bluuebunny/crossref_metadata_2025 huggingface dataset:
Apical Secretion of a Pathogen-Elicited Epithelial Chemoattractant Activity in Response to Surface Colonization of Intestinal Epithelia by Salmonella typhimurium
Authors: McCormick Beth A. , Parkos Charles A. , Colgan Sean P. , Carnes Denice K. , Madara James L.
Published: January 2023

Modeling Salmonella-epithelial cell interaction in vitro has led to the realization that epithelial cells are crucial in orchestrating neutrophil (PMN) responses, in part by stimulating basolateral release of epithelial chemokines, including IL-8. However, such basolaterally released chemokines, while likely important in orchestration of PMN movement across the subepithelial matrix, are unlikely to be responsible for the final step of transepithelial migration of PMN and entry into the apical compartment. We now show that S. typhimurium attachment to T84 cell apical epithelial membranes induces polarized apical secretion of a pathogen-elicited epithelial chemoattractant (PEEC) bioactivity. Experiments employing semipurified PEEC indicate that it is released in a polarized apical fashion and is sufficient to explain the observed final step of transepithelial migration of PMN induced by Salmonella-apical membrane interaction. By preliminary physical characterization and profiles of PMN activation, PEEC appears to be a novel PMN chemotactic bioactivity. This 1- to 3-kDa nominal molecular mass chemokine-like bioactivity directly stimulates PMN via a pertussis toxin-sensitive receptor and elicits a Ca2+ signal. While these latter features are shared by most other chemokines, analysis of PEEC-elicited PMN activation reveals that, unlike these other agonists, PEEC, even at saturating concentrations, elicits chemotactic activity in the absence of stimulation of superoxide production and/or release of primary and/or secondary granules. These data suggest that the apically released PEEC activity appears to represent a novel epithelial-derived chemoattractant that directs PMN movement across epithelial monolayers.
CDR3 Size Spectratyping and Sequencing of Spectratype-Derived TCR of Spinal Cord T Cells in Autoimmune Encephalomyelitis
Authors: Kim Giok , Tanuma Naoyuki , Kojima Takashi , Kohyama Kuniko , Suzuki Yoko , Kawazoe Yoko , Matsumoto Yoh
Published: January 2023

To characterize the nature of autoimmune disease-inducing T cells in the target organ, oligoclonal expansion of spinal cord T cells of Lewis rats with experimental autoimmune encephalomyelitis (EAE) was examined by complementarity-determining region 3 (CDR3) size spectratyping. It is known that TCR of in vitro-established myelin basic protein-specific T cell clones and lines have a short CDR3 and that the amino acid sequence in this region is highly preserved. On the basis of these findings, we analyzed 22 spectratypes of the TCR β-chain (Vβ1–20). Among them, only Vβ8.2 and Vβ17 showed oligoclonal expansion of TCR with a short CDR3 at the early stage of EAE. More interestingly, the spectratype profile of Vβ8.2 seen at the early stage was preserved throughout the course of EAE, whereas that of Vβ17 became more diverse at the peak stage of the disease. Analysis of nucleotide and predicted amino acid sequences of Vβ8.2 CDR3 derived from the spectratypes revealed that the clones with CASSDSSYEQYFGPG, which is one of the representative sequences of encephalitogenic T cell clones, constituted the predominant population not only at the early stage but also at the peak and recovery stages (71, 71, and 60%, respectively). These findings imply that although the phenotype of T cells in the target organ diversifies as the autoimmune disease progresses, disease-associated TCR spectratype(s) are preserved throughout the course of the disease. Thus, CDR3 size spectratyping is a powerful tool for the screening of disease-inducing T cells in an autoimmune disease of unknown pathomechanism.
IL-4 Induces Eotaxin: A Possible Mechanism of Selective Eosinophil Recruitment in Helminth Infection and Atopy
Authors: Mochizuki Mitsuru , Bartels Joachim , Mallet Antony I. , Christophers Enno , Schröder Jens-M.
Published: January 2023

A common feature of some parasitic infections and allergic and atopic skin diseases is the involvement of Th2 lymphocytes and the dermal appearance of eosinophils (Eos). Because Th2 lymphocytes apparently do not release Eo attractants, we addressed the question of whether the Th2 cytokine IL-4 induces its production in dermal fibroblasts. We therefore stimulated fibroblasts with IL-4. HPLC investigation of supernatants revealed a single Eo chemotactic protein, which was purified to homogeneity giving a single 13-kDa band upon SDS-PAGE analyses. Peptide mapping with subsequent amino acid sequencing revealed an Eo-selective chemotaxin, which consists of a mixture of N-terminally truncated and O-glycosylated forms of the chemokine eotaxin. Other chemokines such as RANTES, MCP-3, MCP-4, or MIP-1α were not detected as Eo chemotaxins under these conditions. Using reverse transcriptase-PCR techniques, we found that IL-4 dose and time dependently induces eotaxin mRNA in dermal fibroblasts. Stimulation with IL-4 and TNF-α caused a 10- to 20-fold increase of the release of three biochemically different eotaxin forms, each consisting of a mixture of N-terminally truncated and O-glycosylated variants having the same backbone amino acid sequence but different specific activities. Our findings support the hypothesis that eosinophil recruitment seen in IL-4-mediated skin reactions, at least in part, may be due to Th2 cytokine-mediated induction of eotaxin in dermal fibroblasts.
Naturally Occurring Low Affinity Peptide/MHC Class I Ligands Can Mediate Negative Selection and T Cell Activation
Authors: Motyka Bruce , Teh Hung-Sia
Published: January 2023

