bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of the IL-2 Receptor γ-Chain (γc) in the Control by IL-4 of Human Monocyte and Macrophage Proinflammatory Mediator Production
Authors: Bonder Claudine S. , Dickensheets Harold L. , Finlay-Jones John J. , Donnelly Raymond P. , Hart Prue H.
Published: January 2023

IL-4 has potent anti-inflammatory properties on monocytes and suppresses both IL-1β and TNF-α production. Well-characterized components of the IL-4 receptor on monocytes include the 140-kDa α-chain and the IL-2R γ-chain, γc, which normally dimerize 1:1 for signaling from the receptor. However, mRNA levels for γc were very low in 7-day-cultured monocytes. As mRNA levels for γc declined with culture, so too did the ability of IL-4 to down-regulate LPS-induced TNF-α production. In contrast, IL-4 consistently down-regulated IL-1β production by cultured monocytes. Immunoprecipitation and Western blot analyses demonstrated that 7-day-cultured monocytes do not express the functionally active 64-kDa γc protein. This was associated with decreased STAT6 activation by IL-4. Studies with Abs to γc and an IL-4 mutant that is unable to bind to γc showed that IL-4 can suppress IL-1β but not TNF-α production by LPS-stimulated monocytes in the presence of little or no functioning γc. IL-4 also suppressed IL-1β but not TNF-α production by Mono Mac 6 cells, which express minimal levels of γc. For γc-expressing LPS/PMA-activated U937 cells, IL-4 decreased both TNF-α and IL-1β production. These results suggest that functional γc is not present on in vitro-derived macrophages, and that while some anti-inflammatory responses to IL-4 are lost with this down-regulation of functional γc, others are retained. We conclude that different functional responses to IL-4 by human monocytes and macrophages are regulated by different IL-4 receptor configurations.
A p55 TNF Receptor Immunoadhesin Prevents T Cell-Mediated Intestinal Injury by Inhibiting Matrix Metalloproteinase Production
Authors: Pender Sylvia L. F. , Fell John M. E. , Chamow Steven M. , Ashkenazi Avi , MacDonald Thomas T.
Published: January 2023

Anti-TNF-α Ab therapy has been shown to be of benefit in the treatment of active Crohn’s disease, but the tissue-injuring processes in the gut mediated by TNF-α that might be inhibited by neutralizing Ab are unknown. In this work, we have used a p55 TNF receptor-human IgG fusion protein (TNFR-IgG) to prevent the severe mucosal injury that ensues when lamina propria T cells in explant cultures of human fetal small intestine are directly activated with the lectin PWM. Following T cell activation and associated with mucosal injury, there is a marked elevation of soluble TNF-α in organ culture supernatants and a large increase in TNF-α mRNA transcripts. The addition of TNFR-IgG at the onset of cultures greatly reduced PWM-induced tissue injury, without inhibiting the increase in TNF-α and IFN-γ transcripts seen following T cell activation. Mucosal injury in this model is mediated by endogenously-produced matrix metalloproteinases (MMPs). When TNFR-IgG was added to PWM-stimulated explants, there was a reduction in MMPs in the explant culture supernatants, especially stromelysin-1. Recombinant TNF-α and IL-1β added directly to mucosal mesenchymal cell lines also caused an increase in MMP production, but only the former was inhibited by the TNFR-IgG. These results suggest that one of the ways in which TNF-α causes tissue injury in the gut is by stimulating mucosal mesenchymal cell to secrete matrix-degrading metalloproteinases. Neutralization of this activity should help maintain tissue integrity.
Complement-Fixing Elicited Antibodies Are a Major Component in the Pathogenesis of Xenograft Rejection
Authors: Miyatake Tsukasa , Sato Koichiro , Takigami Ko , Koyamada Nozomi , Hancock Wayne W. , Bazin Herve , Latinne Dominique , Bach Fritz H. , Soares Miguel P.
Published: January 2023

Hamster to rat cardiac xenografts undergo delayed rejection as compared with the hyperacute rejection of discordant xenografts. Elicited xenoreactive Abs (EXA) are thought to initiate hamster to rat cardiac xenograft rejection. In this study, we demonstrate that following transplantation of a hamster heart, rats generated high levels of EXA. Adoptive transfer into naive recipients of purified IgM, IgG2b, or IgG2c, but not IgG1 or IgG2a EXA, induced xenograft rejection in a complement-dependent manner. Ability of EXA to cause rejection correlated with complement activation, platelet aggregation, and P-selectin expression in the xenograft endothelium. Cyclosporin A (CyA) administration, after transplantation, totally suppressed IgG1, IgG2a, IgG2b, and IgG2c EXA, and inhibited IgM EXA production, but failed to overcome rejection. Administration of cobra venom factor (CVF), 1 day before and at the time of transplantation, resulted in complement inhibition during 3 days after transplantation, which failed to overcome rejection. Combination of CyA and CVF, which we have previously shown to overcome rejection, resulted in suppression of IgG EXA production and in the return of IgM XNA to preimmunization serum levels, 3 to 7 days after xenotransplantation, while complement remained inhibited. Thus, under CyA/CVF treatment, complement activation by hamster cells was suppressed following xenotransplantation, and presumably for this reason xenograft rejection did not occur. In conclusion, our data demonstrate that EXA play a pivotal role in the pathogenesis of xenograft rejection and that CyA and CVF suppress xenograft rejection by preventing exposure of xenograft endothelial cells to complement activation by EXA.
Technical Note: Aberrant Detection of Cell Surface Fas Ligand with Anti-Peptide Antibodies
Authors: Smith Dawn , Sieg Scott , Kaplan David
Published: January 2023

