bluuebunny/crossref_metadata_2025 huggingface dataset:
Cutting Edge: Receptor-Mediated Endocytosis of Heat Shock Proteins by Professional Antigen-Presenting Cells
Authors: Arnold-Schild Danièle , Hanau Daniel , Spehner Danièle , Schmid Claudia , Rammensee Hans-Georg , de la Salle Henri , Schild Hansjörg
Published: January 2023

Immunization with heat shock proteins (HSPs) induces Ag-specific CTL responses. The specificity of the immune response is based on peptides associated with HSPs. To investigate how exogenous HSP/peptide complexes gain access to the MHC class I-restricted Ag presentation pathway, we incubated the monocytic cell line P388D1 and the dendritic cell line D2SC/1 with gold-labeled HSPs gp96 and HSC70. We show that HSPs bind specifically to the surface of these APCs and are internalized spontaneously by receptor-mediated endocytosis, demonstrating the existence of specific receptors for HSPs on these cells. In addition, we observe colocalization of internalized HSPs and surface MHC class I molecules in early and late endosomal structures. These findings provide possible explanations for the immunogenicity of HSP/peptide complexes and for the transfer of HSP-associated peptides onto MHC class I molecules.
Cutting Edge: A Test of the Dominant Negative Signal Model for TCR Antagonism
Authors: Daniels Mark A. , Schober Sonya L. , Hogquist Kristin A. , Jameson Stephen C.
Published: January 2023

The mechanism by which TCR antagonists interfere with T cell activation is unclear. One popular hypothesis is that incomplete early signaling events induced by these ligands dominantly inhibit the T cell’s ability to respond to a copresented agonist ligand. Here we test this “dominant negative” signal hypothesis by studying T cells expressing two distinct MHC class I-restricted TCRs (2C and OT-I). Although responses through each TCR can be efficiently inhibited by their specific antagonists, we found no evidence for “cross-antagonism” in which an antagonist for receptor “A” blocks responses through receptor “B.” Such inhibition would have been expected were the dominant negative signaling hypothesis correct, and alternative models for TCR antagonism are discussed.
Cutting Edge: SOCS-1 Is a Potent Inhibitor of IL-4 Signal Transduction
Authors: Losman J. A. , Chen X. P. , Hilton D. , Rothman P.
Published: January 2023

IL-4 is an important regulator of the activation, proliferation, and differentiation of many hematopoetic cells. Many of these biological effects result from the activation of Janus kinases (JAK)1 and JAK3 and the transcription factor Stat6. Recent data suggest that members of the SOCS (suppressor of cytokine signaling) family of proteins can inhibit JAK-STAT signaling. We have examined the ability of SOCS family members to suppress IL-4 signaling, and we have found that SOCS-1 potently inhibits the activation of JAK1 kinase and Stat6 in response to IL-4. Furthermore, SOCS-1 can inhibit the induction of CD23 expression by IL-4. SOCS-2 does not inhibit induction of signaling by IL-4, while inhibition of IL-4 signaling by SOCS-3 can be detected in transient transfection systems, but not in stable cell lines. These studies implicate SOCS-1 in modulation of IL-4 signaling and suggest that SOCS-1 may play a role in regulating the immune response.
Survival of Naive CD4 T Cells: Roles of Restricting Versus Selecting MHC Class II and Cytokine Milieu
Authors: Boursalian Tamar E. , Bottomly Kim
Published: January 2023

The diversity of naive CD4 T cells plays an important role in the adaptive immune response by ensuring the capability of responding to novel pathogens. In the past, it has been generally accepted that naive CD4 T cells are intrinsically long-lived; however, there have been studies suggesting some CD4 T cells are short-lived. In this report, we identify two populations of naive CD4 T cells: a long-lived population as well as a short-lived population. In addition, we identify two factors that contribute to the establishment of long-lived naive CD4 T cells. We confirm earlier findings that MHC class II interaction with the TCR on CD4 T cells is important for survival. Furthermore, we find that MHC class II alleles with the correct restriction element for Ag presentation mediate the peripheral survival of naive CD4 T cells more efficiently than other positively selecting alleles, regardless of the selecting MHC in the thymus. The second component contributing to the survival of naive CD4 T cells is contact with the cytokines IL-4 and IL-7. We find that the physiological levels of IL-4 and IL-7 serve to enhance the MHC class II-mediated survival of naive CD4 T cells in vivo.
Factors Regulating Stem Cell Recruitment to the Fetal Thymus
Authors: Wilkinson B. , Owen J. J. T. , Jenkinson E. J.
Published: January 2023

Colonization of the thymic rudiment during development is initiated before vascularization so that hemopoietic precursors must leave the pharyngeal vessels and migrate through the perithymic mesenchyme to reach the thymus, suggesting that they may be responding to a gradient of chemoattractant factors. We report that diffusible chemoattractants are produced by MHC class II+ epithelial cells of the fetal thymus, and that the response of precursors to these factors is mediated via a G protein-coupled receptor, consistent with factors being members of the chemokine family. Indeed, a number of chemokine receptors are expressed by thymic precursors, and several chemokines are also expressed by thymic epithelial cells. However, these chemokines are also expressed in a tissue that is unable to attract precursors, although the thymus expressed chemokine, TECK, is expressed at higher levels in thymic epithelial cells and we show that it has chemotactic activity for isolated thymic precursors. Neutralizing Ab to TECK, however, did not prevent thymus recolonization by T cell precursors, suggesting that other novel chemokines might be involved in this process. In addition, we provide evidence for the involvement of matrix metalloproteinases in chemoattractant-mediated T cell precursor recruitment to the thymus during embryogenesis.
Cross-Recognition of Two Middle T Protein Epitopes by Immunodominant Polyoma Virus-Specific CTL
Authors: Wilson Christopher S. , Moser Janice M. , Altman John D. , Jensen Peter E. , Lukacher Aron E.
Published: January 2023

