bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Presentation of the Same MHC Class I Epitopes by Fibroblasts and Dendritic Cells
Authors: Butz Eric A. , Bevan Michael J.
Published: January 2023

Ag is presented to CTL as peptide associated with MHC class I molecules, which are present on most types of cells. We have investigated the presentation of Db-restricted lymphocytic choriomeningitis virus (LCMV) peptides by a fibroblast line (MC57) and a dendritic cell line (JawsII) to splenocytes from LCMV-immune C57BL/6 mice. We found that when LCMV-infected MC57 were used to restimulate the spleen cells, the resulting CTL line lost its ability to respond to the two dominant epitopes of the immune response to LCMV glycoprotein (gp)33 and nucleoprotein (np)396 but remained strongly lytic for targets coated with the subdominant gp276 epitope. In contrast, when LCMV-infected JawsII cells were used to restimulate the splenocytes, the resulting line continued to target gp33 and np396 but lost reactivity to gp276. When uninfected JawsII or MC57 cells were coated with peptides and used as stimulators, the resulting CTL lines continued to recognize all three epitopes, indicating that costimulatory or other potential innate differences in Ag presentation between the two cell lines are unlikely to account for the selective expansion of CTL specificities. When infected, both cell types produce similar levels of infectious LCMV, have similar levels of the NP and GP proteins from which np396 and gp33 are derived, and can be recognized by CTL specific for each of the three epitopes. These data indicate that in the generation of peptides for MHC-I binding and presentation to CTL, MC57 and JawsII process the same set of virus proteins in quantitatively different ways.
Mutation of Proline 211 Reduces Shedding of the Human p75 TNF Receptor
Authors: Herman Charlotte , Chernajovsky Yuti
Published: January 2023

We have identified a residue necessary for the cleavage of human p75 TNF-R from the cell surface by deletion and mutagenesis analysis of the membrane-proximal domain between amino acids 147 and 221. Deletion analysis of this area showed that residues between amino acids 207 and 216 are required for shedding. Site-directed mutagenesis of proline 211 to glycine reduced PMA-induced shedding of human p75 TNF-R from COS-7 or Raw 264.7 cells. Mutation of glycine 210 to aspartic acid did not affect receptor shedding. Mutation of serine 212 to leucine did not affect the PMA-induced shedding from the surface of COS-7 cells, but reduced the efficiency of shedding from the surface of Raw 264.7 macrophages by PMA or LPS. Deletion of putative elastase cleavage sites at amino acids 151 to 153, 162 to 163, and 177 to 178 (also a putative metalloprotease site similar to the cleavage site of TNF-α) or mutation of a serine-threonine-serine motif resembling a similar motif at the site of L-selectin cleavage at amino acids 202 to 204 did not reduce shedding of p75 TNF-R after treatment of cells with PMA. This work shows that a single amino acid mutation at proline 211 of human p75 TNF-R can prevent shedding from the cell surface, and that deletion of other previously proposed putative cleavage sites of the human p75 TNF-R does not prevent its shedding.
Mouse CD1-Autoreactive T Cells Have Diverse Patterns of Reactivity to CD1+ Targets
Authors: Brossay Laurent , Tangri Shabnam , Bix Mark , Cardell Susanna , Locksley Richard , Kronenberg Mitchell
Published: January 2023

Humans and mice contain significant populations of T cells that are reactive for autologous CD1 molecules. Using a panel of five mouse CD1 (mCD1)-autoreactive T cell hybridomas, we show here that this autoreactivity does not correlate with the level of CD1 expression. In some cases, these autoreactive T cells can distinguish between different cell types that express the same CD1 molecule, suggesting that some factor in addition to CD1 expression is critical for autoreactive T cell stimulation. To determine whether a CD1-bound ligand may be required, we expressed mutant mCD1 molecules that are defective for the putative endosomal localization sequence in the cytoplasmic domain. We demonstrate that mCD1, like its human CD1 homologues, is found in endosomes, and that it colocalizes extensively with the DM molecule. We further demonstrate, by site-directed mutagenesis, that the tyrosine in the cytoplasmic sequence is required for this endosomal localization. A T cell hybrid expressing Vβ8 and Vα14, the major TCR expressed by NK1+ T cells, exhibited greatly diminished reactivity to mutant CD1 molecules that do not traffic through endosomes, although the reactivity of other T cell hybrids to this mutant was not greatly affected. Therefore, we propose that at least some of the autoreactive T cells require endosomally derived CD1-bound ligands, and that they are capable of distinguishing between a diverse set of such self-ligands, which might be either autologous lipoglycans or peptides.
Induction of a Th1 Immune Response and Simultaneous Lack of Activation of a Th2 Response Are Required for Generation of Immunity to Leishmaniasis
Authors: Sjölander Anders , Baldwin Tracey M. , Curtis Joan M. , Handman Emanuela
Published: January 2023

Experimental systems based on immunization with plasmid DNA or immune-stimulating complexes were used to delineate the requirements for generation of protective immunity against murine leishmaniasis. Vaccination with plasmid DNA encoding the host-protective Leishmania major parasite surface Ag-2 primed for an essentially exclusive Th1 response that protected mice against L. major infection. In contrast, parasite surface Ag-2 in immune-stimulating complexes generated an immune response with mixed Th1-like and Th2-like properties that was not protective despite the activation of large numbers of CD4+ T cells secreting IFN-γ. These results indicate that a Th1 response is sufficient to protect against cutaneous leishmaniasis, but the induction of a simultaneous Th2 response abrogates the Th1 effector function. DNA vaccines may therefore have an advantage for diseases in which protection depends on the induction of Th1 responses.
Immunopathology of Herpetic Stromal Keratitis: Discordance in CD4+ T Cell Function Between Euthymic Host and Reconstituted SCID Recipients
Authors: Thomas Johnson , Rouse Barry T.
Published: January 2023