Polyclonal rabbit Abs raised against peptides from the C-terminal region (the extracellular domain) of human Fas ligand were produced for the detection of the molecule in Western blot analysis and immunohistochemistry. These Abs have been used by several groups of investigators to assess cell surface Fas ligand via flow cytometry, but we show that these polyclonal rabbit Abs do not detect cell surface Fas ligand by that technique.
Hybrid Membrane IgM with the Transmembrane Region of I-Aα Facilitates Enhanced Presentation of Distinct Epitopes to T Cells
Authors: Liu Ko-Jiunn , Schwen Michael , Tucker Philip W. , Kim Byung S.
Published: January 2023

The role of B cell Ag receptors (membrane Ig or mIg) in the efficient Ag presentation to T cells, including the requirement of mIgM-associated Igα/Igβ, remains unclear. We report here that mIgM, substituted with greater than two-thirds of the NH2-terminal Aα transmembrane (TM) regions of the MHC class II molecule, are capable of mediating the efficient presentation of specific Ag to some (Group 1) but not all (Group 2) T cell hybridomas. In contrast, the generation of epitopes recognized by the Group 2 hybridomas can be mediated only by the wild-type mIgM. Tyrosine phosphorylation appears to be necessary for the enhanced Ag presentation to Group 2 hybridomas, while it does not for Group 1 hybridomas. In addition, differential sensitivity of Ag processing to leupeptin, different duration required for epitope generation/presentation, as well as the involvement of distinct epitopes for stimulation of these groups of T cell hybridomas were observed. These results suggest that transport of the mIgM/Ag complexes to an endocytic compartment(s) for generation of certain T cell epitopes may be mediated by the N-terminal TM sequence of mIgM, independent of Igα/Igβ association. This function can be replaced by two-thirds of the NH2-terminal TM region of Aα chain of class II molecules.
Polymorphism at the HLA-DQ Locus Determines Susceptibility to Experimental Autoimmune Myasthenia Gravis
Authors: Raju Raghavan , Zhan Wen-Zhi , Karachunski Peter , Conti-Fine Bianca , Sieck Gary C. , David Chella
Published: January 2023

Studies in myasthenia gravis (MG) patients demonstrate that polymorphism at the HLA-DQ locus influences the development of MG. Several studies using the mouse models also demonstrate the influence of class II molecules, especially the H2-A, which is the mouse homologue of HLA-DQ, in experimental autoimmune myasthenia gravis (EAMG). We used transgenic mice expressing two different DQ molecules, DQ8 (DQA1*0301/B1*0302) and DQ6 (DQA1*0103/B1*0601), to evaluate the role of HLA-DQ genes in MG. These mice do not express endogenous mouse class II molecules since they contain the mutant H2-Aβ0 gene. The mice were immunized with Torpedo acetylcholine receptor, and EAMG was assessed by clinical evaluation and was confirmed by electrophysiology. Clinical scores for EAMG were highest in HLA-DQ8 transgenic mice, whereas the scores of HLA-DQ6 mice rarely exceeded grade 1. There was no incidence of EAMG in class II-deficient (H2-Aβ0) mice. These results demonstrate that polymorphism at the HLA-DQ locus affects the incidence and the severity of EAMG. The manifestation of susceptibility to EAMG in the context of human class II molecules underscores the important roles of these molecules in the initiation and perpetuation of EAMG.
Costimulation via TCR and IL-1 Receptor Reveals a Novel IL-1α-Mediated Autocrine Pathway of Th2 Cell Proliferation
Authors: Huber Magdalena , Beuscher Horst U. , Rohwer Peter , Kurrle Roland , Röllinghoff Martin , Lohoff Michael
Published: January 2023

Previous studies have shown that triggering of Th2 cells via the TCR is sufficient for production of IL-4 but not for proliferation of these cells. Proliferation of Th2 cells occurs only in the additional presence of a costimulatory signal delivered by IL-1. For the majority of Th2 cell clones, this type of proliferation was found to be independent of IL-4. Here, we further investigated the mechanism of IL-4-independent proliferation. We demonstrate that, after costimulation via TCR and IL-1R, but not via either receptor alone, Th2 cells are triggered to produce cell-associated IL-1α, as detected at the level of function, protein, and mRNA expression. In the presence of the TCR signal, autocrine IL-1α is then able to costimulate IL-4-independent proliferation of Th2 cells and to further enhance its own production. Thus, our results point to a novel, IL-4-independent, self-amplifying autocrine pathway of Th2 cell proliferation that requires a signal via the TCR and a costimulatory signal via IL-1R. This pathway may explain frustrating results in experimental models that attempted to treat established Th2-mediated diseases in vivo with IL-4-neutralizing agents alone.
Ultraviolet Light-Induced Immune Tolerance Is Mediated via the Fas/Fas-Ligand System
Authors: Schwarz Agatha , Grabbe Stephan , Grosse-Heitmeyer Karin , Roters Berthold , Riemann Helge , Luger Thomas A. , Trinchieri Giorgio , Schwarz Thomas
Published: January 2023

Hapten sensitization through UV-exposed skin induces tolerance that is mediated via the induction of hapten-specific T suppressor cells. However, the detailed mechanisms underlying tolerance induction remain unclear to date. We show here that the apoptosis-related surface Ag Fas (APO-1, CD95) and its ligand, Fas ligand (FasL) are critically involved, since Fas-deficient lpr mice and FasL-deficient gld mice do not develop UV-induced tolerance. Adoptive transfer experiments revealed that the mediation of tolerance does not require the expression of Fas or FasL by the T suppressor cells but does require the expression of both molecules by the cells of mice receiving the T suppressor cells. To identify the mechanisms involved, the effect of suppressor cells on Ag-presenting dendritic cells (DC) was studied. Coincubation of hapten-pulsed DC with T cells that were obtained from UV-tolerized mice resulted in an enhanced death rate of DC, and this cell death was dependent upon Fas expression. The addition of IL-12, which recently was found to break established tolerance in vivo, prevented DC death. Moreover, IL-12 did not only rescue DC from T suppressor cell-induced death but also from apoptosis induced by rFasL, suggesting that IL-12 may interfere with the Fas/FasL system. Together, these data indicate a crucial role for the Fas/FasL system in UV-induced tolerance, and suggest that UV-induced T suppressor cells may act by inducing the cell death of APCs via the Fas pathway. The ability of IL-12 to break established tolerance may be due to the prevention of DC death induced by T suppressor cells.
TNF-α Messenger RNA and Protein Expression in the Uteroplacental Unit of Mice with Pregnancy Loss
Authors: Gorivodsky Marat , Zemlyak Ilona , Orenstein Hasida , Savion Shoshana , Fein Amos , Torchinsky Arkady , Toder Vladimir
Published: January 2023

