vector system. The episomal Igκ was regulated by LPS and TGF-β like the endogenous gene and established a paused pol II, whereas a construct with a deletion of the intron enhancer and the C region did not establish a paused pol II. Two distinct functions can therefore be assigned to the deleted DNA elements: loading of pol II to its pause site and induction of processive transcription upon LPS stimulation. It had been proposed that somatic hypermutation of Ig genes is connected to transcription. The pause site of pol II described in this work resides upstream of the previously defined 5′ boundary of mutator activity at Igκ genes. The possible role of pausing of pol II for somatic hypermutation is discussed.
Thermolabile H-2Kb Molecules Expressed by Transporter Associated with Antigen Processing-Deficient RMA-S Cells Are Occupied by Low-Affinity Peptides
Authors: De Silva A. Dharshan , Boesteanu Alina , Song Rui , Nagy Nancy , Harhaj Edward , Harding Clifford V. , Joyce Sebastian
Published: December 2022

RMA-S cells do not express functional TAP, yet they express MHC class I molecules at the cell surface, especially at reduced temperatures (26°C). It is generally assumed that such class I molecules are “empty,” devoid of any associated peptide. A radiochemical approach was used to label class I-associated peptides and to determine the extent to which Kb molecules in RMA-S cells are associated with peptides. These studies revealed that at 26°C Kb molecules in RMA-S cells are occupied with self-peptides. Such peptides stably associate with Kb at 26°C but easily dissociate from them at 37°C, suggesting low-affinity interactions between Kb and the associated peptides. At 26°C, at least some of these Kb molecules are stably expressed in a peptide-receptive state on the cell surface, whereas at 37°C they are short lived and are only transiently capable of binding and presenting exogenously supplied OVA 257–264 peptide for presentation to CD8+ Kb-restricted T lymphocytes. Thus contrary to current models of class I assembly in TAP-deficient RMA-S cells, the presumably “empty” molecules are in fact associated with peptides at 26°C. Together, our data support the existence of an alternative mechanism of peptide binding and display by MHC class I molecules in TAP-deficient cells that could explain their ability to present Ag.
Effective DNA Vaccination Against Listeriosis by Prime/Boost Inoculation with the Gene Gun
Authors: Fensterle Joachim , Grode Leander , Hess Jürgen , Kaufmann Stefan H. E.
Published: December 2022

Protective immunity against Listeria monocytogenes strongly depends on CD8+ T lymphocytes, and both IFN-γ secretion and target cell killing are considered relevant to protection. We analyzed whether we could induce a protective type 1 immune response by DNA vaccination with the gene gun using plasmids encoding for two immunodominant listerial Ags, listeriolysin and p60. To induce a Th1 response, we 1) coprecipitated a plasmid encoding for GM-CSF, 2) employed a prime/boost vaccination schedule with a 45-day interval, and 3) coinjected oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs. DNA immunization of BALB/c mice with plasmids encoding for listeriolysin (pChly) and p60 (pCiap) efficiently induced MHC class I-restricted, Ag-specific CD8+ T cells that produced IFN-γ. Coinjection of CpG-ODN significantly increased the frequency of specific IFN-γ-secreting T cells. Although pChly induced specific CD8+ T cells expressing CTL activity, it failed to stimulate CD4+ T cells. Only pCiap induced significant CD4+ T cell and humoral responses, which were predominantly of Th2 type. Vaccination with either plasmid induced protective immunity against listerial challenge, and coinjection of CpG ODN improved vaccine efficacy in some situations. This study demonstrates the feasibility of gene gun administration of plasmid DNA for inducing immunity against an intracellular pathogen for which protection primarily depends on type 1 CD8+ T cells.
TH Cells Primed During Influenza Virus Infection Provide Help for Qualitatively Distinct Antibody Responses to Subsequent Immunization
Authors: Marshall Dana , Sealy Robert , Sangster Mark , Coleclough Christopher
Published: December 2022

The quality of the primary Ab-forming cell (AFC) response in cervical lymph nodes and mediastinal lymph nodes of mice to intranasal influenza virus was strongly influenced by viral replicative capacity. IgA secretors were prominent in the early AFC response to infectious virus in mediastinal lymph nodes, while IgG expression was more frequent among isotypically switched AFC in cervical lymph nodes of the same mice; this pattern was reversed in the response to inactivated virus. Influenza viruses A/PuertoRico/8/34 (A/PR8) and A/X-31 share six of eight genome segments, differing only in hemagglutinin (H1 in A/PR8, H3 in A/X-31) and neuraminidase (N1 in A/PR8, N2 in A/X-31) genes. These viruses therefore elicit extensively cross-reactive TH populations, though their glycoproteins are serologically unrelated. Mice recovered from an A/X-31 infection thus mount a primary B cell response against A/PR8 glycoproteins, when challenged with the latter virus, though this response can call upon memory TH cells. To assess the impact of memory TH populations on a primary Ab response, we compared the AFC response to inactivated A/PR8 in naive mice and mice that had cleared an A/X-31 infection. A/X-31 immune mice mounted a more vigorous AFC response against A/PR8 H1 and N1 glycoproteins than naive animals, when immunized intranasally with inactivated A/PR8. However the distribution of isotypes among H1/N1-specific AFC in lymph nodes of A/X-31-primed mice resembled that of naive mice. Evidently, in this functional context, memory TH cells retained the ability to help Ab responses different in quality from that generated during their primary reaction.