bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Polysaccharide-Specific IgG Isotype Responses to IntactStreptococcus pneumoniaeAre T Cell Dependent and Require CD40- and B7-Ligand Interactions
Authors: Wu Zheng-Qi , Vos Quirijn , Shen Yi , Lees Andrew , Wilson Samuel R. , Briles David E. , Gause William C. , Mond James J. , Snapper Clifford M.
Published: December 2022

In vivo Ig responses to soluble, haptenated polysaccharide (PS) Ags are T cell independent and do not require CD40 ligand (CD40L). However, little is known regarding the regulation of in vivo PS-specific Ig responses to intact bacteria. We immunized mice with a nonencapsulated, type 2 Streptococcus pneumoniae (R36A) and compared the parameters that regulated in vivo Ig isotype responses to the bacterial cell wall C-PS determinant, phosphorylcholine (PC), relative to Ig responses to the cell wall protein, pneumococcal surface protein A. Consistent with previous reports using soluble PS and protein Ags, the anti-PC and anti-pneumococcal surface protein A responses differed in that the anti-PC response was induced more rapidly, had a distinctive Ig isotype profile, and failed to demonstrate boosting upon secondary challenge with R36A. However, in contrast to previous studies, the IgG anti-PC response was TCR-αβ+ T cell dependent, required CD40L, and was blocked by administration of CTLA4 Ig. The nature of the T cell help for the anti-PC response had distinct features in that it was only partially blocked by CTLA4 Ig and was dependent upon both CD4+ and CD8+ T cells. Surprisingly, whereas the IgM anti-PC response was largely T cell independent, a strong requirement for CD40L was still observed, suggesting the possibility of an in vivo T cell-independent source for CD40L-dependent help. These data suggest that the regulatory parameters that govern in vivo Ig responses to purified, soluble PS Ags may not adequately account for PS-specific Ig responses to intact bacteria.
Induction of Antitumor Immunity with Dendritic Cells Transduced with Adenovirus Vector-Encoding Endogenous Tumor-Associated Antigens
Authors: Kaplan Johanne M. , Yu Queendy , Piraino Susan T. , Pennington Sarah E. , Shankara Srinivas , Woodworth Lisa A. , Roberts Bruce L.
Published: December 2022

Dendritic cells (DCs) are professional Ag-presenting cells that are being considered as potential immunotherapeutic agents to promote host immune responses against tumor Ags. In this study, recombinant adenovirus (Ad) vectors encoding melanoma-associated Ags were used to transduce murine DCs, which were then tested for their ability to activate CTL and induce protective immunity against B16 melanoma tumor cells. Immunization of C57BL/6 mice with DCs transduced with Ad vector encoding the hugp100 melanoma Ag (Ad2/hugp100) elicited the development of gp100-specific CTLs capable of lysing syngeneic fibroblasts transduced with Ad2/hugp100, as well as B16 cells expressing endogenous murine gp100. The induction of gp100-specific CTLs was associated with long term protection against lethal s.c. challenge with B16 cells. It was also possible to induce effective immunity against a murine melanoma self Ag, tyrosinase-related protein-2, using DCs transduced with Ad vector encoding the Ag. The level of antitumor protection achieved was dependent on the dose of DCs and required CD4+ T cell activity. Importantly, immunization with Ad vector-transduced DCs was not impaired in mice that had been preimmunized against Ad to mimic the immune status of the general human population. Finally, DC-based immunization also afforded partial protection against established B16 tumor cells, and the inhibition of tumor growth was improved by simultaneous immunization against two melanoma-associated Ags as opposed to either one alone. Taken together, these results support the concept of cancer immunotherapy using DCs transduced with Ad vectors encoding tumor-associated Ags.
Tumor Infiltration by Adoptively Transferred T Cells Is Independent of Immunologic Specificity but Requires Down-Regulation of L-Selectin Expression
Authors: Kjaergaard Jørgen , Shu Suyu
Published: December 2022

Adoptive immunotherapy with anti-CD3/IL-2 activated tumor-draining lymph node (LN) T cells is capable of eradicating tumor established at various histological sites. Tumor-specific effector lymphocytes have recently been identified to be LN T cells with down-regulated L-selectin (L-selectin−). Using fluorochrome labeling, the present study determined the early trafficking pattern of systemically transferred cells. In mice with 10-day established pulmonary 3-methylcholanthrene (MCA) 205 metastases, accumulation of cells in tumors was evident as early as 2 h after i.v. cell transfer, and, by 24 h, >50-fold higher numbers of cells were seen in metastases than in normal tissues. Similarly, transferred cells selectively infiltrated s.c. tumors, albeit at a lower rate. Analysis of the transferred cells isolated from recipient mice revealed that tumor-infiltrating cells were mostly L-selectin− (>95%). By contrast, only 24% and 58% L-selectin− cells were found in the LN and spleen, respectively. The ability of L-selectin− cells to accumulate at tumor sites was confirmed by the transfer of purified cell populations. Despite this selective tumor infiltration, the trafficking pattern did not reflect antigenetic specificity, and tumor regression occurred only after the transfer of tumor-specific effector cells. These results, thus, suggest that there are two distinct mechanisms operative in successful adoptive immunotherapy. Early infiltration of tumors by transferred cells is dictated by the physiological properties of cells and is independent on their immunologic specificity. Tumor regression, however, requires immunologically specific interactions at the site of tumor.
Complex Regulation of Ly-6E Gene Transcription in T Cells by IFNs
Authors: Khodadoust Mehran M. , Khan Khuda Dad , Bothwell Alfred L. M.
Published: December 2022