An elevated expression of TNF-α in embryonic microenvironment was found to be associated with postimplantation loss. In this work, we examined the pattern of TNF-α expression at both the mRNA and the protein level as well as the distribution of TNF-α receptor mRNA in the uteroplacental unit of mice with induced (cyclophosphamide-treated) or spontaneous (CBA/J × DBA/2J mouse combination) pregnancy loss. RNase protection analysis demonstrated an increase in TNF-α mRNA expression in the placentae of mice with pregnancy loss compared with that in control mice. TNF-α messages were localized to the uterine epithelium and stroma as well as the giant and spongiotrophoblast cells of the placenta. The intensity of the hybridization signal in placentae of mice with pregnancy loss was substantially higher than that in control mice. The up-regulation of TNF-α mRNA was accompanied by an increase in the expression of TNF-α receptor I mRNA in the same cell populations. The elevation of TNF-α production was also demonstrated at the protein level. Western blot analysis showed an increased level of the 18- and 26-kDa TNF-α protein species in the uteroplacental unit of mice with pregnancy loss. Immunostaining revealed TNF-α-positive leukocytes located in the uterus and placenta. Finally, we found that immunization of mice with cyclophosphamide-induced pregnancy loss while decreasing the resorption rate in these females resulted in a decline in TNF-α expression at the fetomaternal interface. These data clearly suggest an involvement of TNF-α in pathways leading to both spontaneous and induced placental death.
Induction of Fas Ligand in Murine Bone Marrow NK Cells by Bacterial Polysaccharides
Authors: Halaas Øyvind , Vik Randi , Espevik Terje
Published: January 2023

Bacterial polysaccharides have a wide range of activities in mammals. We have studied the effect of LPS and poly-β-(1→4)-d-mannuronate (mannuronan, poly-M), an exopolysaccharide from Pseudomonas aeruginosa, on the cytotoxicity mediated by murine bone marrow cells (BMC). Addition of LPS or mannuronan to BMC induced a time- and dose-dependent cytotoxicity against Jurkat cells. The LPS- or mannuronan-induced cytotoxicity was due to increased Fas ligand (FasL) expression by BMC, since 1) Fas-transfected L1210-Fas target cells were more susceptible to lysis than the Faslow-expressing parent L1210 cells, 2) stimulated BMC from FasL-defective gld/gld mice were not cytolytic and, 3) the cytolytic activity of normal BMC was inhibited by a Fas-Fc fusion protein. Flow cytometry showed an increase in surface FasL in LPS-stimulated BMC. RT-PCR analysis of BMC revealed constitutive expression of FasL mRNA, which was increased after LPS stimulation. Immunomagnetic depletion of NK1.1-, CD2-, or CD32/16-expressing cells from BMC abrogated the LPS-induced BMC cytotoxicity against L1210-Fas cells, suggesting that NK cells were the cytotoxic effector cells. Depletion of CD45R/B220-, Gr-1-, or CD11b/Mac-1-expressing cells only partially decreased BMC-mediated cytotoxicity, and depletion of CD4- or CD8a-expressing cells had no effect. The results support the conclusion that LPS and mannuronan induce expression of cytotoxic FasL on bone marrow NK cells.
Regulation of Transcription of the TATA-less Human Complement Component C4 Gene
Authors: Vaishnaw Akshay K. , Mitchell Tracey J. , Rose S. Jane , Walport Mark J. , Morley Bernard J.
Published: January 2023

The 5′-sequences flanking the human complement component C4 genes (C4A and C4B) have been analyzed for their ability to direct expression of a reporter gene in cell lines that constitutively express or do not express C4. No difference in the level of reporter gene expression was detected in cells transfected with C4A- or C4B-specific constructs. A series of reporter constructs containing progressively truncated C4 promoter fragments transfected into the hepatocyte Hep G2 cell line, identified the sequence contained within the region −178 to −39 as that associated with maximal reporter gene expression. This region contains consensus binding motifs for nuclear factor 1 (−110 to −97), Sp1 (−57 to −49), and three basic helix-loop-helix (−137 to −132, −98 to −93, and −78 to −73)-like transcription factors. Electromobility shift assays and DNase I footprinting analysis showed specific DNA-protein interactions of the C4 promoter at the nuclear factor 1, two E box (−98 to −93 and −78 to −73), and Sp1 binding domains. Site-directed mutagenesis of the Sp1 binding site resulted in total abrogation of reporter gene expression and mutation of the E box (−78 to −73) resulted in a 8-fold reduction in expression. We conclude that the Sp1 binding site at position −57 to −49 is critical for accurately initiated, basal transcription of C4.
MHC Class I/Peptide Stability: Implications for Immunodominance, In Vitro Proliferation, and Diversity of Responding CTL
Authors: Busch Dirk H. , Pamer Eric G.
Published: January 2023