Infection of the mouse cornea with herpes simplex virus (HSV) results in an immunopathologic disease of the eye termed herpetic stromal keratitis (HSK), in which the principal orchestrator is the CD4+ T cell. The mouse genotype largely determines susceptibility or resistance to HSK. BALB/c mice (H2dIgh-1a) are susceptible, while its congenic C.B-17 strain (H2dIgh-1b), which differs only in the Ig heavy chain locus, is resistant to HSK. As the magnitude and duration of viral replication as well as anti-HSV immune responses were similar in both strains, it was determined whether resistance was due to failure of CD4+ T cells to organize the immunopathologic reaction. Adoptive transfer of HSV-primed or naive CD4+ T cells from resistant C.B-17 strain into HSV-infected SCID mice resulted in HSK lesions indistinguishable from those caused by similar transfers of BALB/c CD4+ T cells. Similar results were obtained with transfers of whole T cell populations as well as with unfractionated splenocytes from the resistant mice. These results show that while intact C.B-17 mice exhibit resistance to HSK, they possess potentially pathogenic CD4+ T cells in their repertoire. The data suggest that the HSV-infected SCID mouse provides a proinflammatory microenvironment that overrides regulatory controls and/or cause activation of quiescent cells into aggressive effector T cells that orchestrate HSK.
Hydrogen Peroxide Induces Up-Regulation of Fas in Human Endothelial Cells
Authors: Suhara Toshimitsu , Fukuo Keisuke , Sugimoto Tomosada , Morimoto Shigeto , Nakahashi Takeshi , Hata Shigeki , Shimizu Masumi , Ogihara Toshio
Published: January 2023

Hydrogen peroxide (H2O2), an oxidant generated by inflammatory cells, is an important mediator of injury of endothelial cells (ECs). Here we show that H2O2 induces up-regulation of the expression of Fas, a death signal, in human ECs in culture. Flow cytometric analysis with a mAb against human Fas showed that incubation for 24 h with H2O2 induced a dose-dependent increase in the level of Fas in ECs. Coincubation with catalase, which rapidly degrades H2O2, inhibited H2O2-induced up-regulation of Fas. H2O2 also induced a dose-dependent increase in Fas mRNA level. A significant increase in Fas mRNA levels was observed from 6 h after stimulation with H2O2. Vanadate, a protein phosphatase inhibitor, significantly enhanced Fas mRNA and protein levels in H2O2-treated ECs. On the other hand, genistein, a tyrosine kinase inhibitor, inhibited H2O2-induced Fas mRNA expression. Furthermore, a flow cytometric method with propidium iodide staining and electron microscopic analysis showed that incubation with an agonistic Ab against Fas (anti-Fas IgM) induced apoptosis in H2O2-treated cells. These findings suggest that H2O2 induces up-regulation of Fas in ECs and that activation of protein tyrosine kinase may be involved in the mechanism of H2O2-induced Fas expression. Therefore, Fas-mediated apoptosis may have a pathologic role in H2O2-induced EC injury and thereby provide a new therapeutic target.
Differential Requirements for LFA-1 Binding to ICAM-1 and LFA-1-Mediated Cell Aggregation
Authors: Petruzzelli Lilli , Maduzia Lisa , Springer Timothy A.
Published: January 2023

Cellular adhesion through the β2 integrin lymphocyte function-associated Ag (LFA)-1 is a complex event involving activation, ligand binding, and cell shape changes that ultimately result in enhanced adhesion. In this report we define requirements for ligand binding and post receptor signaling by comparing two mechanisms of activation of LFA-1: 1) inside-out signaling and 2) direct activation by the β2 Ab, CBR LFA-1/2. Our results demonstrate that activation of LFA-1 binding to ICAM-1 by CBR LFA-1/2, in contrast to inside-out signaling mechanisms, does not require protein kinase C activation or protein phosphatase 2A activity nor is it affected by agents that interfere with reorganization of the cytoskeleton. Inhibition of protein tyrosine kinase activity does not affect ICAM-1 binding by either mechanism of activation. However, activation by either mode does require the presence of the β cytoplasmic domain; deletion of the C-terminal phenylalanine or the five amino acid stretch between 756–762 abolished activation of LFA-1. This, combined with the observation that intracellular energy pools must be preserved, implicates the β cytoplasmic domain in a key energy-dependent conformational change in LFA-1 that is required to achieve enhanced ligand binding. Post ligand binding events induced by both PMA and Ab stimulation, as measured by homotypic aggregation, require protein tyrosine kinase, phosphatase, and RhoA activities. By examining both ligand binding and aggregation, we have been able to dissect the signaling components critical in the multistep process of LFA-1-mediated cellular adhesion.
Involvement of CD40 Ligand-CD40 and CTLA4-B7 Pathways in Murine Acute Graft-Versus-Host Disease Induced by Allogeneic T Cells Lacking CD28
Authors: Saito Kiyoshi , Sakurai Jinkyo , Ohata Junko , Kohsaka Takao , Hashimoto Hiroshi , Okumura Ko , Abe Ryo , Azuma Miyuki
Published: January 2023

The blockade of B7, using B7 antagonists such as anti-CD80 and/or -CD86 mAbs or CTLA4Ig in vivo, has been shown to induce an efficient suppression of T cell-mediated immune responses in allograft, allergy, and autoimmune models. However, this treatment does not result in complete tolerance. In this study, we examined CD28-B7-independent activation pathways in the pathogenesis of graft-vs-host disease (GVHD) using allogeneic T cells from CD28-deficient mice. Acute GVHD was induced in the absence of CD28 on donor T cells and its manifestations were obvious in the lymphoid tissues. The CD28-independent GVHD was significantly improved by treatment with anti-CD40 ligand (CD40L) mAb. In contrast, treatment with anti-CD80 plus anti-CD86 mAbs exacerbated the clinical manifestations of GVHD and increased the T cell response against host alloantigen, resulting in the expression of CTLA4, CD40L, and CD25 on splenic T cells. These data suggested that the CD40L-CD40 pathway significantly contributed to the CD28-independent pathogenesis of acute GVHD, whereas the CTLA4-B7 pathway acted protectively in the development of GVHD. These results imply that selectively blockading CD28, instead of disrupting both CD28 and CTLA4, would be a better therapeutic strategy for GVHD. Additionally, the simultaneous use of CD40 antagonists may be advantageous.
Promiscuous Binding of Synthetic Copolymer 1 to Purified HLA-DR Molecules
Authors: Fridkis-Hareli Masha , Strominger Jack L.
Published: January 2023

