bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of the Second Heparin-Binding Domain in Human Complement Factor H
Authors: Blackmore Timothy K. , Hellwage Jens , Sadlon Tania A. , Higgs Naomi , Zipfel Peter F. , Ward Helena M. , Gordon David L.
Published: December 2022

Complement factor H (fH) regulates activation of the alternative pathway of C, reducing the amount of C3b deposited on sialic acid-rich surfaces. Heparin binding has been used as a model for examining the sialic acid-binding characteristics of fH. We have previously shown that of the 20 short consensus repeat (SCR) modules of fH, SCR 7 contains an important heparin binding site, but other SCRs also play a role in heparin binding. To localize the other sites, we prepared recombinant truncated and SCR deletion mutants of fH and tested them by heparin-agarose affinity chromatography. The 5 C-terminal SCRs were found to contain a heparin binding site as an SCR 7 deletion mutant of the N terminal 15 SCRs did not bind heparin, but a construct consisting of SCRs 16–20 was shown to bind heparin. Double deletion of SCRs 7 and 20 from fH abrogated binding to heparin, indicating that SCR 20 contains a heparin binding site. This finding was confirmed with the observation that attachment of SCR 20 to a group of nonbinding SCRs produced a heparin-binding protein. A protein consisting of SCRs 19 and 20 did not bind heparin, whereas SCRs 18–20 did, indicating that, although SCR 20 contains a heparin binding site, at least two nonspecific adjacent SCRs are required. fH-related protein-3 (FHR-3) possesses an SCR homologous to SCR 7 of fH and bound heparin, whereas FHR-4, which lacks such an SCR, did not. Thus, fH contains two separate heparin binding sites, which are located in SCRs 7 and 20.
Spectratyping of TCR Expressed by CTL-Infiltrating Male Antigen (HY)-Disparate Allografts
Authors: Johnston Sean L. , Wettstein Peter J.
Published: December 2022

Minor histocompatibility Ags (HA) play prominent roles in stimulating allograft rejection and are recognized by CTLs that mediate this process. There is limited information regarding the sequences of minor HA peptides and the diversity of minor HA-specific TCRs. In the case of the male minor HA (HY), a peptide presented by H2Db molecules has been sequenced. We have used spectratyping to study the diversities of Vβ usage and β complementarity-determining region 3 (CDR3) lengths of TCRs expressed by CTLs that infiltrate HY-disparate skin allografts during rejection. Spectratyping of RNA from second- and third-set male allografts on CD4-depleted, female recipients showed a reduction in Vβ usage and β CDR3 length diversity with prominent representation of Vβ8 genes. CDR3 sequences, as a group, were characterized by net negative charges resulting from negatively charged residues at positions 5–6 and 10–11. The effects of in vivo anti-Vβ8 Ab treatment on rejection of second-set male allografts were investigated. This Ab treatment had no effect on allograft rejection time and resulted in increased Vβ7 usage in recipients with complete Vβ8 depletion. More interestingly, the net charges of β CDR3s derived from Vβ8-depleted recipients were altered by the inclusion of positively charged and polar residues at positions 4–6. These results indicate that Vβ-specific T cell depletion has no effect on HY-disparate allograft survival, but it alters Vβ usage and changes the characteristics of β CDR3s that facilitate class I:peptide recognition.
Activation of STAT Proteins and Cytokine Genes in Human Th1 and Th2 Cells Generated in the Absence of IL-12 and IL-4
Authors: Moriggl Richard , Kristofic Colette , Kinzel Bernd , Volarevic Sinisa , Groner Bernd , Brinkmann1 Volker
Published: December 2022

We have shown previously that human CD4+45RO− T cells could be primed for a Th2 phenotype independent of IL-4 if they were activated by anti-CD28 mAb plus IL-2. If additional TCR signals were provided, the cells differentiated toward Th1 independent of IL-12. Here we show that anti-CD28/IL-2-primed Th2 cells expressed high levels of activated STAT6, but no cytokine mRNA. Moreover, both Th1 and Th2 cells expressed active STAT1 and -3, but not STAT2, -4, and -5. Restimulation of Th1 or Th2 cells via CD3 plus CD28 induced production of IFN-γ or IL-4, respectively, but did not alter the activation status/DNA binding activity of STATs. Addition of IL-4 (or anti-IL-4 mAb) to restimulated Th2 cells did not modulate STAT6 activation or IL-4 expression, confirming the full commitment. However, Th2 cells remained responsive to IL-12, which repressed STAT6 DNA binding but activated STAT4, and this coincided with a suppression of IL-4/IL-5 and an induction of IFN-γ. In Th1 cells, IL-12 activated both STAT6 and STAT4, and IL-4 activated STAT6, but in both cases the Th1 phenotype remained. Together the data show that CD28/IL-2-dependent Th2 priming activated STAT6 without inducing IL-4 expression. The primed Th cells resembled memory cells and produced IL-4 upon the first CD3/CD28 costimulus without detectable modulation of STATs. Th2 cells remained responsive to IL-12, which repressed STAT6 DNA binding and activated STAT4, and switched the cells to Th1. The effects of IL-12 may depend on the commitment of the cells, since IL-12 phosphorylated STAT6 in Th1 and dephosphorylated STAT6 in Th2 cells.
αβ T Cell Response toToxoplasma gondiiin Previously Unexposed Individuals
Authors: Subauste Carlos S. , Fuh Franklin , de Waal Malefyt Rene , Remington Jack S.
Published: December 2022