Infection of BALB/c mice with Listeria monocytogenes primes CD8+ cytotoxic T cells specific for four different H2-Kd-restricted peptides. In vitro restimulation of L. monocytogenes immune splenocytes with each of these peptides resulted in larger T cell responses to p60 217–225 and mpl 84–92 than to LLO 91–99 and p60 449–457. Direct frequency analyses of immune splenocytes, however, revealed that LLO 91–99 and p60 217–225 elicit dominant T cell responses, while p60 449–457 and mpl 84–92 elicit minor, subdominant responses. Restimulation of immune splenocytes with a range of peptide concentrations revealed that T cells with dominant specificities respond optimally to low peptide concentrations, while T cells specific for subdominant epitopes expand maximally to high peptide concentrations. This disparity correlates with the stability of H2-Kd/epitope complexes: the two dominant epitopes form stable complexes, while the subdominant epitopes form less stable complexes with H2-Kd. Interestingly, T cells specific for LLO 91–99 and p60 217–225 express more complex TCR-Vβ repertoires than p60 449–457- and mpl 84–92-specific T cells. Thus, in our system, dominant T cell responses have relatively diverse TCR repertoires and are specific for peptides that form stable complexes with MHC class I molecules. Determining the precise roles of epitope/MHC class I stability and TCR repertoire in the generation of dominant T cell responses will require further investigation.
Enhancement of Tumor Outgrowth Through CTL Tolerization After Peptide Vaccination Is Avoided by Peptide Presentation on Dendritic Cells
Authors: Toes René E. M. , van der Voort Ellen I. H. , Schoenberger Stephen P. , Drijfhout Jan Wouter , van Bloois Louis , Storm Gert , Kast W. Martin , Offringa Rienk , Melief Cornelis J. M.
Published: January 2023

Synthetic peptide-based vaccines have been shown to induce potent protective and therapeutic T cell-mediated immunity in preclinical animal models and are now being evaluated in clinical phase I/II studies for their efficacy against tumors or infectious diseases. However, such vaccines might also specifically tolerize T cells causing enhanced tumor outgrowth, as shown by vaccination with two CTL epitopes derived from the adenovirus type 5 early region 1 (Ad5E1) oncogenes. We now report that modification of the Ad5E1 peptide vaccine either through incorporation of the peptides into liposomes or by ligation of the peptides to lipid tails, another vaccine formulation being tested in the clinic, fails to convert immunosuppression into effective antitumor vaccination. Inclusion of a helper T cell epitope into the vaccine likewise induces enhanced tumor outgrowth and thus does not diminish the capacity of the peptides to tolerize Ad5E1-specific CTL. In contrast, the Ad5E1-derived peptides evoke a strong tumor-protective CTL response when presented on dendritic cells (DC), indicating that the in vivo CTL-tolerizing potential of these peptides is converted to specific immunostimulation when presented on DC. These findings have important implications for the development of peptide-based immune intervention strategies and emphasize the superior nature of Ag-pulsed DC over other peptide-based vaccination protocols as well as the crucial importance of the mode of peptide-Ag delivery in setting the balance between T cell stimulation and tolerization.
TNF-α Convertase Enzyme from Human Arthritis-Affected Cartilage: Isolation of cDNA by Differential Display, Expression of the Active Enzyme, and Regulation of TNF-α
Authors: Patel Indravadan R. , Attur Mukundan G. , Patel Rajesh N. , Stuchin Steven A. , Abagyan Ruben A. , Abramson Steven B. , Amin Ashok R.
Published: January 2023

A snake venom-like protease isolated by a differential display screen between normal and osteoarthritis (OA)-affected cartilage (designated as cSVP) has a cDNA sequence identical to TNF-α convertase enzyme (TACE). TACE shows the presence of an unknown prodomain, a cysteine switch, a catalytic domain, a zinc binding region, a disintegrin region, an EGF-like domain, a transmembrane domain, and a unique cytoplasmic region. A TACE construct harboring the signal + prodomain + catalytic region (TACE-SPCΔDETCy), expressed in baculovirus could cleave preferentially (∼12-fold) the TNF-specific peptide over the matrix metalloproteases peptide in vitro. This recombinant protein also cleaved the natural substrate GST-ProTNF-α to TNF-α (17 kDa) in vitro. The mRNA for TACE, which is broadly distributed and differentially expressed in a variety of human tissues, is up-regulated in arthritis-affected cartilage, but not normal cartilage. OA-affected cartilage also expressed TNF-α mRNA that was not detected in normal cartilage. The OA-affected cartilage (in explant assays) spontaneously released TNF-α and IL-8 in ex vivo conditions. Addition of TNF-αR fused to IgG Fc fragment (TNF-αR:Fc) in the presence or absence of soluble IL-1R (with which it acted additively) significantly attenuated the spontaneous/autocrine release of articular IL-8 in this assay. These experiments demonstrate a functional paracrine/autocrine role of TNF-α in OA-affected cartilage that may depend, in part, on up-regulated levels of chondrocyte-derived TACE.
Cutting Edge: TRANK, a Novel Cytokine That Activates NF-κB and c-Jun N-Terminal Kinase
Authors: Haridas Valsala , Ni Jian , Meager Anthony , Su Jeffery , Yu Guo-Liang , Zhai Yifan , Kyaw Hla , Akama Keith T. , Hu Jingru , Van Eldik Linda J. , Aggarwal Bharat B.
Published: January 2023

