bluuebunny/crossref_metadata_2025 huggingface dataset:
RT1-U: Identification of a Novel, Active, Class Ib Alloantigen of the Rat MHC
Authors: Leong Louise Y. W. , Le Rolle Anne-France , Deverson Edward V. , Powis Simon J. , Larkins Audrey P. , Vaage John T. , Stokland Aasa , Lambracht-Washington Doris , Rolstad Bent , Joly Etienne , Butcher Geoffrey W.
Published: December 2022

In common with other mammalian species, the laboratory rat (Rattus norvegicus) expresses MHC class I molecules that have been categorized as either classical (class Ia) or nonclassical (class Ib). This distinction separates the class Ia molecules that play a conventional role in peptide Ag presentation to CD8 T cells from the others, whose function is unconventional or undefined. The class Ia molecules are encoded by the RT1-A region of the rat MHC, while the RT1-C/E/M region encodes up to 60 other class I genes or gene fragments, a number of which are known to be expressed (or to be expressible). Here we report upon novel MHC class Ib genes of the rat that we have expression cloned using new monoclonal alloantibodies and which we term RT1-U. The products detected by these Abs were readily identifiable by two-dimensional analysis of immunoprecipitates and were shown to be distinct from the class Ia products. Cellular studies of these molecules indicate that they function efficiently as targets for cytotoxic killing by appropriately raised polyclonal alloreactive CTL populations. The sequences of these class Ib genes group together in phylogenetic analysis, suggesting a unique locus or family. The combined serological, CTL, and sequence data all indicate that these products are genetically polymorphic.
Evidence That an OX-2-Positive Cell Can Inhibit the Stimulation of Type 1 Cytokine Production by Bone Marrow-Derived B7-1 (and B7-2)-Positive Dendritic Cells
Authors: Gorczynski Laura , Chen Z. , Hu J. , Kai Y. , Lei J. , Ramakrishna V. , Gorczynski R. M.
Published: December 2022

We reported that hepatic mononuclear, nonparenchymal cells (NPC) can inhibit the immune response seen when allogeneic C57BL/6 dendritic cells (DC) are incubated with C3H spleen responder cells. Cells derived from these cultures transfer increased survival of C57BL/6 renal allografts in C3H mice. We also found that increased expression of OX-2 on DC was associated with inhibition of cytokine production and renal allograft rejection. We explored whether inhibition by hepatic NPC was a function of OX-2 expression by these cells. Fresh C57BL/6 spleen-derived DC were cultured with C3H spleen responder cells and other putative coregulatory cells. The latter were derived from fresh C3H or C57BL/6 liver NPC, or from C3H or C57BL/6 mice treated for 10 days by i.v. infusion of human Flt3 ligand. Different populations of murine bone marrow-derived DC from cultures of bone marrow with IL-4 plus granulocyte-macrophage-CSF were also used as a source of putative regulator cells. Supernatants of all stimulated cultures were examined for functional expression of different cytokines (IL-2, IL-4, IFN-γ, and TGFβ). We found that fresh C57BL/6 splenic DC induced IL-2, not IL-4, production. Cells from the sources indicated inhibited IL-2 and IFN-γ production and promoted IL-4 and TGFβ production. Inhibition was associated with increased expression of OX-2 on these cells, as defined by semiquantitative PCR and FACS analysis. By size fractionation, cells expressing OX-2 were a subpopulation of NLDC145+ cells. Our data imply a role for cells expressing OX-2 in the regulation of induction of cytokine production by conventional allostimulatory DC.
Orientation of the Ig Domains of CD8αβ Relative to MHC Class I
Authors: Devine Lesley , Sun Jiaren , Barr Mark R. , Kavathas Paula B.
Published: December 2022

The cell surface glycoprotein CD8 functions as a coreceptor with the TCR for interaction with MHC class I. The cocrystal structure of the CD8αα-MHC complex showed that one CD8 Ig domain provided the majority of the contact with MHC class I and that residue R4 of that domain contacted the α2 domain of MHC class I. We previously showed by mutational analysis that this residue was critical for binding to MHC class I. To determine which of the Ig domains for the CD8αβ heterodimer would make the most contact with class I MHC, we expressed single-chain or dimeric forms of CD8 on COS-7 cells and measured the adhesion of MHC class I positive cells. We found that when one of the R4 residues was mutated in a CD8αα homodimer binding comparable to that of wild type was observed, whereas a double R4 mutant severely impaired binding. However, when mutant CD8α (R4K) was coexpressed with wild-type CD8β, binding was not observed. These results support the model in which it is CD8α, not CD8β, that is making the most of the contact with MHC class I, including the α2 domain. In addition, they demonstrate that a single-chain form of CD8αα can bind to MHC class I.
Perforin-Deficient CD8+ T Cells: In Vivo Priming and Antigen-Specific Immunity AgainstListeria monocytogenes
Authors: White Douglas W. , MacNeil Adam , Busch Dirk H. , Pilip Ingrid M. , Pamer Eric G. , Harty John T.
Published: December 2022