The mechanisms by which T cells from previously unexposed hosts respond in vitro to certain intracellular pathogens remain to be fully understood. We report and characterize the in vitro reactivity to Toxoplasma gondii of human αβ T cells from T. gondii-seronegative individuals. Resting αβ T cells from these individuals proliferated in response to PBMC infected with T. gondii or pulsed with T. gondii lysate Ags. This was accompanied by an increase in the percentage of CD4+ αβ T cells. Purified CD4+ αβ T cells but not CD8+ αβ T cells proliferated in response to these T. gondii preparations. Both CD4+ αβ T cells with naive (CD45RA+) and memory (CD45RO+) phenotypes from adults as well as αβ T cells from T. gondii-seronegative newborns proliferated after incubation with T. gondii. This αβ T cell response to the parasite was inhibited by anti-HLA-DR mAb and to a lesser degree by anti-HLA-DQ mAb. Use of paraformaldehyde-fixed PBMC completely abrogated the proliferation of αβ T cells, indicating the need for processing of T. gondii Ags. Analysis of the TCR Vβ expression did not show evidence for restriction in TCR Vβ usage during T. gondii stimulation of αβ T cells. αβ T cells secreted significant amounts of IFN-γ after incubation with T. gondii-infected monocytes. This rapid and remarkable αβ T cell response may play an important role in the early events of the immune response to T. gondii.
The Multiligand-Binding Protein gC1qR, Putative C1q Receptor, Is a Mitochondrial Protein
Authors: Dedio Jürgen , Jahnen-Dechent Willi , Bachmann Michael , Müller-Esterl Werner
Published: December 2022

A protein of 33 kDa (p33) that tightly binds to the globular domains of the first complement component, C1q, is thought to serve as the major C1q receptor (gC1qR) on B cells, neutrophils, and mast cells. However, the cellular routing and the subcellular localization of p33/gC1qR are unknown. We have performed confocal laser-scanning microscopy and found that p33/gC1qR is present in intracellular compartments, where it colocalizes with the mitochondrial marker protein, pyruvate dehydrogenase. No surface staining for p33/gC1qR on endothelial EA.hy926 cells was observed. A fusion protein of the p33/gC1qR presequence with green fluorescent protein translocated to the mitochondria of transfected COS-7 cells. Concomitantly, a 6-kDa portion of the fusion protein was proteolytically removed. The 33 amino-terminal residues of the presequence proved sufficient to direct reporter constructs to mitochondria. Association of p33/gC1qR with mitoplasts indicated that the mature protein of 209 residues resides in the matrix and/or the inner membrane of mitochondria. Immunocytochemistry of fetal mice tissues revealed a ubiquitous expression of p33/gC1qR, most prominently in tissues that are rich in mitochondria. Thus, the candidate complement receptor p33/gC1qR of intact cells cannot interact with plasma C1q due to mutually exclusive localizations of the components. The functional role of p33/gC1qR needs to be reconsidered.
The Receptor for Complement Anaphylatoxin C3a Is Expressed by Myeloid Cells and Nonmyeloid Cells in Inflamed Human Central Nervous System: Analysis in Multiple Sclerosis and Bacterial Meningitis
Authors: Gasque Philippe , Singhrao Sim K. , Neal Jim W. , Wang Piao , Sayah Sakina , Fontaine Marc , Morgan B. Paul
Published: December 2022

The complement anaphylatoxins C5a and C3a are released at the inflammatory site, where they contribute to the recruitment and activation of leukocytes and the activation of resident cells. The distribution of the receptor for C5a (C5aR) has been well studied; however, the receptor for C3a (C3aR) has only recently been cloned, and its distribution is uncharacterized. Using a specific affinity-purified anti-C3aR peptide Ab and oligonucleotides for reverse transcriptase-PCR analysis, C3aR expression was characterized in vitro on myeloid and nonmyeloid cells and in vivo in the brain. C3aR was expressed by adult astrocytes, astrocyte cell lines, monocyte lines THP1 and U937, neutrophils, and monocytes, but not by K562 or Ramos. C3aR staining was confirmed by flow cytometry, confocal imaging, and electron microscopy analysis. A 65-kDa protein was immunoprecipitated by the anti-C3aR from astrocyte and monocyte cell lysates. Our results at the protein level were confirmed at the mRNA level. Using reverse transcriptase-PCR, Southern blot, and sequencing we found that C3aR mRNA was expressed by fetal astrocytes, astrocyte cell lines, and THP1, but not by K562 or Ramos. The astrocyte C3aR cDNA was identical with the reported C3aR cDNA. C3aR expression was not detected in normal brain sections. However, a strong C3aR staining was evident in areas of inflammation in multiple sclerosis and bacterial meningitis. In meningitis, C3aR was abundantly expressed by reactive astrocytes, microglia, and infiltrating cells (macrophages and neutrophils). In multiple sclerosis, infiltrating lymphocytes did not express C3aR, but a strong staining was detected on smooth muscle cells (pericytes) surrounding blood vessels.
Cutting Edge: Structural Requirements for Galactosylceramide Recognition by CD1-Restricted NK T Cells
Authors: Brossay Laurent , Naidenko Olga , Burdin Nicolas , Matsuda Jennifer , Sakai Teruyuki , Kronenberg Mitchell
Published: December 2022

The reactivity of a group of mouse Vα14+ NK T cell hybridomas was tested with a panel of analogs of the glycolipid α-galactosylceramide (α-GalCer). Interestingly, the nearly complete truncation of the acyl chain from 24 to 2 carbons does not significantly affect the mouse NK T cell response to glycolipid presented by either mouse CD1 (mCD1) or its human homolog CD1d (hCD1d). Therefore, we propose that only one of the two hydrophobic pockets of the CD1 Ag-binding groove needs to be filled by Ag. In terms of the sphingosine base, the mCD1 binding groove has less-demanding structural requirements for presentation to NK T cells than hCD1d. Tests of NK T cell reactivity to analogs presented by hCD1d demonstrates that the invariant TCRs expressed by mouse and human NK T cells are surprisingly similar in their requirements for glycolipid recognition.
Cutting Edge: Passive But Not Active CD8+ T Cell-Based Immunotherapy Interferes with Liver Tumor Progression in a Transgenic Mouse Model
Authors: Romieu Raphaëlle , Baratin Myriam , Kayibanda Michèle , Lacabanne Valérie , Ziol Marianne , Guillet Jean-Gérard , Viguier Mireille
Published: December 2022

