bluuebunny/crossref_metadata_2025 huggingface dataset:
Trophoblast Cell Line Resistance to NK Lysis Mainly Involves an HLA Class I-Independent Mechanism
Authors: Avril Tony , Jarousseau Annie-Claude , Watier Herve , Boucraut Jose , Le Bouteiller Philippe , Bardos Pierre , Thibault Gilles
Published: January 2023

The lack of classical HLA molecules on trophoblast prevents allorecognition by maternal T lymphocytes, but poses the problem of susceptibility to NK lysis. Expression of the nonclassical class I molecule, HLA-G, on cytotrophoblast may provide the protective effect. However, the class I-negative syncytiotrophoblast escapes NK lysis by maternal PBL. In addition, while HLA-G-expressing transfectants of LCL.721.221 cells are protected from lymphokine-activated killer lysis, extravillous cytotrophoblast cells and HLA-G-expressing choriocarcinoma cells (CC) are not. The aim of this work was therefore to clarify the role of HLA class I expression on trophoblast cell resistance to NK lysis and on their susceptibility to lymphokine-activated killer lysis. Our results showed that both JAR (HLA class I-negative) and JEG-3 (HLA-G- and HLA-Cw4-positive) cells were resistant to NK lysis by PBL and were equally lysed by IL-2-stimulated PBL isolated from a given donor. In agreement, down-regulating HLA class I expression on JEG-3 cells by acid treatment, masking these molecules or the putative HLA-G (or HLA-E) receptor CD94/NKG2 and the CD158a/p58.1 NKR with mAbs, and inducing self class I molecule expression on JAR cells did not affect NK or LAK lysis of CC. These results demonstrate that the resistance of CC to NK lysis mainly involves an HLA class I-independent mechanism(s). In addition, we show that the expression of a classical class I target molecule (HLA-B7) on JAR cells is insufficient to induce lysis by allospecific polyclonal CTL.
T21/DP107, A Synthetic Leucine Zipper-Like Domain of the HIV-1 Envelope gp41, Attracts and Activates Human Phagocytes by Using G-Protein-Coupled Formyl Peptide Receptors
Authors: Su Shao Bo , Gao Ji-liang , Gong Wang-hua , Dunlop Nancy M. , Murphy Philip M. , Oppenheim Joost J. , Wang Ji Ming
Published: January 2023

A leucine zipper-like domain, T21/DP107, located in the amino terminus of the ectodomain of gp41, is crucial to the formation of fusogenic configuration of the HIV-1 envelope protein gp41. We report that the synthetic T21/DP107 segment is a potent stimulant of migration and calcium mobilization in human monocytes and neutrophils. The activity of T21/DP107 on phagocytes was pertussis toxin-sensitive, suggesting this peptide uses Gi-coupled seven-transmembrane receptor(s). Since the bacterial chemotactic peptide fMLP partially desensitized the calcium-mobilizing activity of T21/DP107 in phagocytes, we postulated that T21/DP107 might preferentially use a lower affinity fMLP receptor. By using cells transfected to express cloned prototype chemotactic N-formyl peptide receptor (FPR) or its variant, FPR-like 1 (FPRL1), we demonstrate that T21/DP107 activates both receptors but has a much higher efficacy for FPRL1. In addition, T21/DP107 at nM concentrations induced migration of FPRL1-transfected human embryonic kidney 293 cells. In contrast, fMLP did not induce significant chemotaxis of the same cells at a concentration as high as 50 μM. Although a lipid metabolite, lipoxin A4, was a high-affinity ligand for FPRL1, it was not reported to induce Ca2+ mobilization or chemotaxis in FPRL1-transfected cells. Therefore, T21/DP107 is a first chemotactic peptide agonist identified thus far for FPRL1. Our results suggest that this peptide domain of the HIV-1 gp41 may have the potential to activate host innate immune response by interacting with FPR and FPRL1 on phagocytes.
Recognition of an MHC Class I-Restricted Antigenic Peptide Can Be Modulated by para-Substitution of Its Buried Tyrosine Residues in a TCR-Specific Manner
Authors: Saito Naoyuki G. , Chang Hsiu-Ching , Paterson Yvonne
Published: January 2023

Conformational dependence of TCR contact residues of the H-2Kb molecule on the two buried tyrosine side chains of the vesicular stomatitis virus (VSV)-8 peptide was investigated by systematic substitutions of the tyrosines with phenylalanine, p-fluorophenylalanine (pFF), or p-bromophenylalanine (pBrF). The results of peptide competition CTL assays revealed that all of the peptide variants, except for the pBrF analogues, had near-native binding to the H-2Kb molecule. Epitope-mapped anti-H-2Kb mAbs detected conformational differences among H-2Kb molecules stabilized with these VSV-8 variants on RMA-S cells. Selective recognition of the VSV-8 analogues was displayed by a panel of three H-2Kb-restricted, anti-VSV-8 TCRs. Thus, these substitutions result in an antigenically significant conformational change of the MHC molecular surface structure at both C and D pockets, and the effect of this change on cognate T cell recognition is dependent on the TCR structure. Our results confirm that the structure of buried peptide side chains can determine the surface conformation of the MHC molecule and demonstrate that even a very subtle structural nuance of the buried side chain can be incorporated into the surface conformation of the MHC molecule. The ability of buried residues to modulate this molecular surface augments the number of residues on the MHC-peptide complex that can be recognized as “foreign” by the CD8+ T cell repertoire and allows for a higher level of antigenic discrimination. This may be an important mechanism to expand the total number of TCR specificities that can respond to a single peptide determinant.
Signaling Through the Lymphotoxin-β Receptor Stimulates HIV-1 Replication Alone and in Cooperation with Soluble or Membrane-Bound TNF-α
Authors: Marshall William L. , Brinkman Brigitta M. N. , Ambrose Christine M. , Pesavento Patricia A. , Uglialoro Adele M. , Teng Edna , Finberg Robert W. , Browning Jeffrey L. , Goldfeld Anne E.
Published: January 2023