CD8+ T cells require perforin to mediate immunity against some, but not all, intracellular pathogens. Previous studies with H-2b MHC perforin gene knockout (PO) mice revealed both perforin-dependent and perforin-independent pathways of CD8+ T cell-mediated immunity to Listeria monocytogenes (LM). In this study, we address two previously unresolved issues regarding the requirement for perforin in antilisterial immunity: 1) Is CD8+ T cell-mediated, perforin-independent immunity specific for a single Ag or generalizable to multiple Ags? 2) Is there a deficiency in the priming of the CD8+ T cell compartment of PO mice following an immunizing challenge with LM? We used H-2d MHC PO mice to generate CD8+ T cell lines individually specific for three known Ags expressed by a recombinant strain of virulent LM. Adoptive transfer experiments into BALB/c host mice revealed that immunity can be mediated by PO CD8+ T cells specific for all Ags examined, indicating that perforin-independent immunity is not limited to CD8+ T cells that recognize listeriolysin O. Analysis of epitope-specific CD8+ T cell expansion by MHC class I tetramer staining and ELISPOT revealed no deficiency in either the primary or secondary response to LM infection in PO mice. These results demonstrate that the perforin-independent pathway of antilisterial resistance mediated by CD8+ T cells is generalizable to multiple epitopes. Furthermore, the results show that reduced antilisterial resistance observed with polyclonal PO CD8+ T cells is a consequence of a deficiency in effector function and not a result of suboptimal CD8+ T cell priming.
High Avidity CTLs for Two Self-Antigens Demonstrate Superior In Vitro and In Vivo Antitumor Efficacy
Authors: Zeh Herbert J. , Perry-Lalley Donna , Dudley Mark E. , Rosenberg Steven A. , Yang James C.
Published: December 2022

A majority of the human tumor-associated Ags characterized to date are derived from nonmutated “self”-proteins. Little is currently understood about the nature of the self-reactive lymphocytes that recognize these Ags. We recently characterized two nonmutated tumor-associated Ags for the B16 murine melanoma: tyrosinase-related protein-2 (TRP-2) and the endogenous retroviral envelope protein, p15E. We previously reported that both TRP-2 and p15E reactive CTL could be detected in the spleens of naive animals after a single in vitro stimulation using 10−5–10−6 M of the appropriate Kb-binding 9-amino acid epitope. In this report we show that the CTL found in naive animals are low avidity lymphocytes, that respond only to high concentrations of peptide in vitro. We demonstrate that titration of in vitro-stimulating peptide to limiting concentrations distinguishes qualitative differences in the lymphocyte reactivity to these two Ags between vaccinated and unvaccinated animals. We further demonstrate that in vitro expansion of CTL in either high or low concentrations of stimulating peptide generated CTL cultures with different avidities for the relevant epitopes. CTL expanded in low concentrations demonstrated higher avidity for peptide-pulsed targets and better tumor recognition, when compared to CTL generated in the presence of high concentrations of Ag. More importantly, high avidity CTL demonstrated superior in vivo antitumor activity. These results demonstrate that qualitative differences in the CTL that recognize these two self-Ags are critically important to their in vitro and in vivo anti-tumor efficacy.
Cutting Edge: Dominant Effect of Ile50Val Variant of the Human IL-4 Receptor α-Chain in IgE Synthesis
Authors: Mitsuyasu Hiromichi , Yanagihara Yukiyoshi , Mao Xiao-Quan , Gao Pei-Sun , Arinobu Yojiro , Ihara Kenji , Takabayashi Akira , Hara Toshiro , Enomoto Tadao , Sasaki Sei , Kawai Minoru , Hamasaki Naotaka , Shirakawa Taro , Hopkin Julian M. , Izuhara Kenji
Published: December 2022

Two variants of the IL-4R α-chain (IL-4Rα) gene have been recently identified in association with different atopic disorders. To clarify the etiological relationship between the two variants, we analyzed responsiveness to IL-4 of transfectants with four kinds of IL-4Rα carrying either Val or Ile at 50 and either Gln or Arg at 551. The substitution of Ile for Val augmented STAT6 activation, proliferation, and transcription activity of the Iε promoter by IL-4, whereas that of Arg for Gln did not change these IL-4 signals. Arg551 was not associated with atopic asthma in the Japanese population. CD23 expression and IgE synthesis by IL-4 were augmented in Ile50-bearing PBMC, compared with those bearing Val50. Taken together, substitution of Arg551 does not enhance the IL-4 signal for generation of germline ε transcript, whereas the substitution of Ile50 contributes to enhancement of IgE synthesis.
Regulation of Cytotoxic T Lymphocyte-Associated Molecule-4 by Src Kinases
Authors: Chuang Ellen , Lee Kyung-Mi , Robbins Michael D. , Duerr James M. , Alegre Maria-Luisa , Hambor John E. , Neveu Mark J. , Bluestone Jeffrey A. , Thompson Craig B.
Published: December 2022

Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) is a cell surface receptor expressed on activated T cells that can inhibit T cell responses induced by activation of the TCR and CD28. Studies with phosphorylated peptides based on the CTLA-4 intracellular domain have suggested that tyrosine phosphorylation of CTLA-4 may regulate its interactions with cytoplasmic proteins that could determine its intracellular trafficking and/or signal transduction. However, the kinase(s) that phosphorylate CTLA-4 remain uncharacterized. In this report, we show that CTLA-4 can associate with the Src kinases Fyn and Lck and that transfection of Fyn or Lck, but not the unrelated kinase ZAP70, can induce tyrosine phosphorylation of CTLA-4 on residues Y201 and Y218. A similar pattern of tyrosine phosphorylation was found in pervanadate-treated Jurkat T cells stably expressing CTLA-4. Phosphorylation of CTLA-4 Y201 in Jurkat cells correlated with cell surface accumulation of CTLA-4. CTLA-4 phosphorylation induced the association of CTLA-4 with the tyrosine phosphatase SHP-2, but not with phosphatidylinositol 3-kinase. In contrast, Lck-induced phosphorylation of CD28 resulted in the recruitment of phosphatidylinositol 3-kinase, but not SHP-2. These findings suggest that phosphorylation of CD28 and CTLA-4 by Lck activates distinct intracellular signaling pathways. The association of CTLA-4 with Src kinases and with SHP-2 results in the formation of a CTLA-4 complex with the potential to regulate T cell activation.
CD40-CD154 Interaction and IFN-γ Are Required for IL-12 But Not Prostaglandin E2 Secretion by Microglia During Antigen Presentation to Th1 Cells
Authors: Aloisi Francesca , Penna Giuseppe , Polazzi Elisabetta , Minghetti Luisa , Adorini Luciano
Published: December 2022