To evaluate tumor immunotherapies, we used transgenic mice that harbor a progressive liver tumor associated with the expression of the SV40 large tumor T oncoprotein (SV40-T). To induce “self” tumor Ag-specific CD8+ T cells, mice were injected with an immunodominant SV40-T CTL epitope mixed with a heterologous helper peptide. Despite repeated injections, this vaccine failed to raise a tumor-specific CD8+ T cell response that was efficient enough to counteract tumors. Although coimmunization with SV40-T CTL epitope and heterologous helper peptide efficiently recruited the respective Th cells, only low-avidity SV40-T-specific CD8+ T cells were activated. Furthermore, major alterations in SV40-T-specific B and Th cell responses were characterized. In contrast, transfers of higher-avidity CTLs specific for the same SV40-T epitope were effective in counteracting tumors. These results suggest that passive therapies targeted to self tumor Ag may be more suitable than active immunization in the treatment of spontaneous tumors.
Cutting Edge: Generation of a Novel Stem Cell Factor-Dependent Mast Cell Progenitor
Authors: Yuan Qian , Gurish Michael F. , Friend Daniel S. , Austen K. Frank , Boyce Joshua A.
Published: December 2022

Tissue mast cell development requires stem cell factor (SCF), whereas helminth-induced intestinal mucosal mast cell hyperplasia also requires T cell-derived factors such as IL-3. We generated progenitor mast cells (PrMC) from mouse bone marrow cells (BMC) in vitro with a triad of SCF, IL-6, and IL-10 that exhibit IL-3-mediated mitogenic and maturation responses. SCF/IL-6/IL-10 transiently elicited a cell subpopulation with the phenotype (c-kithighThy-1low) of fetal blood promastocytes at 3 wk of culture that progressed within 1 wk to FcεRI-bearing PrMC, designated PrMCTriad. PrMCTriad lacked mouse mast cell carboxypeptidase A (mMC-CPA) protein, required SCF for IL-3-driven thymidine incorporation, and responded to SCF plus IL-3 with strong mMc-CPA immunoreactivity, clarifying distinct sequential roles for SCF and IL-3 in mast cell development. PrMCTriad, arising from BMC through promastocytes, are metamastocytes that acquire microenvironmentally determined phenotypic features.
Age-Associated Decline incdk1Activity Delays Cell Cycle Progression of Human T Lymphocytes
Authors: Quadri Rafael A. , Arbogast Agnès , Phelouzat Marie-Anne , Boutet Stéphane , Plastre Olivier , Proust Jacques J.
Published: December 2022

Despite the repeatedly observed impaired proliferative response of T lymphocytes from aged donors, the precise molecular basis underlying such a defect is still poorly understood. The aim of this study was to determine whether cyclin-dependent kinase 1 (cdk1), a serine-threonine kinase required for entry into mitosis, is implicated in this age-associated dysregulation of the cell cycle. T lymphocytes derived from young and elderly donors were blocked in S phase by hydroxyurea after a 48-h activation by anti-CD3 Abs. Under these experimental conditions, only the cells that were already located beyond the S phase were able to complete the cell cycle, decreasing their DNA content from 4n to 2n chromosomes. Using this procedure, a delay in the accomplishment of mitosis could be observed in cells from elderly individuals, as evidenced by propidium iodide staining. In this age group, only a minimal cdk1 activity could be immunoprecipitated from cells sorted in G2/M after nocodazole block. The decrease in cdk1 activity observed in T lymphocytes from aged donors could be accounted for by at least three mechanisms: 1) a failure of these cells to express a sufficient amount of cdk1, 2) a reduced level of the associated cyclin B1, and 3) an incomplete dephosphorylation of the kinase on tyrosine. This low cdk1 activity is likely to postpone the progression through the G2/M transition and participates in the dysfunction of the cell cycle during the process of aging.
Requirements for Stimulating Naive CD8+ T Cells via Signal 1 Alone
Authors: Luxembourg Alain T. , Brunmark Anders , Kong Yan , Jackson Michael R. , Peterson Per A. , Sprent Jonathan , Cai Zeling
Published: December 2022

In the absence of costimulation, TCR recognition of peptide/MHC complexes is generally considered to be nonimmunogenic. In agreement with this view, naive TCR transgenic CD8+ cells failed to respond to specific peptides presented by MHC class I (Ld) molecules bound to mouse RBC. However, peptide/Ld complexes presented by cell-sized beads or bound to plastic led to overt proliferative responses in the absence of added cytokines. Significantly, equivalent strong proliferative responses occurred when mouse RBC were fixed with glutaraldehyde before Ld coupling. The implication therefore is that the intensity of signaling via the TCR is a reflection of the mobility of the ligand being recognized; TCR signaling is weak when the ligand can move laterally on the cell membrane but strong when the ligand is immobilized.
Type 2 Immune Deviation Has Differential Effects on Alloreactive CD4+ and CD8+ T Cells
Authors: Matesic Damir , Valujskikh Anna , Pearlman Eric , Higgins Alan W. , Gilliam Anita C. , Heeger Peter S.
Published: December 2022