The affinity/avidity model for T cell development postulates that ligands with high affinity for the TCR are efficient in negative selection, whereas those with lower affinity/avidity favor positive selection. Using the 2C TCR transgenic model, we evaluated the efficacy of ligands with widely differing affinity for the TCR (3 × 103 to 2 × 106 M−1) in mediating thymocyte deletion. The relative affinities of the 2C TCR for the p2Ca/Ld, dEV-8/Kb, p2Ca-A3/Ld, and p2Ca/Kb ligands are approximately 1000:50:10:1, respectively. Here we show, using an in vitro assay, that the deletion of 2C CD4+CD8+ thymocytes is mediated not only by p2Ca/Ld, but also by the lower affinity ligands dEV-8/Kb, p2Ca-A3/Ld, and p2Ca/Kb, albeit at relatively higher peptide concentrations. Deletion mediated by low affinity ligands required CD8, whereas high affinity ligand-mediated deletion was CD8 independent. The p2Ca/Kb and dEV-8/Kb ligands are naturally occurring in H-2b mice, and others have shown that p2Ca/Kb can induce the maturation of CD4−CD8+2C-TCRhigh thymocytes in fetal thymic organ culture. In this study we showed that in addition to deletion, the p2Ca/Kb and dEV-8/Kb ligands, in the presence of exogenous IL-2, induced mature 2C T cell proliferation, albeit at a lower level than that induced by the high affinity p2Ca/Ld ligand. Thus, the same low affinity ligands that can effect negative selection and, in the case of p2Ca/Kb, the maturation of CD8 single-positive thymocytes can also induce the activation of mature CD8 T cells.
Cutting Edge: Identification of Novel T Cell Epitopes in Lol p5a by Computational Prediction
Authors: de Lalla Claudia , Sturniolo Tiziana , Abbruzzese Laura , Hammer Juergen , Sidoli Alessandro , Sinigaglia Francesco , Panina-Bordignon Paola
Published: January 2023

Although atopic allergy affects ≤20% of the total population, the relationship between the protein structure and immunogenic activity of the allergens is still largely unknown. We observed that group 5 grass allergens are characterized by repeated structural motifs. Using a new algorithm, TEPITOPE, we predicted promiscuous HLA-DR ligands within the repeated motifs of the Lol p5a allergen from rye grass. In vitro binding studies confirmed the promiscuous binding characteristics of these peptides. Moreover, most of the predicted ligands were novel T cell epitopes that were able to stimulate T cells from atopic patients. We generated a panel of Lol p5a-specific T cell clones, the majority of which recognized the peptides in a cross-reactive fashion. The computational prediction of DR ligands might thus allow the design of T cell epitopes with potential useful application in novel immunotherapy strategies.
Cutting Edge: Differential Effect of Apoptotic Versus Necrotic Tumor Cells on Macrophage Antitumor Activities
Authors: Reiter Isabella , Krammer Barbara , Schwamberger Günter
Published: January 2023

Macrophages (Mφ) play essential roles both in tumor defense and normal tissue homeostasis by removal of transformed as well as damaged and disintegrating cells. Whereas tissue necrosis is known to provoke inflammatory responses, removal of apoptotic cells has been assumed to be immunologically inert. We now show that while Mφ exposure to necrotized tumor cells causes pronounced stimulation of Mφ antitumor activity, exposure of Mφ to apoptotic tumor cells in contrast results in impairment of Mφ-mediated tumor defense and even support of tumor cell growth. Given the fact that apoptosis is a consequence of various cancer treatment modalities, this may lead to a suppression of local antitumor reactions and thus actually counteract endogenous immune-mediated tumor defense mechanisms.
Cutting Edge: IL-4 Production by Mast Cells Does Not Require c-maf
Authors: Sherman Melanie A. , Nachman Tammy Y. , Brown Melissa A.
Published: January 2023

The c-maf transcription factor is selectively expressed in IL-4-producing Th2 cells. It has been implicated in IL-4 gene transcription based on its ability to directly activate the IL-4 gene in nonexpressing B cells and to promote IL-4-induced Th2 differentiation. However, it has not been definitively shown that IL-4 production by other cells is dependent on the presence of c-maf. Here, we show that IL-4-producing mast cells do not express the c-maf factor. Furthermore, mutation of a defined c-maf binding site within the proximal IL-4 promoter, which profoundly affects transcription in T cells, has no effect on expression of a reporter gene driven by the IL-4 promoter in mast cells. These results demonstrate that c-maf and its target binding site are not required for IL-4 production in all cell types and delineate additional cis- and trans-acting elements that contribute to the cell-type specific transcriptional regulation of IL-4.
IL-2-Independent Activation and Proliferation in Human T Cells Induced by CD28
Authors: Boulougouris George , McLeod Julie D. , Patel Yusuf I. , Ellwood Claire N. , Walker Lucy S. K. , Sansom David M.
Published: January 2023

Although the role of CD28 in T cell costimulation is firmly established, the mechanisms by which it exerts its costimulatory actions are less clear. In many circumstances it is difficult to distinguish the effects of CD28 from subsequent actions of cytokines, such as IL-2, on T cell proliferation. Here, we report a model of CD28 costimulation using PMA plus the natural ligand CD80 that resulted in very limited stimulation of IL-2, as evidenced by both cytokine production and IL-2 promoter stimulation. Promoter assays revealed CD28-dependent effects on both NF-κB and AP-1, but not on NF-AT or the intact IL-2 promoter. In addition, T cell proliferation was completely resistant to the actions of the immunosuppressant cyclosporin A (CsA). Moreover T cell proliferation was unaffected by the addition of blocking Abs to both IL-2 and the IL-2 receptor, demonstrating that this form of costimulation by CD28 was independent of IL-2. We also investigated the effects of stimulating T cell blasts with CD80 alone and found that there was a limited requirement for IL-2 in this system. We conclude that CD28 costimulation can cause substantial T cell proliferation in the absence of IL-2, which is driven by a soluble factor independent of NF-AT transactivation.
Identification of Domains in IL-16 Critical for Biological Activity
Authors: Nicoll John , Cruikshank William W. , Brazer William , Liu Yu , Center David M. , Kornfeld Hardy
Published: January 2023