We recently identified the immunodominant epitope for polyoma virus-specific CTL as the Dk-associated peptide MT389–397 derived from the middle T (MT) viral oncoprotein. Another Dk-restricted peptide corresponding to residues 236–244 of MT was recognized by nearly all MT389–397-reactive CTL clones, but required concentrations at least 2 logs higher to sensitize syngeneic target cells for lysis. Except for identity at the three putative Dk-peptide anchor residues, MT236–244 shares no homology with MT389–397. Using a novel europium-based class I MHC-peptide binding immunoassay, we determined that MT236–244 bound Dk 2–3 logs less well than MT389–397. Infection with a mutant polyoma virus whose MT is truncated just before the MT389–397 epitope or immunization with MT389–397 or MT236–244 peptides elicited CTL that recognized both MT389–397 and MT236–244. Importantly, infection with a polyoma virus lacking MT389–397 and mutated in an MT236–244 Dk anchor position induced polyoma virus-specific CTL recognizing neither MT389–397 nor MT236–244 epitopes. Despite predominant usage of the Vβ6 gene segment, MT389–397/MT236–244 cross-reactive CTL clones possess diverse complementarity-determining region 3β domains; this is functionally reflected in their heterogeneous recognition patterns of alanine-monosubstituted MT389–397 peptides. Using Dk/MT389–397 tetramers, we directly visualized MT236–244 peptide-induced TCR down-modulation of virtually all MT389–397-specific CD8+ T cells freshly explanted from polyoma-infected mice, suggesting that a single TCR recognizes both Dk-restricted epitopes. The availability of immunodominant epitope-specific CTL capable of recognizing a second epitope in MT, a viral protein essential for tumorigenesis, may serve to amplify the CTL response to the immunodominant epitope and prevent the emergence of immunodominant epitope-loss viruses and virus-induced tumors.
Adjuvant-Guided Type-1 and Type-2 Immunity: Infectious/Noninfectious Dichotomy Defines the Class of Response
Authors: Yip Hualin C. , Karulin Alexey Y. , Tary-Lehmann Magdalena , Hesse Maike D. , Radeke Heinfried , Heeger Peter S. , Trezza Richard P. , Heinzel Frederick P. , Forsthuber Thomas , Lehmann Paul V.
Published: January 2023

Traditionally, protein Ags have been injected in CFA (oil with inactivated mycobacteria) to induce immunity and with IFA (oil alone) to induce tolerance. We report here that injection of hen eggwhite lysozyme, a prototypic Ag, in CFA-induced and IFA-induced pools of hen eggwhite lysozyme-specific memory T cells of comparable fine specificity, clonal size, and avidity spectrum, but with type-1 and type-2 cytokine signatures, respectively. This adjuvant-guided induction of virtually unipolar type-1 and type-2 immunity was observed with seven protein Ags and in a total of six mouse strains. Highly polarized type-1 and type-2 immunity are thus readily achievable through the choice of adjuvant, irrespective of the genetic bias of the host and of the nature of the protein Ag. This finding should have far-reaching implications for the development of vaccines against infectious and autoimmune diseases. Furthermore, our demonstration that Ag injected with IFA is as strongly immunogenic for T cells as it is with CFA shows that the presence of the mycobacteria determines not the priming of naive T cells through the second-signal link but the path of downstream differentiation toward CD4 memory cells that express either type-1 or type-2 cytokines.
Recapitulation of Normal and Abnormal BioBreeding Rat T Cell Development in Adult Thymus Organ Culture
Authors: Whalen Barbara J. , Weiser Peter , Marounek Jan , Rossini Aldo A. , Mordes John P. , Greiner Dale L.
Published: January 2023

Congenitally lymphopenic diabetes-prone (DP) BioBreeding (BB) rats develop spontaneous T cell-dependent autoimmunity. Coisogenic diabetes-resistant (DR) BB rats are not lymphopenic and are free of spontaneous autoimmune disease, but become diabetic in response to depletion of RT6+ T cells. The basis for the predisposition to autoimmunity in BB rats is unknown. Abnormal T cell development in DP-BB rats can be detected intrathymically, and thymocytes from DR-BB rats adoptively transfer diabetes. The mechanisms underlying these T cell developmental abnormalities are not known. To study these processes, we established adult thymus organ cultures (ATOC). We report that cultured DR- and DP-BB rat thymi generate mature CD4 and CD8 single-positive cells with up-regulated TCRs. DR-BB rat cultures also generate T cells that express RT6. In contrast, DP-BB rat cultures generate fewer CD4+, CD8+, and RT6+ T cells. Analysis of the cells obtained from ATOC suggested that the failure of cultured DP-BB rat thymi to generate T cells with a mature phenotype is due in part to an increased rate of apoptosis. Consistent with this inference, we observed that addition of the general caspase inhibitor Z-VAD-FMK substantially increases the number of both mature and immature T cells produced by DP-BB rat ATOC. We conclude that cultured DR-BB and DP-BB rat thymi, respectively, recapitulate the normal and abnormal T cell developmental kinetics and phenotypes observed in these animals in vivo. Such cultures should facilitate identification of the underlying pathological processes that lead to immune dysfunction and autoimmunity in BB rats.
Failure of Measles Virus to Activate Nuclear Factor-κB in Neuronal Cells: Implications on the Immune Response to Viral Infections in the Central Nervous System
Authors: Dhib-Jalbut Suhayl , Xia Jane , Rangaviggula Himabindu , Fang Yu-Yan , Lee Terry
Published: January 2023