Allograft rejection has been associated with detection of the type 1 lymphokines, IFN-γ and IL-2. The role of type 2 cytokines (IL-4 and IL-5) remains controversial, as is whether alloreactive CD4+ and CD8+ T cells behave similarly when exposed to type 2 cytokine-enhancing manipulations. We studied the characteristics of alloreactive CD4+ and CD8+ T cells before and after type 2 immune deviation induced by IL-4 plus anti-IFN-γ Ab. Alloreactive T cells from naive mice were low in frequency, produced only IL-2, and were predominantly CD4+, while alloreactive T cells from allograft-primed mice were high in frequency, produced IFN-γ, IL-2, and IL-4, and were predominantly CD8+. Type 2 immune deviation of allospecific CD4+ T cells resulted in IL-4 and IL-5 production without IFN-γ, consistent with unipolar type 2 immunity. These T cells mediated delayed-type hypersensitivity, but not cytotoxicity. Under identical type 2 cytokine-inducing conditions, allospecific CD8+ T cells were primed to become IL-4, IL-5, and IFN-γ producers, and exhibited cytotoxicity, but not classic delayed-type hypersensitivity. Adoptive transfer of either cell population into SCID recipients of allogeneic skin resulted in graft rejection, with stable allospecific type 2 cytokine production in vivo. Adoptive transfer of the IL-4/IL-5-producing CD4+ T cells, but not the CD8+ T cells, induced a distinct histopathology characterized by marked eosinophilic infiltration of the skin. We conclude that type 2 immune deviation has differential effects on CD4+ and CD8+ T cells and results in emergence of alternate effector mechanisms capable of destroying allografts.
Tolerant CD8 T Cells Induced by Multiple Injections of Peptide Antigen Show Impaired TCR Signaling and Altered Proliferative Responses In Vitro and In Vivo
Authors: Dubois Patrice M. , Pihlgren Maria , Tomkowiak Martine , Van Mechelen Marcelle , Marvel Jacqueline
Published: December 2022

The mechanisms responsible for peripheral CD8 T cell tolerance to foreign Ags remain poorly understood. In this study we have characterized the state of CD8 T cell tolerance induced in F5 TCR transgenic mice by multiple peptide injections in vivo. The tolerant state of CD8 T cells is characterized by impaired proliferative responses, increased sensitivity to cell death, and failure to acquire cytotoxic effector function after in vitro antigenic challenge. In vivo monitoring of CD8 T cell proliferation using 5-carboxyfluorescein diacetate succinimidyl ester showed that a large subset of the tolerant T cell population failed to divide in response to peptide. TCR down-regulation could not account for this loss of responsiveness to Ag since recombination-activating gene-1 (RAG-1)−/−F5 CD8 T cell responses were similar to those of RAG-1−/−F5 × RAG-1−/− F1 T lymphocytes, which express lower levels of the transgenic TCR. Analysis of early signal transduction in tolerant CD8 T cells revealed high basal levels of cytoplasmic calcium as well as impaired calcium mobilization and tyrosine phosphorylation after cross-linking of CD3ε and CD8α. Together these data indicate that repeated exposure to soluble antigenic peptide in vivo can induce a state of functional tolerance characterized by defective TCR signaling, impaired proliferation, and increased sensitivity to cell death.
Syk- and Lyn-Dependent Phosphorylation of Syk on Multiple Tyrosines Following B Cell Activation Includes a Site That Negatively Regulates Signaling
Authors: Keshvara Lakhu M. , Isaacson Christina C. , Yankee Thomas M. , Sarac Radmila , Harrison Marietta L. , Geahlen Robert L.
Published: December 2022

The Syk protein tyrosine kinase is an essential component of the B cell Ag receptor signaling pathway. Syk is phosphorylated on tyrosine following B cell activation. However, the sites that are modified and the kinases responsible for these modifications have yet to be determined. To approach this problem, we used a mapping strategy based on the electrophoretic separation of peptides on alkaline polyacrylamide gels to identify the tryptic phosphopeptides derived from metabolically labeled Syk. In this work, we report that Syk from activated B cells is phosphorylated principally on six tyrosines: one located between the tandem SH2 domains (Tyr130); three in the linker region (Tyr317, Tyr342, and Tyr346); and two in the catalytic domain (Tyr519 and Tyr520). The linker region sites are the primary targets of the Src family protein tyrosine kinase, Lyn, and include a site that negatively (Tyr317) regulates receptor signaling. Efficient phosphorylation of the catalytic domain and inter-SH2 domain tyrosines is catalyzed primarily by Syk itself, but only occurs to an appreciable extent in cells that express Lyn. We propose that these sites are phosphorylated following the binding of Syk to immunoreceptor tyrosine-based activation motif.
Impaired Cutaneous Immune Responses in Thy-1-Deficient Mice
Authors: Beissert Stefan , He Hai-Tao , Hueber Anne-Odile , Lellouch Annemarie C. , Metze Dieter , Mehling Annette , Luger Thomas A. , Schwarz Thomas , Grabbe Stephan
Published: December 2022

Thy-1 is a cell surface glycoprotein expressed mainly on brain and lymphoid tissue. Although the functions of Thy-1 are incompletely understood, evidence exists that Thy-1 participates in T cell activation. To examine the functional role of Thy-1 in cutaneous immune responses in vivo, Thy-1 gene-targeted mice (Thy-1−/−) and wild-type mice (Thy-1+/+) were immunized with the hapten oxazolone. After challenge with oxazolone, contact hypersensitivity responses in Thy-1−/− mice were reduced by 25% compared with Thy-1+/+ mice. Likewise, irritant dermatitis induced by croton oil was also decreased. In addition, Thy-1−/− mice showed a significantly reduced delayed-type hypersensitivity response after injection of allogeneic spleen cells into the hind footpads of allosensitized animals when compared with Thy-1+/+ mice. Moreover, proliferative responses to immobilized anti-CD3 were decreased in peripheral Thy-1−/− lymphocytes; this decrease was associated with a significantly reduced intracellular Ca2+ influx and protein tyrosine phosphorylation, indicating impairment of early lymphocyte activation. In contrast, the T cell proliferation induced by mitogens was normal, suggesting that Thy-1 expression weakly contributes to TCR-mediated T cell activation. Epidermal Langerhans cells and bone marrow-derived dendritic cells from Thy-1−/− mice exhibited a normal expression of costimulatory surface molecules as well as an unaltered ability to stimulate allogeneic T cells. Taken together, these findings demonstrate that a lack of Thy-1 expression does not generally compromise the immune system; however, Thy-1 expression may be involved in the fine-tuning of T cell-mediated immune responses.
Induction and Inhibition of CD40-CD40 Ligand Interactions: A New Strategy Underlying Host-Virus Relationships
Authors: Sharma Madhav D. , Leite de Moraes Maria , Zavala Flora , Pontoux Christiane , Papiernik Martine
Published: December 2022