IL-16 is a proinflammatory cytokine implicated in the pathogenesis of asthma and other conditions characterized by recruitment of CD4+ T cells to sites of disease. It is postulated that CD4 is an IL-16 receptor, although other receptors or coreceptors may exist. Among several known functions, IL-16 is a chemoattractant factor for CD4+ T cells and it inhibits MLR. We previously reported that an oligopeptide corresponding to the 16 C-terminal residues of human IL-16 inhibits chemoattractant activity. To identify functional domains with greater precision, shorter oligonucleotides containing native or mutated C-terminal IL-16 sequences were tested for IL-16 inhibition. Within the 16 C-terminal residues, the minimal peptide RRKS (corresponding to Arg106 to Ser109) was shown to mediate inhibition of IL-16 chemoattractant activity. Inhibition was lost when either arginine was substituted with alanine. Point mutations in IL-16 revealed that Arg107 is critical for chemoattractant activity, but MLR inhibition was unaffected by mutation of Arg107 or even deletion of the C-terminal tail through Arg106. Deletion of 12 or 22 N-terminal residues of IL-16 had no impact on chemoattractant activity, but MLR inhibition was reduced. Deletion of 16 C-terminal plus 12 N-terminal residues abolished both chemoattractant and MLR-inhibitory activity of IL-16. These data indicate that receptor interactions with IL-16 that activate T cell migration are not identical with those required for MLR inhibition, and suggest that both N-terminal and C-terminal domains in IL-16 participate in receptor binding or activation.
Phospho-LAT-Independent Activation of the Ras-Mitogen-Activated Protein Kinase Pathway: A Differential Recruitment Model of TCR Partial Agonist Signaling
Authors: Chau Luan A. , Madrenas Joaquín
Published: January 2023

Stimulation of mature T cells with agonist ligands of the Ag receptor (TCR) causes rapid phosphorylation of tyrosine-based activation motifs in the intracellular portion of TCR-ζ and CD3 and activation of several intracellular signaling cascades. Coordinate activation of these pathways is dependent on Lck- and ZAP-70-mediated tyrosine phosphorylation of a 36-kDa linker for activation of T cells and subsequent recruitment of phospholipase C-γ1, Grb2-SOS, and SLP-76-vav. Here, we show that TCR partial agonist ligands can selectively activate one of these pathways, the Ras-mitogen-activated protein kinase pathway, by inducing recruitment of Grb2-SOS complexes to incompletely phosphorylated p21 phospho-TCR-ζ. This bypasses the need for activation of Lck and ZAP-70, and for phosphorylation of the linker for activation of T cells to activate Ras. We propose a general model in which differential recruitment of activating complexes away from transmembrane linker proteins may determine selective activation of a given signaling pathway.
In Vivo Overexpression of Dad1, the Defender Against Apoptotic Death-1, Enhances T Cell Proliferation But Does Not Protect Against Apoptosis
Authors: Hong N. A. , Kabra N. H. , Hsieh S. N. , Cado D. , Winoto A.
Published: January 2023

The Dad1 protein has been shown to play a role in prevention of apoptosis in certain cell types. Dad1 is also a subunit of the oligosaccharyltransferase enzyme complex that initiates N-linked glycosylation. It is encoded by a gene located adjacent to the TCR α and δ genes on mouse chromosome 14. We have investigated the role of Dad1 during T cell development and activation. We observe that endogenous Dad1 levels are modulated during T cell development to reach maximal expression in mature thymocytes. Transgenic mice that overexpress Dad1 in both the thymus and peripheral immune system have been generated. Apoptosis of thymocytes from such mice is largely unaffected, but peripheral T cells display hyperproliferation in response to stimuli. Therefore, the linkage between the TCR and Dad1 genes may have important consequences for T cell function.
Intracellular Rate-Limiting Steps in MHC Class I Antigen Processing
Authors: Montoya María , Del Val Margarita
Published: January 2023

Quantitative aspects of the endogenous pathway of Ag processing and presentation by MHC class I molecules to CD8+ CTL were analyzed over a wide range of Ag expression in recombinant vaccinia virus-infected cells expressing β-galactosidase as model Ag. Only the amount of starting Ag was varied, leaving other factors unaltered. Below a certain level of Ag synthesis, increasing protein amounts led to a sharp rise in recognition by CTL. Higher levels of Ag expression led to a saturation point, which intracellularly limited the number of naturally processed peptides bound to MHC and thereby also CTL recognition. The rate-limiting step was located at the binding of the antigenic peptide to MHC inside the vaccinia virus-infected cell or before this event.
IL-4-Transduced Tumor Cell Vaccine Induces Immunoregulatory Type 2 CD8 T Lymphocytes That Cure Lung Metastases Upon Adoptive Transfer
Authors: Rodolfo Monica , Zilocchi Chiara , Accornero Paola , Cappetti Barbara , Arioli Ivano , Colombo Mario P.
Published: January 2023

Vaccinations with tumor cells engineered to produce IL-4 prolonged survival and cured 30% of mice bearing pulmonary metastases, an effect abrogated by in vivo depletion of T cells. Vaccination induced type 2 T cell polarization in both CD4 and CD8 T lymphocyte subsets. We focused on the antitumor activity exerted by type 2 CD8+ T cells (Tc2) activated by IL-4 tumor cell vaccination. Tc2 lymphocytes lacked in vitro tumor cytotoxicity, but released IL-4 upon stimulation with tumor cells, as shown by limiting dilution analysis of the frequencies of tumor-specific pCTL and of CD8 cells producing the cytokine. In vivo fresh purified CD8+ T lymphocytes from IL-4-vaccinated mice eliminated 80–100% of lung metastases when transferred into tumor-bearing mice. CD8+ lymphocytes from IL-4-vaccinated IFN-γ knockout (KO), but not from IL-4 KO, mice cured lung metastases, thus indicating that IL-4 produced by Tc2 cells was instrumental for tumor rejection. The antitumor effect of adoptively transferred Tc2 lymphocytes needed host CD8 T cells and AsGM1 leukocyte populations, and partially granulocytes. These data indicate that Tc2 CD8+ T cells exert immunoregulatory functions and induce tumor rejection through the cooperation of bystander lymphoid effector cells. Tumor eradication is thus not restricted to a type 1 response, but can also be mediated by a type 2 biased T cell response.
Mouse Dendritic Cells Express the P2X7 Purinergic Receptor: Characterization and Possible Participation in Antigen Presentation
Authors: Mutini Carmela , Falzoni Simonetta , Ferrari Davide , Chiozzi Paola , Morelli Anna , Baricordi O. Roberto , Collo Ginetta , Ricciardi-Castagnoli Paola , Di Virgilio Francesco
Published: January 2023