Copolymer 1 (Cop 1) is a random synthetic amino acid copolymer of l-alanine, l-glutamic acid, l-lysine, and l-tyrosine, effective both in suppression of experimental allergic encephalomyelitis and in the treatment of relapsing forms of multiple sclerosis. Cop 1 binds promiscuously and very efficiently to living APCs of various HLA haplotypes. In the present study, a substantial part of the whole mixture of random polypeptides that compose Cop 1 was shown to bind to purified human HLA-DR1, DR2, and DR4 with high affinity in a temperature- and time (and, in the case of DR4, pH)-dependent manner, and was competitively inhibited by DR-restricted peptides, but not by peptide derivatives that bind with low affinity. Bacterial superantigens inhibited Cop 1 binding only at very high concentrations. The formation of the Cop 1-DR1 complex was also shown by SDS-PAGE. These findings represent the first direct evidence for interactions of Cop 1 with purified DR molecules, and suggest that its effectiveness in experimental allergic encephalomyelitis and multiple sclerosis may be directly related to its binding in the groove of HLA-DR proteins.
IL-10 Regulates Liver Pathology in Acute MurineSchistosomiasis mansoniBut Is Not Required for Immune Down-Modulation of Chronic Disease
Authors: Wynn Thomas A. , Cheever Allen W. , Williams Megan E. , Hieny Sara , Caspar Pat , Kühn Ralf , Müller Werner , Sher Alan
Published: January 2023

We have used IL-10 gene knockout mice (IL-10T) to examine the role of endogenous IL-10 in the down-modulation of hepatic granuloma formation and lymphocyte responses that occurs in chronic infection with the helminth parasite Schistosoma mansoni. Although IL-10-deficient animals showed 20 to 30% mortality between 8 and 14 wk postinfection, they displayed no alterations in their susceptibility to infection and produced similar numbers of eggs as their wild-type littermates. The IL-10T mice displayed a significant increase in hepatic granuloma size at the acute stage of infection, which was associated with increased IFN-γ, IL-2, IL-1β, and TNF-α mRNA expression in liver and elevated Th1-type cytokine production by lymphoid cells. Despite developing an enhanced Th1-type cytokine response, the IL-10T mice showed no consistent decrease in their Th2-type cytokine profile. Surprisingly, although granulomatous inflammation was enhanced at the acute stage of infection, the livers of IL-10T mice displayed no significant increase in fibrosis and underwent normal immune down-modulation at the chronic stage of infection. Moreover, the down-modulated state could be induced in IL-10T mice by sensitizing the animals to schistosome eggs before infection, further demonstrating that the major down-regulatory mechanism is not dependent upon IL-10. We conclude that while IL-10 plays an important role in controlling acute granulomatous inflammation, it plays no essential role in the process of immune down-modulation in chronic schistosome infection.
Enhanced Expression of B7.2 (CD86) in Patients with Atopic Dermatitis: A Potential Role in the Modulation of IgE Synthesis
Authors: Jirapongsananuruk Orathai , Hofer Michaël F. , Trumble Anne E. , Norris David A. , Leung Donald Y. M.
Published: January 2023

Recent studies have suggested that the accessory molecules B7.1 (CD80) and B7.2 (CD86) differ in their capacity to generate Th1 vs Th2 responses. Atopic dermatitis (AD) is a chronic allergic skin disease associated with increased IgE synthesis. To determine the potential role of B7.2 molecules in AD, the present study was conducted to compare the expression of B7.1 vs B7.2 on B cells from patients with AD vs normal subjects or patients with psoriasis. The expression of B7.2 on B cells of AD patients (53.67 ± 3.10%) was significantly higher than normals (38.02 ± 4.95%; p = 0.02) and psoriasis patients (40.19 ± 2.70%; p = 0.006). In contrast, there was no significant difference in B7.1 expression among the three subject groups. Interestingly, total serum IgE from AD patients and normal subjects correlated significantly with B7.2 expression on B cells (r = 0.68; p = 0.004), suggesting a role for B7.2+ B cells in IgE synthesis. Indeed, purified B7.2+ B cells produced significantly more IgE than B7.2− B cells in vitro (p = 0.04). Anti-human B7.2, but not B7.1, mAb significantly (p < 0.05) decreased IgE production by PBMC stimulated with IL-4 and anti-CD40 mAb. Furthermore, B7.2+ B cells had a significantly higher level of IL-4R and CD23 expression than B7.1+ B cells. These data demonstrate the predominant expression of B7.2 in AD, but not psoriasis, and a novel role for this molecule in IgE synthesis.
Dystrophin Acts as a Transplantation Rejection Antigen in Dystrophin-Deficient Mice: Implication for Gene Therapy
Authors: Ohtsuka Yoshikazu , Udaka Keiko , Yamashiro Yuichiro , Yagita Hideo , Okumura Ko
Published: January 2023