The complexity of IFN-mediated regulation of the murine Ly-6E gene in T cell lines is highlighted by the following observations: 1) multiple regulatory regions are present within different parts of the Ly-6E promoter and are necessary for IFN inducibility of the Ly-6E gene, 2) multiple transcription factors including Oct-1 and Oct-2 and the high mobility group (HMG) protein HMGI(Y) bind to regulatory elements present within the G region required for both IFN-αβ and IFN-γ responses, 3) mutational analysis of the G region reveals that a complex interaction exists between the factors binding to this region as shown by their mutual interdependence for detection in DMSA, and 4) inhibition of expression of HMG proteins by antisense HMGI-C RNA in EL4 cells causes the loss of IFN-αβ and IFN-γ inducibility of the endogenous Ly-6 gene. These findings taken together suggest that, in response to IFN treatment, an HMG protein-dependent complex involving multiple regulatory factors is assembled and is required for IFN inducibility of the Ly-6E gene.
Modulation of Terminal Deoxynucleotidyltransferase Activity by the DNA-Dependent Protein Kinase
Authors: Mickelsen Scott , Snyder Carolyn , Trujillo Kelly , Bogue Molly , Roth David B. , Meek Katheryn
Published: December 2022

Rare Ig and TCR coding joints can be isolated from mice that have a targeted deletion in the gene encoding the 86-kDa subunit of the Ku heterodimer, the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). However in the coding joints isolated from Ku86−/− animals, there is an extreme paucity of N regions (the random nucleotides added during V(D)J recombination by the enzyme TdT). This finding is consistent with a decreased frequency of coding joints containing N regions isolated from C.B-17 SCID mice that express a truncated form of the catalytic subunit of the DNA-PK (DNA-PKCS). This finding suggests an unexpected role for DNA-PK in addition of N nucleotides to coding ends during V(D)J recombination. In this report, we establish that TdT forms a stable complex with DNA-PK. Furthermore, we show that DNA-PK modulates TdT activity in vitro by limiting both the length and composition of nucleotide additions.
Neutrophil Defensins Induce Histamine Secretion from Mast Cells: Mechanisms of Action
Authors: Befus A. Dean , Mowat Connie , Gilchrist Mark , Hu Jing , Solomon Samuel , Bateman Andrew
Published: December 2022

Defensins are endogenous antimicrobial peptides stored in neutrophil granules. Here we report that a panel of defensins from human, rat, guinea pig, and rabbit neutrophils all have histamine-releasing activity, degranulating rat peritoneal mast cells with EC50 ranging from 70 to 2500 nM, and between 45 and 60% of the total histamine released. The EC50 for defensin-induced histamine secretion correlates with their net basic charge at neutral pH. There is no correlation between histamine release and antimicrobial potency. Degranulation induced by defensins has characteristics similar to those of activation by substance P. The maximum percent histamine release is achieved in <10 s, and it can be markedly inhibited by pertussis toxin (100 ng/ml) and by pretreatment of mast cells with neuraminidase. These properties differ from those for degranulation induced by IgE-dependent Ag stimulation and by the calcium ionophore A23187. GTPase activity, a measure of G protein activation, was induced in a membrane fraction from mast cells following treatment with defensin. Thus, neutrophil defensins are potent mast cell secretagogues that act in a manner similar to substance P and 48/80, through a rapid G protein-dependent response that is mechanistically distinct from Ag/IgE-dependent mast cell activation. Defensins may provide important pathways for communication between neutrophils and mast cells in defenses against microbial agents and in acute inflammatory responses.
Effects of CD18 Deficiency on the Emigration of Murine Neutrophils During Pneumonia
Authors: Mizgerd Joseph P. , Horwitz Bruce H. , Quillen Henry C. , Scott Martin L. , Doerschuk Claire M.
Published: December 2022

We hypothesized that CD18 deficiency would impair the ability of neutrophils to emigrate from pulmonary blood vessels during certain pneumonias. To directly compare the abilities of wild-type (WT) and CD18-deficient neutrophils to emigrate, mice with both types of leukocytes in their blood were generated by reconstituting the hemopoietic systems of lethally irradiated C57BL/6 mice with mixtures of fetal liver cells from WT and CD18-deficient mice. Percentages of CD18-deficient neutrophils in the circulating and emigrated pools were compared during experimental pneumonias. Similar percentages were observed in the blood and bronchoalveolar lavage fluid 6 or 24 h after intratracheal instillation of Streptococcus pneumoniae, demonstrating that no site on the CD18 molecule was required for either its adhesive or its signaling functions during neutrophil emigration. However, 6 h after instillation of Escherichia coli LPS or Pseudomonas aeruginosa, the percentage of CD18-deficient neutrophils in the bronchoalveolar lavage fluid was only about one-fourth of that observed in the blood. This difference persisted for at least 24 h after instillation of E. coli LPS. Thus, neutrophil emigration elicited by the Gram-negative stimuli E. coli LPS or P. aeruginosa was compromised by deficiency of CD18. These data, based on comparing WT and gene-targeted CD18-deficient neutrophils within the same animals, provide evidence for molecular pathways regulating neutrophil emigration, which could not be appreciated in previous studies with pharmacological blockade or genetic deficiency of CD18.
Cutting Edge: Myelin Basic Protein-Specific Cytotoxic T Cell Tolerance Is Maintained In Vivo by a Single Dominant Epitope in H-2k Mice
Authors: Huseby Eric S. , Öhlén Claes , Goverman Joan
Published: December 2022