Immune cells express P2 purinoceptors of the P2Y and P2X subtypes. In the present work, we show that three dendritic cell (DC) lines, D2SC/1, CB1, and FSDC, representative of immature DCs, express the P2X7 (formerly P2Z) receptor, as judged from RT-PCR amplification, reactivity to a specific antiserum, and pharmacological and functional evidence. Receptor expression is higher in FSDC cells, a cell line that is functionally more mature than D2SC/1 and CB1. From the wild-type DC population, we selected cell clones lacking the P2X7R (P2X7less). We also used a P2XR blocker, oxidized ATP, to irreversibly inhibit the P2X7R. Ability of P2X7less FSDCs or of oxidized ATP-inhibited FSDCs to stimulate Ag-specific TH lymphocytes was severely decreased although Ag endocytosis was minimally affected. During coculture with TH lymphocytes, wild-type FSDC secreted large amounts of IL-1β. Release of this cytokine was reduced in P2X7less DCs. These data show that DCs express the P2X7 purinoceptor and suggest a correlation between P2X7R expression and Ag-presenting activity.
Accelerated Development and Aging of the Immune System in p53-Deficient Mice
Authors: Ohkusu-Tsukada Kozo , Tsukada Teruyo , Isobe Ken-ichi
Published: January 2023

Development and aging of the immune system lead to an accumulation of memory T cells over the long term. The predominance of T cells of the memory phenotype in the T cell population induces an age-related decline in protective immune responses. We found that development and aging of the immune system were accelerated in p53-deficient (p53−/−) mice; the accumulation of memory T cells was spontaneously accelerated, and a strong T cell-dependent Ab response and Th2 cytokine expression (IL-4, IL-6, and IL-10) were induced by Ag stimulation in young p53−/− mice in the developmental stage. The high T cell proliferative response in the young mice rapidly progressed to a depressed proliferative response in adult mice. It was suggested that the loss of regulation of the cell cycle, DNA repair, and apoptosis by p53 deficiency potentially leads to immunosenescence with the accumulation of memory T cells.
Roles of IFN Consensus Sequence Binding Protein and PU.1 in Regulating IL-18 Gene Expression
Authors: Kim Yong-Man , Kang Hyung-Sik , Paik Sang-Gi , Pyun Kwang-Ho , Anderson Karen L. , Torbett Bruce E. , Choi Inpyo
Published: January 2023

IL-18 is expressed from a variety of cell types. Two promoters located upstream of exon 1 (5′-flanking region) and upstream of exon 2 (intron 1) regulate its expression. Both promoter regions were cloned into pCAT-Basic plasmid to yield p1-2686 for the 5′-flanking promoter and p2-2.3 for the intron 1 promoter. Both promoters showed basal constitutive activity and LPS inducibility when transfected into RAW 264.7 macrophages. To learn the regulatory elements of both promoters, 5′-serial deletion and site-directed mutants were prepared. For the activity of the p1-2686 promoter, the IFN consensus sequence binding protein (ICSBP) binding site between −39 and −22 was critical. EMSA using an oligonucleotide probe encompassing the ICSBP binding site showed that LPS treatment increased the formation of DNA binding complex. In addition, when supershift assays were performed, retardation of the protein-DNA complex was seen after the addition of anti-ICSBP Ab. For the activity of the p2-2.3 promoter, the PU.1 binding site between −31 and −13 was important. EMSA using a PU.1-specific oligonucleotide demonstrated that LPS treatment increased PU.1 binding activity. The addition of PU.1-specific Ab to LPS-treated nuclear extracts resulted in the formation of a supershifted complex. Furthermore, cotransfection of ICSBP or PU.1 expression vector increased p1 promoter activity or IL-18 expression, respectively. Taken together, these results indicate that ICSBP and PU.1 are critical elements for IL-18 gene expression.
Point Mutation Causing Constitutive Signaling of CXCR2 Leads to Transforming Activity Similar to Kaposi’s Sarcoma Herpesvirus-G Protein-Coupled Receptor
Authors: Burger Meike , Burger Jan A. , Hoch Robert C. , Oades Zenaida , Takamori Hiroshi , Schraufstatter Ingrid U.
Published: January 2023

The chemokine receptor CXCR2 is the closest homologue to Kaposi’s sarcoma herpesvirus-G protein-coupled receptor (KSHV-GPCR), which is known to be constitutively activated and able to cause oncogenic transformation. Among G protein-coupled receptors, a DRY sequence in the second intracellular loop is highly conserved. However, the KSHV-GPCR shows a VRY sequence instead. In this study, we exchanged Asp138 of the DRY sequence in the CXCR2 with a Val (D138V), the corresponding amino acid in KSHV-GPCR, or with a Gln (D138Q), and investigated the functional consequences of these mutations. In focus formation and soft agar growth assays in NIH 3T3 cells, the D138V mutant exhibited transforming potential similar to the KSHV-GPCR. Surprisingly, the CXCR2 wild type itself showed transforming activity, although not as potently, due to continuous autocrine stimulation, whereas the D138Q mutant formed no foci. In agreement with these results were high levels of inositol phosphate accumulation in the D138V mutant and the KSHV-GPCR, indicating constitutive activity. These data emphasize the importance of the DRY sequence for G protein-coupled signaling of the CXCR2. Either constitutive activation or persistent autocrine stimulation of the CXCR2 causes transformation similar to KSHV-GPCR-transfected cells, probably activating the same signal transduction cascade that can abrogate normal growth control mechanisms.
Little Evidence of HLA-G mRNA Polymorphism in Caucasian or Afro-Caribbean Populations
Authors: Bainbridge David R. J. , Ellis Shirley  A. , Sargent Ian L.
Published: January 2023