Duchenne muscular dystrophy is a lethal and common X-linked recessive disease caused by a defect in dystrophin. Normal myoblast transplantation and dystrophin gene transfer have been expected to correct the deficiency in the muscles, but their clinical application has been hampered by the limited preservation of dystrophin-positive myofibers. In this study we investigated the mechanism for immunologic rejection of normal C57BL/10 (B10) myoblasts transplanted into dystrophin-deficient mdx mice, an animal model of Duchenne muscular dystrophy. We found that mdx mice develop CTL specific for dystrophin itself, which were CD8 dominant and restricted by H-2Kb. We identified several antigenic peptides derived from dystrophin that bind to H-2Kb and are recognized by the mdx anti-B10 CTL. Immunologic tolerance against dystrophin was successfully induced by i.v. injection of these peptides before B10 myoblast transplantation, which resulted in sustained preservation of dystrophin-expressing myofibers in mdx mice. These results demonstrate that dystrophin is antigenic in dystrophin-deficient mice and that immunologic regimen would be necessary to achieve the persistent expression of introduced dystrophin in the muscles of dystrophin-deficient individuals.
Differences in the Immune Response During the Acute Phase of E-55+ Murine Leukemia Virus Infection in Progressor BALB and Long Term Nonprogressor C57BL Mice
Authors: Panoutsakopoulou Vily , Little C. Scott , Sieck Thomas G. , Blankenhorn Elizabeth P. , Blank Kenneth J.
Published: January 2023

E-55+ murine leukemia virus infection of both progressor (BALB) and long term nonprogressor (C57BL) mouse strains is characterized by an acute and a persistent phase of infection. During the acute phase, progressor strains require CD8+ T cells to decrease virus burden, whereas the long term nonprogressor strains do not. In the present studies the immune response in BALB and C57BL mice during the acute phase of E-55+ murine leukemia virus infection was examined. The results demonstrate that BALB mice produce both IL-4 and IFN-γ, in contrast to C57BL mice, which produce only IFN-γ. In BALB mice, IL-4 production results in the absolute requirement for CD8+ T cells to reduce the virus burden during the acute phase of infection. The anti-virus immune response in these mice is IFN-γ dependent. On the other hand, C57BL mice do not produce IL-4 and, in the absence of both CD8+ T cells and IFN-γ, still generate an effective anti-virus immune response. Genetic studies suggest that these distinct immune responses are regulated by more than one non-MHC-linked gene. Two candidate regions that may encode this gene(s), located on chromosomes 7 and 19, respectively, were identified by recombinant inbred strain linkage analysis.
The Cytoplasmic Domain of CD8β Regulates Lck Kinase Activation and CD8 T Cell Development
Authors: Irie Hanna Yoko , Mong Mimi S. , Itano Andrea , Crooks M. E. Casey , Littman Dan R. , Burakoff Steven J. , Robey Ellen
Published: January 2023

Previous studies have shown that CD8β plays a role in both enhancing CD8α-associated Lck kinase activity and promoting the development of CD8-lineage T cells. To examine the role of this enhancement in the maturation of CD8-lineage cells, we assessed CD8α-associated Lck kinase activity in both T cell hybridomas and thymocytes of mice expressing CD8β mutations known to impair CD8 T cell development. Lack of CD8β expression or expression of a cytoplasmic domain-deleted CD8β resulted in a severalfold reduction in CD8α-associated Lck kinase activity compared with that observed with cells expressing wild-type CD8β chain. This analysis indicated a critical role for the cytoplasmic domain of CD8β in the regulation of CD8α-associated Lck activity. Decreased CD8α-associated Lck activity observed with the various CD8β mutations also correlated with diminished in vivo cellular tyrosine phosphorylation. In addition, analysis of CD8β mutant mice (CD8β−/− or cytoplasmic domain-deleted CD8β transgenic) indicated that the degree of reduction in CD8α-associated Lck activity associated with each mutation correlated with the severity of developmental impairment. These results support the importance of CD8β-mediated enhancement of CD8α-associated Lck kinase activity in the differentiation of CD8 single-positive thymocytes.
Fas/Fas Ligand Interactions Are Involved in Ultraviolet-B-Induced Human Lymphocyte Apoptosis
Authors: Caricchio Roberto , Reap Elizabeth A. , Cohen Philip L.
Published: January 2023

We wondered whether the apoptosis known to occur after UV-B irradiation might involve the Fas/Fas ligand (FasL) signaling pathway. We exposed PBLs from normal individuals, and also the Jurkat (E6-1) and U937 cell lines, to graded doses of UV-B irradiation and observed a prompt and marked increase in Fas expression at doses as low as 0.5 mJ/cm2. Increased Fas expression did not require new protein synthesis, since cycloheximide-treated cells also showed an increase in Fas after UV-B. UV-B-irradiated cells cultured in the presence of zinc showed inhibition of apoptosis coincident with a marked increase in Fas+ cells, apparently indicating the accumulation of Fas-bearing cells unable to undergo apoptosis. After UV-B irradiation, PBLs showed increased expression of Fas ligand; the E6-1 lymphocytic cell line also released soluble FasL. UV-B induced apoptosis could be partially blocked by neutralizing FasL Abs, and a FasL-resistant variant of E6-1 cell line showed reduced apoptosis after UV-B irradiation, implying that the increase in Fas expression signified a role for Fas in UV-induced apoptosis. UV-induced Fas expression may serve to target stress-injured cells for removal by FasL-bearing cells or by FasL produced by the cells themselves in response to the stimuli, and may represent a general function of the Fas/FasL pathway in facilitating the apoptosis and elimination of undesirable or harmful cells.
Role of μ Heavy Chain in B Cell Development. I. Blocked B Cell Maturation But Complete Allelic Exclusion in the Absence of Igα/β
Authors: Cronin Frank E. , Jiang Ming , Abbas Abul K. , Grupp Stephan A.
Published: January 2023

