bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulated Association Between the Tyrosine Kinase Emt/Itk/Tsk and Phospholipase-Cγ1 in Human T Lymphocytes
Authors: Perez-Villar Juan J. , Kanner Steven B. 
Published: December 2022

AbstractThe Emt/Itk/Tsk tyrosine kinase is involved in intracellular signaling events induced by several lymphocyte surface receptors. Modulation of TCR/CD3-induced phospholipase-Cγ1 (PLCγ1) activity by the tyrosine kinase Emt/Itk/Tsk has been demonstrated based on studies of Itk-deficient murine T lymphocytes. Here we report a TCR/CD3-regulated association between Emt and PLCγ1 in both normal and leukemic T cells. In addition, this association was enhanced following independent ligation of the CD2, CD4, or CD28 costimulatory molecules, but not of CD5 or CD6 surface receptors, correlating to the induced tyrosine phosphorylation of Emt. Before Ab-induced T cell activation, we found that the Emt-SH3 domain was crucial for the constitutive Emt/PLCγ1 association; however, upon TCR/CD3 engagement, the Emt-SH2 domain was more efficient in mediating the enhanced Emt/PLCγ1 interaction. Furthermore, the PLCγ1-SH3 domain, but not the two PLCγ1-SH2 domains, contributed to formation of the protein complex. Thus, we describe a regulated interaction between Emt and PLCγ1, and based on our studies with individual Emt and PLCγ1 SH2/SH3 domains, we propose a mechanism for this association.
Aluminium Hydroxide Adjuvant Initiates Strong Antigen-Specific Th2 Responses in the Absence of IL-4- or IL-13-Mediated Signaling
Authors: Brewer James M. , Conacher Margaret , Hunter Christopher A. , Mohrs Markus , Brombacher Frank , Alexander James 
Published: December 2022

AbstractPrevious studies demonstrate that aluminium hydroxide adjuvant (alum) produces increased Th1 responses in IL-4-deficient mice compared with wild-type animals, although the continued production of IL-5 by spleen cells from these mice also indicates that Th2 responses are induced. In the present study, we demonstrate that alum can induce Th2-associated IL-4 and IL-5 production in the absence of IL-4 signaling in mice deficient in either IL-4Rα or Stat6. The Th2 responses observed could not be due to IL-13 as IL-13 responses are also impaired in IL-4Rα- and Stat6-deficient mice. We also detected higher levels of IL-4 in IL-4Rα gene-deficient, though not Stat6-deficient, mice compared with their wild-type counterparts. The increased levels of IL-4 could be explained by the IL-4R being unavailable to neutralize this cytokine in IL-4Rα-deficient mice. While levels of IL-5 production in IL-4Rα- or Stat6-deficient mice were similar to IL-4-deficient and wild-type mice, other type 2-associated responses, which are largely or wholly IL-4 dependent, such as the production of IgG1 or IgE Abs, were either reduced or absent. We conclude that alum adjuvants can induce IL-4 production and Th2 responses independently of IL-4 or IL-13, negating the requirement for an early source of IL-4 in the Th2 response induced by this adjuvant.
The In Vivo Fate of APCs Displaying Minor H Antigen and/or MHC Differences Is Regulated by CTLs Specific for Immunodominant Class I-Associated Epitopes
Authors: Loyer Véronique , Fontaine Pierre , Pion Stéphane , Hétu Francis , Roy Denis-Claude , Perreault Claude 
Published: December 2022