HLA-G is a nonclassical class I MHC molecule of unknown function expressed on human trophoblast. The level of polymorphism at the HLA-G locus is of considerable importance, since the paternally inherited gene product is exposed to the maternal immune system during pregnancy. However, previous studies of HLA-G polymorphism using genomic DNA samples have produced conflicting results. Our aim was to investigate polymorphism in trophoblast HLA-G mRNA from pregnancies in ten Caucasian and twelve Afro-Caribbean women by RT-PCR. A similar PCR protocol was also applied to umbilical cord blood genomic DNA from two Caucasian and two Afro-Caribbean neonates. Caucasian cDNA yielded only two different sequences: G*01011, and one containing a previously reported synonymous substitution. Afro-Caribbean samples yielded these sequences as well as one previously reported conservative (leucine-to-isoleucine) substitution. PCR amplification from genomic DNA samples from both populations using previously published primer pairs generated sequences containing multiple substitutions, many of which were nonsynonymous. More than two sequences were produced from genomic DNA from each individual. In contrast, amplification from the same genomic DNA using new primers complementary to exons of the HLA-G gene yielded the same few sequences generated from cDNA. These results suggest that polymorphism at the HLA-G locus is extremely limited in Caucasian and Afro-Caribbean populations. This suggests that spurious polymorphism has been reported in African Americans due to the use of intron-complementary PCR primers on genomic DNA samples. The monomorphic nature of HLA-G may allow trophoblast to carry out the immunological functions of class I-bearing tissues without compromising successful pregnancy.
Mycobacterium avium Infection of Mouse Macrophages Inhibits IFN-γ Janus Kinase-STAT Signaling and Gene Induction by Down-Regulation of the IFN-γ Receptor
Authors: Hussain Shabbir , Zwilling Bruce S. , Lafuse William P.
Published: January 2023

Macrophage activation is required to control the growth of intracellular pathogens. Recent data indicate that macrophages become functionally deactivated during mycobacterial infection. We studied macrophage deactivation by examining the expression of a panel of IFN-γ-inducible genes and activation of Janus Kinase (JAK)-STAT pathway in Mycobacterium avium-infected macrophages. Reduced expression of IFN-γ-inducible genes—MHC class II gene Eβ; MHC class II transactivator; IFN regulatory factor-1; and Mg21, a gene coding for a GTP-binding protein—was observed in M. avium-infected macrophages. Decreased tyrosine phosphorylation and DNA binding activity of STAT1 in M. avium-infected macrophages stimulated with IFN-γ was observed. Tyrosine phosphorylation of JAK1, JAK2, and IFN-γRα was also reduced in infected cells. Northern and Western blot analyses showed that a down-regulation of IFN-γR α- and β-chain mRNA and protein occurred in M. avium-infected macrophages. The down-regulation of IFN-γR and inhibition of STAT1 activation were time dependent and required 4 h of infection for down-regulation of the IFN-γR and 8 h for STAT1 inhibition. These findings suggest that M. avium infection inhibits induction of IFN-γ-inducible genes in mouse macrophages by down-regulating IFN-γR, resulting in reduced phosphorylation of IFN-γRα, JAK1, JAK2, and STAT1.
Activation of Human Monocytic Cells by Borrelia burgdorferi and Treponema pallidum Is Facilitated by CD14 and Correlates with Surface Exposure of Spirochetal Lipoproteins
Authors: Sellati Timothy J. , Bouis Deborah A. , Caimano Melissa J. , Feulner J. Amelia , Ayers Christopher , Lien Egil , Radolf Justin D.
Published: January 2023

Here we examined the involvement of CD14 in monocyte activation by motile Borrelia burgdorferi and Treponema pallidum. B. burgdorferi induced secretion of IL-8 by vitamin D3-matured THP-1 cells, which was inhibited by a CD14-specific mAb known to block cellular activation by LPS and the prototypic spirochetal lipoprotein, outer surface protein A. Enhanced responsiveness to B. burgdorferi also was observed when THP-1 cells were transfected with CD14. Because borreliae within the mammalian host and in vitro-cultivated organisms express different lipoproteins, experiments also were performed with “host-adapted” spirochetes grown within dialysis membrane chambers implanted into the peritoneal cavities of rabbits. Stimulation of THP-1 cells by host-adapted organisms was CD14 dependent and, interestingly, was actually greater than that observed with in vitro-cultivated organisms grown at either 34°C or following temperature shift from 23°C to 37°C. Consistent with previous findings that transfection of Chinese hamster ovary cells with CD14 confers responsiveness to LPS but not to outer surface protein A, B. burgdorferi failed to stimulate CD14-transfected Chinese hamster ovary cells. T. pallidum also activated THP-1 cells in a CD14-dependent manner, although its stimulatory capacity was markedly less than that of B. burgdorferi. Moreover, cell activation by motile T. pallidum was considerably less than that induced by treponemal sonicates. Taken together, these findings support the notion that lipoproteins are the principle component of intact spirochetes responsible for monocyte activation, and they indicate that surface exposure of lipoproteins is an important determinant of a spirochetal pathogen’s proinflammatory capacity.
Murine Neutrophil Stimulation byToxoplasma gondiiAntigen Drives High Level Production of IFN-γ-Independent IL-12
Authors: Bliss Susan K. , Zhang Yin , Denkers Eric Y.
Published: January 2023