Neurons are postmitotic cells that foster virus persistence. These cells lack the HLA class I molecules required for clearance of infected cells. Previously, we showed that HLA class I is induced by measles virus (MV) on glial cells, which is primarily mediated by IFN-β. In contrast, MV was unable to induce HLA class I or IFN-β in neuronal cells. This failure was associated with lack of NF-κB binding to the positive regulatory domain II element of the IFN-β promoter, which is essential for virus-induced IFN-β gene activity. In this study, we demonstrate that the failure to activate NF-κB in neuronal cells is due to the inability of MV to induce phosphorylation and degradation of IκB, the inhibitor of NF-κB. In contrast, TNF-α induced degradation of IκBα in the neuronal cells, suggesting that failure to induce IκBα degradation is likely due to a defect in virus-mediated signaling rather than to a defect involving neuronal IκBα. Like MV, mumps virus and dsRNA failed to induce IκBα degradation in the neuronal cells, suggesting that this defect may be specific to viruses. Autophosphorylation of the dsRNA-dependent protein kinase, a kinase possibly involved in virus-mediated IκBα phosphorylation, was intact in both cell types. The failure of virus to induce IκBα phosphorylation and consequently to activate NF-κB in neuronal cells could explain the repression of IFN-β and class I gene expression in virus-infected cells. These findings provide a potential mechanism for the ability of virus to persist in neurons and to escape immune surveillance.
Expression of Murine IL-12 Is Regulated by Translational Control of the p35 Subunit
Authors: Babik Jennifer M. , Adams Elizabeth , Tone Yukiko , Fairchild Paul J. , Tone Masahide , Waldmann Herman
Published: January 2023

IL-12 is a heterodimer of two subunits, p35 and p40, encoded by separate genes that are regulated independently. To investigate the mechanisms underlying the regulation of the p35 gene, we characterized murine p35 expression in the B cell lymphoma line A20 and in bone marrow-derived dendritic cells. Multiple transcription start sites were identified in both cell types, resulting in four p35 mRNA isoforms (types I–IV) that differ in the number and position of upstream ATGs in their 5′ untranslated regions. In nonstimulated cells, the predominant forms of p35 message (types II and IV) contained an additional upstream ATG, whose presence was shown to inhibit the downstream translation of the p35 subunit. After LPS stimulation, however, transcription initiated from alternate positions, so that the proportion of transcripts not containing this upstream ATG (types I and III) was significantly increased in the population of p35 mRNA. These type I and type III transcripts readily supported translation of the p35 subunit and its incorporation into bioactive IL-12. Furthermore, p35 mRNA levels were substantially up-regulated after LPS stimulation in both cell types. Thus, our results show that p35 gene expression is highly regulated by both transcriptional and translational mechanisms.
Scanning a DRB3*0101 (DR52a)-Restricted Epitope Cross-Presented by DR3: Overlapping Natural and Artificial Determinants in the Human Acetylcholine Receptor
Authors: Nagvekar Nita , Corlett Louise , Jacobson Leslie W. , Matsuo Hidenori , Chalkley Robert , Driscoll Paul C. , Deshpande Shrikant , Spack Edward G. , Willcox Nicholas
Published: January 2023

A recurring epitope in the human acetylcholine receptor (AChR) α subunit (α146–160) is presented to specific T cells from myasthenia gravis patients by HLA-DRB3*0101—“DR52a”—or by DR4. Here we first map residues critical for DR52a in this epitope by serial Ala substitution. For two somewhat similar T cells, this confirms the recently deduced importance of hydrophobic “anchor” residues at peptide p1 and p9; also of Asp at p4, which complements this allele’s distinctive Arg74 in DRβ. Surprisingly, despite the 9 sequence differences in DRβ between DR52a and DR3, merely reducing the bulk of the peptide’s p1 anchor residue (Trp149→Phe) allowed maximal cross-presentation to both T cells by DR3 (which has Val86 instead of Gly). The shared K71G73R74N77 motif in the α helices of DR52a and DR3 thus outweighs the five differences in the floor of the peptide-binding groove. A second issue is that T cells selected in vitro with synthetic AChR peptides rarely respond to longer Ag preparations, whereas those raised with recombinant subunits consistently recognize epitopes processed naturally even from whole AChR. Here we compared one T cell of each kind, which both respond to many overlapping α140–160 region peptides (in proliferation assays). Even though both use Vβ2 to recognize peptides bound to the same HLA-DR52a in the same register, the peptide-selected line nevertheless proved to depend on a recurring synthetic artifact—a widely underestimated problem. Unlike these contaminant-responsive T cells, those that are truly specific for natural AChR epitopes appear less heterogeneous and therefore more suitable targets for selective immunotherapy.
In Vitro Analysis of Complement-Dependent HIV-1 Cell Infection Using a Model System
Authors: Tacnet-Delorme Pascale , Boyer Véronique , Thielens Nicole M. , Hernandez Jean-François , Bally Isabelle , Sim Robert B. , Desgranges Claude , Arlaud Gérard J.
Published: January 2023

Previous studies based on the use of human serum as a source of C have provided evidence for the C-dependent enhancement of cell infection by HIV-1. The present study was undertaken to distinguish C from other serum factors and to identify the proteins and the mechanisms involved in C-dependent cell infection by HIV-1. The classical C activation pathway was reconstituted from the proteins C1q, C1r, C1s, C4, C2, C3, factor H, and factor I; each were purified to homogeneity. A mixture of these proteins at physiological concentrations was shown to reproduce the ability of normal human serum to enhance the infection of MT2 cells by HIV-1 at low doses of virus. This enhancing effect was abolished when heat-inactivated serum and C2- or C3-depleted serum were used, and was restored upon addition of the corresponding purified proteins. A mixture of two synthetic peptides corresponding to positions 10–15 and 90–97 of human C receptor type 2 (CD21) as well as soluble CD4 both inhibited the C-dependent infection process. These data provide unambiguous evidence that HIV-1 triggers a direct activation of the classical C pathway in vitro and thereby facilitates the infection of MT2 cells at low doses of virus. These findings are consistent with a mechanism involving increased interaction between the virus opsonized by C3b-derived fragment(s) and the CD21 cell receptors and subsequent virus entry through CD4 receptors.
Ectopic Expression of DNA Encoding IFN-α1 in the Cornea Protects Mice from Herpes Simplex Virus Type 1-Induced Encephalitis
Authors: Noisakran Sansanee , Campbell Iain L. , Carr Daniel J. J.
Published: January 2023