IL-12 and PGE2 promote and inhibit, respectively, the development of Th1 responses. Production of these mediators by APC residing in the central nervous system (CNS) may be involved in the local regulation of the T cell phenotype during infectious and autoimmune CNS diseases. In the present study we have examined IL-12 and PGE2 secretion by cultured microglia and astrocytes from the mouse brain upon Ag-dependent interaction with I-Ad-restricted, OVA323–339 specific TCR transgenic Th1 and Th2 cell lines. We show that microglia, which restimulate efficiently both Th1 and Th2 cells, secrete IL-12 upon Ag-dependent interaction with Th1, but not with Th2 cells. Th1-driven IL-12 production depends on TCR ligation by MHC class II/peptide complexes, CD40 engagement on microglia, and IFN-γ secretion by activated Th1 cells. Th1 and, to a lesser extent, Th2 cells also stimulate the production of PGE2 by microglia. T cell-mediated induction of PGE2 requires MHC class II/peptide/TCR interactions but does not depend on CD40 engagement or on the presence of IFN-γ. Astrocytes, which preferentially activate Th2 cells, fail to produce IL-12 and secrete negligible amounts of PGE2 upon interaction with either Th1 or Th2 cells. These results suggest that during CNS infection or immunopathology, IL-12 produced by microglia upon Ag-specific interaction with Th1 cells may further skew the immune response to Th1, whereas the T cell-dependent production of PGE2 by microglia may represent a negative feedback mechanism, limiting the propagation of Th1 responses.
T Cell Epitopes of a Lipocalin Allergen Colocalize with the Conserved Regions of the Molecule
Authors: Zeiler Thomas , Mäntyjärvi Rauno , Rautiainen Jaakko , Rytkönen-Nissinen Marja , Vilja Pekka , Taivainen Antti , Kauppinen Juha , Virtanen Tuomas
Published: December 2022

In this study we characterized the human T cell-reactive sites of the major cow dander allergen, Bos d 2, a member of the lipocalin protein family. We showed that Bos d 2 contains only a limited number of epitopes. This is in contrast to many other allergens, which usually contain multiple T cell epitopes throughout the molecule. The epitopes of Bos d 2 were primarily concentrated in the conserved regions of the molecule. One of the epitopes was recognized by all the cow-asthmatic individuals regardless of their HLA phenotype. Computer-predicted T cell epitopes on Bos d 2, other lipocalin allergens, and human endogenous lipocalins were situated in similar locations on these molecules and corresponded to experimentally identified epitopes on Bos d 2. The results suggest that human endogenous lipocalins could be involved in the modulation of immune responses against exogenous lipocalin allergens. In addition, our findings are likely to facilitate the development of new forms of immunotherapy against allergies induced by the important group of lipocalin allergens.
Class II-Associated Invariant Chain Peptide-Independent Binding of Invariant Chain to Class II MHC molecules
Authors: Thayer Wesley P. , Ignatowicz Leszek , Weber Dominique A. , Jensen Peter E.
Published: December 2022

The class II-associated invariant chain peptide (CLIP) region of invariant chain (Ii) is believed to play a critical role in the assembly and transport of MHC class II αβIi complexes through its interaction with the class II peptide-binding site. The role of the CLIP sequence was investigated by using mutant Ii molecules with altered affinity for the DR1 peptide-binding site. Both high- and low-affinity mutants were observed to efficiently assemble with DR1 and mediate transport to endosomal compartments in COS cell transfectants. Using N- and C-terminal truncations, a region adjacent to CLIP within Ii(103–118) was identified that can complement loss of affinity for the peptide-binding site in mediating efficient assembly of αβIi. A C-terminal fragment completely lacking the CLIP region, Ii(103–216), was observed binding stably to class II molecules in immunoprecipitation studies and experiments with purified proteins. The Ii(103–118) region was required for this binding, which occurs through interactions outside of the αβ peptide-binding groove. We conclude that strong interactions involving Ii(103–118) and other regions of Ii cooperate in the assembly of functional αβIi under conditions where CLIP has little or no affinity for the class II peptide-binding site. Our results support the hypothesis that the CLIP sequence has evolved to avoid high-stability interactions with the peptide-binding sites of MHC class II molecules rather than as a promiscuous binder with moderate affinity for all class II molecules.
TFEC Is a Macrophage-Restricted Member of the Microphthalmia-TFE Subfamily of Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
Authors: Rehli Michael , Lichanska Agnieszka , Cassady A. Ian , Ostrowski Michael C. , Hume David A.
Published: December 2022

The murine homologue of the TFEC was cloned as part of an analysis of the expression of the microphthalmia-TFE (MiT) subfamily of transcription factors in macrophages. TFEC, which most likely acts as a transcriptional repressor in heterodimers with other MiT family members, was identified in cells of the mononuclear phagocyte lineage, coexpressed with all other known MiT subfamily members (Mitf, TFE3, TFEB). Northern blot analysis of several different cell lineages indicated that the expression of murine TFEC (mTFEC) was restricted to macrophages. A 600-bp fragment of the TATA-less putative proximal promoter of TFEC shares features with many known macrophage-specific promoters and preferentially directs luciferase expression in the RAW264.7 macrophage cell line in transient transfection assays. Five of six putative Ets motifs identified in the TFEC promoter bind the macrophage-restricted transcription factor PU.1 under in vitro conditions and in transfected 3T3 fibroblasts; the minimal luciferase activity of the TFEC promoter could be induced by coexpression of PU.1 or the related transcription factor Ets-2. The functional importance of the tissue-restricted expression of TFEC and a possible role in macrophage-specific gene regulation require further investigation, but are likely to be linked to the role of the other MiT family members in this lineage.
Measles Virus Infection Synergizes with IL-4 in IgE Class Switching
Authors: Imani Farhad , Proud David , Griffin Diane E.
Published: December 2022