The level of ongoing HIV-1 replication within an individual is critical to HIV-1 pathogenesis. Among host immune factors, the cytokine TNF-α has previously been shown to increase HIV-1 replication in various monocyte and T cell model systems. Here, we demonstrate that signaling through the TNF receptor family member, the lymphotoxin-β (LT-β) receptor (LT-βR), also regulates HIV-1 replication. Furthermore, HIV-1 replication is cooperatively stimulated when the distinct LT-βR and TNF receptor systems are simultaneously engaged by their specific ligands. Moreover, in a physiological coculture cellular assay system, we show that membrane-bound TNF-α and LT-α1β2 act virtually identically to their soluble forms in the regulation of HIV-1 replication. Thus, cosignaling via the LT-β and TNF-α receptors is probably involved in the modulation of HIV-1 replication and the subsequent determination of HIV-1 viral burden in monocytes. Intriguingly, surface expression of LT-α1β2 is up-regulated on a T cell line acutely infected with HIV-1, suggesting a positive feedback loop between HIV-1 infection, LT-α1β2 expression, and HIV-1 replication. Given the critical role that LT-α1β2 plays in lymphoid architecture, we speculate that LT-α1β2 may be involved in HIV-associated abnormalities of the lymphoid organs.
Covalent Linkage to β2-Microglobulin Enhances the MHC Stability and Antigenicity of Suboptimal CTL Epitopes
Authors: Uger Robert A. , Chan Steven M. , Barber Brian H.
Published: January 2023

Many CTL epitopes of clinical importance, particularly those derived from tumor Ags, display relatively poor MHC binding affinity and stability. Because in vivo immunogenicity, and thus the efficacy of peptide-based vaccines, is thought to be determined by MHC/peptide complex stability, there is a need to develop a simple strategy for enhancing the binding of suboptimal epitopes. Toward this goal, the ability to enhance suboptimal peptides through covalent linkage to β2-microglobulin (β2m) was explored. Two suboptimal variants of a high-affinity Db-restricted influenza nucleoprotein peptide were covalently linked, via a polypeptide spacer, to the amino terminus of human β2m and the recombinant fusion proteins expressed in Escherichia coli. When compared with their uncoupled counterparts, the β2m-linked epitopes display enhanced MHC stabilization and antigenicity. Thus, tethering epitopes to β2m provides a simple method for augmenting the biological activity of suboptimal peptides and could be useful in the design of peptide-based vaccines or immunotherapeutics.
Evidence That the Igκ Gene MAR Regulates the Probability of Premature V-J Joining and Somatic Hypermutation
Authors: Yi Ming , Wu Peiqing , Trevorrow Kenneth W. , Claflin Latham , Garrard William T.
Published: January 2023

The Igκ gene contains an evolutionarily conserved nuclear matrix association region (MAR) adjacent to the intronic enhancer. To test for the function of this MAR, we created mouse lines with a targeted MAR deletion. In MAR knockout animals, the immune system was normal in nearly all respects, including the distributions of various B cell populations and Ab levels. However, in pro-B cells, enhanced rearrangement was noted on the MAR− allele in heterozygotes. In addition, the efficiencies for targeting and generating somatic mutations were reduced on MAR-deleted alleles. These results provide evidence for the MAR negatively regulating the probability of premature rearrangement and positively regulating the probability of somatic hypermutation.
Fine Specificity of Ligand-Binding Domain 1 in the Polymeric Ig Receptor: Importance of the CDR2-Containing Region for IgM Interaction
Authors: Røe Målfrid , Norderhaug Inger N. , Brandtzaeg Per , Johansen Finn-Eirik
Published: January 2023

The human polymeric Ig receptor (pIgR), also called transmembrane secretory component, is expressed basolaterally on exocrine epithelia, and mediates specific external transport of dimeric IgA and pentameric IgM. The extracellular part of pIgR consists of five Ig-like domains (D1-D5), and a highly conserved D1 region appears to mediate the initial noncovalent ligand interaction. While the human pIgR binds both dimeric IgA and pentameric IgM with high affinity, the rabbit counterpart has virtually no binding capacity for pentameric IgM. This remarkable disparity constitutes evidence that the binding site of the two ligands differs with regard to essential receptor contact elements. Therefore, we expressed human/rabbit chimeric pIgRs in Madin-Darby canine kidney cells and found that human pIgR D1 is crucial for the interaction with pentameric IgM when placed in the context of a full-length receptor regardless of its backbone species. D1 contains three complementarity-determining region-like loops (CDR1–3), and to further map human D1 regions involved in pentameric IgM binding, we transfected Madin-Darby canine kidney cells with human/rabbit chimeric receptors in which the regions containing the CDR-like loops had been interchanged. Our results showed that the region containing the CDR2-like loop is the most essential for pentameric IgM binding. The region containing the CDR1-like loop also contributed substantially to this interaction, whereas only little contribution was provided by the region containing the CDR3-like loop, although it appeared to be necessary for maximal pentameric IgM binding.
TRAIL (Apo-2L) and TRAIL Receptors in Human Placentas: Implications for Immune Privilege
Authors: Phillips Teresa A. , Ni Jian , Pan Guohua , Ruben Steven M. , Wei Ying-Fei , Pace Judith L. , Hunt Joan S.
Published: January 2023