AbstractThe goal of this work was to evaluate the fate of APCs following interactions with T cells in unprimed mice with a normal T cell repertoire. We elaborated a model in which male adherent peritoneal mononuclear cells were injected into the foreleg footpads of naive female recipients mismatched for either minor or major histocompatibility Ags. At various times after injection, APC numbers in the draining (axillary and brachial) lymph nodes were assessed using a Ube1y gene-specific PCR assay. Our experimental model was designed so that the number of APCs expressing the priming epitope was similar to what is observed under real life conditions. Thus, early after injection, the frequency of afferent lymph-derived APCs expressing the priming epitope was in the range of 101–102/106 lymph node cells. We found that APCs presenting some, but not all, nonself epitopes were killed rapidly after entrance into the lymph nodes. Rapid elimination of APCs occurred following interactions with MHC class I-restricted, but not class II-restricted, T cells and was observed when APCs presented an immunodominant (B6dom1/H7a), but not a nondominant (HY), epitope. Killing of APCs was mediated partly, but not exclusively, by perforin-dependent process. We propose that killing of APCs by CTLs specific for immunodominant MHC class I-restricted epitopes may be instrumental in regulating the intensity, duration, and diversity of T cell responses.
MHC Class I-Restricted Cytotoxic Lymphocyte Responses Induced by Enterotoxin-Based Mucosal Adjuvants
Authors: Simmons Cameron P. , Mastroeni Pietro , Fowler Ray , Ghaem-maghami Marjan , Lycke Nils , Pizza Mariagrazia , Rappuoli Rino , Dougan Gordon 
Published: December 2022

AbstractThe ability of enterotoxin-based mucosal adjuvants to induce CD8+ MHC class I-restricted CTL responses to a codelivered bystander Ag was examined. Escherichia coli heat-labile toxin (LT), or derivatives of LT carrying mutations in the A subunit (LTR72, LTK63), were tested in parallel with cholera toxin (CT) or a fusion protein consisting of the A1 subunit of CT fused to the Ig binding domain of Staphylococcus aureus protein A (called CTA1-DD). Intranasal (i.n.) immunization of C57BL/6 mice with CT, CTA1-DD, LT, LTR72, LTK63, but not rLT-B, elicited MHC class I-restricted CD8+ T cell responses to coadministered OVA or the OVA CTL peptide SIINFEKL (OVA257–264). CT, LT, and LTR72 also induced CTL responses to OVA after s.c. or oral coimmunization whereas LTK63 only activated responses after s.c. coimmunization. rLT-B was unable to adjuvant CTL responses to OVA or OVA257–264 administered by any route. Mice treated with an anti-CD4 mAb to deplete CD4+ T cells mounted significant OVA-specific CTL responses after i.n. coadministration of LT with OVA or OVA257–264. Both 51Cr release assays and IFN-γ enzyme-linked immunospot assays indicated that IFN-γ−/− and IL-12 p40−/− gene knockout mice developed CTL responses equivalent to those detected in normal C57BL/6 mice. The results highlight the versatility of toxin-based adjuvants and suggest that LT potentiates CTL responses independently of IL-12 and IFN-γ and probably by a mechanism unrelated to cross-priming.
Central Nervous System-Targeted Expression of the Complement Inhibitor sCrry Prevents Experimental Allergic Encephalomyelitis
Authors: Davoust Nathalie , Nataf Serge , Reiman Rachael , V. Holers Michael , Campbell Iain L. , Barnum Scott R. 
Published: December 2022

AbstractAlthough generally thought of as a T cell-driven autoimmune disease, recent studies in experimental allergic encephalomyelitis (EAE), the animal model of multiple sclerosis, suggest a significant role for innate immune mechanisms. To address the possibility that the complement system plays a central role in these diseases, we developed a transgenic mouse with astrocyte-targeted production of a soluble inhibitor of complement activation, complement receptor-related protein y (sCrry). Here, we show that sCrry transgenic mice are either fully protected against EAE or develop significantly delayed clinical signs. These results indicate that complement activation may have an essential role in the pathogenesis of the disease and that complement-mediated events may occur early during the effector phase of EAE. Furthermore, this work underscores the importance of humoral immunity in amplifying a T cell-initiated pathogenic process.
TNF and Lymphotoxin β Cooperate in the Maintenance of Secondary Lymphoid Tissue Microarchitecture But Not in the Development of Lymph Nodes
Authors: Kuprash23 Dmitry V. , Alimzhanov2 Marat B. , Tumanov2 Alexei V. , Anderson Arthur O. , Pfeffer Klaus , Nedospasov Sergei A. 
Published: December 2022