Successful immunity to Toxoplasma gondii requires a strong cell-mediated immune response. Neutrophils possess the ability to rapidly migrate into tissues in response to microbial stimuli. Therefore, we sought to determine whether murine neutrophils could respond to T. gondii by producing immunoregulatory cytokines. We show that murine neutrophils produce high levels of IL-12 and low, but significant, levels of TNF-α when stimulated with T. gondii Ag. Both cytokines are produced in the absence of IFN-γ. Production of IL-12 does not require TNFR p55, and release of TNF-α occurs independently of IL-12. We show that there is an influx of neutrophils into the peritoneal cavity that peaks at ∼8 h in response to injection of live tachyzoites and that this is correlated with increased transcription of IL-12 p40. Our results establish that murine neutrophils possess the ability to produce immunoregulatory cytokines during T. gondii infection and suggest that this response may be important in early host defense and in triggering cell-mediated immunity to the parasite.
Toxoplasma gondii and Schistosoma mansoni Synergize to Promote Hepatocyte Dysfunction Associated with High Levels of Plasma TNF-α and Early Death in C57BL/6 Mice
Authors: Marshall Anthony J. , Brunet Laura Rosa , van Gessel Yvonne , Alcaraz Ana , Bliss Susan K. , Pearce Edward J. , Denkers Eric Y.
Published: January 2023

To address the question of how the murine host responds to a prototypic type 1 cytokine inducer while concurrently undergoing a helminth-induced type 2 cytokine response, C57BL/6 strain animals with patent schistosomiasis mansoni were orally infected with the cystogenic Toxoplasma gondii strain ME49. Schistosoma mansoni infection resulted in a significantly higher mortality rate when mice were subsequently orally infected with ME49, and these animals displayed a defective IFN-γ and NO response relative to animals infected with T. gondii alone. Plasma levels of TNF-α and aspartate transaminase in double-infected mice were greatly elevated relative to mice infected with either parasite alone. Consistent with the latter observation, these animals exhibited severe liver pathology, with regions of coagulative necrosis and hepatocyte vacuolization unapparent in mice carrying either infection alone. Interestingly, mean egg granuloma size was ∼50% of that in mice with S. mansoni infection alone. The exacerbated liver pathology in coinfected mice did not appear to be a result of uncontrolled tachyzoite replication, because both parasite-specific RT-PCR analysis and immunohistochemical staining demonstrated a low number of tachyzoites in the liver. We hypothesize that mortality in these animals results from the high level of systemic TNF-α, which mediates a severe liver pathology culminating in death of the animal.
Pretransplant Frequency of Donor-Specific, IFN-γ-Producing Lymphocytes Is a Manifestation of Immunologic Memory and Correlates with the Risk of Posttransplant Rejection Episodes
Authors: Heeger Peter S. , Greenspan Neil S. , Kuhlenschmidt Shannon , Dejelo Cora , Hricik Donald E. , Schulak James A. , Tary-Lehmann Magdalena
Published: January 2023

While matching for MHC Ags improves renal allograft survival, closely matched grafts sometimes fail due to rejection, and poorly matched allografts are often well tolerated by the recipient. The severity of the rejection process may partially depend on the presence of environmentally primed T cells in the recipient that cross-react with donor Ags. To test for the presence of primed, donor-specific T cells in humans before transplantation, we used an enzyme-linked immunospot assay for detection of allospecific cytokines produced by individual human PBLs. We demonstrate that this approach detects cytokine production at single cell resolution and detects production of IFN-γ only when there is defined immunologic priming, thus representing a measure of primed donor-specific immunity. Because the environmental Ag exposure of the recipient is not a function of the HLA mismatch between donor and potential recipient, the number of HLA mismatches may not correlate with the frequency of pretransplant, donor-specific IFN-γ-producing PBLs. Studies of donor-specific IFN-γ-producing lymphocytes in a cohort of patients being evaluated for renal transplantation corroborated this hypothesis. Moreover, for recipients of both living and cadaver renal allografts, the pretransplant frequency of donor-specific memory cells correlated with the posttransplant risk of developing acute rejection episodes. This improved ability to define the strength of the allospecific immune response by enzyme-linked immunospot assay may allow improved pairing of recipients with donors and identification of kidney allograft donor-recipient pairs at high risk for acute rejection, thus permitting targeted interventions aimed at prolonging graft survival.
Coexpression of CCR5 and IL-2 in Human Genital But Not Blood T Cells: Implications for the Ontogeny of the CCR5+ Th1 Phenotype
Authors: Hladik Florian , Lentz Gretchen , Delpit Elizabeth , McElroy Ami , McElrath M. Juliana
Published: January 2023

Memory T cells that home to inflamed tissues typically express the β-chemokine receptor CCR5 and exhibit a Th1 cytokine profile. The migration of these cells into the genital tract following antigenic exposure has particular relevance to acquisition of HIV-1 infection, because CCR5 functions as the coreceptor for most sexually transmitted HIV-1 strains. We recently established methodology to purify and culture mononuclear cells from the female reproductive tract, and here we analyzed the phenotype, CCR5 expression, and cytokine production of cervicovaginal T cells in up to 16 donors. The proportion of mucosal T cells expressing CCR5 was markedly expanded as compared with peripheral blood (mean 88% vs 24% in 13 donors), but the receptor density on individual CCR5+ T cells was only slightly increased (mean 5837 vs 4191 MEPE (molecules of equivalent PE) units in 6 of 7 donors). Intracellular costaining for IL-2, IFN-γ, IL-4, and IL-5 revealed a Th1-type pattern in cervical T cells, with significantly higher percentages of IL-2- and IFN-γ-producing T cells in the mucosa than in blood (mean 67% vs 29%). Coexpression of surface CCR5 with intracellular IL-2 and IFN-γ was observed only among T cells in the mucosa, but not among those in circulation. Thus, we postulate that T cell homing to the genital mucosa leads to differentiation into the combined CCR5+ Th1 phenotype. Moreover, the predominance of CCR5+ Th1-type T cells in normal cervical mucosa provides targets accessible for the efficient transmission of macrophage-tropic HIV-1 variants in women following sexual exposure.
The Human Liver Contains Multiple Populations of NK Cells, T Cells, and CD3+CD56+ Natural T Cells with Distinct Cytotoxic Activities and Th1, Th2, and Th0 Cytokine Secretion Patterns
Authors: Doherty Derek G. , Norris Suzanne , Madrigal-Estebas Laura , McEntee Gerry , Traynor Oscar , Hegarty John E. , O’Farrelly Cliona
Published: January 2023