Multiple sclerosis (MS) is believed to be an autoimmune disease mediated by T cells specific for CNS Ags. MS lesions contain both CD4+ and CD8+ T lymphocytes. The contribution of CD4+ T cells to CNS autoimmune disease has been extensively studied in an animal model of MS, experimental autoimmune encephalomyelitis. However, little is known about the role of autoreactive CD8+ cytotoxic T cells in MS or experimental autoimmune encephalomyelitis. We demonstrate here that myelin basic protein (MBP) is processed in vivo by the MHC class I pathway leading to a MBP79–87/Kk complex. The recognition of this complex by MBP-specific cytotoxic T cells leads to a high degree of tolerance in vivo. This study is the first to show that the pool of self-reactive lymphocytes specific for MBP contain MHC class I-restricted T cells whose response is regulated in vivo by the induction of tolerance.
Generation of Nitric Oxide by the Inducible Nitric Oxide Synthase Protects γδ T Cells fromMycobacterium tuberculosis-Induced Apoptosis
Authors: Sciorati Clara , Rovere Patrizia , Ferrarini Marina , Paolucci Clara , Heltai Silvia , Vaiani Roberto , Clementi Emilio , Manfredi Angelo A.
Published: December 2022

γδ T cells are early recruited into mycobacterial lesions. Upon microbial Ag recognition, γδ cells secrete cytokines and chemokines and undergo apoptosis via CD95/CD95 ligand (CD95L) interaction, possibly influencing the outcome of infection and the characteristics of the disease. In this paper we show that activated phagocytes acquire, upon challenge with Mycobacterium tuberculosis, the ability to inhibit M. tuberculosis-induced γδ cell apoptosis. Apoptosis protection was due to NO because it correlated with NO synthase (NOS)-2 induction and activity in scavenger cells and was abrogated by NOS inhibitors. Furthermore, the NO donor S-nitrosoacetylpenicillamine mimicked the effect of enzyme induction. NO left unaffected the expression of CD95 and CD95L, suggesting interference with an event ensuing CD95/CD95L interaction. NO was found to interfere with the intracellular accumulation of ceramide and the activation of caspases, which were involved in γδ T cells apoptosis after M. tuberculosis recognition. We propose that NO generated by infected macrophages determines the life span and therefore the function of lymphocytes at the infection site, thus linking innate and adaptive immunity.
Pancreatic IL-4 Expression Results in Islet-Reactive Th2 Cells That Inhibit Diabetogenic Lymphocytes in the Nonobese Diabetic Mouse
Authors: Gallichan W. Scott , Balasa Balaji , Davies Joanna D. , Sarvetnick Nora
Published: December 2022

When immunological tolerance breaks down, autoimmune destruction of insulin-producing β cells in the pancreas can cause insulin-dependent diabetes mellitus. We previously showed that transgenic nonobese diabetic (NOD) mice expressing IL-4 in the pancreas (NOD-IL-4 mice) were protected from insulitis and diabetes. Here we have characterized the avoidance of pathological autoimmunity in these mice. The absence of disease did not result from a lack of T cell priming, because T cells responding to dominant islet Ags were present. These islet Ag-specific T cells displayed a Th2 phenotype, indicating that Th2 responses could account for the observed tolerance. Interestingly, islet Ag-specific Th1 T cells were present and found to be functional, because neutralization of the Th2 effector cytokines IL-4 and IL-10 resulted in diabetes. Histological examination revealed that NOD-IL-4 splenocytes inhibited diabetogenic T cells in cotransfer experiments by limiting insulitis and delaying diabetes. Neutralization of IL-4 in this system abrogated the ability of NOD-IL-4 splenocytes to delay the onset of diabetes. These results indicate that IL-4 expressed in the islets does not prevent the generation of pathogenic islet responses but induces islet Ag-specific Th2 T cells that block the action of diabetogenic T cells in the pancreas.
Cutting Edge: N-Hydroxy Peptides: A New Class of TCR Antagonists
Authors: Hin Sascha , Zabel Claus , Bianco Alberto , Jung Günther , Walden Peter
Published: December 2022

TCR antagonists are altered T cell epitopes that specifically inactivate T cells. Commonly, they are derived from agonists by amino acid side chain replacement at positions accessible to the TCR. In this paper we report for the first time that a main chain N-hydroxylation, which is not exposed at the surface of the MHC peptide complex, renders an agonist into an antagonist. These mimotopes are a new, yet undescribed class of TCR antagonists. The antagonists are about 100 times more potent than an unrelated peptide that competes for binding to the MHC molecule. The novel main chain modification enhances biostability and maintains side chain constitution and thus opens new prospects for the use of TCR antagonists in the treatment of pathological immune reactions.
Macrophage Colony-Stimulating Factor Induces the Expression of Mitogen-Activated Protein Kinase Phosphatase-1 Through a Protein Kinase C-Dependent Pathway
Authors: Valledor Annabel F. , Xaus Jordi , Marquès Laura , Celada Antonio
Published: December 2022

M-CSF triggers the activation of extracellular signal-regulated protein kinases (ERK)-1/2. We show that inhibition of this pathway leads to the arrest of bone marrow macrophages at the G0/G1 phase of the cell cycle without inducing apoptosis. M-CSF induces the transient expression of mitogen-activated protein kinase phosphatase-1 (MKP-1), which correlates with the inactivation of ERK-1/2. Because the time course of ERK activation must be finely controlled to induce cell proliferation, we studied the mechanisms involved in the induction of MKP-1 by M-CSF. Activation of ERK-1/2 is not required for this event. Therefore, M-CSF activates ERK-1/2 and induces MKP-1 expression through different pathways. The use of two protein kinase C (PKC) inhibitors (GF109203X and calphostin C) revealed that M-CSF induces MKP-1 expression through a PKC-dependent pathway. We analyzed the expression of different PKC isoforms in bone marrow macrophages, and we only detected PKCβI, PKCε, and PKCζ. PKCζ is not inhibited by GF109203X/calphostin C. Of the other two isoforms, PKCε is the best candidate to mediate MKP-1 induction. Prolonged exposure to PMA slightly inhibits MKP-1 expression in response to M-CSF. In bone marrow macrophages, this treatment leads to a complete depletion of PKCβI, but only a partial down-regulation of PKCε. Moreover, no translocation of PKCβI or PKCζ from the cytosol to particulate fractions was detected in response to M-CSF, whereas PKCε was constitutively present at the membrane and underwent significant activation in M-CSF-stimulated macrophages. In conclusion, we remark the role of PKC, probably isoform ε, in the negative control of ERK-1/2 through the induction of their specific phosphatase.
Disruption of CD154:CD40 Blocks Generation of Allograft Immunity Without Affecting APC Activation
Authors: Shepherd David M. , Kerkvliet Nancy I.
Published: December 2022