Interaction between CD40 and the CD40 ligand (CD40L) is required for mouse mammary tumor virus (MMTV) propagation. We found that Fas was expressed on B cells and CD40L on a small subset of viral superantigen-cognate T cells 12 h after MMTV(SW) infection. CD40L and Fas were down-regulated after 24 h. All CD4 T cells then became resistant to anti-CD3-induced CD40L induction in vitro for 2 wk. Initiation of CD40L expression and its rapid shut-off was associated with IL-12 production and was controlled by IFN-γ and shedding of soluble CD40. These results suggest that a rapid, transient CD40-CD40L interaction involving a small number of cells is sufficient for MMTV propagation. Modulation of CD40L expression may be a major mechanism regulating the balance between viral propagation and host defenses, allowing mutual survival.
Attenuation of Inducible Th2 Immunity with Autoimmune Disease Progression
Authors: Tian Jide , Kaufman Daniel L.
Published: December 2022

Autoantigen-based immunotherapeutics have been shown to activate regulatory responses capable of inhibiting T cell-mediated autoimmune disease in animal models. However, their efficacy generally declines, as treatment occurs later in the disease process, and their mechanism of action is a matter of intense debate. Here, we report that the early administration of β cell autoantigens (βCAAs) to nonobese diabetic (NOD) mice broadly diverts the natural development of potentially pathogenic Th1-biased autoimmune responses toward the Th2 phenotype through Th2 spreading. With disease progression, there was a steady decline in the ability of βCAA treatment to promote Th2-type cellular and humoral autoimmunity. Late in the disease process, some βCAAs were still able to induce Th2 responses and Th2 spreading (although to a much lesser extent), while other autoantigens were not. This attenuation of inducible Th2 immunity with disease progression is likely to reflect a reduction in the availability of uncommitted autoantigen-reactive precursor T cells. These findings suggest that there are inherent differences in the frequency of βCAA-reactive T cells and that, in advanced stages of autoimmune disease, regulatory responses may be best elicited with target tissue Ags against which large uncommitted T cell pools are still available. Since individuals presenting the first signs of autoimmune disease are likely to already have an advanced disease process, these findings may be useful for the rational design of Ag-based immunotherapeutics.
Phosphatidylinositol 3-Kinase Is Required for CD28 But Not CD3 Regulation of the TEC Family Tyrosine Kinase EMT/ITK/TSK: Functional and Physical Interaction of EMT with Phosphatidylinositol 3-Kinase
Authors: Lu Yiling , Cuevas Bruce , Gibson Spencer , Khan Humera , LaPushin Ruth , Imboden John , Mills Gordon B.
Published: December 2022

Ligation of the TCR or CD28 induces activation of phosphatidylinositol 3-kinase (PI3K), the TEC family protein tyrosine kinase, EMT/ITK/TSK (EMT), and the SRC family tyrosine kinase, LCK. LCK is required for the activation and phosphorylation of EMT induced by ligation of the TCR or CD28 placing LCK upstream of EMT in T cell signaling cascades. We report herein that inhibition of PI3K activity with the specific inhibitors LY294002 and wortmannin markedly decreased EMT activation induced by CD28 cross-linking but not by CD3 cross-linking. Further, inhibition of PI3K markedly decreased EMT in vitro autokinase activity induced by activated LCK. In contrast, PI3K inhibitors did not alter CD28 or CD3 cross-linking or LCK-induced EMT phosphorylation. Consistent with the requirement of PI3K activity for CD28 but not CD3-induced stimulation of the EMT in vitro autokinase activity, a small but significant portion of cellular EMT associates with PI3K following CD28 cross-linking but not following CD3 cross-linking. CD28-induced association of EMT with PI3K also requires functional expression of LCK. Fusion proteins containing the SRC homology 2 domain of EMT interact with PI3K or a PI3K-associated molecule in a tyrosine phosphorylation-dependent manner. Taken together, the data suggest that EMT is differentially regulated and recruited to different signaling complexes following ligation of CD28 or the TCR complex, perhaps contributing to the disparate roles that EMT appears to play downstream of CD28 and the TCR.
Secretory Component Delays the Conversion of Secretory IgA into Antigen-Binding Competent F(ab′)2: A Possible Implication for Mucosal Defense
Authors: Crottet Pascal , Corthésy Blaise
Published: December 2022