Increasing evidence suggests that viral infections are associated with the induction and exacerbation of asthma. One characteristic of human asthma is an increase in the levels of circulating IgE. Previous studies have shown that circulating IgE levels are elevated during the early phase of infection with measles virus (MV). We have shown previously that one mechanism by which viral infections can increase IgE levels is via an induction of IgE class switching through the activation of the antiviral protein kinase (dsRNA-activated protein kinase), leading to the activation of multiple NF-κB complexes. Therefore, to determine whether infection with MV can also induce IgE class switching, we infected the human Ramos B cell line with the Edmonston strain of MV. Infecting Ramos cells with MV did not result directly in either the activation of dsRNA-activated protein kinase or IgE class switching. However, a synergistic effect on IgE class switching was observed when Ramos cells were infected with MV before IL-4 treatment. Ab cross-linking of the MV receptor, CD46, mimicked the effects of MV infection in synergizing with IL-4 to induce IgE class switching, suggesting that viral hemagglutinin is involved in this synergistic effect. These data provide the first indication of a potential mechanism for MV-induced IgE up-regulation and suggest a model for a viral-induced exacerbation of IgE-mediated disorders such as asthma.
Vascular Endothelial Cell Expression of ICAM-1 and VCAM-1 at the Onset of Eliciting Contact Hypersensitivity in Mice: Evidence for a Dominant Role of TNF-α
Authors: McHale Julie F. , Harari Olivier A. , Marshall Diane , Haskard Dorian O.
Published: December 2022

We have studied vascular endothelial activation and increased expression of ICAM-1 and VCAM-1 at the onset of the elicitation phase of oxazolone contact hypersensitivity in mice. By measuring the local uptake of i.v. administered radiolabeled anti-ICAM-1 and anti-VCAM-1 mAb, we found that endothelial ICAM-1 and VCAM-1 was increased by 4 h after challenge, 2 h later than the first peak of ear swelling and 125I-labeled human serum albumen uptake. Increased expression of endothelial ICAM-1 and VCAM-1 was significantly greater in sensitized animals than in naive animals. Anti-TNF-α antiserum significantly inhibited both the increase in ear thickness (p < 0.01), and the up-regulation of ICAM-1 and VCAM-1 expression (p < 0.01 for both) at 4 h. In contrast, the combination of anti-IL-1α and IL-1β had only a small inhibitory effect on ICAM-1 expression (p < 0.05) and no significant effect on increased ear thickness or on VCAM-1 expression. A mixture of anti-TNF-α, anti-IL-1α, and IL-1β was no more inhibitory for endothelial ICAM-1 and VCAM-1 expression than anti-TNF-α alone. ICAM-1 and VCAM-1 expression at 4 h was unaffected by a combination of mAb against α4 and β2 integrins, whereas expression at 24 h was significantly inhibited (p < 0.05), suggesting that the release of TNF-α and other cytokines involved in the initiation of the response may not require leukocyte traffic or other leukocyte functions involving these integrins. We conclude that the early up-regulation of endothelial ICAM-1 and VCAM-1 during the elicitation of contact hypersensitivity is primarily due to the immune-dependent local release of TNF-α.
Role of p38-Mitogen-Activated Protein Kinase in Spontaneous Apoptosis of Human Neutrophils
Authors: Aoshiba Kazutetsu , Yasui Shuji , Hayashi Mitsutoshi , Tamaoki Jun , Nagai Atsushi
Published: December 2022

Neutrophils constitutively undergo apoptosis at both normal and inflamed sites: an important process that limits the toxic potential of the neutrophil. However, the signal pathway for neutrophil apoptosis is currently unknown. In this study, we evaluated the role of p38-mitogen-activated protein kinase (MAPK) in the spontaneous apoptosis of neutrophils in vitro. We found that p38-MAPK was constitutively tyrosine phosphorylated and activated during spontaneous apoptosis of neutrophils. Inhibition of p38-MAPK by SB203580 and an antisense oligonucleotide delayed apoptosis by approximately 24 h. The antioxidants catalase and N-acetylcysteine delayed neutrophil apoptosis, but failed to inhibit phosphorylation and activation of p38-MAPK. Granulocyte-macrophage CSF and anti-Fas Ab, which altered the rate of apoptosis, did not affect phosphorylation and activation of p38-MAPK. These results suggest that the constitutive phosphorylation and activation of p38-MAPK are involved in the program of spontaneous apoptosis in neutrophils.
Negative Feedback Between Prostaglandin and α- and β-Chemokine Synthesis in Human Microglial Cells and Astrocytes
Authors: Janabi Nazila , Hau Isabelle , Tardieu Marc
Published: December 2022

The understanding of immune surveillance and inflammation regulation in cerebral tissue is essential in the therapy of neuroimmunological disorders. We demonstrate here that primary human glial cells were able to produce α- and β-chemokines (IL-8 > growth related protein α (GROα) ≫ RANTES > microphage inflammatory protein (MIP)-1α and MIP-1β) in parallel to PGs (PGE2 and PGF2α) after proinflammatory cytokine stimulation: TNF-α + IL-1β induced all except RANTES, which was induced by TNF-α + IFN-γ. Purified cultures of astrocytes and microglia were also induced by the same combination of cytokines, to produce all these mediators except MIP-1α and MIP-1β, which were produced predominantly by astrocytes. The inhibition of PG production by indomethacin led to a 37–60% increase in RANTES, MIP-1α, and MIP-1β but not in GROα and IL-8 secretion. In contrast, inhibition of IL-8 and GRO activities using neutralizing Abs resulted in a specific 6-fold increase in PGE2 but not in PGF2α production by stimulated microglial cells and astrocytes, whereas Abs to β-chemokines had no effect. Thus, the production of PGs in human glial cells down-regulates their β-chemokine secretion, whereas α-chemokine production in these cells controls PG secretion level. These data suggest that under inflammatory conditions, the intraparenchymal production of PGs could control chemotactic gradient of β-chemokines for an appropriate effector cell recruitment or activation. Conversely, the elevated intracerebral α-chemokine levels could reduce PG secretion, preventing the exacerbation of inflammation and neurotoxicity.
A Transgenic Model to Analyze the Immunoregulatory Role of IL-10 Secreted by Antigen-Presenting Cells
Authors: Groux Hervé , Cottrez Françoise , Rouleau Matthieu , Mauze Smita , Antonenko Svetlana , Hurst Stephen , McNeil Tom , Bigler Mike , Roncarolo Maria-Grazia , Coffman Robert L.
Published: December 2022