CD154 (CD40 ligand, gp39) interaction with its receptor CD40 has been shown to be critically important for the generation of cell-mediated as well as humoral immunity. It has been proposed that ligation of CD40 on APCs, presumably by activated Th cells, leads to increased APC function as defined by up-regulation of costimulatory molecules and enhancement of IL-12 production. In this report, we directly examined the contribution of the CD154:CD40 pathway in a murine model of allograft rejection. Generation of both the CTL and alloantibody responses following injection with allogeneic P815 tumor cells was severely compromised in CD154 knockout mice and wild-type C57BL/6 mice treated with the anti-CD154 mAb, MR1. Splenic production of IL-2, IFN-γ, and TNF was significantly suppressed from CD154-deficient mice, indicating a lack of T cell priming. However, splenic cells from CD154 knockout mice induced comparable levels of CD86 expression and IL-12 production when compared with their wild-type littermates. The treatment of CD154−/− mice with the agonistic anti-CD40 mAb, FGK45, generated activated APCs yet failed to restore either the CTL or alloantibody responses to P815. Likewise, immunization with B7-transfected P815 tumor cells failed to generate expansion of the CTL effector population in CD154−/− mice. These results suggest that the generation of allograft immunity is dependent on the interaction of CD154 with CD40 but not primarily for the activation of APCs.
Polarized Transport of MHC Class II Molecules in Madin-Darby Canine Kidney Cells Is Directed by a Leucine-Based Signal in the Cytoplasmic Tail of the β-Chain
Authors: Simonsen Anne , Pedersen Ketil Winther , Nordeng Tommy W. , von der Lippe Alexander , Stang Espen , Long Eric O. , Bakke Oddmund
Published: December 2022

MHC class II molecules are found on the basolateral plasma membrane domain of polarized epithelial cells, where they can present Ag to intraepithelial lymphocytes in the vascular space. We have analyzed the sorting information required for efficient intracellular localization and polarized distribution of MHC class II molecules in stably transfected Madin-Darby canine kidney cells. These cells were able to present influenza virus particles to HLA-DR1-restricted T cell clones. Wild-type MHC class II molecules were located on the basolateral plasma membrane domain, in basolateral early endosomes, and in late multivesicular endosomes, the latter also containing the MHC class II-associated invariant chain and an HLA-DM fusion protein. A phenylalanine-leucine residue within the cytoplasmic tail of the β-chain was required for basolateral distribution, efficient internalization, and localization of the MHC class II molecules to basolateral early endosomes. However, distribution to apically located, late multivesicular endosomes did not depend on signals in the class II cytoplasmic tails as both wild-type class II molecules and mutant molecules lacking the phenylalanine-leucine motif were found in these compartments. Our results demonstrate that sorting information in the tails of class II dimers is an absolute requirement for their basolateral surface distribution and intracellular localization.
Signals Transduced by CD3ε, But Not by Surface Pre-TCR Complexes, Are Able to Induce Maturation of an Early Thymic Lymphoma In Vitro
Authors: Carleton Michael , Ruetsch Norman R. , Berger Marc A. , Rhodes Michele , Kaptik Steven , Wiest David L.
Published: December 2022

Development of immature CD4−CD8− (double-negative) thymocytes to the CD4+CD8+ (double-positive) stage is linked to productive rearrangement of the TCRβ locus by signals transduced through the pre-TCR. However, the mechanism whereby pre-TCR signaling is initiated remains unclear, in part due to the lack of an in vitro model system amenable to both biochemical and genetic analysis. In this study, we establish the thymic lymphoma Scid.adh as such a model system. Scid.adh responds to Ab engagement of surface IL-2Ra (TAC):CD3ε molecules (a signaling chimera that mimics pre-TCR signaling in vivo) by undergoing changes in gene expression observed following pre-TCR activation in normal thymocytes. These changes include down-regulation of CD25, recombinase-activating gene (RAG)-1, RAG-2, and pTα; and the up-regulation of TCRα germline transcripts. We term this complete set of changes in gene expression, in vitro maturation. Interestingly, Scid.adh undergoes only a subset of these changes in gene expression following Ab engagement of the pre-TCR. Our findings make two important points. First, because TAC:CD3ε stimulation of Scid.adh induces physiologically relevant changes in gene expression, Scid.adh is an excellent cellular system for investigating the molecular requirements for pre-TCR signaling. Second, Ab engagement of CD3ε signaling domains in isolation (TAC:CD3ε) promotes in vitro maturation of Scid.adh, whereas engagement of CD3ε molecules contained within the complete pre-TCR fails to do so. Our current working hypothesis is that CD3ε fails to promote in vitro maturation when in the context of an Ab-engaged pre-TCR because another pre-TCR subunit(s), possibly TCRζ, qualitatively alters the CD3ε signal.
Lymphoid Transcription of the Murine CD21 Gene Is Positively Regulated by Histone Acetylation
Authors: Zabel Mark D. , Weis Janis J. , Weis John H.
Published: December 2022