There is good evidence for a signaling role played by Ig heavy chain in the developmental transition through the pre-B cell stage. We have previously described signal-capable or signal-incapable mutants of μ heavy chain in which a signaling defect is caused by failure to associate with the Igα/β heterodimer. To further characterize the role of Ig heavy chain-mediated signaling in vivo, as well as in B cell development and allelic exclusion, we have created transgenic mice in which the B cells express these signal-capable and signal-incapable mutant μ chains. Failure of μ to signal via Igα/β results in a block in B cell development in mice expressing the signal-incapable μ. A small number of B cells in these animals do escape the developmental block and are expressed in the spleen and the periphery as B220+ transgenic IgM+ cells. These cells respond to LPS by proliferating but show no response to T-independent-specific Ag. In contrast, B cells expressing the signal-capable B cell receptor show a strong signaling response to Ag-specific stimulus. There is no Igα seen in association with signal-deficient IgM. Thus, the B cell receptor complex is not assembled, and no signal can be delivered. Despite the block in developmental signaling, allelic exclusion is complete. There is no detectable coexpression of transgenic IgM and endogenous murine IgM, nor is there rearrangement of the endogenous heavy chain genes. This suggests that differing signaling mechanisms are responsible for the developmental transition and allelic exclusion and thus allows for separate examination of these signaling mechanisms.
Expression and Characterization of Recombinant Subunits of Human Complement Component C8: Further Analysis of the Function of C8α and C8γ
Authors: Schreck Steven F. , Plumb Mnason E. , Platteborze Peter L. , Kaufman Kenneth M. , Michelotti Gregory A. , Letson Carole S. , Sodetz James M.
Published: January 2023

Human C8 is composed of three nonidentical subunits (C8α, C8β, and C8γ) that are encoded in separate genes. In C8 isolated from serum, these are arranged as a disulfide-linked C8α-γ dimer that is noncovalently associated with C8β. In this study, a recombinant form of C8α-γ was expressed independently of C8β in insect cells and COS-7 cells and was shown to be equivalent to serum-derived C8α-γ with respect to its ability to combine with C8β and form functional C8. Also expressed separately were mutant (mut) forms of C8α and C8γ in which the single interchain disulfide bond was eliminated. MutC8α exhibited the ability to combine with C8β and express hemolytic activity, although at a lower level than human C8. Addition of purified mutC8γ increased this activity, presumably by binding to mutC8α. A possible role for C8γ as a retinol binding protein was also investigated. Absorbance spectroscopy and fluorescence emission and quenching revealed no specific binding of retinol to mutC8γ. Together, these results indicate that 1) the biosynthesis and secretion of C8α-γ is not dependent on C8β, which is consistent with in vivo observations in C8β-deficient humans; 2) C8α can be synthesized independently of C8γ; therefore, protection of C8α from premature membrane interactions during biosynthetic processing is not a likely function of C8γ; 3) C8γ enhances but is not required for expression of C8 activity; and 4) C8γ does not bind retinol; therefore, it cannot function as a retinol transport protein.
A Longitudinal Study of Type-Specific Antibody Responses toPlasmodium falciparumMerozoite Surface Protein-1 in an Area of Unstable Malaria in Sudan
Authors: Cavanagh David R. , Elhassan Ibrahim M. , Roper Cally , Robinson V. Jane , Giha Haider , Holder Anthony A. , Hviid Lars , Theander Thor G. , Arnot David E. , McBride Jana S.
Published: January 2023

Merozoite surface protein-1 (MSP-1) of Plasmodium falciparum is a malaria vaccine candidate Ag. Immunity to MSP-1 has been implicated in protection against infection in animal models. However, MSP-1 is a polymorphic protein and its immune recognition by humans following infection is not well understood. We have compared the immunogenicity of conserved and polymorphic regions of MSP-1, the specificity of Ab responses to a polymorphic region of the Ag, and the duration of these responses in Sudanese villagers intermittently exposed to P. falciparum infections. Recombinant Ags representing the conserved N terminus (Block 1), the conserved C terminus, and the three main types of the major polymorphic region (Block 2) of MSP-1 were used to determine the specificity and longitudinal patterns of IgG Ab responses to MSP-1 in individuals. Abs from 52 donors were assessed before, during, and after malaria transmission seasons for 4 yr. Ags from the Block 1 region were rarely recognized by any donor. Responses to the C-terminal Ag occurred in the majority of acutely infected individuals and thus were a reliable indicator of recent clinical infection. Ags from the polymorphic Block 2 region of MSP-1 were recognized by many, although not all individuals after clinical malaria infections. Responses to Block 2 were type specific and correlated with PCR typing of parasites present at the time of infection. Responses to all of these Ags declined within a few months of drug treatment and parasite clearance, indicating that naturally induced human Ab responses to MSP-1 are short lived.
Lymphocyte Activation Gene-3 (LAG-3) Expression and IFN-γ Production Are Variably Coregulated in Different Human T Lymphocyte Subpopulations
Authors: Scala Enrico , Carbonari Maurizio , Del Porto Paola , Cibati Marina , Tedesco Tiziana , Mazzone Anna Maria , Paganelli Roberto , Fiorilli Massimo
Published: January 2023

We evaluated the relationship between cytokine profile and the expression of the lymphocyte activation gene-3 (LAG-3) in both T cell clones and polyclonal T cell lines; LAG-3 is a CD4-like protein whose expression is reportedly restricted to Th1/0 cells and dependent upon IFN-γ. We found that, while LAG-3 was expressed only by CD4+ T cell clones producing IFN-γ, most CD8+ clones producing IL-4 but not IFN-γ (i.e., with a T cytotoxic-2-like profile) were LAG-3+. The intensity of LAG-3 expression by CD8+ clones correlated with the amount of released IFN-γ, suggesting that this cytokine is not required for expression but rather for the up-regulation of LAG-3. Flow cytometric analyses of polyclonal T cell lines confirmed that LAG-3 could be expressed by both CD4+ and CD8+ cells that did not contain cytoplasmic IFN-γ. In these cell lines, large proportions of CD4+ and CD8+ cells coexpressed LAG-3 and CD30, a putative marker of Th2-like cells. Overall, our data do not support the earlier suggestion that LAG-3 and CD30 are selective markers of T cells with type-1 and type-2 cytokine profiles, respectively.
Polyclonality and Multispecificity of the CTL Response to a Single Viral Epitope
Authors: Ishikawa T. , Kono D. , Chung J. , Fowler P. , Theofilopoulos A. , Kakumu S. , Chisari F. V.
Published: January 2023