IL-10 is a cytokine secreted by a wide variety of cells type that has pleiotropic stimulatory and suppressive activities on both lymphoid and myeloid cells in vitro. To analyze the consequences of high IL-10 secretion by APCs in immune responses, we produced transgenic mice expressing human IL-10 directed by the MHC class II Ea promoter. Despite alterations in the development of T and B cells, no gross abnormalities were detected in peripheral lymphocyte populations or serum Ig levels. However, when immunized using conditions that give either a Th2-type or a Th1-type response, IL-10 transgenic mice failed to mount a significant T or B cell immune response to OVA. IL-10 transgenic mice were also highly susceptible to infection with intracellular pathogens like Listeria monocytogenes or Leishmania major, in contrast to IL-10 transgenic mice, where the transgene was express in T cells. Finally, the recently described stimulatory effect of IL-10 on CD8+ T cells was confirmed by the ability of IL-10 transgenic mice to limit the growth of immunogenic tumors by a CTL-mediated mechanism. These results demonstrate, that, depending on the type of immune response, IL-10 can mediate immunosuppressive or immunostimulatory activities in vivo.
A hsp70-2 Mutation Recognized by CTL on a Human Renal Cell Carcinoma
Authors: Gaudin Catherine , Kremer Florence , Angevin Eric , Scott Véronique , Triebel Frédéric
Published: December 2022

We performed T cell cloning experiments with a tumor-infiltrating lymphocyte subpopulation derived from a renal cell carcinoma tumor site (RCC-7) in which the TCR clonotypic repertoire had been analyzed in terms of TCRBV complementarity-determining region 3 size distribution. We report in this work the characterization of one of the five RCC-specific MHC class I-restricted CTL clones isolated in RCC-7. This TCRBV6J1S1 CTL recognized only the autologous RCC-7 tumor cell line in the context of HLA-A*0201, and the Ag is encoded by a mutated form of the hsp70-2 gene found in the tumor cells, but not in autologous PBLs nor in 47 other tumors. The identification of this gene was achieved by cotransfecting into COS cells a cDNA library of RCC-7 together with HLA-A*0201. Transfectants expressing the Ag were identified by their ability to stimulate TNF release by the CTL clone. The antigenic peptide is a decamer with a mutated residue at position 8. Half-maximal lysis was obtained with only 5 × 10−11 M of decapeptide in target sensitization assays compared with 5 × 10−8 M for the wild-type decapeptide. This difference in recognition was not related to difference in binding HLA-A*0201-presenting molecules, as assessed in an immunofluorescence-based peptide-binding assay using T2 cells. Constitutive hsp70 expression in various tumors suggests that this stress-induced protein may be recognized in situ by tumor-infiltrating lymphocytes. The finding in the tumor of a mutated form of the stress-induced hsp70-2 gene whose product is specifically recognized by TILs with high avidity is discussed in view of the present use of mycobacteria or heterologous heat-shock proteins as immunomodulators or as subunit vaccine candidates.
Integrin Engagement Regulates Proliferation and Collagenase Expression of Rheumatoid Synovial Fibroblasts
Authors: Sarkissian Madathia , Lafyatis Robert
Published: December 2022

Growth of and metalloproteinase production by fibroblast-like synoviocytes (FLSs) in patients with rheumatoid arthritis (RA) contribute to cartilage and bone destruction associated with development of the expanding inflammatory tissue referred to as pannus. Increased levels of extracellular matrix (ECM) proteins in the pannus suggest that intracellular signals generated through integrin receptors might control these processes. We developed a cell culture system permitting accurate assessment of the effect of cell adhesion to various ECM proteins on FLS phenotype. We show that FLS proliferation to platelet-derived growth factor requires a second signal provided by adhesion to an ECM protein. Fibronectin, vitronectin, collagen, or laminin could provide the second signal and was similarly required for the proliferation of FLSs from RA or osteoarthritis patients. Adhesion to fibronectin, collagen, or Arg-Gly-Asp peptide down-regulated collagenase expression. Primarily αv integrin receptors mediated this down-regulation upon adhesion to fibronectin. Loss of cell adhesion and TNF-α stimulation synergistically increased collagenase expression. Increased collagenase expression upon nonadherence was mimicked by treatment with cytochalasin B, suggesting that the loss of cytoskeletal structure associated with a change in cell shape mediates increased collagenase in nonadherent cells. Thus, although increased fibronectin in the lining layer in RA might be expected to inhibit collagenase expression, the change in cell shape associated with this multilayer structure might actually lead to increased collagenase expression.
Mimicry Between the Hepatitis B Virus DNA Polymerase and the Antigenic Targets of Nuclear and Smooth Muscle Antibodies in Chronic Hepatitis B Virus Infection
Authors: Gregorio Germana V. , Choudhuri Kaushik , Ma Yun , Vegnente Angela , Mieli-Vergani Giorgina , Vergani Diego
Published: December 2022