Secretory component (SC) represents the soluble ectodomain of the polymeric Ig receptor, a membrane protein that transports mucosal Abs across epithelial cells. In the protease-rich environment of the intestine, SC is thought to stabilize the associated IgA by unestablished molecular mechanisms. To address this question, we reconstituted SC-IgA complexes in vitro by incubating dimeric IgA (IgAd) with either recombinant human SC (rSC) or SC isolated from human colostral milk (SCm). Both complexes exhibited an identical degree of covalency when exposed to redox agents, peptidyl disulfide isomerase, and temperature changes. In cross-competition experiments, 50% inhibition of binding to IgAd was achieved at ∼10 nM SC competitor. Western blot analysis of IgAd digested with intestinal washes indicated that the α-chain in IgAd was primarily split into a 40-kDa species, a phenomenon delayed in rSC- or SCm-IgAd complexes. In the same assay, either of the SCs was resistant to degradation only if complexed with IgAd. In contrast, the κ light chain was not digested at all, suggesting that the F(ab′)2 region was left intact. Accordingly, IgAd and SC-IgAd digestion products retained functionality as indicated by Ag reactivity in ELISA. Size exclusion chromatography under native conditions of digested IgAd and rSC-IgAd demonstrates that SC exerts its protective role in secretory IgA by delaying cleavage in the hinge/Fc region of the α-chain, not by holding together degraded fragments. The function of integral secretory IgA and F(ab′)2 is discussed in terms of mucosal immune defenses.
Alterations in TCR-MHC Contacts Subsequent to Cross-Recognition of Class I MHC and Singly Substituted Peptide Variants
Authors: Ono Toshiro , DiLorenzo Teresa P. , Wang Fuming , Kalergis Alexis M. , Nathenson Stanley G.
Published: December 2022

Vesicular stomatitis virus (VSV) elicits H-2Kb-restricted CTLs specific for the immunodominant VSV octapeptide RGYVYQGL. To study the structural features important for interaction between the TCR β-chain and the peptide/MHC complex, we immunized TCR α-chain transgenic mice with the VSV peptide and raised a panel of anti-VSV CTL clones with identical TCR α-chains. Consistent with our previous analysis of uncloned populations of primary CTLs, the anti-VSV CTL clones were all Vβ13+ and expressed TCR β-chains with highly homologous complementarity-determining region 3 (CDR3) loops. Although the clones expressed similar TCRs, they differed in their ability to cross-react with VSV peptide variants singly substituted at TCR contact positions 4 and 6. These findings allowed us to identify short stretches of amino acids in the C-terminal region of the CDR3β loop that, when altered, modify the cross-reaction capability of the TCR to position 4 and position 6 variant peptides. To further probe the structural correlates of biologic cross-reactivity, we used cross-reactive CTL clones and cell lines expressing point mutations in H-2Kb to investigate the effect of single amino acid changes in the peptide on the pattern of recognition of the TCR for the peptide/MHC complex. Single conservative substitutions in the peptide were sufficient to alter the recognition contacts between a cross-reactive TCR and the MHC molecule, supporting the idea that the TCR can make overall structural adjustments in MHC contacts to accommodate single amino acid changes in the peptide.
The Charge of Endotoxin Molecules Influences Their Conformation and IL-6-Inducing Capacity
Authors: Schromm Andra B. , Brandenburg Klaus , Loppnow Harald , Zähringer Ulrich , Rietschel Ernst Th. , Carroll Stephen F. , Koch Michel H. J. , Kusumoto Shoichi , Seydel Ulrich
Published: December 2022

The activation of cells by endotoxin (LPS) is one of the early host responses to infections with Gram-negative bacteria. The lipid A part of LPS molecules is known to represent the endotoxic principle; however, the specific requirements for the expression of biologic activity are still not fully understood. We previously found that a specific molecular conformation (endotoxic conformation) is a prerequisite for lipid A to be biologically active. In this study, we have investigated the interdependence of molecular charge and conformation of natural and chemically modified LPS and lipid A and its transport and intercalation into phospholipid membranes mediated by human LPS-binding protein, as well as IL-6 production after stimulation of whole blood or PBMCs. We found that the number, nature, and location of negative charges strongly modulate the molecular conformation of endotoxin. In addition, the LPS-binding protein-mediated transport of LPS into phospholipid membranes depends on the presence of net negative charge, yet charge is only a necessary, but not a sufficient, prerequisite for transport and intercalation. The biologic activity is determined mainly by the molecular conformation: only conical molecules are highly biologically active, whereas cylindrical ones are largely inactive. We could demonstrate that the net negative charge of the lipid A component and its distribution within the hydrophilic headgroup strongly influence the molecular conformation and, therefore, also the biologic activity.
A Site for CD4 Binding in the β1 Domain of the MHC Class II Protein HLA-DR1
Authors: Brogdon Jennifer , Eckels David D. , Davies Christopher , White Stephen , Doyle Carolyn
Published: December 2022

Using a lymphocyte binding assay, we have previously demonstrated that the CD4 protein can mediate cell adhesion by direct interaction with MHC class II molecules. In this report, we have used this assay to test whether synthetic peptides, corresponding to DRβ sequences, could inhibit CD4-class II adhesion. A peptide derived from sequences within the β1 domain (DRβ41–55), as well as two peptides derived from sequences within the β2 domain (DRβ121–135 and DRβ141–155), were shown to inhibit CD4-class II adhesion. Inasmuch as a site for CD4 binding in the β2 domain had been previously documented, these studies were designed to investigate the role of the β1 domain as an additional site of interaction with CD4. Sixteen site-specific mutations were engineered within the β1 domain of DRβ1*0101. Several mutations were shown to disrupt CD4-dependent T cell activation. Based on these results, we propose a model for the molecular interaction of CD4 with MHC class II proteins in which both the β1 and β2 domains of class II interact with the two amino-terminal Ig-like domains of CD4.
IL-12 Enhances Antibody Responses to T-Independent Polysaccharide Vaccines in the Absence of T and NK Cells
Authors: Buchanan Renee M. , Arulanandam Bernard P. , Metzger Dennis W.
Published: December 2022