A novel approach to combat acute herpes simplex virus type 1 (HSV-1) infection has recently been developed by administration with a plasmid DNA construct encoding cytokine genes. Cytokines, especially type I IFNs (IFN-α and IFN-β) play an important role in controlling acute HSV-1 infection. The purpose of the present study was to investigate the potential efficacy of ectopically expressed IFN-α1 against ocular HSV-1 infection following in situ transfection of mouse cornea with a naked IFN-α1-containing plasmid DNA. Topical administration of the IFN-α1 plasmid DNA exerted protection against ocular HSV-1 challenge in a time- and dose-dependent manner and antagonized HSV-1 reactivation. In addition, IFN-α1-transfected eyes expressed a fivefold increase in MHC class I mRNA over vector-treated controls. The protective efficacy of the IFN-α1 transgene antagonized viral replication, as evidenced by the reduction of the viral gene transcripts (infected cell polypeptide 27, thymidine kinase, and viral protein 16) and viral load in eyes and trigeminal ganglia during acute infection. The administration of neutralizing Ab to IFN-αβ antagonized the protective effect of the IFN-α1 transgene in mice. Collectively, these findings demonstrate the potential of using naked plasmid DNA transfection in the eye to achieve ectopic gene expression of therapeutically active agents.
Nerve Growth Factor Modifies the Expression of Inflammatory Cytokines by Mast Cells Via a Prostanoid-Dependent Mechanism
Authors: Marshall Jean S. , Gomi Kaede , Blennerhassett Michael G. , Bienenstock John
Published: January 2023

Nerve growth factor (NGF) is well recognized to have a number of potent effects on mast cells, including increasing mast cell numbers in vivo and inducing mast cell degranulation in vitro. More recently, NGF has been demonstrated to induce PGD2 production by mast cells through the induction of mast cell cyclooxygenase expression. We have observed that NGF at doses as low as 10 ng/ml will induce IL-6 production and inhibit TNF-α release from rat peritoneal mast cells in the presence of lysophosphatidylserine as a cofactor. NGF synergizes with LPS treatment of peritoneal mast cells (PMC) for the induction of IL-6. Examination of the mechanism of this phenomenon has revealed that NGF can induce both rat PMC and mouse bone marrow-derived cultured mast cells to produce substantial levels of PGE2. This response is maximal at later time points 18–24 h after NGF activation. The ability of NGF to induce PGE2 is not dependent on mast cell degranulation. Other stimuli capable of inducing IL-6, such as LPS, do not induce production of this prostanoid. Inhibition of cyclooxygenase activity by PMC using either flurbiprofen or indomethacin inhibited both the NGF-induced PGE2 synthesis and the NGF-induced alterations in TNF-α and IL-6 production. These results suggest a role for mast cell-derived prostanoids in the regulation of local inflammatory responses and neuronal degeneration after tissue injury involving induction of NGF production.
Eotaxin Activates T Cells to Chemotaxis and Adhesion Only if Induced to Express CCR3 by IL-2 Together with IL-4
Authors: Jinquan Tan , Quan Sha , Feili Gong , Larsen Christian Grønhøj , Thestrup-Pedersen Kristian
Published: January 2023

The transmigration and adherence of T lymphocytes through microvascular endothelium are essential events for their recruitment into inflammatory sites. In the present study, we investigated the expression of CC chemokine receptor CCR3 on T lymphocytes and the capacities of the CC chemokine eotaxin to induce chemotaxis and adhesion in T lymphocytes. We have observed a novel phenomenon that IL-2 and IL-4 induce the expression of CCR3 on T lymphocytes. We also report that CC chemokine eotaxin is a potent chemoattractant for IL-2- and IL-4-stimulated T lymphocytes, but not for freshly isolated T lymphocytes. Eotaxin attracts T lymphocytes via CCR3, documented by the fact that anti-CCR3 mAb blocks eotaxin-mediated T lymphocyte chemotaxis. In combination with IL-2 and IL-4, eotaxin enhances the expression of adhesion molecules such as ICAM-1 and several integrins (CD29, CD49a, and CD49b) on T lymphocytes and thus promotes adhesion and aggregation of T lymphocytes. The eotaxin-induced T lymphocyte adhesion could be selectively blocked by a specific cAMP-dependent protein kinase inhibitor, H-89, indicating that eotaxin activates T lymphocytes via a special cAMP-signaling pathway. Our new findings all point toward the fact that eotaxin, in association with the Th1-derived cytokine IL-2 and the Th2-derived cytokine IL-4, is an important T lymphocyte activator, stimulating the directional migration, adhesion, accumulation, and recruitment of T lymphocytes, and paralleled the accumulation of eosinophils and basophils during the process of certain types of inflammation such as allergy.
Molecular Characterization of American Cockroach Tropomyosin (Periplaneta americana Allergen 7), a Cross-Reactive Allergen
Authors: Asturias Juan A. , Gómez-Bayón Nuria , Arilla M. Carmen , Martínez Alberto , Palacios Ricardo , Sánchez-Gascón Fernando , Martínez Jorge
Published: January 2023