AbstractInactivation of genes encoding members of TNF and TNF receptor families reveal their divergent roles in the formation and function of secondary lymphoid organs. Most lymphotoxin α (ltα)- and all lymphotoxin β receptor (ltβr)-deficient mice are completely devoid of lymph nodes (LNs); however, most lymphotoxin β (ltβ)-deficient mice develop mesenteric LNs. Tnf- and tnfrp55-deficient mice develop a complete set of LNs, while ltβ/tnfrp55 double-deficient mice lack all LNs, demonstrating cooperation between LTβ and TNFRp55 in LN development. Now we report that ltβ/tnf double-deficient mice develop the same set of mucosal LNs as do ltβ-deficient mice, suggesting that ligands other than TNF signal through TNFRp55 during LN development. These LNs retain distinct T and B cells areas; however, they lack follicular dendritic cell networks. Structures resembling germinal centers can be found in the LNs from immunized ltβ-deficient mice but not in ltβ/tnf double-deficient mice. Additionally, stromal components of the spleen and LNs appear to be more severely disturbed in ltβ/tnf double-deficient mice as compared with ltβ-deficient mice. We conclude that LTβ and TNF cooperate in the establishment of the correct microarchitecture of lymphoid organs.
Commitment to the CD4 Lineage Mediated by Extracellular Signal-Related Kinase Mitogen-Activated Protein Kinase and Lck Signaling
Authors: Sharp Leslie L. , Hedrick Stephen M. 
Published: December 2022

AbstractThe development of T cells results in a concordance between the specificity of the TCR for MHC class I and class II molecules and the expression of CD8 and CD4 coreceptors. Based on analogy to simple metazoan models of organ development and lineage commitment, we sought to determine whether extracellular signal-related kinase (Erk) mitogen-activated protein (MAP) kinase pathway signaling acts as an inductive signal for the CD4 lineage. Here, we show that, by altering the intracellular signaling involving the Erk/MAP kinase pathway, T cells with specificity for MHC class I can be diverted to express CD4, and, conversely, T cells with specificity for MHC class II can be diverted to express CD8. Furthermore, we find that activation of the src-family tyrosine kinase, p56lck is an upstream mediator of lineage commitment. These results suggest a simple mechanism for lineage commitment in T cell development.
Analysis of the Defect in IFN-γ Induction of MHC Class II Genes in G1B Cells: Identification of a Novel and Functionally Critical Leucine-Rich Motif (62-LYLYLQL-68) in the Regulatory Factor X 5 Transcription Factor
Authors: Brickey W. June , Wright Kenneth L. , Zhu Xin-Sheng , P.-Y. Ting Jenny 
Published: December 2022

AbstractMHC class II deficiency found in bare lymphocyte syndrome patients results from the absence or dysfunction of MHC class II transcriptional regulators, such as regulatory factor X (RFX) and class II transactivator (CIITA). Understanding the roles of these factors has been greatly facilitated by the study of genetic defects in cell lines of bare lymphocyte syndrome patients, as well as in cell lines that have been generated by chemical mutagenesis in vitro. The latter group includes MHC class II-deficient lines that are no longer responsive to induction by IFN-γ. Here, we show that the defect in G1B, one such cell line, is attributed to the lack of functional RFX5, the largest subunit of RFX. The RFX5 gene isolated from G1B cells contains two separate single-base pair mutations. One alteration does not exhibit a phenotype, whereas a leucine-to-histidine mutation eliminates DNA-binding and transactivating functions. This mutation lies outside of previously defined functional domains of RFX5 but within an unusual, leucine-rich region (62-LYLYLQL-68). To further investigate the significance of the leucine-rich region, we targeted all neighboring leucine residues for mutagenesis. These mutants were also unable to transactivate a MHC class II reporter gene, confirming that these leucine residues play an essential role in RFX activity and characterize a novel leucine-rich motif.
Staphylococcal Enterotoxin H Displays Unique MHC Class II-Binding Properties
Authors: Nilsson Helen , Björk Per , Dohlsten Mikael , Antonsson Per 
Published: December 2022