Mechanisms accounting for protection of the fetal semiallograft from maternal immune cells remain incompletely understood. In other contexts, interactions between TRAIL (TNF-related apoptosis-inducing ligand/Apo-2L) and its receptors kill activated lymphocytes. The purpose of this study was therefore to investigate the potential of the TRAIL/TRAIL-R system to protect the placenta against immune cell attack. Analysis by Northern blotting demonstrated mRNAs encoding TRAIL as well as the four TRAIL receptors (DR4, DR5, DcR1/TRID, DcR2/TRUNDD) in human placentas. Immunohistochemical experiments demonstrated that TRAIL protein is prominent in syncytiotrophoblast, an uninterrupted placental cell layer that is continuously exposed to maternal blood, as well as in macrophage-like placental mesenchymal cells (Hofbauer cells). Studies on cell lines representing trophoblasts (Jar, JEG-3 cells) and macrophages (U937, THP-1 cells) showed that both lineages contained TRAIL mRNA and that steady state levels of transcripts were increased 2- to 11-fold by IFN-γ. By contrast, cell lineage-specific differences were observed in expression of the TRAIL-R genes. Although all four lines contained mRNA encoding the apoptosis-inducing DR5 receptor, only trophoblast cells contained mRNA encoding the DcR1 decoy receptor and only macrophages contained DcR2 decoy receptor transcripts. DR4 mRNA was present only in THP-1 cells and was the only TRAIL-R transcript increased by IFN-γ. Cytotoxicity assays revealed that the two trophoblast cell lines were resistant, whereas the two macrophage lines were partially susceptible to killing by rTRAIL. Collectively, the results are consistent with a role for the TRAIL/TRAIL-R system in the establishment of placental immune privilege.
Fas-Independent Cytotoxicity Mediated by Human CD4+ CTL Directed Against Herpes Simplex Virus-Infected Cells
Authors: Yasukawa Masaki , Ohminami Hideki , Yakushijin Yoshihiro , Arai Junko , Hasegawa Atsuhiko , Ishida Yasushi , Fujita Shigeru
Published: January 2023

The present study was undertaken to clarify the mechanisms of cytotoxicity mediated by virus-specific human CD4+ CTLs using the lymphocytes of family members with a Fas gene mutation. CD4+ CTL bulk lines and clones directed against HSV-infected cells were established from lymphocytes of a patient with a homozygous Fas gene mutation and of the patient’s mother. HSV-specific CD4+ CTLs generated from lymphocytes of the patient and her mother exerted cytotoxicity against HSV-infected cells from the patient (Fas−/−) and from her mother (Fas+/−) to almost the same degree in an HLA class II-restricted manner. mRNAs for the major mediators of CTL cytotoxicity, Fas ligand, perforin, and granzyme B, were detected in these CD4+ CTLs using the RT-PCR and flow cytometry. The cytotoxicity of the HSV-specific CD4+ CTLs appeared to be Ca2+-dependent and was almost completely inhibited by concanamycin A, a potent inhibitor of the perforin-based cytotoxic pathway. Although the Fas/Fas ligand system has been reported to be the most important mechanism for CD4+ CTL-mediated cytotoxicity in the murine system, the present findings strongly suggest that granule exocytosis, not the Fas/Fas ligand system, is the main pathway for the cytotoxicity mediated by HSV-specific human CD4+ CTLs.
TNF-α and IL-10 Modulate the Induction of Apoptosis by Virulent Mycobacterium tuberculosis in Murine Macrophages
Authors: Rojas Mauricio , Olivier Martin , Gros Philippe , Barrera Luis F. , García Luis F.
Published: January 2023

The Bcg/Nramp1 gene controls early resistance and susceptibility of macrophages to mycobacterial infections. We previously reported that Mycobacterium tuberculosis-infected (Mtb) B10R (Bcgr) and B10S (Bcgs) macrophages differentially produce nitric oxide (NO−), leading to macrophage apoptosis. Since TNF-α and IL-10 have opposite effects on many macrophage functions, we determined the number of cells producing TNF-α and IL-10 in Mtb-infected or purified protein derivative-stimulated B10R and B10S macrophages lines, and Nramp1+/+ and Nramp1−/− peritoneal macrophages and correlated them with Mtb-mediated apoptosis. Mtb infection and purified protein derivative treatment induced more TNF-α+Nramp1+/+ and B10R, and more IL-10+Nramp1−/− and B10S cells. Treatment with mannosylated lipoarabinomannan, which rescues macrophages from Mtb-induced apoptosis, augmented the number of IL-10 B10R+ cells. Anti-TNF-α inhibited apoptosis, diminished NO− production, p53, and caspase 1 activation and increased Bcl-2 expression. In contrast, anti-IL-10 increased caspase 1 activation, p53 expression, and apoptosis, although there was no increment in NO− production. Murine rTNF-α induced apoptosis in noninfected B10R and B10S macrophages that was reversed by murine rIL-10 in a dose-dependent manner with concomitant inhibition of NO− production and caspase 1 activation. NO− and caspase 1 seem to be independently activated in that aminoguanidine did not affect caspase 1 activation and the inhibitor of caspase 1, Tyr-Val-Ala-Asp-acylooxymethylketone, did not block NO− production; however, both treatments inhibited apoptosis. These results show that Mtb activates TNF-α- and IL-10-dependent opposite signals in the induction of macrophage apoptosis and suggest that the TNF-α-IL-10 ratio is controlled by the Nramp1 background of resistance/susceptibility and may account for the balance between apoptosis and macrophage survival.
IL-4- and IL-4 Receptor-Deficient BALB/c Mice Reveal Differences in Susceptibility to Leishmania major Parasite Substrains
Authors: Noben-Trauth Nancy , Paul William E. , Sacks David L.
Published: January 2023