We searched the expressed sequence tag database using sequence homology and identified a novel cytokine, which we have named TRANK (thioredoxin peroxidase-related activator of NF-κB and c-Jun N-terminal kinase). The predicted amino acid sequence of TRANK was highly homologous to that of the thiol-specific antioxidant proteins. Unlike these proteins, however, TRANK had a putative secretory signal polypeptide and was found to be secreted by cells. TRANK was expressed in most tissues and cell lines, and the gene that encodes it was mapped to chromosome Xp21–22.1. TRANK activated NF-κB and induced the degradation of the inhibitory subunit of NF-κB. In addition, TRANK up-regulated the expression of NF-κB-dependent gene products, ICAM-1, and inducible nitric oxide synthase. TRANK also activated c-Jun N-terminal kinase and induced the proliferation of normal human foreskin fibroblasts. Its homology with antioxidant proteins, wide distribution in tissues, and ability to activate NF-κB and c-Jun N-terminal kinase suggest that TRANK plays an important role in inflammation.
In Vivo Expression of a TCR Antagonist: T Cells Escape Central Tolerance But Are Antagonized in the Periphery
Authors: Williams Calvin B. , Vidal Karine , Donermeyer David , Peterson Daniel A. , White J. Michael , Allen Paul M.
Published: January 2023

Transgenic 3.L2 T cells are stimulated by Hb(64–76)/I-Ek and are positively selected on I-Ek plus self-peptides. To this pool of self-peptides we have added a single, well-defined 3.L2 TCR antagonist (A72) in vivo. We find that mice expressing both the 3.L2 TCR and A72 have a minimal loss of T cells expressing the clonotypic TCR in the thymus and spleen. Importantly, the proliferative response of 3.L2 × A72 splenocytes is significantly reduced compared with splenocytes from 3.L2 mice. This reduced response can be attributed to peripheral antagonism. Thus we have identified a new class of self-ligands whose predominant effect is constitutive peripheral antagonism rather than negative selection. The net effect of these ligands is to avoid potential self-reactivity while maintaining as large a repertoire as possible.
Characterization of Human Fibroleukin, a Fibrinogen-Like Protein Secreted by T Lymphocytes
Authors: Marazzi Susanna , Blum Stephanie , Hartmann Reto , Gundersen Doris , Schreyer Magali , Argraves Scott , von Fliedner Vladimir , Pytela Robert , Rüegg Curzio
Published: January 2023

We have recently cloned the human homologue of the murine pT49 cDNA (hpT49h), a transcript encoding a protein homologous to the β- and γ-chains of fibrinogen. Here, we report the identification of the hpT49h gene product using mAbs generated against a peptide corresponding to the carboxyl-terminal end of the deduced protein and a recombinant protein fragment expressed in Escherichia coli. mAbs 23A6, 7B12, and 3F4 specifically recognized a protein of 70 kDa in reducing SDS-PAGE in the culture supernatant of 293T cells transiently transfected with the full length hpT49h cDNA and freshly isolated PBMC. Under nonreducing conditions, the material migrated with a molecular mass of 250 to 300 kDa, indicating that the 70-kDa protein forms a disulfide bonded complex. Because of its homology with fibrinogen, we have termed this protein fibroleukin. Fibroleukin is spontaneously secreted in vitro by freshly isolated CD4+ and CD8+ T lymphocytes. RT-PCR analysis revealed preferential expression of fibroleukin mRNA in memory T lymphocytes (CD3+/CD45R0+) compared with naive T lymphocytes (CD3+/CD45RA+). Fibroleukin production by PBMC was rapidly lost in culture. Production could be partially maintained in the presence of IFN-γ, while T lymphocyte activation had no effect. To demonstrate fibroleukin production in vivo, we analyzed colon mucosa by immunohistology. Fibroleukin staining was detected in the extracellular matrix of the T lymphocyte-rich upper portion of the lamina propria mucosa. While the exact function of fibroleukin remains to be defined, these data suggest that fibroleukin may play a role in physiologic lymphocyte functions at mucosal sites.
Strong Alloantigenicity of the α-Helices Residues of the MHC Class I Molecule
Authors: Noun Ghada , Reboul Murielle , Abastado Jean-Pierre , Kourilsky Philippe , Sigaux François , Pla Marika
Published: January 2023

To evaluate the role of single residues of a MHC class I molecule in the induction of a primary allogeneic response, we have tested the ability of various point mutants (of the α-helices or β-sheet of the α1 and α2 domains) of the Kd molecule to induce a primary cytotoxic T cell response in mice carrying the wild-type molecule. For that, we have used an in vivo model in which cells expressing mutant molecules were injected into the hind footpads of mice carrying wild-type Kd, and the recipient graft-draining popliteal lymph nodes were tested for the presence of alloreactive CTL. Under these experimental conditions, only 7 of the 25 mutant Kd molecules induced a primary allogeneic response. All of these mutations (positions 62, 65, 69, 72, 155, 163, 166) concern residues of the α-helices, demonstrating that very small variances from self in a class I molecule, located outside the peptide-binding groove, can be antigenic. To determine the peptide requirements for the generation of a primary allogeneic response, we have analyzed the repertoire of peptides selected by individual mutant molecules shown to be able or unable to induce a CTL response. No correlation was observed between the peptidic make-up presented by a given mutant and its capacity to induce a primary allogeneic response. On the whole, our data point to the alloantigenicity of potentially TCR-contacting surface residues of the MHC class I molecules.
Induction of Oral Tolerance in TGF-β1 Null Mice
Authors: Barone K. Siobhan , Tolarova Dana D. , Ormsby Ilona , Doetschman Thomas , Michael J. Gabriel
Published: January 2023