Inhalation of allergens produced by the American cockroach (Periplaneta americana) induces IgE Ab production and the development of asthma in genetically predisposed individuals. The cloning and expression in Escherichia coli of P. americana tropomyosin allergen have been achieved. The protein shares high homology with other arthropod tropomyosins (80% identity) but less homology with vertebrate ones (50% identity). The recombinant allergen was produced in E. coli as a nonfusion protein with a yield of 9 mg/l of bacterial culture. Both natural and recombinant tropomyosins were purified by isoelectric precipitation. P. americana allergen 1 (Per a 1) and Per a 7 (tropomyosin) are to date the only cross-reacting allergens found in cockroaches. ELISA and Western blot inhibition experiments, using natural and recombinant purified tropomyosins from shrimp and cockroach, showed that tropomyosin induced cross-reactivity of IgE from patients allergic to these allergens, suggesting that this molecule could be a common allergen among invertebrates.
Production of High Affinity Autoantibodies in Autoimmune New Zealand Black/New Zealand White F1 Mice Targeted with an Anti-DNA Heavy Chain
Authors: Friedmann Dinorah , Yachimovich Nurit , Mostoslavsky Gustavo , Pewzner-Jung Yael , Ben-Yehuda Arie , Rajewsky Klaus , Eilat Dan
Published: January 2023

Lupus-prone, anti-DNA, heavy (H) chain “knock-in” mice were obtained by backcrossing C57BL/6 mice, targeted with a rearranged H chain from a VH11(S107)-encoded anti-DNA hybridoma (D42), onto the autoimmune genetic background of New Zealand Black/New Zealand White (NZB/NZW) F1 mice. The targeted female mice developed typical lupus serologic manifestations, with the appearance of transgenic IgM anti-DNA autoantibodies at a young age (2–3 mo) and high affinity, somatically mutated IgM and IgG anti-DNA Abs at a later age (6–7 mo). However, they did not develop clinical, lupus-associated glomerulonephritis and survived to at least 18 mo of age. L chain analysis of transgenic anti-DNA Abs derived from diseased NZB/NZW mouse hybridomas showed a very restricted repertoire of Vκ utilization, different from that of nonautoimmune (C57BL/6 × BALB/c)F1 transgenic anti-DNA Abs. Strikingly, a single L chain was repetitively selected by most anti-DNA, transgenic NZB/NZW B cells to pair with the targeted H chain. This L chain had the same Vκ-Jκ rearrangement as that expressed by the original anti-DNA D42 hybridoma. These findings indicate that the kinetics of the autoimmune serologic manifestations are similar in wild-type and transgenic lupus-prone NZB/NZW F1 mice and suggest that the breakdown of immunologic tolerance in these mice is associated with the preferential expansion and activation of B cell clones expressing high affinity anti-DNA H/L receptor combinations.
TNF Receptor-Associated Factor-2 Is Involved in Both IL-1β and TNF-α Signaling Cascades Leading to NF-κB Activation and IL-8 Expression in Human Intestinal Epithelial Cells
Authors: Jobin Christian , Holt Lisa , Bradham Cynthia A. , Streetz Konrad , Brenner David A. , Sartor R. Balfour
Published: January 2023

Cytokine signaling involves the participation of many adaptor proteins, including the docking protein TNF receptor-associated factor-2 (TRAF-2), which is believed to transmit the TNF-α signal through both the IκB/NF-κB and c-Jun N-terminal kinase (JNK)/stress-related protein kinase (SAPK) pathways. The physiological role of TRAF proteins in cytokine signaling in intestinal epithelial cells (IEC) is unknown. We characterized the effect of a dominant-negative TRAF-2 delivered by an adenoviral vector (Ad5dnTRAF-2) on the cytokine signaling cascade in several IEC and also investigated whether inhibiting the TRAF-2-transmitting signal blocked TNF-α-induced NF-κB and IL-8 gene expression. A high efficacy and level of Ad5dnTRAF-2 gene transfer were obtained in IEC using a multiplicity of infection of 50. Ad5dnTRAF-2 expression prevented TNF-α-induced, but not IL-1β-induced, IκBα degradation and NF-κB activation in NIH-3T3 and IEC-6 cells. TNF-α-induced JNK activation was also inhibited in Ad5dnTRAF-2-infected HT-29 cells. Induction of IL-8 gene expression by TNF-α was partially inhibited in Ad5dnTRAF-2-transfected HT-29, but not in control Ad5LacZ-infected, cells. Surprisingly, IL-1β-mediated IL-8 gene expression was also inhibited in HT-29 cells as measured by Northern blot and ELISA. We concluded that TRAF-2 is partially involved in TNF-α-mediated signaling through IκB/NF-κB in IEC. In addition, our data suggest that TRAF-2 is involved in IL-1β signaling in HT-29 cells. Manipulation of cytokine signaling pathways represents a new approach for inhibiting proinflammatory gene expression in IEC.
T Cell Immunity Induced by Allogeneic Microglia in Relation to Neuronal Retina Transplantation
Authors: Ma Naili , Streilein J. Wayne
Published: January 2023

Microglia share a lineage relationship with bone marrow-derived monocytes/macrophages and dendritic cells, and their inclusion in retinal and brain transplants may function as “passenger leukocytes.” In other solid allografts, passenger leukocytes are the primary sources of immunogenicity, triggering alloimmune rejection. We have conducted a series of in vitro and in vivo studies examining the capacity of microglia cultured from forebrain to activate alloreactive T cells and to induce and elicit alloimmunity. Cultured microglia expressed class II MHC molecules and costimulatory molecules (B7-1, B7-2, and CD40), and they secreted IL-12. Cultured microglia injected s.c. into naive recipients induced allospecific delayed hypersensitivity and elicited delayed hypersensitivity directed at alloantigens. Cultured microglia differed from conventional APCs by secreting significant amounts of mature TGF-β2, but smaller amounts of IL-12. Moreover, while both cultured microglia and conventional APC stimulated T cell proliferation in vitro, microglia directed the responding T cells toward the Th2 pathway in which IL-4, but not IL-2 and IFN-γ, was secreted. The abilities of microglia to secrete TGF-β2, to stimulate alloreactive Th2 cells, and to induce anterior chamber associated immune deviation when injected into the eye of naive allogeneic mice suggest that they are not typical passenger leukocytes. The unique functional properties of cultured microglia may account for the capacity of neonatal retinal tissue transplanted into the eye to alter the systemic alloimmune response in a manner that delays, but does not prevent, graft rejection.
Biochemical and Immunogenetic Analysis of an Immunodominant Peptide (B6dom1) Encoded by the Classical H7 Minor Histocompatibility Locus
Authors: Eden Peter A. , Christianson Gregory J. , Fontaine Pierre , Wettstein Peter J. , Perreault Claude , Roopenian Derry C.
Published: January 2023