The human liver contains significant numbers of T cells, NK cells, and lymphocytes that coexpress T and NK cell receptors. To evaluate their functional activities, we have compared the cytotoxic activities and cytokines produced by normal adult hepatic CD3+CD56− (T) cells, CD3−CD56+ (NK) cells, and CD3+CD56+ (natural T (NT)) cells. In cytotoxicity assays using immunomagnetic bead-purified NK cell, T cell, and NT cell subpopulations as effectors, fresh hepatic NK cells lysed K562 targets, while NT cells could be induced to do so by culturing with IL-2. Both NT and T cells were capable of redirected cytolysis of P815 cells using Abs to CD3. Flow cytometric analysis of cytokine production by fresh hepatic lymphocyte subsets activated by CD3 cross-linking or PMA and ionomycin stimulation indicated that NT cells and T cells could produce IFN-γ, TNF-α, IL-2, and/or IL-4, but little or no IL-5, while NK cells produced IFN-γ and/or TNF-α only. The majority of NT cells produced inflammatory (Th1) cytokines only; however, ∼6% of all hepatic T cells, which included 5% of Vα24 TCR-bearing NT cells and 2% of γδTCR+ cells, simultaneously produced IFN-γ and IL-4. The existence of such large numbers of cytotoxic lymphocytes with multiple effector functions suggests that the liver is an important site of innate immune responses, early regulation of adaptive immunity, and possibly peripheral deletion of autologous cells.
Expression of Stromal Cell-Derived Factor-1/Pre-B Cell Growth-Stimulating Factor Receptor, CXC Chemokine Receptor 4, on CD34+ Human Bone Marrow Cells Is a Phenotypic Alteration for Committed Lymphoid Progenitors
Authors: Ishii Takefumi , Nishihara Masamichi , Ma Feng , Ebihara Yasuhiro , Tsuji Kohichiro , Asano Shigetaka , Nakahata Tatsutoshi , Maekawa Taira
Published: January 2023

We found that the stromal cell-derived factor-1/pre-B cell growth-stimulating factor receptor, CXC chemokine receptor 4 (CXCR4), is expressed on human CD34+ bone marrow (BM) cells. Stringently FACS-sorted CD34+CXCR4+ BM cells completely lack myeloid, erythroid, megakaryocytic, and mixed colony-forming potential (myeloid progenitors), but give rise to B and T lymphoid progenitors, whereas CD34+CXCR4− BM cells can generate colonies formed by myeloid progenitors and can also develop into these lymphoid progenitors. Therefore, expression of CXCR4 on CD34+ BM cells can allow lymphoid progenitors to be discriminated from myeloid progenitors. Because CD34+CXCR4+ cells are differentiated from CD34+CXCR4− cells, multipotential progenitors located in the BM are likely to be negative for CXCR4 expression. CXCR4 seems to be expressed earlier than the IL-7R and terminal deoxynucleotidyl transferase during early lymphohemopoiesis. These results suggest that the expression of CXCR4 on CD34+ BM cells is one of the phenotypic alterations for committed lymphoid progenitors.
MHC Class II Engagement in Brain Endothelial Cells Induces Protein Kinase A-Dependent IL-6 Secretion and Phosphorylation of cAMP Response Element-Binding Protein
Authors: Etienne Sandrine , Bourdoulous Sandrine , Strosberg A. Donny , Couraud Pierre-Olivier
Published: January 2023

Activated endothelial cells can directly participate in immune responses by interacting with immunocompetent cells via class II MHC proteins. We show here that, after induction of MHC class II molecule expression by IFN-γ, rat brain endothelial cells responded to MHC class II ligands, anti-MHC class II Abs, or superantigens by expression of IL-6 transcript and IL-6 secretion. This response was not affected by protein kinase C depletion but was mimicked by the cAMP-elevating agent forskolin and completely blocked by H89, an inhibitor of cAMP-dependent protein kinase (PKA). Involvement of a cAMP/PKA signaling pathway in response to MHC class II ligands was further demonstrated by measure of a dose-dependent increase in cAMP level and phosphorylation of the transcription factor cAMP response element-binding protein (CREB). Our results indicate that MHC class II engagement in brain endothelial cells is directly coupled to IL-6 production via a cAMP/PKA-dependent intracellular pathway.
Modulating Dendritic Cells to Optimize Mucosal Immunization Protocols
Authors: Williamson Eilidh , Westrich Gina M. , Viney Joanne L.
Published: January 2023