The molecular anatomy of an immunodominant, Ld restricted CTL epitope located between residues 28–39 in hepatitis B surface Ag was defined to explore the immunologic constraints on mutational escape from the CTL response during a viral infection. Using a panel of hepatitis B surface Ag residue 28–39-specific CTL clones, the response to this epitope was found to be extremely diverse at the level of TCR fine specificity and β-chain usage. Although each clone recognized shared as well as unique residues within the epitope as TCR contact sites, even the shared residues were recognized differently by different TCRs. Despite these differences, all clones were comparably cytolytic following Ag stimulation and produced similar amounts of antiviral cytokines previously shown to inhibit HBV replication. These results demonstrate that the CTL response to individual viral epitopes can be markedly polyclonal and multispecific, such that mutational inactivation of a single TCR contact site will not usually lead to viral escape from all CTL clones of the same epitope specificity. Given these constraints and the fact that the CTL response is usually directed against several different epitopes during most viral infections, mutational inactivation of a single epitope is not likely to be sufficient to cause viral persistence.
A Limited Role for β-Selection During γδ T Cell Development
Authors: Wilson Anne , MacDonald H. Robson
Published: January 2023

T cells belong to two distinct lineages expressing either αβ or γδ TCR. During αβ T cell development, it is clearly established that productive rearrangement at the TCR β locus in immature precursor cells leads to the expression of a pre-TCR complex. Signaling through the pre-TCR results in the selective proliferation and maturation of TCR β+ cells, a process that is known as β-selection. However, the potential role of β-selection during γδ T cell development is controversial. Whereas PCR-RFLP and sequencing techniques have provided evidence for a bias toward in-frame VDJβ rearrangements in γδ cells (consistent with β-selection), γδ cells apparently develop normally in mice that are unable to assemble a pre-TCR complex due to a deficiency in TCR β or pTα genes. In this report, we have directly addressed the physiologic significance of β-selection during γδ cell development in normal mice by quantitating intracellular TCR β protein in γδ cells and correlating its presence with cell cycle status. Our results indicate that β-selection plays a significant (although limited) role in γδ cell development by selectively amplifying a minor subset of γδ precursor cells with productively rearranged TCR β genes.
Regulation of NF-κB, AP-1, NFAT, and STAT1 Nuclear Import in T Lymphocytes by Noninvasive Delivery of Peptide Carrying the Nuclear Localization Sequence of NF-κB p50
Authors: Torgerson Troy R. , Colosia Ann D. , Donahue John P. , Lin Yao-Zhong , Hawiger Jacek
Published: January 2023

Activation of T lymphocytes by Ags or cytokines results in translocation of the transcription factors NF-κB, AP-1, NFAT, and STAT from the cytoplasm into the nucleus. The first step in the nuclear import process is recognition of a nuclear localization sequence (NLS) within the karyophilic protein by a cytoplasmic receptor such as the importin (karyopherin)-α subunit. The NLSs of NF-κB, AP-1, and NFAT differ and the NLS of STAT1 has not yet been identified. Herein we demonstrate that the inducible nuclear import of NF-κB, AP-1, NFAT, and STAT1 in Jurkat T lymphocytes is significantly inhibited by a cell-permeable peptide carrying the NLS of the NF-κB p50 subunit. NLS peptide-mediated disruption of the nuclear import of these transcription factors results in inhibition of IκBα and IL-2 gene expression, processes dependent on NF-κB or the combination of NF-κB, AP-1, and NFAT. Further, we show that inhibitory NLS peptide interacts in vitro with a cytoplasmic NLS receptor complex comprised of the Rch1/importin (karyopherin)-β heterodimer expressed in Jurkat T cells. Taken together, these data indicate that the inducible nuclear import of NF-κB, AP-1, NFAT, and STAT1 in Jurkat T cells can be regulated by NLS peptide delivered noninvasively to the cytoplasm of Jurkat T cells to target members of the importin (karyopherin)-αβ NLS receptor complex.
Mice withSTAT6-Targeted Gene Disruption Develop a Th1 Response and Control Cutaneous Leishmaniasis
Authors: Stamm Luisa M. , Räisänen-Sokolowski Anne , Okano Mitsuhiro , Russell Mary E. , David John R. , Satoskar Abhay R.
Published: January 2023

The cutaneous growth of Leishmania mexicana was measured in STAT6-deficient mice (STAT6−/−) and compared with that in similarly infected wild-type (STAT6+/+) mice. Following s.c. inoculation with 5 × 106 amastigotes of L. mexicana into the shaven rump, STAT6+/+ mice developed large, nonhealing cutaneous lesions, while STAT6−/− mice failed to develop detectable lesions during most of the course of study. As infection progressed, STAT6+/+ mice infected with L. mexicana displayed significantly higher titers of Leishmania-specific IgG1 and IgE compared with STAT6−/− mice, which conversely produced significantly higher titers of Leishmania-specific IgG2a, indicating development of a Th1-like response in the latter group. At 12 wk postinfection, Leishmania Ag-stimulated lymph node cells from STAT6−/− mice produced significantly higher amounts of IL-12 and IFN-γ than those from STAT6+/+ mice as measured by ELISA. However, there was no significant difference in IL-4 production between the two groups. Semiquantitative RT-PCR of transcript levels in intact draining lymph nodes and skin from inoculation sites confirmed a similar pattern of cytokines in vivo as that observed in stimulated lymph node cells in vitro. These results indicate that STAT6-mediated IL-4 signaling is critical for progression of L. mexicana infection in genetically susceptible mice and demonstrate that in the absence of STAT6, susceptible mice default toward a Th1-like response and control cutaneous L. mexicana infection.
Experimental MurineTrypanosoma congolenseInfections. II. Role of Splenic Adherent CD3+ Thy1.2+ TCR-αβ− γδ− CD4+8− and CD3+ Thy1.2+ TCR-αβ− γδ− CD4−8− Cells in the Production of IL-4, IL-10, and IFN-γ and in Trypanosome-Elicited Immunosuppression
Authors: Uzonna Jude E. , Kaushik Radhey S. , Zhang Ying , Gordon John R. , Tabel Henry
Published: January 2023