Autoantibodies to nuclear and smooth muscle are common in hepatitis B virus (HBV) infection. To understand their origin, we scanned protein databases and found that HBV-DNA polymerase (HBV-pol) shares 7–9 amino acid sequences with nuclear (MHC II trans-activator, nuclear pore core protein, nuclear mitotic apparatus, and polymyositis sclerosis Ag) and smooth muscle proteins (caldesmon and myosin). Twenty-mer peptides with relevant homologues and an irrelevant control peptide were constructed and ELISAs were established. Sixty-five children with chronic HBV infection, 104 patients with other chronic liver diseases (CLD), 36 patients with extrahepatic autoimmune diseases, and 24 healthy controls were investigated. Double reactivity to HBV-pol peptides and corresponding self homologues was observed in 40% of HBV-positive patients as compared with four (4%) with other chronic liver diseases, two (6%) with extrahepatic autoimmune diseases, and in none of the healthy controls (p < 0.001 for all). Double reactivity to myosin or caldesmon peptides and their HBV-pol homologues was associated with anti-smooth muscle Ab positivity by immunofluorescence (p < 0.05 for both). HBV-positive sera double reactive for myosin or caldesmon and their homologous HBV-pol peptides also reacted with the native proteins on immunoblot. Fifty to ninety percent Ab inhibition to individual HBV-pol and HBV-pol99–118 peptides was noted by preincubation with individual HBV-pol/self homologue peptide and native proteins, respectively, but not with control peptide. Our results show that cross-reactive immunity targeting homologous sequences of viral and self proteins may partly account for autoantibody production in HBV infection.
Engagement of B Cell Receptor Regulates the Invariant Chain-Dependent MHC Class II Presentation Pathway
Authors: Zimmermann Valérie S. , Rovere Patrizia , Trucy Jeannine , Serre Karine , Machy Patrick , Forquet Frédérique , Leserman Lee , Davoust Jean
Published: December 2022

The intracellular sites in which Ags delivered by the B cell receptor (BCR) are degraded and loaded onto class II molecules remain poorly defined. To address this issue, we generated wild-type and invariant chain (Ii)-deficient H-2k mice bearing BCR specific for hen egg lysozyme. Our results show that, 1) unlike Ags taken up from the fluid phase, Ii is required for presentation of hen egg lysozyme internalized through the BCR in a manner independent of the peptide analyzed; 2) BCR ligation induces intracellular accumulation of MHC class II molecules only in Ii-positive B cells; and 3) these class II molecules reach intracellular compartments where BCR targets exogenous Ag. No differences in expression of adhesion and costimulatory molecules or in the presentation of soluble peptides were detectable between Ii-positive and -negative B cells. Therefore, the BCR delivers its ligand to compartments containing MHC class II-Ii complexes and bypasses the Ii-independent presentation pathway. The linked roles of Ag internalization and B cell activation of the BCR leads to potent Ii-dependent presentation in splenic B cells.
B Cell Response After MMTV Infection: Extrafollicular Plasmablasts Represent the Main Infected Population and Can Transmit Viral Infection
Authors: Ardavín Carlos , Martín Pilar , Ferrero Isabel , Azcoitia Iñigo , Anjuère Fabienne , Diggelmann Heidi , Luthi Frédéric , Luther Sanjiv , Acha-Orbea Hans
Published: December 2022

The immune response to mouse mammary tumor virus (MMTV) relies on the presentation of an MMTV-encoded superantigen by infected B cells to superantigen-specific T cells. The initial extrafollicular B cell differentiation involved the generation of B cells expressing low levels of B220. These B220low B cells corresponded to plasmablasts that expressed high levels of CD43 and syndecan-1 and were CD62 ligand− and IgD−. Viral DNA was detected nearly exclusively in these B220low B cells by PCR, and retroviral type-A particles were observed in their cytoplasm by electron microscopy. An MMTV transmission to the offspring was also achieved after transfer of B220low CD62 ligand− CD43+ plasmablasts into noninfected females. These data suggest that B220low plasmablasts, representing the bulk of infected B cells, are capable of sustaining viral replication and may be involved in the transmission of MMTV.
Overlapping Roles for L-Selectin and P-Selectin in Antigen-Induced Immune Responses in the Microvasculature
Authors: Kanwar Samina , Steeber Douglas A. , Tedder Thomas F. , Hickey Michael J. , Kubes Paul
Published: December 2022

Although L-selectin mediates lymphocyte attachment to endothelial venules of peripheral lymph nodes, its role in leukocyte recruitment into tissues following Ag challenge is less well established. The objective of this study was to systematically examine the role of L-selectin in leukocyte rolling in the peripheral microvasculature during the first 24 h of an immune response. A type I hypersensitivity response was elicited in wild-type (C57BL/6) and L-selectin-deficient mice by systemic (i.p.) sensitization and intrascrotal challenge with chicken egg OVA. The cremaster microcirculation was observed in untreated and sensitized mice 4, 8, and 24 h post-Ag challenge by intravital microscopy. Leukocyte recruitment in L-selectin-deficient mice and wild-type mice treated with an L-selectin function-blocking mAb was examined at each time point. Ag challenge induced a significant increase in leukocyte rolling (60 cells/min/venule to ∼300 cells/min/venule) in wild-type mice at 4–24 h. This response was reduced by approximately 60–70% in L-selectin-deficient mice and in wild-type mice treated with an L-selectin-blocking mAb. P-selectin blockade by Ab completely inhibited leukocyte rolling at 4–24 h in wild-type animals and also blocked the residual rolling seen in L-selectin-deficient mice. Blocking E-selectin function had no effect on leukocyte rolling flux at any time point in wild-type or L-selectin-deficient mice. Despite reduced rolling, leukocyte adhesion and emigration were not measurably reduced in the L-selectin-deficient mice in this vascular bed. In conclusion, leukocyte rolling is L-selectin-dependent post-Ag challenge with L-selectin and P-selectin sharing overlapping functions.
Lymphocyte Activation Gene-3, a MHC Class II Ligand Expressed on Activated T Cells, Stimulates TNF-α and IL-12 Production by Monocytes and Dendritic Cells
Authors: Avice Marie-Noëlle , Sarfati Marika , Triebel Frederic , Delespesse Guy , Demeure Christian E.
Published: December 2022