Of the many minor histocompatibility (H) Ags that have been detected in mice, the ability to induce graft vs host disease (GVHD) after bone marrow transplantation is restricted to a limited number of immunodominant Ags. One such murine Ag, B6dom1, is presented by the H2-Db MHC class I molecule. We present biochemical evidence that the natural B6dom1 peptide is indistinguishable from AAPDNRETF, and we show that this peptide can be isolated from a wide array of tissues, with highest levels from the lymphoid organs and lung. Moreover, we employ a novel, somatic cell selection technique involving CTL-mediated immunoselection coupled with classical genetics, to show that B6dom1 is encoded by the H7 minor H locus originally discovered ∼40 years ago. These studies provide a molecular genetic framework for understanding B6dom1, and exemplify the fact that mouse minor H loci that encode immunodominant CTL epitopes can correspond to classical H loci originally identified by their ability to confer strong resistance to tumor transplantation. Additionally, these studies demonstrate the utility of somatic cell selection approaches toward resolving H Ag immunogenetics.
Bystander Virus Infection Prolongs Activated T Cell Survival
Authors: Mitchell Tom , Kappler John , Marrack Philippa
Published: January 2023

In animals, T cells often die rapidly after activation, unless activation occurs in the presence of inflammatory factors. To understand how such activated cells survive to participate in immune responses, we studied the effects of viral infection on T cells responding to an unrelated superantigen. Normal T cells activated by superantigen in uninfected mice died as a result of their activation, whereas T cells that were activated during vaccinia infection survived longer in vivo and in culture. This bystander effect of viral infection on activated T cells was independent of effects on the magnitude of the initial T cell response, on induction of Bcl-2 and Bcl-x, on T cell proliferation, and on Fas killing. The failure of such effects to predict the fate of activated T cells in vivo indicates that virus infections shape T cell responses via mechanisms that differ from those described previously. These mechanisms may contribute to the ability of viral infections to induce autoimmunity.
In Vivo Selection of Neutralization-Resistant Virus Variants But No Evidence of B Cell Tolerance in Lymphocytic Choriomeningitis Virus Carrier Mice Expressing a Transgenic Virus-Neutralizing Antibody
Authors: Seiler Peter , Senn Beatrice M. , Bründler Marie-Anne , Zinkernagel Rolf M. , Hengartner Hans , Kalinke Ulrich
Published: January 2023

B cell tolerance is maintained by active deletion and functional anergy of self-reactive B cells depending on the time, amount, and site of the self-antigen expression. To study B cell tolerance toward a transplacentally transmitted viral Ag, we crossed transgenic mice expressing the μ heavy and the κ light chain of the lymphocytic choriomeningitis virus (LCMV)-neutralizing mAb KL25 (HL25-transgenic mice) with persistently infected LCMV carrier mice. Although HL25-transgenic LCMV carrier mice exhibited the same high virus titers as nontransgenic LCMV carrier mice, no evidence for B cell tolerance was found. In contrast, enhanced LCMV-neutralizing Ab titers were measured that, however, did not clear the virus. Instead, LCMV isolates from different tissues turned out to be neutralization resistant Ab escape variants expressing different substitutions of amino acid Asn119 of the LCMV-glycoprotein 1 that displays the neutralizing B cell epitope. Virus variants with the same mutations were also selected in vitro in the presence of the transgenic mAb KL25 confirming that substitutions of Asn119 have been selected by LCMV-neutralizing Abs. Thus, despite abundant expression of viral neo-self-antigen in HL25-transgenic LCMV carrier mice, transgenic B cells expressing LCMV-neutralizing Abs were rather stimulated than tolerized and neutralization resistant Ab escape variants were selected in vivo.
Unique Superantigen Activity of Staphylococcal Exfoliative Toxins
Authors: Monday Steven R. , Vath Gregory M. , Ferens Witold A. , Deobald Claudia , Rago James V. , Gahr Pamala J. , Monie Dileep D. , Iandolo John J. , Chapes Stephen K. , Davis William C. , Ohlendorf Douglas H. , Schlievert Patrick M. , Bohach Gregory A.
Published: January 2023

Certain strains of Staphylococcus aureus express one or both of two related, but immunologically distinct, exfoliative toxins (ETA and ETB). These toxins induce the symptoms associated with staphylococcal scalded skin syndrome. Both ETs have been shown to stimulate T cell proliferation. Recently, it was reported that ETA is a superantigen that stimulates T cells bearing human Vβ2 or several murine Vβs. However, other investigators have proposed that the superantigenicity reported for ETA resulted from contaminants in commercial preparations. This present study addresses those conflicting reports by assessing the biological and immunologic activities of highly purified rETs. ETA and ETB required APCs to induce selective polyclonal expansion of several human Vβs (huVβs), although, neither toxin expanded huVβ2. ETB induced expansion of murine T cells bearing Vβs 7 and 8, those that have the highest homology to the huVβs expanded by ETA and ETB. Although flow cytometry of ETB-stimulated T cells matched PCR results, stimulation by ETA reduced percentages of T cells positive for several huVβs that had been shown to have increased levels of mRNA transcripts. ETA and ETB induced contrasting reactions in vivo. In rabbits, ETB was moderately pyrogenic and enhanced susceptibility to lethal shock, while ETA lacked both activities. Predictions based on comparisons with other superantigens suggest molecular regions potentially involved in receptor binding in the ETA crystal structure and a modeled ETB three-dimensional structure. These results show that ETs are superantigens with unique properties that could account for the discrepancies reported.
Autoantigen-Independent Deletion of Diabetogenic CD4+ Thymocytes by Protective MHC Class II Molecules
Authors: Schmidt Dennis , Amrani Abdelaziz , Verdaguer Joan , Bou Sonny , Santamaria Pere
Published: January 2023