Oral administration of soluble protein Ag induces tolerance, a phenomenon that has hampered mucosal vaccine design. To provoke active immunity, orally administered Ag must be fed together with a mucosal adjuvant such as cholera toxin (CT). Unfortunately, CT is not suitable for clinical use because of its associated toxicity. There is, therefore, a need to develop alternative mucosal immunization regimens. Here we have attempted to alter the intrinsically tolerogenic nature of the intestine and improve immunization potential by expanding and activating intestinal APC in vivo. Previous studies have indicated that intestinal dendritic cells (DC) present oral Ag, but do so in a tolerogenic manner. In the present study we investigated whether DC can be converted from tolerogenic into immunogenic APC by treating mice with Flt3 ligand (Flt3L), a DC growth factor, and then immunizing with CT. We observed increased local and systemic responses to CT in the presence of elevated numbers of intestinal DC. In parallel, CT induced up-regulation of CD80 and CD86 on these Flt3L-expanded DC. In an attempt to develop a toxin-free adjuvant system, we investigated whether IL-1 could be used as an alternative DC-activating stimulus. Using a combination of Flt3L and IL-1α, we observed a potent active response to fed soluble Ag, rather than the tolerogenic response normally observed. These data suggest that Flt3L-expanded DC are well positioned to regulate intestinal responses depending on the presence or the absence of inflammatory signals. Flt3L may therefore be a reagent useful for the design of mucosal immunization strategies.
T Cell Memory Against Colon Carcinoma Is Long-Lived in the Absence of Antigen
Authors: Xiang Rong , Lode Holger N. , Gillies Stephen D. , Reisfeld Ralph A.
Published: January 2023

Eradication of established colon carcinoma metastases is a major goal for adjuvant immunotherapy of this disease. This was accomplished in a murine model by targeting IL-2 to the tumor microenvironment with a recombinant Ab-IL-2 fusion protein (huKS1/4-IL-2). The generation of a long-lived protective immunity was demonstrated by a 10- to 14-fold increase in CTL precursor (pCTL) frequency and induction of genes encoding Th1 cytokines, followed by the generation of tumor-specific CD8+ T effector cells, some of which differentiated into long-lived T memory cells. The frequency of pCTL correlated with enhanced immune protection against tumor cell challenge, and long-lived T cell memory was maintained in syngeneic SCID mice in the absence of tumor Ag. Tumor cell challenge of these SCID mice, concomitant with a boost of two noncurative doses of huKS1/4-IL-2 fusion protein, resulted in the generation of primed CD8+ T effector cells with concurrent release of Th1 cytokines. These events culminated in the complete rejection of the tumor cell challenge and prevention of pulmonary metastases. Taken together, the data suggest that T cell memory against colon carcinoma can be maintained in the absence of Ag.
Contact Hypersensitivity Responses Following Ribavirin Treatment In Vivo Are Influenced by Type 1 Cytokine Polarization, Regulation of IL-10 Expression, and Costimulatory Signaling
Authors: Tam Robert C. , Lim Charmaine , Bard Josie , Pai Bharati
Published: January 2023

We previously described the promotion of type 1 cytokine responses by the nucleoside analogue, ribavirin, in human T cells in vitro. In this study, we examined whether type 1 cytokine polarization by ribavirin in vivo could promote contact hypersensitivity (CHS) responses to dinitrofluorobenzene, a type 1 cytokine-mediated immune response. Unexpectedly, although type 1 cytokine responses were enhanced following ribavirin treatment in vitro and in vivo, the magnitude of CHS responses in BALB/c and C57BL/6 mice was influenced more by a second ribavirin-regulated pathway. The key regulatory molecule in this pathway was IL-10. Ribavirin-mediated suppression of IL-10 in BALB/c mice was associated with increased B7-2 expression and enhanced CHS responses, whereas enhanced IL-10 levels, following ribavirin administration, led to increased B7-1 expression and impaired CHS responses in C57BL/6 mice. The effect of ribavirin on the expression of B7 molecules and on CHS responses was neutralized by IL-10 administration in BALB/c and by anti-IL-10 Ab in C57BL/6. Thus, ribavirin controlled CHS responses directly through the modulation of IL-10 expression, and in vivo outcome was dictated by the preferential expression of either B7-1, an inappropriate costimulatory molecule in CHS, or B7-2, the predominant costimulatory molecule in CHS. Replacing dinitrofluorobenzene priming with IFN-α stimulation, we showed that the ribavirin-regulated pathway could function independent of Ag priming. Altogether, these data showed that, although ribavirin treatment induced a type 1 cytokine bias in contact allergen-primed BALB/c and C57BL/6 mice, in vivo CHS responses were dependent on ribavirin-mediated regulation of both IL-10 and preferential costimulatory signaling.
Th1/Th2-Regulated Expression of Arginase Isoforms in Murine Macrophages and Dendritic Cells
Authors: Munder Markus , Eichmann Klaus , Morán José M. , Centeno Francisco , Soler Germán , Modolell Manuel
Published: January 2023

Activated murine macrophages metabolize arginine by two alternative pathways involving the enzymes inducible NO synthase (iNOS) or arginase. The balance between the two enzymes is competitively regulated by Th1 and Th2 T helper cells via their secreted cytokines: Th1 cells induce iNOS, whereas Th2 cells induce arginase. Whereas the role of macrophages expressing iNOS as inflammatory cells is well established, the functional competence of macrophages expressing arginase remains a matter of speculation. Two isoforms of mammalian arginases exist, hepatic arginase I and extrahepatic arginase II. We investigated the regulation of arginase isoforms in murine bone marrow-derived macrophages (BMMΦ) in the context of Th1 and Th2 stimulation. Surprisingly, in the presence of either Th2 cytokines or Th2 cells, we observe a specific induction of the hepatic isoform arginase I in BMMΦ. Induction of arginase I was shown on the mRNA and protein levels and obeyed the recently demonstrated synergism among the Th2 cytokines IL-4 and IL-10. Arginase II was detectable in unstimulated BMMΦ and was not significantly modulated by Th1 or Th2 stimulation. Similar to murine BMMΦ, murine bone marrow-derived dendritic cells, as well as a dendritic cell line, up-regulated arginase I expression and arginase activity upon Th2 stimulation, whereas arginase II was never detected. In addition to revealing the unexpected expression of arginase I in the macrophage/monocyte lineage, these results uncover a further intriguing parallelism between iNOS and arginase: both have a constitutive and an inducible isoform, the latter regulated by the Th1/Th2 balance.