Previous studies have suggested that oral tolerance induction by low doses of Ag is mediated by inhibitory cytokines, particularly TGF-β1. To examine the roles of TGF-β1 and other inhibitory cytokines in the induction of oral tolerance, TGF-β1 null mice and controls were gavaged with 10 to 20 mg (high dose) or 1 mg (low dose) of OVA for 3 days. After immunization with OVA, the in vitro proliferative response of OVA-specific popliteal lymph node cells was assessed. Lymphocytes from all TGF-β1 null mice fed high doses of OVA exhibited highly significant suppression compared with controls. A weaker, but still significant, suppression was observed in lymphocytes from the majority of TGF-β1 null mice fed low doses of OVA. In addition, supernatants from these lymphocytes exhibited lower levels of IL-4, IL-10, and IFN-γ than those from water-fed control animals. These results indicate that while TGF-β1 may play a role in suppression, inhibitory cytokines are not the exclusive mechanism by which low dose oral tolerance is induced.
V7 (CD101) Ligation Inhibits TCR/CD3-Induced IL-2 Production by Blocking Ca2+ Flux and Nuclear Factor of Activated T Cell Nuclear Translocation
Authors: Soares Luis R. B. , Tsavaler Larisa , Rivas Alberto , Engleman Edgar G.
Published: January 2023

Ligation of the V7 (CD101) molecule on T cells with anti-V7 mAb blocks TCR/CD3-induced proliferation by inhibiting IL-2 transcription. To explore the basis for this observation, we analyzed the effects of V7 ligation on CD3/TCR-induced changes in intracellular free Ca2+ and Ca2+-dependent nuclear factor of activated T cells (NF-AT) translocation to the nucleus, which is required for IL-2 transcription. T cells exposed to anti-V7 mAb fluxed Ca2+ transiently, but did not flux Ca2+ in response to subsequent treatment with anti-CD3; however, they recovered the capacity to flux Ca2+ after treatment with pervanadate, indicating that tyrosine dephosphorylation of a critical V7-related substrate is required in the desensitization process. One such substrate, phospholipase C (PLC)-γ1, becomes tyrosine phosphorylated on CD3/TCR activation and mediates inositol triphosphate-dependent Ca2+ flux. Co-cross-linking of T cells with anti-CD3 and anti-V7 resulted in selective inhibition of PLC-γ1 tyrosine phosphorylation, which may explain V7-mediated blockade of anti-CD3-induced Ca2+ flux. Moreover, anti-CD3-induced binding of transcription factors to a consensus NF-AT-binding oligonucleotide, which is dependent on Ca2+, was blocked completely by treatment of the cells with anti-V7, whereas binding to a consensus-activating protein-1 oligonucleotide was unaffected. Western blot analysis of cytoplasmic and nuclear extracts confirmed that anti-V7 prevented nuclear translocation of NF-ATc induced by anti-CD3. We conclude that V7 ligation interferes with T cell activation and IL-2 secretion through a Ca2+ and tyrosine kinase-dependent pathway that inhibits PLC-γ1 phosphorylation and prevents NF-AT translocation to the nucleus.
Differential Regulation of the Janus Kinase-STAT Pathway and Biologic Function of IL-13 in Primary Human NK and T Cells: A Comparative Study with IL-4
Authors: Yu Cheng-Rong , Kirken Robert A. , Malabarba M. Grazia , Young Howard A. , Ortaldo John R.
Published: January 2023

IL-13, a cytokine similar to IL-4, is a regulator of human B cell and monocyte functions. Biologic effects of IL-13 on primary human NK and T cells have not been well defined. We demonstrate that, in primary NK cells, IL-13, but not IL-4, may induce low levels of IFN-γ secretion. When NK cells were costimulated with IL-13 and IL-2, IL-13 generally resulted in two types of reactivity: IL-13 synergized with IL-2 to stimulate IFN-γ production or it modestly inhibited IL-2-mediated IFN-γ production. In both types of donors, the effect of IL-13 on IL-2-induced IFN-γ production was in marked contrast to the strong inhibition seen with IL-4 in NK cells. Additionally, IL-13 suppresses IL-2-induced NK cytolytic and proliferative activities although less efficiently than IL-4. In T cells, IL-13 inhibits anti-CD3 mAb/IL-2- or PHA-mediated IFN-γ production and enhances cytolytic potential. Furthermore, we demonstrate that IL-13, like IL-4, induces distinct STAT6-DNA binding complexes and tyrosine phosphorylation of STAT6 and Janus kinase 3 (JAK3) in NK and T cells. We observed that Abs directed against unique domains of STAT6 have differential effects on complexes in T cells but not in NK cells, suggesting different STAT6 isoforms. These findings show that IL-13 and IL-4 have the ability to regulate NK and T cell activation and that IL-13 is a potent regulator of STAT6 and JAK3 in these cell types.
Development of Human Peripheral TCRBJ Gene Repertoire
Authors: Nanki Toshihiro , Kohsaka Hitoshi , Miyasaka Nobuyuki
Published: January 2023

Previous studies of TCRBJ gene repertoires of human peripheral T lymphocytes showed that all TCRBV family transcripts had some common features in BJ gene usage, and nevertheless, transcripts of each BV family gene had a distinct pattern. To discern how the development of the peripheral BJ repertoire is controlled, the effects of preferential BJ gene rearrangement, thymic selection, and peripheral stimulation on the repertoire formation were investigated. A PCR-ELISA technique was used to examine the immature CD3−CD4+CD8−, and mature CD3+CD4+CD8− and CD3+CD4−CD8+ thymocytes, and peripheral CD4+ and CD8+ T lymphocytes for their BJ gene repertoires. Analogous to the peripheral repertoire, the BJ gene repertoires of the immature thymocytes displayed common features, and each BV transcript had a distinct pattern. All features were conserved well by those of mature thymocytes and peripheral T lymphocytes. In addition, the BJ gene repertoires of mature CD4 and CD8 thymocytes and peripheral lymphocytes with the same coreceptors were apparently different in a few BV-BJ combinations. The results showed that the overall BJ gene repertoire pattern was developed before antigenic selection. Thus, the preferential BJ gene expression, primarily based on preferential use of certain BJ gene rearrangements, dictates the peripheral BJ gene repertoire, which is then further modified by thymic selection and peripheral stimulation.
The Prototypic Th2 Autoimmunity Induced by Mercury Is Dependent on IFN-γ and Not Th1/Th2 Imbalance
Authors: Kono Dwight H. , Balomenos Dimitri , Pearson Deborah L. , Park Miyo S. , Hildebrandt Bernhard , Hultman Per , Pollard K. Michael
Published: January 2023