Trypanosome-induced suppression of T and B cell responses to parasite-related and -unrelated Ags is considered a major mechanism of evasion of the host’s immune defenses by the parasite. Reduced T and B cell responses have been attributed to suppressor T cells, suppressor macrophages, or both. We have previously shown that endogenously produced IL-10 and IFN-γ mediate the suppression of T cell responses in Trypanosoma congolense-infected mice. Here, we show for the first time that splenic CD3+ Thy1.2+ αβ− γδ− CD4+8− and CD3+ Thy1.2+ αβ− γδ− CD4−8− cells that copurify with plastic-, nylon wool-, or Sephadex G-10-adherent cell populations, in synergy with adherent Thy1.2− cells, are the major producers of IL-4, IL-10, and IFN-γ in T. congolense-infected mice. We further demonstrate the involvement of these cells in the suppression of T cell proliferative responses to mitogen and in B cell responses to a parasite-unrelated Ag.
Identification of a Truncated Form of the CC Chemokine CKβ-8 Demonstrating Greatly Enhanced Biological Activity
Authors: Macphee Colin H. , Appelbaum Edward R. , Johanson Kyung , Moores Kitty E. , Imburgia Christina S. , Fornwald Jim , Berkhout Theo , Brawner Mary , Groot Pieter H. E. , O’Donnell Kevin , O’Shannessy Daniel , Scott Gil , White John R.
Published: January 2023

A new CC chemokine, designated CKβ-8 or myeloid progenitor inhibitor factor-1, was recently identified in a large scale sequencing effort and was cloned from a human aortic endothelial library. CKβ-8 cDNA encodes a signal sequence of 21 amino acids, followed by a 99-amino acid predicted mature form. CKβ-8 was expressed and purified from a baculovirus insect cell expression system, which resulted in the identification of different N-terminal variants of the secreted chemokine. The three major forms (containing amino acids 1–99, 24–99, and 25–99 of the secreted chemokine) showed a large variation in potency. CKβ-8 activated both monocytes and eosinophils to mobilize intracellular calcium; however, the shortest form of CKβ-8 (25–99) was >2 orders of magnitude more potent than the longest form. Cross-desensitization experiments in both monocytes and eosinophils suggested that the CCR1 receptor was probably the predominant receptor that mediates this chemokine’s physiologic response. However, incomplete desensitization was encountered in both cell systems, suggesting involvement of an additional receptor(s). Interestingly, the short form of CKβ-8 was the most potent chemotactic chemokine that we have ever evaluated in the monocyte system (EC50 = 54 pM). However, in contrast to its action on monocytes, CKβ-8 was a very poor chemotactic factor for eosinophils.
A High Endothelial Cell-Derived Chemokine Induces Rapid, Efficient, and Subset-Selective Arrest of Rolling T Lymphocytes on a Reconstituted Endothelial Substrate
Authors: Tangemann Kirsten , Gunn Michael D. , Giblin Patricia , Rosen Steven D.
Published: January 2023

The homing of lymphocytes to secondary lymphoid organs is thought to involve the action of chemokines. Secondary lymphoid- tissue chemokine (SLC), a high endothelial venule (HEV)-associated chemokine, has emerged as a candidate for participating in this process. We now show that immobilized SLC strongly induces β2 integrin-mediated binding of T lymphocytes of naive phenotype and B lymphocytes to ICAM-1 under static conditions. This effect is not mediated by β2 integrin affinity modulation, because SLC does not elicit a β2 integrin activation epitope (mAb24) on naive T lymphocytes. In a parallel plate flow chamber, lymphocytes rolling via L-selectin are rapidly arrested through β2 integrins in a pertussis toxin-sensitive manner on a substrate consisting of L-selectin ligands (peripheral lymph node addressins) together with ICAM-1 and SLC. Naive T lymphocytes are arrested on the HEV substrate with sixfold higher efficiency than memory cells. Neutrophils roll, but are not arrested by SLC, whereas they respond to immobilized IL-8 with rapid arrest. Thus, our artificial HEV system recapitulates critical features of lymphocyte interactions with HEV in vivo. These observations strongly point to the participation of SLC in homing of lymphocytes to secondary lymphoid organs.
Mechanisms of Nasal Tolerance Induction in Experimental Autoimmune Myasthenia Gravis: Identification of Regulatory Cells
Authors: Shi Fu-Dong , Li Hulun , Wang Huabing , Bai Xuefeng , van der Meide Peter H. , Link Hans , Ljunggren Hans-Gustaf
Published: January 2023