Using genetically pure BALB/c mice deficient in IL-4 (IL-4−/−) or IL-4 receptor α-chain (IL-4Rα−/−), we have observed different disease outcomes to Leishmania major infection depending on the parasite substrain. Infection with L. major LV39 caused progressive, nonhealing ulcers and uncontrolled parasite growth in both IL-4−/− and IL-4Rα−/− mice. In contrast, infection with L. major IR173 was partially controlled in IL-4−/− mice but efficiently controlled in IL-4Rα−/− mice. Both IL-4−/− and IL-4Rα−/− mice infected with either substrain displayed reduced Th2 responses. Surprisingly, IFN-γ secretion was not up-regulated in the mutant mice, even in the IL-4Rα−/− mice, which were resistant to L. major IR173. The lack of increased IFN-γ production suggests that cytokine cross-regulation may not be operating in this model and that the effective ratios of Th1/Th2 cytokines become more indicative of disease outcome. The partial vs complete resistance to IR173 in IL-4−/− or IL-4Rα−/− mice implies that, in addition to IL-4, IL-13 may be involved in disease progression during L. major infection. The results with LV39 infection indicate that yet another unidentified factor is capable of causing susceptibility to L. major in the absence of IL-4 or IL-4 signaling.
Regulation of the Macrophage Vacuolar ATPase and Phagosome-Lysosome Fusion by Histoplasma capsulatum
Authors: Strasser Jane E. , Newman Simon L. , Ciraolo Georgianne M. , Morris Randal E. , Howell Michael L. , Dean Gary E.
Published: January 2023

Histoplasma capsulatum (Hc) maintains a phagosomal pH of about 6.5. This strategy allows Hc to obtain iron from transferrin, and minimize the activity of macrophage (Mø) lysosomal hydrolases. To determine the mechanism of pH regulation, we evaluated the function of the vacuolar ATPase (V-ATPase) in RAW264.7 Mø infected with Hc yeast or the nonpathogenic yeast Saccharomyces cerevisae (Sc). Incubation of Hc-infected Mø with bafilomycin, an inhibitor of the V-ATPase, did not affect the intracellular growth of Hc, nor did it affect the intraphagosomal pH. In contrast, upon addition of bafilomycin, phagosomes containing Sc rapidly changed their pH from 5 to 7. Hc-containing phagosomes had 5-fold less V-ATPase than Sc-containing phagosomes as quantified by immunoelectron microscopy. Furthermore, Hc-containing phagosomes inhibited phagolysosomal fusion as quantified by the presence of acid phosphatase, accumulation of LAMP2, and fusion with rhodamine B-isothiocyanate-labeled dextran-loaded lysosomes. Finally, in Hc-containing phagosomes, uptake of ferritin was equivalent to phagosomes containing Sc, indicating that Hc-containing phagosomes have full access to the early “bulk flow” endocytic pathway. Thus, Hc yeasts inhibit phagolysosomal fusion, inhibit accumulation of the V-ATPase in the phagosome, and actively acidify the phagosomal pH to 6.5 as part of their strategy to survive in Mø phagosomes.
Central Role of Transcription Factor NF-IL6 for Cytokine and Iron-Mediated Regulation of Murine Inducible Nitric Oxide Synthase Expression
Authors: Dlaska Margit , Weiss Günter
Published: January 2023

We have previously shown that iron regulates the transcription of inducible nitric oxide synthase (iNOS). To elucidate the underlying mechanisms we performed a series of transient transfections of murine fibroblast (NIH-3T3) and macrophage-like cells (J774.A1) with reporter plasmids containing the iNOS promoter and deletions thereof. By means of this and subsequent DNase I footprinting analysis we identified a regulatory region between −153 and −142 bp upstream of the transcriptional start site of the iNOS promoter that was sensitive to regulation by iron perturbation. Gel shift and supershift assays revealed that the responsible protein for this observation is NF-IL6, a member of the CCAAT/enhancer binding protein family of transcription factors. Binding of NF-IL6 to its consensus motif within the iNOS promoter was inducible by IFN-γ and/or LPS, was reduced by iron, and was enhanced by the iron chelator desferrioxamine. Introduction of a double mutation into the NF-IL6 binding site (−153/−142) of an iNOS promoter construct resulted in a reduction of IFN-γ/LPS inducibility by >90% and also impaired iron mediated regulation of the iNOS promoter. Our results provide evidence that this NF-IL6 binding site is of central importance for maintaining a high transcriptional rate of the iNOS gene after IFN-γ/LPS stimulation, and that NF-IL6 may cooperate with hypoxia inducible factor-1 in the orchestration of iron-mediated regulation of iNOS.
Mechanisms of Selective Leukocyte Recruitment from Whole Blood on Cytokine-Activated Endothelial Cells Under Flow Conditions
Authors: Patel Kamala D.
Published: January 2023

Selective recruitment of eosinophils to sites of allergic and parasitic inflammation involves specific adhesion and activation signals expressed on or presented by stimulated endothelial cells. Here we examined leukocyte recruitment on cytokine-activated HUVEC under flow conditions. We perfused whole blood through a flow chamber to examine mechanisms of selective leukocyte recruitment. Although there was substantial recruitment of leukocytes on TNF-α-stimulated HUVEC, we found no selective accumulation of any particular leukocyte subpopulations. In contrast, fewer leukocytes were recruited to IL-4-stimulated HUVEC, but the recruitment was selective for eosinophils. We examined the role of adhesion molecules in these interactions and found that eosinophil recruitment was completely blocked with an α4 integrin mAb at the shear rates examined. A significant number of neutrophils were also recruited to IL-4-stimulated HUVEC, and these interactions required P-selectin and P-selectin glycoprotein ligand-1. Thus, whole blood perfusion over cytokine-activated endothelium revealed that IL-4-stimulated HUVEC support selective recruitment of eosinophils, whereas TNF-α-stimulated HUVEC lack selectivity for any leukocyte subclass.
Oral Administration of Myelin Basic Protein Is Superior to Myelin in Suppressing Established Relapsing Experimental Autoimmune Encephalomyelitis
Authors: Benson Jacqueline M. , Stuckman Scott S. , Cox Karen L. , Wardrop Richard M. , Gienapp Ingrid E. , Cross Anne H. , Trotter John L. , Whitacre Caroline C.
Published: January 2023