Lymphocyte activation gene-3 (LAG-3) is an MHC class II ligand structurally and genetically related to CD4. Although its expression is restricted to activated T cells and NK cells, the functions of LAG-3 remain to be elucidated. Here, we report on the expression and function of LAG-3 on proinflammatory bystander T cells that are activated in the absence of TCR engagement. LAG-3 is expressed at high levels on human T cells cocultured with autologous monocytes and IL-2 and synergizes with the low levels of CD40 ligand (CD40L) expressed on these cells to trigger TNF-α and IL-12 production by monocytes. Indeed, anti-LAG-3 mAb inhibits both IL-12 and IFN-γ production in IL-2-stimulated cocultures of T cells and autologous monocytes. Soluble LAG-3Ig fusion protein markedly enhances IL-12 production by monocytes stimulated with infra-optimal concentrations of sCD40L, whereas it directly stimulates monocyte-derived dendritic cells (DC) for the production of TNF-α and IL-12, unravelling an enhanced responsiveness to MHC class II engagemenent in DC as compared with activated monocytes. Thus similar to CD40L, LAG-3 may be involved in the proinflammatory activity of cytokine-activated bystander T cells and most importantly it may directly activate DC.
γδ T Cells Contribute to Control of Chronic Parasitemia inPlasmodium chabaudiInfections in Mice
Authors: Seixas Elsa M. G. , Langhorne Jean
Published: December 2022

During a primary infection of mice with Plasmodium chabaudi, γδ T cells are stimulated and their expansion coincides with recovery from the acute phase of infection in normal mice or with chronic infections in B cell-deficient mice (μ-MT). To determine whether the large γδ T cell pool observed in female B cell-deficient mice is responsible for controlling the chronic infection, studies were done using double-knockout mice deficient in both B and γδ cells (μ-MT × δ−/−TCR) and in γδ T cell-depleted μ-MT mice. In both types of γδ T cell-deficient mice, the early parasitemia following the peak of infection was exacerbated, and the chronic parasitemia was maintained at significantly higher levels in the absence of γδ T cells. The majority of γδ T cells in C57BL/6 and μ-MT mice responding to infection belonged predominantly to a single family of γδ T cells with TCR composed of Vγ2Vδ4 chains and which produced IFN-γ rather than IL-4.
IL-5 and Eosinophils Are Essential for the Development of Airway Hyperresponsiveness Following Acute Respiratory Syncytial Virus Infection
Authors: Schwarze Jürgen , Cieslewicz Grzegorz , Hamelmann Eckard , Joetham Anthony , Shultz Leonard D. , Lamers Marinus C. , Gelfand Erwin W.
Published: December 2022

Viral respiratory infections can cause bronchial hyperresponsiveness and exacerbate asthma. In mice, respiratory syncytial virus (RSV) infection, which induces an immune response dominated by IFN-γ, results in airway hyperresponsiveness (AHR) and eosinophil influx into the airways, both of which are prevented by pretreatment with anti-IL-5 Ab. To delineate the role of IL-5, IL-4, and IFN-γ in the development of RSV-induced AHR and lung eosinophilia, we tested the ability of mice deficient in each of these cytokines to develop these symptoms of RSV infection. Mice deficient in either IL-5, IL-4, or IFN-γ were administered infectious RSV intranasally, and 6 days later, airway responsiveness to inhaled methacholine was assessed by barometric body plethysmography, and numbers of lung eosinophils and production of IFN-γ, IL-4, and IL-5 by mononuclear cells from peribronchial lymph nodes were monitored. RSV infection resulted in airway eosinophilia and AHR in both IL-4- and IFN-γ-deficient mice, but not in IL-5-deficient mice. Reconstitution of IL-5-deficient mice with IL-5 restored these responses and enhanced the responses in IL-4-deficient mice. Anti-VLA-4 (very late Ag-4) treatment prevented lung eosinophilia and AHR following RSV infection and IL-5 reconstitution. We conclude that in response to RSV, IL-5 is essential for the influx of eosinophils into the lung and that eosinophils in turn are critical for the development of AHR. IFN-γ and IL-4 are not essential for these responses to RSV infection.
HIV-1-Specific CTL Responses Primed In Vitro by Blood-Derived Dendritic Cells and Th1-Biasing Cytokines
Authors: Wilson Cara C. , Olson Walter C. , Tuting Thomas , Rinaldo Charles R. , Lotze Michael T. , Storkus Walter J.
Published: December 2022

Vaccine strategies designed to elicit strong cell-mediated immune responses to HIV Ags are likely to lead to protective immunity against HIV infection. Dendritic cells (DC) are the most potent APCs capable of priming both MHC class I- and II-restricted, Ag-specific T cell responses. Utilizing a system in which cultured DC from HIV-seronegative donors were used as APC to present HIV-1 Ags to autologous T cells in vitro, the strength and specificity of primary HIV-specific CTL responses generated to exogenous HIV-1 Nef protein as well as intracellularly expressed nef transgene product were investigated. DC expressing the nef gene were able to stimulate Nef-specific CTL, with T cells from several donors recognizing more than one epitope restricted by a single HLA molecule. Primary Nef-specific CTL responses were also generated in vitro using DC pulsed with Nef protein. T cells primed with Nef-expressing DC (via protein or transgene) were able to lyse MHC class I-matched target cells pulsed with defined Nef epitope peptides as well as newly identified peptide epitopes. The addition of Th1-biasing cytokines IL-12 or IFN-α, during priming with Nef-expressing DC, enhanced the Nef-specific CTL responses generated using either Ag-loading approach. These results suggest that this in vitro vaccine model may be useful in identifying immunogenic epitopes as vaccine targets and in evaluating the effects of cytokines and other adjuvants on Ag-specific T cell induction. Successful approaches may provide information important to the development of prophylactic HIV vaccines and are envisioned to be readily translated into clinical DC-based therapeutic vaccines for HIV-1.
Self-Reactive T Cells Selected on Thymic Cortical Epithelium Are Polyclonal and Are Pathogenic In Vivo
Authors: Laufer Terri M. , Fan Lian , Glimcher Laurie H.
Published: December 2022