Imbalances of Th1- and Th2-type responses have been postulated to be a predisposing factor for both humoral and cellular mediated autoimmune diseases. To further define their roles in systemic autoimmunity, IL-4 and IFN-γ gene knockout mice were studied for susceptibility to the prototypic Th2-mediated mercury-induced autoimmunity. A predominant Th2-type response following HgCl2 treatment of wild-type B10.S mice was confirmed by the findings of a significant increase in splenic IL-4 and hypergammaglobulinemia primarily of the IgG1 isotype, without an increase in IFN-γ levels. Paradoxically, IL-4-deficient mice developed the characteristic anti-nucleolar autoantibodies and tissue deposition of immune complexes, while IFN-γ-deficient mice had very low autoantibody levels and essentially normal immunohistology. Studies to define defects in Ab responses of IFN-γ-deficient mice, using the T-dependent Ag (4-hydroxy-3-nitrophenyl)acetyl, revealed an attenuated IgG response to low and to a lesser extent high doses of (4-hydroxy-3-nitrophenyl)acetyl-hemocyanin, but maintenance of affinity maturation. These results indicate that Th1/Th2 imbalance does not directly play a role in susceptibility to mercury-induced autoimmunity, and suggest that the dependence on Th1-type responses in certain autoimmune diseases is due to the requirement for IFN-γ for Ab production to weakly antigenic self molecules.
Two Distinct Pathways Exist for Down-Regulation of the TCR
Authors: Lauritsen Jens Peter H. , Christensen Mette D. , Dietrich Jes , Kastrup Jesper , Ødum Niels , Geisler Carsten
Published: January 2023

TCR down-regulation plays an important role in modulating T cell responses both during T cell development and in mature T cells. Down-regulation of the TCR is induced by engagement of the TCR by specific ligands and/or by activation of protein kinase C (PKC). We report here that ligand- and PKC-induced TCR down-regulation is mediated by two distinct, independent mechanisms. Ligand-induced TCR down-regulation is dependent on the protein tyrosine kinases p56lck and p59fyn but independent of PKC and the CD3γ leucine-based (L-based) internalization motif. In contrast, PKC-induced TCR down-regulation is dependent on the CD3γ L-based internalization motif but independent of p56lck and p59fyn. Finally, our data indicate that in the absence of TCR ligation, TCR expression levels can be finely regulated via the CD3γ L-based motif by the balance between PKC and serine/threonine protein phosphatase activities. Such a TCR ligation-independent regulation of TCR expression levels could probably be important in determining the activation threshold of T cells in their encounter with APC.
Stability of Virus-Specific CD4+ T Cell Frequencies from Acute Infection into Long Term Memory
Authors: Varga Steven M. , Welsh Raymond M.
Published: January 2023

Mice infected with viruses develop long-lasting high frequency memory CD8+ T cell pools, but much less is known about the CD4+ T cell response. FACS analysis revealed the modulation of several activation markers on CD4+ T cells during an acute infection with lymphocytic choriomeningitis virus (LCMV), consistent with an activated cell phenotype. Examination of virus-specific cytokine production using ELISPOT assays showed a significant increase in the number of IFN-γ-secreting cells in the spleen during an acute LCMV infection. CD8+ T cells made up the majority of the IFN-γ-producing cells, but analysis of the cell culture supernatants by ELISA showed that the CD4+ T cells produced more IFN-γ on a per cell basis. Using limiting dilution assays, we examined the CD4+ T cell precursor (Thp) frequency in C57BL/6 mice infected with LCMV. The virus-specific Thp frequency increased from <1/100,000 in uninfected mice to a peak of ∼1/600 in purified splenic CD4+ T cell populations by 10 days postinfection with LCMV. After the peak of the response, the Thp frequency decreased only about twofold per CD4+ T cell to ∼1/1200 and remained stable into long term memory. In contrast to the highly activated CD4+ T cells recovered during the acute LCMV infection, the memory CD4+ T cells were maintained at a lower activation state as judged by cell size and ability to secrete IFN-γ. Thus, like the CD8+ T cell frequencies, the CD4+ T cell frequencies remain elevated after the acute infection subsides and stay elevated throughout long term immunity.
Galectin-3 Down-Regulates IL-5 Gene Expression on Different Cell Types
Authors: Cortegano Isabel , del Pozo Victoria , Cárdaba Blanca , de Andrés Belén , Gallardo Soledad , del Amo Ana , Arrieta Ignacio , Jurado Aurora , Palomino Pilar , Liu Fu-Tong , Lahoz Carlos
Published: January 2023

Galectin-3 is an animal lectin, formerly named ε-binding protein or Mac-2, which has been described to play an important role in some inflammatory processes by the implication of different cells and the increase in cell adhesion functions through laminin binding activity. In this work we analyzed the role of galectin-3 in the modulation of Th2 cytokines that have an important role in the development of the inflammatory response. We have found that the addition of galectin-3 to human eosinophils, the eosinophilic cell line EoL-3, PBMC, and an Ag-specific T cell line (CD4+) produced a selective inhibition of IL-5 transcription. No inhibitory effect was found on the IL-4 mRNA transcription rate. The inhibitory effect on IL-5 transcription was reversed by incubation with lactose and using specific Ab against galectin-3. Galectin-3 is able to induce inhibition of the IL-5 released in the supernatants from PBMC stimulated with phorbol 12,13-dibutyrate and anti-CD3. Similar results were obtained when a T-specific cell line was stimulated with Ag. Also, EoL-3 stimulated with anti-CD32 produced IL-5 protein, the synthesis of which was partially inhibited by galectin-3. The present results demonstrate that galectin-3 induces a selective down-regulation of IL-5 expression in different cell types, opening important new possibilities in the regulation of the allergic reactions.
Cooperation of Both TNF Receptors in Inducing Apoptosis: Involvement of the TNF Receptor-Associated Factor Binding Domain of the TNF Receptor 75
Authors: Declercq Wim , Denecker Geertrui , Fiers Walter , Vandenabeele Peter
Published: January 2023