Oral administration of a myelin component, myelin basic protein (MBP), induces immunological unresponsiveness to CNS Ags and ameliorates murine relapsing experimental autoimmune encephalomyelitis (REAE). However, a recent clinical trial in which multiple sclerosis patients were treated with repeated doses of oral myelin was unsuccessful in reducing disease exacerbations. Therefore, we directly compared the tolerizing capacity of myelin vs MBP during REAE in B10.PL mice. Oral administration of high doses of myelin, either before disease induction or during REAE, did not provide protection from disease or decrease in vitro T cell responses. In contrast, repeated oral administration of high doses of MBP suppressed established disease and MBP-specific T cell proliferation and cytokine responses. The frequency of IL-2-, IFN-γ-, and IL-5-secreting MBP-specific T cells declined with MBP feeding, implicating anergy and/or deletion as the mechanism(s) of oral tolerance after high Ag doses. We have previously shown that the dosage and timing of Ag administration are critical parameters in oral tolerance induction. Studies presented here demonstrate that Ag homogeneity is also important, i.e., homogeneous Ag (MBP) is more effective at inducing oral tolerance than heterogeneous Ag (myelin).
The Role of Environmental Antigens in the Spontaneous Development of Autoimmunity in MRL-lprMice
Authors: Maldonado Michael A. , Kakkanaiah Vellalore , MacDonald Glen C. , Chen Fangqi , Reap Elizabeth A. , Balish Edward , Farkas Walter R. , Jennette J. Charles , Madaio Michael P. , Kotzin Brian L. , Cohen Philip L. , Eisenberg Robert A.
Published: January 2023

It has been proposed that the “normal” stimulation of the immune system that occurs from interactions with environmental stimuli, whether infectious or dietary, is necessary for the initiation and/or continuation of autoimmunity. We tested this hypothesis by deriving a group of MRL-lpr mice into a germfree (GF) environment. At 5 mo of age, no differences between GF and conventional MRL-lpr mice were noted in lymphoproliferation, flow cytometric analysis of lymph node cells (LN), or histologic analysis of the kidneys. Autoantibody levels were comparably elevated in both groups. A second experiment tested the role of residual environmental stimuli by contrasting GF mice fed either a low m.w., ultrafiltered Ag-free (GF-AF) diet or an autoclaved natural ingredient diet (GF-NI). At 4 mo of age, both groups showed extensive lymphoproliferation and aberrant T cell formation, although the GF-AF mice had ∼50% smaller LNs compared with sex-matched GF-NI controls. Autoantibody formation was present in both groups. Histologic analysis of the kidneys revealed that GF-AF mice had much lower levels of nephritis, while immunofluorescence analysis demonstrated no difference in Ig deposits but did reveal a paucity of C3 deposition in the kidneys of GF-AF mice.

These data do not support a role for infectious agents in the induction of lymphoproliferation and B cell autoimmunity in MRL-lpr mice. Furthermore, they suggest that autoantibodies do not originate from B cells that were initially committed to exogenous Ags. They do suggest a possible contributory role for dietary exposure in the extent of lymphoproliferation and development of nephritis in this strain.
Selection and Long-Term Persistence of Reactive CTL Clones During an EBV Chronic Response Are Determined by Avidity, CD8 Variable Contribution Compensating for Differences in TCR Affinities
Authors: Couedel Chrystelle , Bodinier Marie , Peyrat Marie-Alix , Bonneville Marc , Davodeau François , Lang François
Published: January 2023

Recent studies have suggested that the diversity of TCR repertoire after primary immunization is conserved in memory T cells and that a progressive narrowing of this repertoire may take place during recall infections. It now remains to be investigated which parameters determine the repertoire of the memory response and possibly restrict its diversity after subsequent antigenic challenges. To address this question, we took advantage of a panel of CD8+ T cell clones from the joint of a rheumatoid arthritis patient and selected for their reactivity against a single MHC/peptide complex. Characterization of both TCR chains documented a great diversity among those clones and the persistence of clonotypes over a 2-yr period. Strikingly, despite the observed repertoire heterogeneity, all clones displayed a narrow range of MHC/peptide density requirements in cytotoxicity assays (ED50 between 9 and 36 nM). TCR affinities were then indirectly estimated by blocking CD8 interaction with an anti-CD8 mAb. We found a wide range of TCR affinities among the different clonotypes that segregated with Vβ usage. We thus propose that during an in vivo chronic response, a narrow range of avidity of the TCR-CD8 complex conditions long-term clonotype persistence, and that the level of CD8 contribution is adjusted to keep clonotypes with variable TCR affinities within this avidity window.
The Src-Homology Domain 2-Bearing Protein Tyrosine Phosphatase-1 Inhibits Antigen Receptor-Induced Apoptosis of Activated Peripheral T Cells
Authors: Zhang Jinyi , Somani Ally-Khan , Watt Stephen , Mills Gordon B. , Siminovitch Katherine A.
Published: January 2023

Restimulation of Ag receptors on peripheral T lymphocytes induces tyrosine phosphorylation-based signaling cascades that evoke Fas ligand expression and induction of Fas-mediated programmed cell death. In view of the role for the Src homology domain 2-bearing protein tyrosine phosphatase-1 (SHP-1) in modulating TCR signaling, we investigated the influence of SHP-1 on TCR-mediated apoptosis by assaying the sensitivity of peripheral T cells from SHP-1-deficient viable motheaten (mev) mice to cell death following TCR restimulation. The results of these studies revealed mev peripheral T cells to be markedly more sensitive than wild-type cells to induction of cell death following TCR stimulation. By contrast, PMA/ionophore and anti-Fas Ab-induced apoptotic responses were no different in mev compared with wild-type activated cells. Enhanced apoptosis of TCR-restimulated mev lymphocytes was associated with marked increases in Fas ligand expression as compared with wild-type cells, but was almost abrogated in both mev and wild-type cells by Fas-Fc treatment. Thus, the increased sensitivity of mev T cells to apoptosis following TCR restimulation appears to reflect a TCR-driven phenomenon mediated through up-regulation of Fas-Fas ligand interaction and induction of the Fas signaling cascade. These findings, together with the hyperproliferative responses of mev peripheral T cells to initial TCR stimulation, indicate that SHP-1 modulation of TCR signaling translates to the inhibition of both T cell proliferation and activation and, as such, is likely to play a pivotal role in regulating the expansion of Ag-stimulated T cells during an immune response.
Cellular Basis of B Cell Clonal Populations in Old Mice
Authors: LeMaoult Joël , Manavalan John Sanil , Dyall Ruben , Szabo Paul , Nikolic-Zugic Janko , Weksler Marc E.
Published: January 2023