Autoantigen administration via nasal mucosal tissue can induce systemic tolerance more effectively than oral administration in a number of experimental autoimmune diseases, including Ab-mediated experimental autoimmune myasthenia gravis, a murine model of myasthenia gravis. The mechanisms underlying nasal tolerance induction are not clear. In this study, we show that nasal administration of acetylcholine receptor (AChR) in C57BL/6 mice, before immunizations with AChR in adjuvant, results in delayed onset and reduced muscle weakness compared with control mice. The delayed onset and reduced muscle weakness were associated with decreased AChR-specific lymphocyte proliferation and decreased levels of anti-AChR Abs of the IgG2a and IgG2b isotypes in serum. The clinical and immunological changes in the AChR-pretreated C57BL/6 wild-type (wt) mice were comparable with those observed in AChR-pretreated CD8−/− mice, indicating that CD8+ T cells were not required for the generation of nasal tolerance. AChR-pretreated wt and CD8−/− mice showed augmented TGF-β and reduced IFN-γ responses, whereas levels of IL-4 were unaltered. Splenocytes from AChR-pretreated wt and CD8−/− mice, but not from CD4−/− mice, suppressed AChR-specific lymphocyte proliferation. This suppression could be blocked by Abs against TGF-β. Thus, our results demonstrate that the suppression induced in the present model is independent of CD8+ T cells and suggest the involvement of Ag-specific CD4+ Th3 cells producing TGF-β.
IL-18 and CD28 Use Distinct Molecular Mechanisms to Enhance NK Cell Production of IL-12-Induced IFN-γ
Authors: Walker William , Aste-Amezaga Miguel , Kastelein Robert A. , Trinchieri Giorgio , Hunter Christopher A.
Published: January 2023

NK cells play an important role in innate immune resistance, particularly through synthesis of the pro-inflammatory cytokine IFN-γ. This study compares the abilities of the cytokine IL-18 and the costimulatory cell surface molecule CD28 to enhance IL-12-driven IFN-γ production by NK cells. Studies with other cytokines (IL-1β, IL-6, TNF-α, IL-15) showed that IL-18 or anti-CD28 treatments were the most efficient inducers of IFN-γ when combined with IL-12. The ability of IL-18 to enhance IFN-γ was shown to be dependent on the presence of IL-12. Similarly, although anti-CD28 stimulation alone could enhance IFN-γ synthesis, this effect was significantly increased in the presence of IL-12. Although neither method of costimulation required de novo protein synthesis for their effects on IFN-γ mRNA expression, these molecules used distinct mechanisms. Specifically, nuclear run-on analysis revealed that IL-18 in combination with IL-12 enhanced the rate of transcription of the IFN-γ gene. Conversely, treatment with anti-CD28 plus IL-12 did not significantly up-regulate the rate of transcription of the IFN-γ gene, but stabilized IFN-γ mRNA expression within NK cells. These findings illustrate costimulatory pathways that result in potent IFN-γ responses by NK cells and show that although IL-18 and anti-CD28 can enhance the synthesis of IL-12-driven IFN-γ, they employ molecular mechanisms that are distinct from one another.
Expansion of Extrathymic T Cells as Well as Granulocytes in the Liver and Other Organs of Granulocyte-Colony Stimulating Factor Transgenic Mice: Why They Lost the Ability of Hybrid Resistance
Authors: Kawamura Hiroki , Kawamura Toshihiko , Kokai Yasuo , Mori Michio , Matsuura Akihiro , Oya Hiroshi , Honda Shigeru , Suzuki Susumu , Weerashinghe Anura , Watanabe Hisami , Abo Toru
Published: January 2023

When we attempted to characterize the immunological state in G-CSF transgenic mice, a large number of not only granulocytes but also lymphoid cells expanded in various immune organs. Such lymphoid cells were present at unusual sites of these organs, e.g., the parenchymal space in the liver. We then determined the phenotype of these lymphoid cells by immunofluorescence tests. It was demonstrated that CD3intIL-2Rβ+ cells (i.e., extrathymic T cells), including the NK1.1+ subset of CD3int cells (i.e., NKT cells), increased in the liver and all other tested organs. These T cells as well as NK cells mediated NK and NK-like cytotoxicity, especially at youth. However, they were not able to mediate such cytotoxicity in the presence of granulocytes. This result might be associated with deficiency in the hybrid resistance previously ascribed to these mice. In other words, G-CSF transgenic mice had a large number of extrathymic T cells (including NKT cells) and NK cells that mediate hybrid resistance, but their function was suppressed by activated granulocytes. Indeed, these granulocytes showed an elevated level of Ca2+ influx upon stimulation. The present results suggest that, in parallel with overactivation of granulocytes, extrathymic T cells and NK cells are concomitantly activated in number but that their function is suppressed in G-CSF transgenic mice.
Requirement for Efficient Interactions Between CD4 and MHC Class II Molecules for Survival of Resting CD4+ T Lymphocytes In Vivo and for Activation-Induced Cell Death
Authors: Maroto Rosario , Shen Xiaoli , König Rolf
Published: January 2023

Regulation of homeostasis in the immune system includes mechanisms that promote survival of resting T lymphocytes, and others that control activation-induced cell death (AICD). In this study, we report on the use of a transgenic mouse model to test the role of CD4-MHC class II interactions for the susceptibility of CD4+ T lymphocytes to AICD, and for the survival of resting CD4+ T cells in peripheral lymphoid organs. The only I-Aβ gene expressed in these mice is an Aβk transgene with a mutation that prevents MHC class II molecules from interacting with CD4. We show increased apoptosis in CD4+ T lymphocytes derived from wild-type, but not from mutant Aβk transgenic mice following stimulation with staphylococcal enterotoxin A. Therefore, AICD may be impaired in CD4+ T cells derived from mutant Aβk transgenic mice. Importantly, we observed much higher apoptosis in resting CD4+ T cells from mutant Aβk transgenic mice than from wild-type mice. Furthermore, resting CD4+ T cells from mutant Aβk transgenic mice expressed higher levels of cell surface CD95 (Fas, APO-1). Ab-mediated cross-linking of CD95 further increased apoptosis in CD4+ T cells from mutant Aβk transgenic mice, but not from wild-type mice, suggesting apoptosis involved CD95 signaling. When cocultured with APC-expressing wild-type MHC class II molecules, apoptosis in resting CD4+ T lymphocytes from mutant Aβk transgenic mice was reduced. Our results show for the first time that interactions between CD4 and MHC class II molecules are required for the survival of resting CD4+ T cells in peripheral lymphoid organs.