AbstractStaphylococcal enterotoxin H (SEH) has been described as a superantigen by sequence homology with the SEA subfamily and briefly characterized for its in vivo activity. In this study, we demonstrate that SEH is a potent T cell mitogen and inducer of T cell cytotoxicity that possesses unique MHC class II-binding properties. The apparent affinity of SEH for MHC class II molecules is the highest affinity ever measured for a staphylococcal enterotoxin (Bmax1/2 ∼ 0.5 nM for MHC class II expressed on Raji cells). An excess of SEA or SEAF47A, which has reduced binding to the MHC class II α-chain, is able to compete for binding of SEH to MHC class II, indicating an overlap in the binding sites at the MHC class II β-chain. The binding of SEH to MHC class II is like SEA, SED, and SEE dependent on the presence of zinc ions. However, SEH, in contrast to SEA, binds to the alanine-substituted DR1 molecule, βH81A, believed to have impaired zinc-bridging capacity. Furthermore, alanine substitution of residues D167, D203, and D208 in SEH decreases the affinity for MHC class II as well as its in vitro potency. Together, this indicates an MHC class II binding site on SEH with a different topology as compared with SEA. These unique binding properties will be beneficial for SEH to overcome MHC class II isotype variability and polymorphism as well as to allow an effective presentation on APCs also at low MHC class II surface expression.
Mutational Analysis of Avidity and Fine Specificity of Anti-Levan Antibodies
Authors: Brorson Kurt , Thompson Cynthia , Wei George , Krasnokutsky Michael , Stein Kathryn E. 
Published: December 2022

AbstractUsing the polyfructose, bacterial levan, as a model polysaccharide, we analyzed how V regions affect binding in anti-polysaccharide mAbs. Previously, panels of mAb were constructed from bacterial levan-immunized BALB/c and CBA/Ca mice. The BALB/c mAb were mostly germline VHJ606:Vκ11, and a subset contained presumed somatic mutations in the complementarity-determining regions (CDRs) that correlated with increases in avidity for the β(2→1) inulin linkage of levan. The CBA/Ca mAb were more heterogeneous in V gene usage, but a subset of inulin-nonreactive mAb were VHJ606:Vλ and had VH sequence differences in the CDRs from the VHJ606 regions of the BALB/c mAb. In this report, VHJ606 Abs containing various combinations of specifically mutated H and L chains were produced by engineered transfectants and tested for inulin avidity and levan binding. Two presumed somatic mutations seen in CDRs of the BALB/c hybridomas were shown to directly cause marked increases in avidity for inulin (VH N53H, 9-fold; VL N53I, 20-fold; together, 46-fold) but not for β(2→6) levan. Exchange of either positions 50 or 53 in VH or the H3 loop between the BALB/c and CBA/Ca mAb resulted in either fine specificity shift or total loss of bacterial levan binding. Three-dimensional models of the V regions suggested that residues that affect binding to inulin alone are near the edge of the CDR surface, while residues involved with binding both forms of levan and affecting fine specificity are in the VH:VL junctional area.
Induction of Th2 Responses and IgE Is Largely Due to Carbohydrates Functioning as Adjuvants onSchistosoma mansoniEgg Antigens
Authors: Okano Mitsuhiro , Satoskar Abhay R. , Nishizaki Kazunori , Abe Mie , Harn Donald A. 
Published: December 2022