Polysaccharide vaccines to encapsulated bacteria such as Neisseria meningitidis and Streptococcus pneumoniae are weakly immunogenic due to their T-independent (TI) nature. Even when converted to T-dependent forms through conjugation to foreign proteins, polysaccharides induce responses that are deficient in many respects, such as induction of murine IgG2a Ab, the isotype that mediates optimal complement fixation and opsonization. We now show that IL-12 treatment of mice induces significantly increased levels of IgG2a Ab to the model TI-2 Ag, DNP-Ficoll, and to vaccines composed of polysaccharides from pneumococci and meningococci. Use of immunodeficient mice lacking T cells and/or NK cells demonstrated that such cells were not responsible for the observed Ab enhancement. Furthermore, the use of IFN-γ knockout mice showed that stimulation of TI-2 Ab responses by IL-12 was only partially dependent on IFN-γ. The ability of IL-12 to dramatically enhance TI Ab responses suggests that IL-12 will be useful as a powerful vaccine adjuvant to induce protective immune responses against encapsulated pathogens.
Bacterial Lipopolysaccharide Can Enter Monocytes Via Two CD14-Dependent Pathways
Authors: Kitchens Richard L. , Wang Ping-yuan , Munford Robert S.
Published: December 2022

Host recognition and disposal of LPS, an important Gram-negative bacterial signal molecule, may involve intracellular processes. We have therefore analyzed the initial pathways by which LPS, a natural ligand of glycosylphosphatidylinositol (GPI)-anchored CD14 (CD14-GPI), enters CD14-expressing THP-1 cells and normal human monocytes. Exposure of the cells to hypertonic medium obliterated coated pits and blocked 125I-labeled transferrin internalization, but failed to inhibit CD14-mediated internalization of [3H]LPS monomers or aggregates. Immunogold electron microscope analysis found that CD14-bound LPS moved principally into noncoated structures (mostly tubular invaginations, intracellular tubules, and vacuoles), whereas relatively little moved into coated pits and vesicles. When studied using two-color laser confocal microscopy, internalized Texas Red-LPS and BODIPY-transferrin were found in different locations and failed to overlap completely even after extended incubation. In contrast, in THP-1 cells that expressed CD14 fused to the transmembrane and cytosolic domains of the low-density lipoprotein receptor, a much larger fraction of the cell-associated LPS moved into coated pits and colocalized with intracellular transferrin. These results suggest that CD14 (GPI)-dependent internalization of LPS occurs predominantly via noncoated plasma membrane invaginations that direct LPS into vesicles that are distinct from transferrin-containing early endosomes. A smaller fraction of the LPS enters via coated pits. Aggregation, which greatly increases LPS internalization, accelerates its entry into the nonclathrin-mediated pathway.
Regulation of Hepatitis B Virus mRNA Expression in a Hepatitis B Surface Antigen Transgenic Mouse Model by IFN-γ-Secreting T Cells After DNA-Based Immunization
Authors: Mancini Maryline , Hadchouel Michelle , Tiollais Pierre , Michel Marie-Louise
Published: December 2022

The immunotherapeutic effect of DNA-mediated immunization against chronic hepatitis B virus (HBV) infection has been evaluated in transgenic mice expressing the sequences that code for the envelope proteins of HBV in the liver. In this model of HBV chronic carriers, a single i.m. injection of plasmid DNA encoding HBV envelope proteins is sufficient to generate specific immune responses leading to the clearance of the transgene expression product and the control of HBV mRNA. The relative contributions of the T cell subpopulations induced by DNA immunization were examined using adoptive transfer experiments. It was shown that either CD8+ or CD4+ T lymphocytes from immunocompetent DNA-immunized animals were sufficient to control viral gene expression in the livers of the recipient transgenic mice. This effect was mediated by a cytokine-dependent mechanism common to both T cell subpopulations; this mechanism did not require cell lysis, but did involve the production of IFN-γ by the activated T cells.
Evaluation of IL-12 in Immunotherapy and Vaccine Design in ExperimentalMycobacterium aviumInfections
Authors: Silva Regina A. , Pais Teresa F. , Appelberg Rui
Published: December 2022

IL-12 is a pivotal cytokine in the induction of IFN-γ-mediated protective immune responses. We tested the effects of rIL-12 administration to Mycobacterium avium-infected mice and found a limited ability to induce protection against the infection; this ability varied according to the mycobacterial strain studied. IL-12 accelerated the expression and production of IFN-γ in both immunocompetent and immunodeficient SCID or CD4-depleted mice. Evidence of NK cell activation was found as well as an enhancement of the ability to adoptively transfer resistance with T cell-enriched spleen cell populations and an increase in inflammatory cell recruitment in the liver. The protective ability of IL-12 was dependent upon the endogenous production of IFN-γ as evaluated by the use of specific neutralizing Abs or IFN-γ gene-disrupted mice. IL-12 potentiated the protective immunity conferred by a subunit vaccine containing M. avium culture filtrate proteins and dimethyl dioctadecyl ammonium chloride as an adjuvant. Thus, we show limited immunotherapeutic benefits from IL-12 administration in M. avium infections and promising results in its use as a coadjuvant in vaccine design.
Staphylococcal Enterotoxin A-Induced Injury of Human Lung Endothelial Cells and IL-8 Accumulation Are Mediated by TNF-α
Authors: Fujisawa Nobumitsu , Hayashi Shinichiro , Kurdowska Anna , Noble James M. , Naitoh Keiko , Miller Edmund J.
Published: December 2022