Positive selection of CD4+ T cells requires that the TCR of a developing thymocyte interact with self MHC class II molecules on thymic cortical epithelium. In contrast, clonal deletion is mediated by dendritic cells and medullary epithelium. We previously generated K14 mice expressing MHC class II only on thymic cortical epithelium. K14 CD4+ T cells were positively, but not negatively, selected and had significant in vitro autoreactivity. Here, we examine the function of these autoreactive CD4+ T cells in more detail. Analysis of a series of K14-derived T hybrids demonstrated that the autoreactive population of CD4+ T cells is phenotypically and functionally diverse. Purified K14 CD4+ T cells transferred into lethally irradiated wild-type B6 mice cause acute graft vs host disease with bone marrow failure. Further, these autoreactive CD4+ T cells cause hypergammaglobulinemia and the production of autoantibodies when transferred into unirradiated wild-type hosts. Thus, positive selection by normal thymic cortical epithelial cells, unopposed by negative selection, produces polyclonal CD4+ T cells that are pathologic.
Immunization with a Peptide of Sm B/B′ Results in Limited Epitope Spreading But Not Autoimmune Disease
Authors: Mason Lesley J. , Timothy Linda M. , Isenberg David A. , Kalsi Jatinderpal K.
Published: December 2022

An experimental model of systemic lupus erythematosus has recently been described in normal animals. We sought to confirm and extend this model, which involved immunization of normal rabbits and mice with a peptide of Sm B/B′, PPPGMRPP. This peptide is an early target of the immune response in anti-Sm-positive patients with lupus. The peptide was used in a multiple Ag peptide format, with multiple copies of PPPGMRPP bound to an inert lysine backbone. New Zealand White rabbits and A/J and C57BL/10ScSn mouse strains were immunized with PPPGMRPP-MAP. Pepscan assays were used to determine the epitope spreading of the anti-PPPGMRPP-MAP response to other octamers of SmB/B′ following immunization. We obtained high titer anti-PPPGMRPP-MAP IgG responses in the New Zealand White rabbits and A/J mice. The rabbits immunized with PPPGMRPP-MAP showed varying degrees of epitope spreading, while the A/J mice showed no spreading. We observed no autoantibodies to dsDNA or other anti-nuclear autoantibodies in our animals by ELISA or immunofluorescence, although anti-nuclear autoantibodies were found by Western blotting in some of the rabbits. No evidence of clinical disease was seen in our normal animals. These data underline the difficulties often associated with the reproduction of animal models in different laboratories.
A Major Linkage Region on Distal Chromosome 4 Confers Susceptibility to Mouse Autoimmune Gastritis
Authors: Silveira Pablo A. , Baxter Alan G. , Cain Wendy E. , van Driel Ian R.
Published: December 2022

Although much is known about the pathology of human chronic atrophic (type A, autoimmune) gastritis, its cause is poorly understood. Mouse experimental autoimmune gastritis (EAG) is a CD4+ T cell-mediated organ-specific autoimmune disease of the stomach that is induced by neonatal thymectomy of BALB/c mice. It has many features similar to human autoimmune gastritis. To obtain a greater understanding of the genetic components predisposing to autoimmune gastritis, a linkage analysis study was performed on (BALB/cCrSlc × C57BL/6)F2 intercross mice using 126 microsatellite markers covering 95% of the autosomal genome. Two regions with linkage to EAG were identified on distal chromosome 4 and were designated Gasa1 and Gasa2. The Gasa1 gene maps within the same chromosomal segment as the type 1 diabetes and systemic lupus erythematosus susceptibility genes Idd11 and Nba1, respectively. Gasa2 is the more telomeric of the two genes and was mapped within the same chromosomal segment as the type 1 diabetes susceptibility gene Idd9. In addition, there was evidence of quantitative trait locus controlling autoantibody titer within the telomeric segment of chromosome 4. The clustering of genes conferring susceptibility to EAG with those conferring susceptibility to type 1 diabetes is consistent with the coinheritance of gastritis and diabetes within human families. This is the first linkage analysis study of autoimmune gastritis in any organism and as such makes an important and novel contribution to our understanding of the etiology of this disease.
IL-4 Inhibits the Production of TNF-α and IL-12 by STAT6-Dependent and -Independent Mechanisms
Authors: Levings Megan K. , Schrader John W.
Published: December 2022

IL-4 promotes allergic responses and inhibits the production of proinflammatory cytokines by monocytes and macrophages. The promotion of allergic responses by IL-4 has been shown to be absolutely dependent on the transcription factor STAT6. We report here that the inhibitory effects of IL-4 on the production of TNF-α or IL-12 by macrophages had both STAT6-dependent and -independent components, depending on the stimuli. IL-4 failed to inhibit the release of TNF-α or IL-12 from STAT6 null macrophages stimulated with LPS alone. However, IL-4 still induced significant inhibition of the production of TNF-α and IL-12 from STAT6 null macrophages that were stimulated with the more physiologically relevant combination of LPS and IFN-γ. These data show that STAT6 is required for the IL-4-mediated inhibition of the production of TNF-α and IL-12 stimulated by LPS alone, but that IL-4 also activates distinct, STAT6 independent mechanism(s) that inhibit the IFN-γ-mediated enhancement of IL-12 and TNF-α production.