Intronic transcriptional control sequences influence the cell- and tissue-specific expression of the CD21 gene. The interactions of such intronic control sequences, which are physically separated from the gene’s promoters, suggest that factors that alter chromatin structure might be influential in this process. Accordingly, we analyzed the effect of histone acetylation on the expression of CD21 in nonexpressing T and B lymphocytes, respectively. The acetylase inhibitors sodium butyrate and trichostatin A were used to create hyperacetylated histones. The CD21 gene was specifically activated in the previously transcriptionally quiescent cells in a time- and dose-dependent fashion: the expression of a number of other genes was not influenced. These data suggest a model of cell-type-specific deacetylase activity that serves to repress gene transcription when present and active.
Biased VH Gene Usage in Early Lineage Human B Cells: Evidence for Preferential Ig Gene Rearrangement in the Absence of Selection
Authors: Rao Sambasiva P. , Riggs Jeffrey M. , Friedman David F. , Scully Michael S. , LeBien Tucker W. , Silberstein Leslie E.
Published: December 2022

Certain VH genes are predominantly expressed in mature B cells. We hypothesized that several, mutually nonexclusive VH-dependent mechanisms operating at distinct stages during B cell development may be responsible for overrepresentation of these VH genes. In the present study, we have assessed whether one of the mechanisms involves preferential rearrangement at the pro-B cell stage. The frequency of individual VH4 and VH3 genes in rearrangement libraries from FACS-purified human CD34+/CD19+ pro-B and CD34−/CD19+ pre-B cells was assessed. The in-frame and out-of-frame rearrangements from both cell populations were analyzed using a high resolution PAGE system. The frequencies of individual VH gene segments among out-of-frame rearrangements from pro-B cells were determined, because these frequencies should reflect only processes before the translation of the μ-heavy chain and should not be biased by selection mechanisms. Our results demonstrate that, at the pro-B cell stage, the V4–34, V4–39, and V4–59 gene segments are the most frequently rearranged VH4 family genes, and the V3–23 and V3–30 gene segments are the most frequently rearranged VH3 family genes. This finding suggests that the predominant expression of these VH genes in peripheral mature B cells is determined to a significant degree by their preferential rearrangement during V-DJ recombination.
Partial Agonist Effect Influences the CTL Response to a Heterologous Dengue Virus Serotype
Authors: Zivny Jaroslav , DeFronzo Matthew , Jarry William , Jameson Julie , Cruz John , Ennis Francis A. , Rothman Alan L.
Published: December 2022

Activation of dengue serotype-cross-reactive memory CTL during secondary dengue virus (DV) infection is thought to be important in the pathogenesis of dengue hemorrhagic fever. To model this effect, we studied the CTL responses to DV types 2 (D2V) and 3 (D3V) in PBMC from an individual previously infected with D3V. DV-specific CD8+ CTL from this donor recognized two HLA-B62-restricted epitopes on the NS3 protein, aa 71–79 (SVKKDLISY) and 235–243 (AMKGLPIRY). Both D3V-specific and D2V/D3V-cross-reactive CTL clones were detected for each epitope; all D2V-reactive CTL clones could lyse D2V-infected autologous cells. CTL responses to both epitopes were detected in bulk cultures stimulated with D3V, but PBMC stimulated with D2V recognized only the 235–243 epitope. IFN-γ enzyme-linked immunospot assay showed that the D2V (71–79) peptide (DVKKDLISY) did not efficiently activate T cells. Analysis of a CTL clone suggests that the D2V (71–79) peptide acts as a partial agonist, able to sensitize target cells for lysis and inducing only minimal proliferation at high concentrations. These results suggest that variant peptide sequences present in the heterologous DV serotype can influence the CTL response in vivo during secondary DV infection.
Altered Immune Responses and Susceptibility toLeishmania majorandStaphylococcus aureusInfection in IL-18-Deficient Mice
Authors: Wei Xiao-qing , Leung Bernard P. , Niedbala Wanda , Piedrafita David , Feng Gui-jie , Sweet Matt , Dobbie Lorraine , Smith Andrew J. H. , Liew Foo Yew
Published: December 2022

IL-18, formerly designated IFN-inducing factor, is a novel cytokine produced by activated macrophages. It synergizes with IL-12 in the induction of the development of Th1 cells and NK cells. To define the biological role of IL-18 in vivo, we have constructed a strain of mice lacking IL-18. Homozygous IL-18 knockout (−/−) mice are viable, fertile, and without evident histopathologic abnormalities. However, in contrast to the heterozygous (+/−) or wild-type (+/+) mice, which are highly resistant to the infection of the protozoan parasite Leishmania major, the IL-18−/− mice are uniformly susceptible. The infected IL-18−/− mice produced significantly lower levels of IFN-γ and larger amounts of IL-4 compared with similarly infected +/− and +/+ mice. In contrast, when infected with the extracellular Gram-positive bacteria Staphylococcus aureus, the IL-18−/− mice developed markedly less septicemia than similarly infected wild-type (+/+) mice. However, the mutant mice developed significantly more severe septic arthritis than the control wild-type mice. This was accompanied by a reduction in the levels of Ag-induced splenic T cell proliferation, decreased IFN-γ and TNF-α synthesis, but increased IL-4 production by the mutant mice compared with the wild-type mice. These results therefore provide direct evidence that IL-18 is not only essential for the host defense against intracellular infection, but it also plays a critical role in regulating the synthesis of inflammatory cytokines, and therefore could be an important target for therapeutic intervention.
A Novel Function of IL-12p40 as a Chemotactic Molecule for Macrophages
Authors: Ha Sang Jun , Lee Chu Hee , Lee Seong Beom , Kim Chang Min , Jang Kyung Lib , Shin Hee Sup , Sung Young Chul
Published: December 2022