Previous studies from this laboratory have shown that >85% of old mice have stable B cell clonal populations detectable by Ig heavy chain complementary-determining region 3 mRNA size analysis and confirmed by sequence analysis. B cells from the same clone are frequently detected in several lymphoid compartments of the same mouse. We now report the phenotype of all ten stable B cell clonal populations detected in five 20-month-old C57BL/6 mice. These clonal B cells appear to develop in the periphery and nine of the ten B cell clonal populations expressed the CD5 cell surface marker. Stable B cell expansions may be dominated by cells at two stages of differentiation. Some B cell populations were detected with DNA as well as RNA and represent large clonal populations of B cells, detectable in several lymphoid compartments. These populations are found predominantly in B cell populations expressing CD45R/B220 and the mRNA coding for the membrane-bound form of the μ Ig heavy chain, which suggests a predominance of B lymphocytes in these populations. In other cases, smaller clonal populations were detected only in splenic RNA samples. These clonal populations were found predominantly among CD45R/B220− B cells and did not express the membrane-bound form of the μ Ig heavy chain. We offer the hypothesis that the B cell clonal populations present in old mice may be precursors of the two types of B cell neoplasms which are dominated by CD5+ B cells (B cell chronic lymphocytic leukemia) or plasma cells (multiple myeloma).
Expression of Multiple Unique Rejection Antigens on Murine Leukemia BALB/c RL♂1 and the Role of Dominant Akt Antigen for Tumor Escape
Authors: Matsuo Mitsutoshi , Wada Hisashi , Honda Shinichiro , Tawara Isao , Uenaka Akiko , Kanematsu Takashi , Nakayama Eiichi
Published: January 2023

Using the pRL1a Ag-loss RL♂1 tumor variant cell line RM2-1, we demonstrated the presence of tumor Ags other than pRL1a that were recognized by CTLs on RL♂1 cells. Semiallogeneic CB6F1 or syngeneic BALB/c CTLs generated against RM2-1 lysed RM2-1 and RL♂1 cells to a similar extent, but no killing was observed with any other tumor or normal cells examined. Clonal analysis and sensitization with reversed phase-HPLC fractions revealed that there were Dd- and Ld-binding peptides recognized by RM2-1 CTLs. Lysis by bulk CTLs stimulated against RL♂1 and limiting dilution analysis suggested that the pRL1a peptide was dominantly recognized to the RM2-1 peptides by CTLs on RL♂1 cells. The rejection response against the parental RL♂1 tumor was much less than that against RM2-1 cells in either CB6F1 or BALB/c mice, suggesting that the presence of altered Akt molecules from which the dominant pRL1a peptide was derived inhibited the rejection response against RL♂1. Depletion of CD4 T cells caused the regression of RL♂1 at the doses in which the tumor grew in untreated mice. The generation of pRL1a CTLs was inhibited in RL♂1-bearing mice. Thus, immunoregulatory CD4 T cells were most likely activated by the altered Akt molecules and inhibited the efficient generation of CTLs against the dominant pRL1a Ag in RL♂1.
Calreticulin Displays In Vivo Peptide-Binding Activity and Can Elicit CTL Responses Against Bound Peptides
Authors: Nair Smita , Wearsch Pamela A. , Mitchell Duane A. , Wassenberg James J. , Gilboa Eli , Nicchitta Christopher V.
Published: January 2023

Calreticulin is an endoplasmic reticulum (ER) chaperone that displays lectin activity and contributes to the folding pathways for nascent glycoproteins. Calreticulin also participates in the reactions yielding assembly of peptides onto nascent MHC class I molecules. By chemical and immunological criteria, we identify calreticulin as a peptide-binding protein and provide data indicating that calreticulin can elicit CTL responses to components of its bound peptide pool. In an adoptive immunotherapy protocol, dendritic cells pulsed with calreticulin isolated from B16/F10.9 murine melanoma, E.G7-OVA, or EL4 thymoma tumors elicited a CTL response to as yet unknown tumor-derived Ags or the known OVA Ag. To evaluate the relative efficacy of calreticulin in eliciting CTL responses, the ER chaperones GRP94/gp96, BiP, ERp72, and protein disulfide isomerase were purified in parallel from B16/F10.9, EL4, and E.G7-OVA tumors, and the capacity of the proteins to elicit CTL responses was compared. In both the B16/F10.9 and E.G7-OVA models, calreticulin was as effective as or more effective than GRP94/gp96 in eliciting CTL responses. Little to no activity was observed for BiP, ERp72, and protein disulfide isomerase. The observed antigenic activity of calreticulin was recapitulated in in vitro experiments, in which it was observed that pulsing of bone marrow dendritic cells with E.G7-OVA-derived calreticulin elicited sensitivity to lysis by OVA-specific CD8+ T cells. These data identify calreticulin as a peptide-binding protein and indicate that calreticulin-bound peptides can be re-presented on dendritic cell class I molecules for recognition by CD8+ T cells.
The SH2-Containing 5′-Inositol Phosphatase (SHIP) Is Tyrosine Phosphorylated after Fcγ Receptor Clustering in Monocytes
Authors: Maresco Diane L. , Osborne Jeanne M. , Cooney Damon , Coggeshall K. Mark , Anderson Clark L.
Published: January 2023