AbstractInfection with the helminth parasite Schistosoma mansoni induces a pronounced Th2-type response that is associated with significant IgE production. To better understand how the parasite drives these responses, we investigated the relative roles of proteins and carbohydrates in driving Th2-type and/or IgE responses using a murine model of intranasal sensitization with soluble egg Ags (SEA) of Schistosoma mansoni. We found that repeated intranasal sensitization with soluble egg Ags led to the induction of both total and specific IgE production and nasal eosinophilia. By comparing the responses of mice sensitized with SEA or metaperiodate-treated SEA we were able to demonstrate that carbohydrates on SEA are the major inducers of IgE production and nasal recruitment of eosinophils. Mice sensitized with periodate-treated SEA displayed a significant decrease in both total and specific IgE levels in comparison to mice sensitized with native SEA. Furthermore, sensitization of mice with periodate-treated SEA significantly reduced levels of Ag-specific IgG1, but had no effect on IgG2a production. Nasal lymphocytes from mice sensitized with native SEA, but not with periodate-treated SEA, produced IL-4, IL-5, and IL-10 when restimulated with native SEA in vitro. On the other hand, lymphocytes from mice sensitized with periodate-treated SEA did not produce any of these same cytokines following in vitro restimulation, suggesting that carbohydrates were required for in vivo induction of Th2 response and for that of associated cytokine responses in this model. Lastly, competitive inhibition ELISA showed that although carbohydrates are required for SEA-specific IgE induction, they are not targets of the induced IgE response.
The Adjacent Flanking Region Plays a Critical Role in Facilitating the Presentation of theListeria monocytogenesProduct lemA to H2 M3wt-Restricted, Peptide-Specific Murine CD8 Cells
Authors: Kurlander Roger J. , Chao Elizabeth , Fields Janet , Nataraj Chandrasekaran 
Published: December 2022

AbstractMice infected with Listeria monocytogenes (LM) generate CD8 effectors specific for f-MIGWII, the amino terminus of the bacterial product lemA presented by the class Ib MHC molecule H2 M3wt. lemA has several distinctive properties: 1) it is readily presented as an exogenous Ag in the absence of bacterial infection; 2) it is processed by a TAP-independent pathway, which is sensitive to chloroquine, pepstatin, and brefeldin; and 3) the immunogenic portion of the molecule is extremely resistant to proteolytic degradation even by proteinase K. To assess the structural basis for these findings, we expressed a truncated variant (t-lemA) containing the amino-terminal hexapeptide and the subsequent 27 amino acids linked to a histidine tail in Escherichia coli, and purified the product by affinity chromatography. Purified t-lemA could be presented to f-MIGWII-specific effectors by macrophages and fibroblasts at 1–10 nM. Unlike f-MIGWII, which binds directly to H2 M3wt, t-lemA required processing by a chloroquine-, pepstatin-, and brefeldin-sensitive pathway. Brefeldin sensitivity often implies endogenous processing in the cytoplasm, but several lines of evidence suggest translocation to the cytoplasm and proteosomal degradation are not critical for t-lemA presentation. Unlike f-MIGWII, t-lemA was profoundly resistant to proteinase K, and, using 35S-labeled t-lemA, we could identify the region from position 1 to ∼30 as the protease-resistant element. Thus, the hydrophobic peptide sequence following f-MIGWII can account for the unusual properties of lemA noted above. Analogous modification could be used to alter the properties of other peptide Ags presented by class I MHC products.
A DNA Immunization Model Study with Constructs Expressing the Tick-Borne Encephalitis Virus Envelope Protein E in Different Physical Forms
Authors: Aberle Judith H. , Aberle Stephan W. , Allison Steven L. , Stiasny Karin , Ecker Michael , Mandl Christian W. , Berger Rudolf , Heinz Franz X. 
Published: December 2022