IL-12p70 plays a pivotal role in regulating the Th1/Th2 balance in the initial stage of immune responses. In contrast, IL-12p40, which is produced excess over IL-12p70, has been known to down-regulate IL-12p70-mediated responses by acting as an antagonist. To investigate in vivo function of IL-12p40, RH7777 rat hepatoma cells were engineered to inducibly express mouse IL-12p40 under the tight control of doxycycline (dox). In the absence of dox, s.c. injection of these cells into syngeneic rat was shown to generate tumors. However, the induction of IL-12p40 by dox was sufficient for inhibiting tumor formation, as well as for tumor regression. Immunohistochemical analysis showed that macrophages, but not CD4+ T, CD8+ T, and NK cells, were predominantly recruited into tumor sites as early as 3 days after IL-12p40 induction. These results were further supported by the observation that IL-12p40, but not C-terminal deletion mutants by more than 5 amino acids, was able to chemoattract peritoneal macrophages in vitro, suggesting that IL-12p40, when produced in a large excess over IL-12p70 in vivo, can initially amplify the immune responses against tumors by directly recruiting macrophages. Our findings indicate that IL-12p40 may function as an effector molecule as well as an antagonist of IL-12p70.
Role of the Stress Kinase Pathway in Signaling Via the T Cell Costimulatory Receptor 4-1BB
Authors: Cannons Jennifer L. , Hoeflich Klaus P. , Woodgett James R. , Watts Tania H.
Published: December 2022

4-1BB is a member of the TNFR superfamily expressed on activated CD4+ and CD8+ T cells. 4-1BB can costimulate IL-2 production by resting primary T cells independently of CD28 ligation. In this study, we report signaling events following 4-1BB receptor aggregation using an Ak-restricted costimulation-dependent T cell hybridoma, C8.A3. Aggregation of 4-1BB on the surface of C8.A3 cells induces TNFR-associated factor 2 recruitment, which in turn recruits and activates apoptosis signal-regulating kinase-1, leading to downstream activation of c-Jun N-terminal/stress-activated protein kinases (JNK/SAPK). 4-1BB ligation also enhances anti-CD3-induced JNK/SAPK activation in primary T cells. Overexpression of a catalytically inactive form of apoptosis signal-regulating kinase-1 in C8.A3 T cells interferes with activation of the SAPK cascade and with IL-2 secretion, consistent with a critical role for JNK/SAPK activation in 4-1BB-dependent IL-2 production. Given the ability of both CD28 and 4-1BB to induce JNK/SAPK activation, we asked whether hyperosmotic shock, another inducer of this cascade, could function to provide a costimulatory signal to T cells. Osmotic shock of resting primary T cells in conjunction with anti-CD3 treatment was found to costimulate IL-2 production by the T cells, consistent with a pivotal role for JNK/SAPK in T cell costimulation.
Peptide Affinity and Concentration Affect the Sensitivity of M3-Restricted CTLs Induced In Vitro
Authors: Byers Derek E. , Lindahl Kirsten Fischer
Published: December 2022

In vitro stimulation of mouse splenocytes with hemagglutinin (HA) 173–190, a peptide derived from influenza virus hemagglutinin (A/JAP/305/57, H2N2), induces CTLs that are directed to the MHC class Ib molecule, H2-M3. M3 preferably binds peptides bearing an N-terminal formylmethionine. In this study, we show that several related nonformylated peptides can induce anti-HA CTLs in vitro: MLIIW (the minimal epitope), derived from HA186–190 at the C-terminal end of HA173–190; MLIIWG; MLIIWGV; and MLIIWGI, as well as formylated MLIIW. The heptamer peptides correspond to a polymorphism of HA192 in H2 strains of influenza; they have the highest relative affinities for M3 of the nonformylated peptides and higher affinities than some formylated mitochondrial peptides. Depending on the affinity of the peptide, a range of concentrations can be used to induce CTLs. One nanomolar of the high affinity f-MLIIW peptide can induce anti-HA CTLs, whereas 100-fold more of the lower affinity MLIIW peptide is needed. Lines induced with high concentrations (1 μM or greater) of f-MLIIW recognize Ag poorly, and the most efficient CTLs are induced with the lowest concentrations of peptide. Analysis with a panel of anti-TCRVβ Abs shows that different T cells respond to high vs low peptide; the repertoire of cells responding to higher concentrations is more diverse, consistent with the expansion of more, but less efficient, clones. Thus, peptide affinity and concentration should be considered together for generating efficient antipeptide CTLs in vitro.
Human Rheumatoid Factor Production Is Dependent on CD40 Signaling and Autoantigen
Authors: Kyburz Diego , Corr Maripat , Brinson Diana C. , Von Damm Amila , Tighe Helen , Carson Dennis A.
Published: December 2022

High-affinity pathologic rheumatoid factor (RF) B cells occur in autoimmune diseases such as rheumatoid arthritis, but are deleted in healthy individuals. The reasons for the survival and differentiation of these autoreactive B cells in rheumatoid arthritis are not known. Previous studies in mice transgenic for a human IgM RF have shown that peripheral encounter with soluble human IgG leads to deletion of high-affinity RF B cells; however, deletion can be prevented when concomitant T cell help is provided. This study aimed to further discern the minimal factors necessary not only for the in vivo survival of RF B cells, but also for their differentiation into Ab-secreting cells. The combination of MHC class II-reactive T cells and Ag induced the production of RF in human IgM RF transgenic mice, while either stimulus alone was ineffective. Neutralizing Abs against CD40 ligand (CD40L), but not against IL-4 or IL-15, abrogated IgM-RF production. Moreover, blockade of CD40L-CD40 allowed IgG to delete the RF precursor cells. Most importantly, activating Abs to CD40 could substitute entirely for T cell help in promoting the survival of RF precursors and in stimulating RF synthesis in T cell deficient animals. The data indicate that CD40 signaling alone can prevent deletion of RF B cells by Ag and in the presence of IgG is sufficient to trigger RF synthesis. The results suggest that selective induction of apoptosis in high-affinity RF B cells may be achieved by blockade of CD40L-CD40 interaction.
CD40-CD40 Ligand Costimulation Is Required for Generating Antiviral CD4 T Cell Responses But Is Dispensable for CD8 T Cell Responses
Authors: Whitmire Jason K. , Flavell Richard A. , Grewal Iqbal S. , Larsen Christian P. , Pearson Thomas C. , Ahmed Rafi
Published: December 2022