Some MHC class II genes provide dominant resistance to certain autoimmune diseases via mechanisms that remain unclear. We have shown that thymocytes bearing a highly diabetogenic, I-Ag7-restricted β-cell-reactive TCR (4.1-TCR) undergo negative selection in diabetes-resistant H-2g7/x mice by engaging several different antidiabetogenic MHC class II molecules on thymic (but not peripheral) hemopoietic cells, independently of endogenous superantigens. Here we have investigated 1) whether this TCR can also engage protective MHC class II molecules (I-Ab) on cortical thymic epithelial cells in the absence of diabetogenic (I-Ag7) molecules, and 2) whether deletion of 4.1-CD4+ thymocytes in I-Ab-expressing mice might result from the ability of I-Ab molecules to present the target β-cell autoantigen of the 4.1-TCR. We show that, unlike I-Ag7 molecules, I-Ab molecules can restrict neither the positive selection of 4.1-CD4+ thymocytes in the thymic cortex nor the presentation of their target autoantigen in the periphery. Deletion of 4.1-CD4+ thymocytes by I-Ab molecules in the thymic medulla, however, is a peptide-specific process, since it can be triggered by hemopoietic cells expressing heterogeneous peptide/I-Ab complexes, but not by hemopoietic cells expressing single peptide/I-Ab complexes. Thus, unlike MHC-autoreactive or alloreactive TCRs, which can engage deleting MHC molecules in the thymic cortex, thymic medulla, and peripheral APCs, the 4.1-TCR can only engage deleting MHC molecules (I-Ab) in the thymic medulla. We therefore conclude that this form of MHC-induced protection from diabetes is based on the presentation of an anatomically restricted, nonautoantigenic peptide to highly diabetogenic thymocytes.
IFN-γ Regulation of the Type IV Class II Transactivator Promoter in Astrocytes
Authors: Dong Yuanshu , Rohn Wolfgang M. , Benveniste Etty N.
Published: January 2023

The transcriptional activation of class II MHC genes requires the class II transactivator (CIITA) protein, a regulator that is essential for both constitutive and IFN-γ-inducible class II MHC expression. The CIITA gene is controlled by multiple independent promoters; two promoters direct constitutive expression, while another, the type IV CIITA promoter, mediates IFN-γ-induced expression. We investigated the molecular regulation of IFN-γ-induced type IV CIITA promoter activity in astrocytes. IFN-γ inducibility of the type IV CIITA promoter is dependent on three cis-acting elements contained within a 154-bp fragment of the promoter; the proximal IFN-γ activation sequence (GAS) element, the E box, and the proximal IFN regulatory factor (IRF) element. Two IFN-γ-activated transcription factors, STAT-1α and IRF-1, bind the proximal GAS and IRF elements, respectively. The E box binds upstream stimulating factor-1 (USF-1), a constitutively expressed transcription factor. Furthermore, STAT-1α binding to the proximal GAS element is dependent on the binding of USF-1 to the adjacent E box. Functionally, the proximal IRF element is essential for IFN-γ induction of type IV CIITA promoter activity, while the proximal GAS and E box elements contribute to the IFN-γ inducibility of this promoter. In astrocytes, TNF-α enhances IFN-γ-induced class II MHC transcription. Our results demonstrate that TNF-α does not enhance IFN-γ-induced transcriptional activation of the type IV CIITA promoter, indicating that the enhancing effect of TNF-α is mediated downstream of CIITA transcription. These results define the molecular basis of IFN-γ activation of the type IV CIITA promoter in astrocytes.
Persistent HIV-1-Specific CTL Clonal Expansion Despite High Viral Burden Post In Utero HIV-1 Infection
Authors: Brander Christian , Goulder Philip J. R. , Luzuriaga Katherine , Yang Otto O. , Hartman Kelly E. , Jones Norman G. , Walker Bruce D. , Kalams Spyros A.
Published: January 2023

To address the issue of clonal exhaustion in humans, we monitored HLA class I-restricted, epitope-specific CTL responses in an in utero HIV-1-infected infant from 3 mo through 5 years of age. Serial functional CTL precursor assays demonstrated persistent, vigorous, and broadly directed HIV-1 specific CTL activity with a dominant response against an epitope in HIV-1 Gag-p17 (SLYNTVATL, aa 77–85). A clonal CTL response directed against the immunodominant, HLA-A*0201-restricted epitope was found to persist over the entire observation period, as shown by TCR analysis of cDNA libraries generated from PBMC. The analysis of autologous viral sequences did not reveal any escape mutations within the targeted epitope, and viral load measurement indicated ongoing viral replication. Furthermore, inhibition of viral replication assays indicated that the epitope was properly processed from autologous viral protein. These data demonstrate that persistent exposure to high levels of viral Ag does not necessarily lead to clonal exhaustion and that epitope-specific clonal CTL responses induced within the first weeks of life can persist for years without inducing detectable viral escape variants.
Host B7-1 and B7-2 Costimulatory Molecules Contribute to the Eradication of B7-1-Transfected P815 Tumor Cells Via a CD8+ T Cell-Dependent Mechanism
Authors: Motte Ross N. La , Sharpe Arlene H. , Bluestone Jeffrey A. , Mokyr Margalit B.
Published: January 2023