AbstractWe have conducted a DNA immunization study to evaluate how the immune response is influenced by the physical structure and secretion of the expressed Ag. For this purpose, we used a series of plasmid constructs encoding different forms of the envelope glycoprotein E of the flavivirus tick-borne encephalitis virus. These included a secreted recombinant subviral particle, a secreted carboxyl-terminally truncated soluble homodimer, a nonsecreted full-length form, and an inefficiently secreted truncated form. Mice were immunized using both i.m. injection and Gene Gun-mediated application of plasmids. The functional immune response was evaluated by determining specific neutralizing and hemagglutination-inhibiting Ab activities and by challenging the mice with a lethal dose of the virus. As a measure for the induction of a Th1 and/or Th2 response, we determined specific IgG subclasses and examined IFN-γ, Il-4, and Il-5 induction. The plasmid construct encoding a secreted subviral particle, which carries multiple copies of the protective Ag on its surface, was superior to the other constructs in terms of extent and functionality of the Ab response as well as protection against virus challenge. As expected, the type of Th response was largely dependent on the mode of application (i.m. vs Gene Gun), but our data show that it was also strongly influenced by the properties of the Ag. Most significantly, the plasmid encoding the particulate form was able to partially overcome the Th2 bias imposed by the Gene Gun, resulting in a balanced Th1/Th2 response.
Critical Role of CD28 in Protective Immunity AgainstSalmonella typhimurium
Authors: Mittrücker Hans-Willi , Köhler Anne , Mak Tak W. , Kaufmann Stefan H. E. 
Published: December 2022

AbstractEfficient T cell activation requires both TCR signals and costimulatory signals. CD28 is one of the molecules that provide costimulatory signals for T cells. We used mice deficient in CD28 expression (CD28−/− mice) to analyze the role of CD28 in the immune response against the intracellular bacterium Salmonella typhimurium, the causative agent of murine typhoid fever. CD28−/− mice were highly susceptible to infection with wild-type S. typhimurium and even failed to control infection with attenuated aroA− S. typhimurium. More detailed analysis revealed that CD28−/− animals did not mount a T-dependent Ab response and were highly impaired in the production of IFN-γ. Thus, CD28 cosignaling is crucial for immunity against S. typhimurium. To our knowledge, this is the first report describing an essential role for CD28 in protective immunity against an intracellular microbial pathogen.
Protection Against the Mortality Associated with Disease Models Mediated by TNF and IFN-γ in Mice Lacking IFN Regulatory Factor-1
Authors: Senaldi Giorgio , Shaklee Christine L. , Guo Jane , Martin Laura , Boone Thomas , W. Mak Tak , Ulich Thomas R. 
Published: December 2022

AbstractMortality and cytokine production associated with disease models mediated by TNF- and IFN-γ were studied in mice lacking IFN regulatory factor-1 (IRF-1). IRF-1 knockout (KO) mice showed no mortality after the injection of a dose of LPS lethal in intact control mice (LD95). KO mice showed lower circulating levels of TNF and IFN-γ than controls. KO mice also showed lower TNF and IFN-γ mRNA in the spleen or liver than controls. KO mice had smaller spleens than controls, which contained similar percentage but lower absolute count of macrophages and lower percentage and absolute count of NK cells. IRF-1 KO mice survived longer than controls after the coinjection of LPS and galactosamine. IRF-1 KO mice also showed less mortality than controls after the injection of Con A and in a model of cerebral malaria. After the injection of a lethal dose of TNF (LD88), mortality was similar between KO and intact mice. Mortality was also similar after the coinjection of two nonlethal doses of TNF and IFN-γ, a lethal combination (LD100). This study shows that the lack of IRF-1 protects against the mortality associated with disease models mediated by TNF and IFN-γ but has no effect on the mortality directly induced by TNF and IFN-γ. The lack of IRF-1 appears to result in impaired production of TNF and IFN-γ, reflecting a down-regulation of gene expression in the liver and spleen as well as a reduction in the number of splenic cells.