This study documents a striking dichotomy between CD4 and CD8 T cells in terms of their requirements for CD40-CD40 ligand (CD40L) costimulation. CD40L-deficient (−/−) mice made potent virus-specific CD8 T cell responses to dominant as well as subdominant epitopes following infection with lymphocytic choriomeningitis virus. In contrast, in the very same mice, virus-specific CD4 T cell responses were severely compromised. There were 10-fold fewer virus-specific CD4 T cells in CD40L−/− mice compared with those in CD40L+/+ mice, and this inhibition was seen for both Th1 (IFN-γ, IL-2) and Th2 (IL-4) responses. An in vivo functional consequence of this Th cell defect was the inability of CD40L−/− mice to control a chronic lymphocytic choriomeningitis virus infection. This study highlights the importance of CD40-CD40L interactions in generating virus-specific CD4 T cell responses and in resolving chronic viral infection.
Selective Diapedesis of Th1 Cells Induced by Endothelial Cell RANTES
Authors: Kawai Toshihisa , Seki Makoto , Hiromatsu Kenji , Eastcott Jean W. , Watts Gerald F. M. , Sugai Motoyuki , Smith Daniel J. , Porcelli Steven A. , Taubman Martin A.
Published: December 2022

Differentiated CD4 T cells can be divided into Th1 and Th2 types based on the cytokines they produce. Differential expression of chemokine receptors on either the Th1-type or the Th2-type cell suggests that Th1-type and Th2-type cells differ not only in cytokine production but also in their migratory capacity. Stimulation of endothelial cells with IFN-γ selectively enhanced transmigration of Th1-type cells, but not Th2-type cells, in a transendothelial migration assay. Enhanced transmigration of Th1-type cells was dependent on the chemokine RANTES produced by endothelial cells, as indicated by the findings that Ab neutralizing RANTES, or Ab to its receptor CCR5, inhibited transmigration. Neutralizing Ab to chemokines macrophage-inflammatory protein-1α or monocyte chemotactic protein-1 did not inhibit Th1 selective migration. Whereas anti-CD18 and anti-CD54 blocked basal levels of Th1-type cell adherence to endothelial cells and also inhibited transmigration, anti-RANTES blocked only transmigration, indicating that RANTES appeared to induce transmigration of adherent T cells. RANTES seemed to promote diapedesis of adherent Th1-type cells by augmenting pseudopod formation in conjunction with actin rearrangement by a pathway that was sensitive to the phosphoinositol 3-kinase inhibitor wortmannin and to the Rho GTP-binding protein inhibitor, epidermal cell differentiation inhibitor. Thus, enhancement of Th1-type selective migration appeared to be responsible for the diapedesis induced by interaction between CCR5 on Th1-type cells and RANTES produced by endothelial cells. Further evidence that CCR5 and RANTES play a modulatory role in Th1-type selective migration derives from the abrogation of this migration by anti-RANTES and anti-CCR5 Abs.
The Mode of Ligand Recognition by Two Peptide:MHC Class I-Specific Monoclonal Antibodies
Authors: Messaoudi Ilhem , LeMaoult Joël , Nikolić-Z̆ugić Janko
Published: December 2022

The Ig superfamily members TCR and B cell receptor (BCR) share high structural and amino acid homology, yet interact with Ags in a distinct manner. The overall shape of the TCR ligand is rather constant, with the variation coming from the MHC polymorphism and the peptide heterogeneity. Consequently, the TCR α- and β-chains form a relatively flat ligand-binding site that interacts with the peptide:MHC (pep:MHC) ligand in a fixed diagonal orientation relative to the MHC α-helices, with the α- and β-chains of the TCR contacting the N and C termini of the pep:MHC complex, respectively. By contrast, the shape of BCR ligands varies dramatically, and the BCR exhibits much greater variability of the Ag-binding site. The mAbs 25-D1.16 (D1) and 22-C5.9 (C5), specific for the OVA-8:H-2Kb complex, allowed us to directly compare how TCR and BCR approach the same ligand. To that effect, we mapped D1 and C5 footprints over the OVA-8:H-2Kb complex. Using peptide variants and mutant MHC molecules, we show that the D1 and C5 contacts with the OVA-8:Kb complex C terminus overlap with the TCR β-chain footprint, but that this footprint also extends to the regions of the molecule not contacted by the TCR. These studies suggest that D1 and C5 exhibit a hybrid mode of pep:MHC recognition, in part similar to that of the TCR β-chain and in part similar to the conventional anti-MHC Ab.
Role of CD28 in the Generation of Effector and Memory Responses Required for Resistance toToxoplasma gondii
Authors: Villegas Eric N. , Elloso M. Merle , Reichmann Gaby , Peach Robert , Hunter Christopher A.
Published: December 2022