B7-1 (CD80)-transfected P815 tumor cells were previously shown to elicit tumor-eradicating immunity that leads to the regression of B7-1+ P815 tumors after transient growth in normal syngeneic (DBA/2) mice. Here, we show that not only the B7-1 molecule but also the B7-2 (CD86) molecule contributed to the eradication of B7-1+ P815 tumors. The B7-1 molecule that contributed to the eradication of B7-1+ P815 tumors was expressed not only on the tumor cells but also on host APCs, including MAC-1+ cells. The B7-2 molecule that contributed to the eradication of B7-1+ P815 tumors was expressed only on host APCs, such as B220+ cells, and not on the tumor cells. In spite of the fact that B7-expressing host APCs contributed to the eradication of B7-1+ P815 tumors, only CD8+ T cells without help from CD4+ T cells were important for tumor eradication. Taken together, these findings indicate that in addition to the ability of B7-1-transfected tumor cells to stimulate CD8+ T cell-mediated tumor-eradicating immunity directly, such tumor cells can also stimulate CD8+ T cell-mediated tumor-eradicating immunity indirectly as a result of cross-priming through B7-expressing host APCs.
Signal Transduction Pathways Activated in Endothelial Cells Following Infection with Chlamydia pneumoniae
Authors: Krüll Matthias , Klucken Andrea C. , Wuppermann Frederik N. , Fuhrmann Oliver , Magerl Christian , Seybold Joachim , Hippenstiel Stefan , Hegemann Johannes H. , Jantos Christian A. , Suttorp Norbert
Published: January 2023

Chlamydia pneumoniae is an important respiratory pathogen. Recently, its presence has been demonstrated in atherosclerotic lesions. In this study, we characterized C. pneumoniae-mediated activation of endothelial cells and demonstrated an enhanced expression of endothelial adhesion molecules followed by subsequent rolling, adhesion, and transmigration of leukocytes (monocytes, granulocytes). These effects were blocked by mAbs against endothelial and/or leukocyte adhesion molecules (β1 and β2 integrins). Additionally, activation of different signal transduction pathways in C. pneumoniae-infected endothelial cells was shown: protein tyrosine phosphorylation, up-regulation of phosphorylated p42/p44 mitogen-activated protein kinase, and NF-κB activation/translocation occurred within 10–15 min. Increased mRNA and surface expression of E-selectin, ICAM-1, and VCAM-1 were noted within hours. Thus, C. pneumoniae triggers a cascade of events that could lead to endothelial activation, inflammation, and thrombosis, which in turn may result in or may promote atherosclerosis.
Differential Effects of Leukotactin-1 and Macrophage Inflammatory Protein-1α on Neutrophils Mediated by CCR1
Authors: Zhang Shangming , Youn Byung-S. , Gao Ji-Liang , Murphy Philip M. , Kwon Byoung S.
Published: January 2023

The human CC chemokine leukotactin-1 (Lkn-1) is both a strong chemoattractant for neutrophils, monocytes, and lymphocytes and a potent agonist for CCR1 and CCR3. However, human neutrophils do not migrate when the cells are stimulated with other human CC chemokines, such as human macrophage inflammatory protein-1α (hMIP-1α) and eotaxin, which also use the CCR1 and CCR3 as their receptors. In this report, we demonstrate that while hMIP-1α induced a negligible level of calcium flux and chemotaxis, Lkn-1 produced a high level of calcium flux and chemotaxis in human neutrophils. Lkn-1 cross-desensitized hMIP-1α-induced calcium flux, but hMIP-1α had little effect on the Lkn-1-induced response in human neutrophils. The same pattern was observed in peritoneal neutrophils from wild-type mice, whereas neutrophils from CCR1−/− mice failed to respond to either MIP-1α or Lkn-1. Scatchard analysis revealed a single class of receptor for both hMIP-1α and Lkn-1 on human neutrophils with dissociation constants (Kd) of 3.2 nM and 1.1 nM, respectively. We conclude that CCR1 is a receptor mediating responses to both MIP-1α and Lkn-1 on neutrophils and produces different biological responses depending on the ligand bound.
Development of an Animal Model of Autoimmune Thyroid Eye Disease
Authors: Many M.-C. , Costagliola S. , Detrait M. , Denef J.-F. , Vassart G. , Ludgate M.
Published: January 2023

In previous studies we have transferred thyroiditis to naive BALB/c and NOD mice with human thyrotropin (TSH) receptor (TSHR)-primed splenocytes. Because the TSHR has been implicated in the pathogenesis of thyroid eye disease (TED) we have examined the orbits of recipients of TSHR-primed T cells, generated using a TSHR fusion protein or by genetic immunization. In the NOD mice, 25 of 26 animals treated with TSHR-primed T cells developed thyroiditis with considerable follicular destruction, numerous activated and CD8+ T cells, and immunoreactivity for IFN-γ. Thyroxine levels were reduced. Thyroiditis was not induced in controls. None of the NOD animals developed any orbital pathology. Thirty-five BALB/c mice received TSHR-primed spleen cells. Thyroiditis was induced in 60–100% and comprised activated T cells, B cells, and immunoreactivity for IL-4 and IL-10. Autoantibodies to the receptor were induced, including TSH binding inhibiting Igs. A total of 17 of 25 BALB/c orbits displayed changes consisting of accumulation of adipose tissue, edema caused by periodic acid Schiff-positive material, dissociation of the muscle fibers, the presence of TSHR immunoreactivity, and infiltration by lymphocytes and mast cells. No orbital changes or thyroiditis were observed in control BALB/c mice. We have induced orbital pathology having many parallels with human TED, only in BALB/c mice, suggesting that a Th2 autoimmune response to the TSHR may be a prerequisite for the development of TED.