Current models of FcγR signal transduction in monocytes describe a molecular cascade that begins upon clustering of FcγR with the phosphorylation of critical tyrosine residues in the cytoplasmic domains of FcγRIIa or the γ-chain subunit of FcγRI and FcγRIIIa. The cascade engages several other tyrosine-phosphorylated molecules, either enzymes or adapters, to manifest ultimately an array of biological responses, including phagocytosis, cell killing, secretion of a variety of inflammatory mediators, and activation. Continuing to assess systematically the molecules participating in the cascade, we have found that the SH2-containing 5′-inositol phosphatase (SHIP) is phosphorylated on tyrosine early and transiently after FcγR clustering. This molecule in other systems, such as B cells and mast cells, mediates an inhibitory signal. We find that clustering of either FcγRIIa or FcγRI is effective in inducing SHIP phosphorylation, that SHIP binds in vitro to a phosphorylated immunoreceptor tyrosine-based activation motif, peptide from the cytoplasmic domain of FcγRIIa in activation-independent fashion, although SHIP binding increases upon cell activation, and that FcγRIIb and FcγRIIc are not responsible for the observed SHIP phosphorylation. These findings prompt us to propose that SHIP inhibits FcγR-mediated signal transduction by engaging immunoreceptor tyrosine-based activation motif-containing cytoplasmic domains of FcγRIIa and FcγRI-associated γ-chain.
Generation of IL-2-Dependent Cytolytic T Lymphocytes (CTLs) with Altered TCR Responses Derived from Antigen-Dependent CTL Clones
Authors: Gullo Charles A. , Esser Mark T. , Fuller Claudette L. , Braciale Vivian Lam
Published: January 2023

Ag-specific CD8+ CTL clones require TCR stimulation to respond to IL-2 for growth. Because IL-2 may be produced in the vicinity of CD8+ CTLs when Ag is limiting at the end of an immune response, we have examined the effect of culturing viral-specific CTL clones in IL-2 in the absence of antigenic stimulation. Limiting dilution analysis revealed a high precursor frequency for CTL clones derived from IL-2 propagation (termed CTL-factor dependent (FD)) that are dependent upon exogenous IL-2 for growth and survival and no longer require TCR stimulation to proliferate. Culturing CTL-FDs with infected splenocytes presenting Ag and IL-2 did not revert the clones but did lead to a TCR-induced inhibition of proliferation. The derived CTL-FDs have lost the ability to kill via the perforin/granule exocytosis mechanism of killing, although they express similar levels of TCR, CD3ε, CD8αβ, CD45, and LFA-1 compared with the parental clones. The CTL-FDs retain Fas ligand/Fas-mediated cytotoxicity, and IFN-γ production and regulate the expression of CD69 and IL-2Rα when triggered through the TCR. A parental CTL protected BALB/c mice from a lethal challenge of influenza virus, whereas a CTL-FD did not. These findings represent a novel regulatory function of IL-2 in vitro that, if functional in vivo, may serve to down-regulate cellular immune responses.
rC5a Directs the In Vitro Migration of Human Memory and Naive Tonsillar B Lymphocytes: Implications for B Cell Trafficking in Secondary Lymphoid Tissues
Authors: Ottonello Luciano , Corcione Anna , Tortolina Giuseppe , Airoldi Irma , Albesiano Emilia , Favre Anna , D’Agostino Roberto , Malavasi Fabio , Pistoia Vito , Dallegri Franco
Published: January 2023

Human C5a is a potent chemoattractant for granulocytes, monocytes, and dendritic cells. In mice C5a has been shown to be chemotactic for germinal center (GC) B cells. To date, no information is available on the effects of C5a on human B cell locomotion. Here we demonstrate that rC5a increases polarization and migration of human tonsillar B cells. The locomotory response was due to both chemokinetic and chemotactic activities of rC5a. Moreover, memory and, at a lesser extent, naive B cell fractions from purified tonsillar populations displayed rC5a-enhanced migratory properties, whereas GC cells did not. Flow cytometry revealed C5aR (CD88) on approximately 40% memory and 10% naive cells, respectively, whereas GC cells were negative. Immunohistochemistry showed that a few CD88+ cells were of the B cell lineage and localized in tonsillar subepithelial areas, where the majority of memory B cells settle. Pretreatment of memory B cells with the CD88 mAb abolished their migratory responsiveness to rC5a. Finally, the C5 gene was found to be expressed in naive, GC, and memory B lymphocytes at both the mRNA and the protein level. This study delineates a novel role for C5a as a regulator of the trafficking of human memory and naive B lymphocytes and supports the hypothesis that the B cells themselves may serve as source of C5 in secondary lymphoid tissues.
Active Sites in Complement Components C5 and C3 Identified by Proximity to Indels in the C3/4/5 Protein Family
Authors: Low Pamela J. , Ai Rong , Ogata Ronald T.
Published: January 2023

We recently suggested that sites of length polymorphisms in protein families (indels) might serve as useful guides for locating protein:protein interaction sites. This report describes additional site-specific mutagenesis and synthetic peptide inhibition studies aimed at testing this idea for the paralogous complement C3, C4, and C5 proteins. A series of C5 mutants was constructed by altering the C5 sequence at each of the 27 indels in this protein family. Mutants were expressed in COS cells and were assayed for hemolytic activity and protease sensitivity. Mutants at five indels showed relatively normal expression but substantially reduced sp. act., indicating that the mutations damaged sites important for C5 function. Twenty-three synthetic peptides with C5 sequences and 10 with C3 sequences were also tested for the ability to inhibit C hemolytic activity. Three of the C5 peptides and one of the C3 peptides showed 50% inhibition of both C hemolytic and bactericidal activities at a concentration of 100 μM. In several cases both the mutational and peptide methods implicated the same indel site. Overall, the results suggest that regions important for function of both C3 and C5 lie proximal to residues 150–200 and 1600–1620 in the precursor sequences. Additional sites potentially important for C5 function are near residue 500 in the β-chain and at two or three sites between the N-terminus of the α′-chain and the C5d fragment. One of the latter sites, near residue 865, appears to be important for proteolytic activation of C5.
The Tumorigenicity of IL-2 Gene-Transfected Murine M-3D Melanoma Cells Is Determined by the Magnitude and Quality of the Host Defense Reaction: NK Cells Play a Major Role
Authors: Schneeberger Achim , Koszik Frieder , Schmidt Walter , Kutil Raphaela , Stingl Georg
Published: January 2023