CD28 deficient (CD28−/−) mice were used to study the role of costimulation in the T cell-mediated, IFN-γ-dependent mechanism of resistance to Toxoplasma gondii. These mice were resistant to infection with the ME49 strain of T. gondii. Analysis of the immune response of acutely infected CD28−/− mice revealed that IL-12 was required for T cell production of IFN-γ and this was independent of the CD40/CD40 ligand interaction. A similar mechanism of IL-12-dependent, CD28/B7 independent production of IFN-γ by T cells was also observed in wild-type mice. Interestingly, although chronically infected wild-type mice were resistant to rechallenge with the virulent RH strain of T. gondii, chronically infected CD28−/− mice were susceptible to rechallenge with the RH strain. This deficiency in the protective memory response by CD28−/− mice correlated with a lack of IL-2 and IFN-γ in recall responses and reduced numbers of CD4+ T cells expressing a memory phenotype. Together, our findings demonstrate that CD28 is not required for the development of a protective T cell response to T. gondii, but CD28 is required for an optimal secondary immune response.
Receptor-Mediated Modulation of Murine Mast Cell Function by α-Melanocyte Stimulating Hormone
Authors: Adachi Shiro , Nakano Teruaki , Vliagoftis Harrisios , Metcalfe Dean D.
Published: December 2022

The proopiomelanocortin (POMC)-derived neuropeptide α-melanocyte stimulating hormone (α-MSH) is known to modulate some aspects of inflammation through direct effects on T cells, B cells, and monocytes. To determine whether α-MSH might similarly influence mast cell responsiveness, mast cells were examined to see if they expressed the receptor for α-MSH, melanocortin-1 (MC-1), and whether α-MSH altered mast cell function. We thus first identified MC-1 on bone marrow cultured murine mast cells (BMCMC) and a murine mast cell line (MCP-5) employing flow cytometry and through detection of specific binding. Subsequent treatment of mast cells with α-MSH increased the cAMP concentration in a characteristic biphasic pattern, demonstrating that α-MSH could affect intracellular processes. We next examined the effect of α-MSH on mediator release and cytokine expression. IgE/DNP-human serum albumin-stimulated histamine release from mast cells was inhibited by ∼60% in the presence of α-MSH. Although activation of BMCMC induced the expression of mRNAs for the inflammatory cytokines IL-1β, IL-4, IL-6, TNF-α, and the chemokine lymphotactin, mRNAs for IL-1β, TNF-α, and lymphotactin were down-modulated in the presence of α-MSH. Finally, IL-3-dependent proliferative activity of BMCMC was slightly but significantly augmented by α-MSH. Taken together, these observations suggest that α-MSH may exert an inhibitory effect on the mast cell-dependent component of a specific inflammatory response.
Fc-Mediated Nonspecific Binding Between Fibronectin-Binding Protein I ofStreptococcus pyogenesand Human Immunoglobulins
Authors: Medina Eva , Molinari Gabriella , Rohde Manfred , Haase Bernd , Chhatwal Gursharan S. , Guzmán Carlos A.
Published: December 2022

Fibronectin-binding protein I (SfbI) from Streptococcus pyogenes plays a key role in bacterial adhesion to, and invasion of, eukaryotic cells. In addition, SfbI exhibits a considerable potential as mucosal adjuvant and can trigger polyclonal activation of B cells. Here, we report that SfbI is also capable of binding human IgG in a nonimmune fashion. SfbI was reactive with IgG1, IgG2, IgG3, and IgG4 isotypes (type IIo IgG-binding profile). The affinity constant (Kd) of the SfbI-IgG interaction was in the range of 1–2 × 10−5 M. Further studies demonstrated that the SfbI binding was mediated by the Fc component of the IgG molecule. Experiments performed using purified recombinant proteins spanning different domains of SfbI showed that the IgG-binding activity was restricted to the fibronectin-binding domains, and in particular to the fibronectin-binding repeats. Finally, the presence of recombinant SfbI resulted in an impairment of both phagocytosis of IgG-coated RBCs and Ab-dependent cell cytotoxicity by macrophages. These results demonstrated for the first time that, in addition to its major role during the colonization process, SfbI may also favor bacterial immune evasion after the onset of the infection by interfering with host clearance mechanisms.
Molecular Mechanisms Underlying IL-4-Induced Leukocyte Recruitment In Vivo: A Critical Role for the α4 Integrin
Authors: Hickey Michael J. , Granger D. Neil , Kubes Paul
Published: December 2022

IL-4 is known to induce recruitment of eosinophils and mononuclear leukocytes. In vitro this occurs in part by selective expression of VCAM-1, the ligand for the α4 integrin. The objective of this study was to determine the molecular mechanisms that underlie IL-4-induced leukocyte recruitment in vivo. Mice received an intrascrotal injection of IL-4 (100 ng). Twenty-four hours later, leukocyte rolling, adhesion, and emigration in cremasteric postcapillary venules were examined via intravital microscopy, and expression of VCAM-1 and P- and E-selectin was quantitated using a radiolabeled mAb technique. IL-4 increased VCAM-1 expression, but P-selectin and E-selectin remained at constitutive levels. IL-4 induced significant increases in leukocyte adhesion and emigration, with 50% of the emigrated cells being eosinophils and the remainder being mononuclear leukocytes. Leukocyte rolling in IL-4-treated mice was >95% inhibitable using an anti-P-selectin Ab. However, IL-4-induced leukocyte recruitment was unaltered in mice treated chronically with P-selectin Ab or mice deficient in either P-selectin or P- and E-selectin, suggesting that the residual rolling supported all of the IL-4-induced recruitment. In IL-4-treated mice following P-selectin blockade, tethering and rolling were not dependent on L-selectin, but were abolished by α4 integrin blockade. These findings show that the α4 integrin can initiate leukocyte-endothelial cell interactions in the absence of selectins under shear conditions in vivo, and that the absence of selectins does not affect recruitment of eosinophils and mononuclear cells to IL-4-treated tissue.