TNF-R55 is the main receptor mediating TNF-induced cytotoxicity. However, in some cells TNF-R75 also signals cell death. In PC60 cells, the presence of both receptor types is required to induce apoptosis following either specific TNF-R55 or TNF-R75 triggering, pointing to a mechanism of receptor cooperation. In this study, we extend previous observations and show that TNF-R55 and TNF-R75 cooperation in the case of apoptosis in PC60 cells is bidirectional. We also demonstrate ligand-independent TNF-R55-mediated cooperation in TNF-R75-induced granulocyte/macrophage-CSF secretion, but not vice versa. To determine which part of the intracellular TNF-R75 sequence was responsible for the observed receptor cooperation in apoptosis, we introduced different TNF-R75 mutant constructs in PC60 cells already expressing TNF-R55. Our data indicate that an intact TNF-R-associated factors 1 and 2 (TRAF1/TRAF2)-binding domain is required for receptor cooperation. These findings suggest a role for the TRAF complex in TNF-R cooperation in the induction of cell death in PC60 cells. Nevertheless, introduction of a dominant negative (DN) TRAF2 molecule was not able to affect receptor cooperation. Remarkably, TRAF2-DN overexpression, which was found to inhibit the TNF-dependent recruitment of endogenous wild-type TRAF2 to the TNF-R75 signaling complex, could neither block TNF-R55- or TNF-R75-induced NF-κB activation nor granulocyte/macrophage-CSF secretion. Possibly, additional factors different from TRAF2 are involved in TNF-mediated NF-κB activation.
Enhancing Effect of IL-17 on IL-1-Induced IL-6 and Leukemia Inhibitory Factor Production by Rheumatoid Arthritis Synoviocytes and Its Regulation by Th2 Cytokines
Authors: Chabaud Martine , Fossiez François , Taupin Jean-Luc , Miossec Pierre
Published: January 2023

IL-17 is a cytokine produced by CD4 T cells that activates the production of inflammatory mediators by synoviocytes. To study the contribution of soluble factors in the interaction between T cells and synoviocytes in rheumatoid arthritis (RA), we looked at the effect of IL-17 on these cells in the presence of cytokines classified as pro (IL-1)- and anti-inflammatory (IL-4, IL-13, IL-10). Both human rIL-1β and rIL-17 induced IL-6 and leukemia inhibitory factor (LIF) production by synovial fibroblasts in a dose-dependent manner. After 7 days of culture, optimal concentrations of IL-1β increased IL-6 (33-fold) and LIF (10-fold) production by synoviocytes, while IL-17 showed a lesser effect on IL-6 (17-fold) and LIF (4-fold) production. Using low concentrations of IL-17 and IL-1β in combination, a synergistic effect was observed on the production of IL-6, whereas an additive effect was observed for LIF production. Production of biologically active IL-17 was demonstrated in RA synovium supernatants with the use of a blocking anti-IL-17 Ab. Both IL-4 and IL-13 had a modest stimulatory effect on IL-1- and IL-17-induced production of IL-6, but inhibited that of LIF. In contrast, IL-10 had a limited inhibitory effect on IL-6 production and no effect on that of LIF. These findings indicate that low levels of cytokines produced by monocytes (IL-1) and T cells (IL-17) can act together on synoviocytes. Thus, some RA synovium T cells producing IL-17 can activate mesenchymal cells leading to an increased proinflammatory pattern sensitive to Th2 cytokine regulation.
Repertoire Analysis of CD8+ T Cell Responses to Minor Histocompatibility Antigens Involved in Graft-Versus-Host Disease
Authors: Friedman Thea M. , Gilbert Michael , Briggs Constance , Korngold Robert
Published: January 2023

Graft-vs-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation. Experimentally, lethal GVHD can be induced in MHC-matched strain combinations differing in expression of multiple minor histocompatibility Ags (miHA). Recently, the GVHD potential of C57BL/6By (B6) T cells in irradiated BALB.B (both H2b) and related CXB recombinant inbred strains of mice has been studied to determine the scope of the response to miHA in vivo and how it compared with CTL responses to immunodominant miHA in vitro. The GVHD response in these strain combinations appeared to be limited to a few Ags, yet there was no correlation of these miHA with that of in vitro CTL responses. To further investigate the role of CD8+ T cells in GVHD, we analyzed positively selected miHA-specific donor CD8+ thoracic duct lymphocytes (TDL) collected from irradiated BALB.B and CXBE mice, 5 to 6 days after transplantation of B6 T cells. Flow cytometric analysis of B6→BALB.B TDL did not indicate expansion of any particular TCR Vβ family, whereas Vβ10 and Vβ14 families were significantly expanded in the B6->CXBE TDL. However, PCR-based complementarity-determining region 3 size spectratyping revealed overlapping involvement of donor Vβ1, 6, 8, 9, 10, and 14 families in both BALB.B and CXBE recipients and unique utilization of the Vβ4 family in BALB.B mice, suggesting oligoclonal T cell responses to a limited number of miHA. In addition, the injection of CD8+Vβ14+ B6 T cells into irradiated BALB.B and CXBE mice induced lethal GVHD, confirming the involvement of miHA-specific T cells within an individual Vβ family.