Staphylococcal enterotoxin A (SEA), a superantigen produced by some strains of Staphylococcus aureus, causes a variety of clinical manifestations ranging from food poisoning to shock. S. aureus can also be associated with the development of acute respiratory distress syndrome, and SEA has been shown to cause an inflammatory reaction in the lung. Therefore, we examined possible interactions between SEA, PBMCs, polymorphonuclear cells (PMNs), and normal human lung microvascular endothelial cells (HMVEC-L), as well as the role of these interactions on the secretion of IL-8. Injury to HMVEC-L, as measured by the release of 51Cr, increased significantly when HMVEC-L were incubated with SEA and PBMCs. IL-8 was secreted by both PBMCs and HMVEC-L. The accumulation of IL-8 in the culture medium of HMVEC-L was increased by SEA in a dose-dependent manner and was directly related to the number of PBMCs present. Although neither anti-human IL-8 nor IL-1 mAb inhibited HMVEC-L cytotoxicity, anti-human TNF-α mAb inhibited both the cytotoxicity and IL-8 accumulation completely. When HMVEC-L were incubated with supernatants from SEA-treated PBMCs, HMVEC-L cytotoxicity was comparable with HMVEC-L incubated with SEA and PBMCs at the same time. Although high concentrations of purified PMNs induced HMVEC-L lysis in a dose-dependent manner, the effect of PMNs was not changed in the presence of SEA. These findings suggest that TNF-α secreted by SEA-stimulated PBMCs plays a leading role in HMVEC-L injury.
Peroxynitrite Mediates IL-8 Gene Expression and Production in Lipopolysaccharide-Stimulated Human Whole Blood
Authors: Filep János G. , Beauchamp Micheline , Baron Chantal , Paquette Yves
Published: December 2022

Recent evidence indicates that free oxygen radicals, in particular hydroxyl radicals, may act as intracellular second messengers for the induction of IL-8, a potent chemoattractant and activator of neutrophil granulocytes. Here we report that peroxynitrite (ONOO−), formed by a reaction of nitric oxide (NO) with superoxide, mediates IL-8 gene expression and IL-8 production in LPS-stimulated human whole blood. The NO synthase inhibitors aminoguanidine and NG-nitro-l-arginine methyl ester (L-NAME) blocked IL-8 release by ∼90% in response to LPS (1 μg/ml), but did not affect the production of IL-1β or TNF-α. Both aminoguanidine and L-NAME blocked the induction of IL-8 mRNA by LPS. Authentic ONOO− (2.5–80 μM) augmented IL-8 mRNA expression and stimulated IL-8 release in a concentration-dependent manner, whereas the NO-releasing compounds, S-nitroso-N-acetyl-dl-penicillamine and sodium nitroprusside failed to induce cytokine production. Combination of the NO-generating chemicals with a superoxide-generating system (xanthine/xanthine oxidase) markedly increased IL-8 release. Enhanced ONOO− formation was detected in granulocytes, monocytes, lymphocytes, and plasma after challenge with LPS. Furthermore, pyrrolidine dithiocarbamate, an inhibitor of activation of nuclear factor-κB, markedly attenuated the induction of IL-8 mRNA expression and IL-8 release by either LPS or ONOO−. Our study identifies ONOO− as a novel signaling mechanism for IL-8 gene expression and suggests that inhibition of ONOO− formation or scavenging ONOO− may represent a novel therapeutic approach to inhibit IL-8 production that could lead to reduction of neutrophil accumulation and activation.
Inhibition of MAP Kinase Kinase Prevents Cytokine and Prostaglandin E2 Production in Lipopolysaccharide-Stimulated Monocytes
Authors: Scherle Peggy A. , Jones Elizabeth A. , Favata Margaret F. , Daulerio Andrea J. , Covington Maryanne B. , Nurnberg Sherrill A. , Magolda Ronald L. , Trzaskos James M.
Published: December 2022

Activation of the extracellular signal-regulated kinase (ERK) pathway has been shown to occur in monocytes following stimulation with LPS. However, the importance of this event for monocyte function is not clear. To address this issue, we used the novel MAP/ERK kinase (MEK) inhibitor, U0126. Stimulation of monocytes with LPS resulted in activation of the mitogen-activated protein kinase (MAPK) family members ERK, Jun NH2-terminal kinase (JNK), and p38. Treatment of monocytes with LPS in the presence of U0126 blocked the activation of ERK1 and ERK2. However, the activation of Jun NH2-terminal kinase and p38 family members was not affected by the compound, confirming the selectivity of U0126. To examine the effects of MEK inhibition on monocyte function, we measured production of the cytokines IL-1, IL-8, and TNF, as well as PGE2. Monocytes treated with LPS in the presence of U0126 failed to release IL-1, IL-8, TNF, or PGE2. The failure to secrete IL-1 and TNF was due to decreased levels of mRNA. These results demonstrate that activation of MEK/ERK is critical for cytokine and PGE2 production by monocytes in response to LPS.
Lyme Arthritis Synovial γδ T Cells Respond toBorrelia burgdorferiLipoproteins and Lipidated Hexapeptides
Authors: Vincent Michael S. , Roessner Karen , Sellati Timothy , Huston Christopher D. , Sigal Leonard H. , Behar Samuel M. , Radolf Justin D. , Budd Ralph C.
Published: December 2022

Lyme arthritis synovial fluid contains a large proportion of γδ T cells that proliferates upon stimulation with the causative spirochete, Borrelia burgdorferi. A panel of Borrelia-reactive γδ T cell clones was derived from synovial fluid of two patients with Lyme arthritis. Each of six γδ clones from one patient used the Vδ1 TCR segment but had otherwise unique CDR3 sequences and diverse Vγ segment usage. Stimulation of the Vδ1 clones was optimal in the presence of Borrelia, dendritic cells, and exogenous IL-2, which was reflected by proliferation, TCR down-modulation, as well as induction of CD25 and Fas ligand expression. Stimulation by B. burgdorferi-pulsed dendritic cells withstood chemical fixation and was not restricted to class I or class II MHC, CD1a, CD1b, or CD1c. In contrast, anti-γδ antibody potently inhibited proliferation. Extraction of B. burgdorferi lipoproteins with Triton X-114 enriched for the stimulatory component. This was confirmed using lipidated vs nonlipidated hexapeptides of Borrelia outer surface proteins. These observations suggest that synovial Vδ1 T cells may mediate an innate immune response to common lipoprotein products of spirochetes.