Transfection of a variety of tumor lines with the IL-2 gene strongly reduces their tumorigenic potential when applied to either euthymic or athymic animals. To elucidate the mechanisms underlying this phenomenon, we inoculated IL-2-transfected M-3D melanoma (M-3D-IL-2) cells into DBA/2 mice immunosuppressed by γ-irradiation. Animals thus treated developed pigmented tumors, suggesting that IL-2 transfection of melanoma cells, instead of altering their neoplastic growth properties, renders them capable of evoking a tumoricidal host response. To define the critical effector cell, we injected M-3D-IL-2 and, for control purposes, nontransfected M-3D cells into DBA/2 recipients and analyzed the injection site. We found that 1) IL-2-expressing M-3D cells induce a much stronger inflammatory reaction than wild-type cells, 2) in both instances the infiltrate consists mainly of macrophages (40–60%) and granulocytes (30–40%), and 3) only the infiltrate of M-3D-IL-2 cell deposits contains a minor fraction of NK cells (∼1–2%). When we reconstituted sublethally irradiated animals with various leukocyte subsets, we found that unfractionated as well as macrophage-depleted peritoneal lavage cells but not NK cell-depleted peritoneal lavage cells were able to suppress the growth of IL-2-expressing M-3D cells. In vivo leukocyte depletion experiments showed that the NK cell-depleting asialo-GM1 antiserum, but not anti-macrophage and/or anti-granulocyte reagents, restored the tumorigenicity of M-3D-IL-2 cells. Our results indicate that the inflammatory tissue response evoked by IL-2-transfected cancer cells includes the attraction and/or activation of NK cells and that, in the experimental system used, these cells are critically needed for successfully controlling cancer growth in vivo.
Therapeutic Effects of Tumor-Reactive Type 1 and Type 2 CD8+ T Cell Subpopulations in Established Pulmonary Metastases
Authors: Dobrzanski Mark J. , Reome Joyce B. , Dutton Richard W.
Published: January 2023

Cytolytic CD8+ T cells fall into two subpopulations based on cytokine-secretion. Type 1 CD8+ cells (Tc1) characteristically secrete IFN-γ, whereas type 2 CD8+ cells (Tc2) secrete IL-4 and IL-5. We assessed the relative therapeutic effects of adoptively transferred OVA-specific Tc1 and Tc2 CD8+ cells in mice bearing established OVA-transfected B16 melanoma lung metastases. Both Tc1 and Tc2 subpopulations mediated a reduction in lung tumor growth that subsequently prolonged survival times in mice with both early (day 7) and more advanced (day 14) levels of tumor development. CD8+ T cell populations recovered from spleens of tumor-bearing mice receiving Tc1 or Tc2 cells showed markedly enhanced tumor Ag-specific cytolytic and cytokine-releasing activities that correlated with delays in tumor cell growth and progression. Initially, both tumor-reactive Tc1 and Tc2 effector cells accumulated at the tumor site with nearly equal frequency. Tc1 cells persisted, whereas Tc2 cell numbers progressively diminished over time. Titration of Tc1 and Tc2 effector cells showed that protection was dose dependent with the former being 5-fold more effective. Tc2 cells achieved a comparable reduction in lung tumor cell growth at higher concentrations of cell transfer. Tc1 effectors from IFN-γ-deficient mice were less therapeutically effective than wild-type mice, but there was no significant reduction in activity between corresponding Tc2 populations. We speculate that the effectiveness of Tc1 and Tc2 cells may depend on different mechanisms. These studies suggest a potential role for Tc1 and Tc2 CD8+ subpopulations in tumor regression and immunotherapy.
Presence of Effector CD8+ T Cells in Hepatitis C Virus-Exposed Healthy Seronegative Donors
Authors: Scognamiglio Paola , Accapezzato Daniele , Casciaro Marco Antonio , Cacciani Antonella , Artini Marco , Bruno Guglielmo , Chircu Maria Lucia , Sidney John , Southwood Scott , Abrignani Sergio , Sette Alessandro , Barnaba Vincenzo
Published: January 2023

CTL responses against multiple hepatitis C virus (HCV) epitopes were detected in 7 of 29 (24.1%) healthy family members (HFM) persistently exposed to chronically HCV-infected patients (HCV-HFM). These precursor CTL were at very low or undetectable frequencies, as determined by limiting dilution analysis. However, when HCV-specific effector CD8+ T cells, freshly isolated from PBMC of HCV-HFM, were assessed by a sensitive enzyme-linked immunospot assay, their frequencies were severalfold higher than those of precursor CTL. These results indicate that the two assays detect two functionally distinct T cell populations and that the effector cells are not assayed by the 51Cr-release assay. Furthermore, the combination of cell depletion and enzyme-linked immunospot analyses showed that the effector cells were confined into a CD8+ CD45RO+ CD28− population. The persistence of effector CD8+ T cells specific for both the structural and nonstructural viral proteins in uninfected HCV-HFM, suggest that: 1) an immunological memory is established upon a subclinical infection without any evidence of hepatitis, in a large cohort of HCV-exposed individuals; 2) because these cells required neither restimulation nor the addition of particular cytokines in vitro for differentiating in effectors, they should be capable of prompt HCV-specific effector function in vivo, possibly providing antiviral protection; and 3) the maintenance of effector T cell responses may be sustained by persisting low-level stimulation induced by inapparent infections.