bluuebunny/crossref_metadata_2025 huggingface dataset:
The Epitopes Targeted by the Rheumatoid Arthritis-Associated Antifilaggrin Autoantibodies are Posttranslationally Generated on Various Sites of (Pro)Filaggrin by Deimination of Arginine Residues
Authors: Girbal-Neuhauser Elisabeth , Durieux Jean-Jacques , Arnaud Michel , Dalbon Pascal , Sebbag Mireille , Vincent Christian , Simon Michel , Senshu Tatsuo , Masson-Bessière Christine , Jolivet-Reynaud Colette , Jolivet Michel , Serre Guy
Published: December 2022

Antifilaggrin autoantibodies (AFA) are a population of IgG autoantibodies associated to rheumatoid arthritis (RA), which includes the so-called “antikeratin” Abs and antiperinuclear factor. AFA are the most specific serological markers of RA. We previously showed that they recognize human epidermal filaggrin and other profilaggrin-related proteins of various epithelial tissues. Here, we report further characterization of the protein Ags and epitopes targeted by AFA. All the Ags that exhibit numerous neutral/acidic isoelectric variants were immunochemically demonstrated to be deiminated proteins. In vitro deimination of a recombinant human filaggrin by a peptidylarginine deiminase generated AFA epitopes on the protein. Moreover, two of three filaggrin-derived synthetic peptides with a citrulline in the central position were specifically and widely recognized by AFA affinity-purified from a series of RA sera. These results indicate that citrulline residues are constitutive of the AFA epitopes, but only in the context of specific amino acid sequences of filaggrin. In competition experiments, the two peptides abolished the AFA reactivity of RA sera, showing that they present major AFA epitopes. These data should help in the identification of a putative deiminated AFA-inducing or cross-reactive articular autoantigen and provide new insights into the pathogenesis of RA. They could also open the way toward specific immunosuppressive and/or preventive therapy of RA.
In Vitro Intrathymic Differentiation Kinetics of Human Fetal Liver CD34+CD38− Progenitors Reveals a Phenotypically Defined Dendritic/T-NK Precursor Split
Authors: Plum Jean , De Smedt Magda , Verhasselt Bruno , Offner Fritz , Kerre Tessa , Vanhecke Dominique , Leclercq Georges , Vandekerckhove Bart
Published: December 2022

Human CD34+CD38− hematopoietic precursor cells from fetal liver are able to develop into T, NK, and dendritic cells in a hybrid human/mouse fetal thymic organ culture (FTOC). In this report, we pay particular attention to the early events in differentiation of these precursor cells. We show that the CD34+CD38− precursor cells, which are CD4−CD7−cyCD3−HLA-DR−/++ (cy, cytoplasmatic), differentiate into a CD4+ population that remained CD7−cyCD3−HLA-DR++ and a CD4− population that expressed CD7 and cyCD3. The CD4+CD7−cyCD3− cells differentiate into phenotypically and functionally mature dendritic cells, but do not differentiate into T or NK cells. The CD4−CD7+cyCD3+ population later differentiates into a CD4+CD7+cyCD3+HLA-DR− population, which has no potential to differentiate into dendritic cells but is able to differentiate into NK cells and γδ and αβ T lymphocytes. These findings support the notion that the T/NK split occurs downstream of the NK/dendritic split.
Class I MHC Molecule-Mediated Inhibition of Sindbis Virus Replication
Authors: Hahn Young S. , Guanzon Angelo , Rice Charles M. , Hahn Chang S.
Published: December 2022

The threshold for systemic viral infection relies on the amplification of virus at a primary infection site. We have identified that class I MHC molecules can trigger the inhibition of replication of Sindbis virus in a haplotype- and allele-specific manner. Class I MHC molecules of H-2d haplotypes exhibit a strong inhibitory effect whereas H-2k haplotypes show minimal inhibition of Sindbis viral replication. By a single gene transfection of H-2d class I MHC molecules, into cells that express class I MHC molecules of H-2k haplotype and are susceptible to viral replication, these cells became resistant to viral replication. The inhibition of viral replication by class I MHC molecules occurs neither during the stage of virus entry/endocytosis nor during virus maturation. Rather, viral-specific RNA replication, as well as viral gene expression, are inhibited in cells expressing inhibitory class I MHC molecules. This class I MHC molecule-mediated inhibition requires newly synthesized host gene products, implying the activation of an intracellular signaling mechanism that is triggered by specific class I MHC molecules.
Mouse IL-13 Enhances Antibody Production In Vivo and Acts Directly on B Cells In Vitro to Increase Survival and Hence Antibody Production
Authors: Lai Yew Hon , Mosmann Timothy R.
Published: December 2022

IL-13, a Th2 cytokine, exhibits similar functions to IL-4 in stimulating proliferation and class switching of human B cells. Although mouse B cells were reported to be unresponsive to IL-13, we now show that IL-13 directly stimulates mouse B cells, causing extended survival and higher Ab levels. Recombinant mouse IL-13 was administered via osmotic pump during immunization of BALB/c mice with chicken RBCs. IL-13 treatment enhanced not only the plasma levels of total IgG1, IgG2a, and IgG2b but also Ag-specific Ig levels. To examine whether IL-13 acted directly on mouse B cells, B220+ B cells were cultured with fixed, anti-CD3-activated Th2 clones. Production of IgM and IgG1 was enhanced moderately by IL-13 and strongly by IL-4. Anti-CD40-stimulated sIgD+ mouse B cells also responded to IL-13 by producing increased levels of IgM, and to a lesser extent IgG1, IgG2a, IgG2b, and IgG3. No evidence was found for IL-13-induced class switching. Mouse B cells were stimulated directly rather than indirectly via contaminating cells, as IL-13 increased the numbers of both total and Ab-secreting B cells in aliquots of 100 sIgD+ B cells (>99.5% pure) stimulated with anti-CD40 Ab. Stimulation of B cells by IL-13 was unaffected by the addition of anti-IL-4 to the cultures. In contrast to IL-4, IL-13 did not increase CD23 expression or B cell proliferation as measured by dilution of an intracellular fluorescence label. Collectively, these data indicate that IL-13 can enhance mouse B cell Ab production by increasing survival of the B cells.
T Lymphocyte Development in the Absence of CD3ε or CD3γδεζ
Authors: Wang Baoping , Wang Ninghai , Whitehurst Charles E. , She Jian , Chen Jianzhu , Terhorst Cox
Published: December 2022

CD3γ, δ, ε, and ζ proteins together with the pre-TCR α-chain (pTα) and a rearranged TCR β-chain assemble to form the pre-TCR that controls the double negative (DN) to double positive (DP) stages of thymopoiesis. The CD3 proteins are expressed before pTα and TCR β-chains in prothymocytes and are expressed intracellularly in precursor NK cells, suggesting that the CD3 complex may function independent of pTα and TCRβ. In this report, both the role of CD3ε exclusively, and the role of CD3 proteins collectively, in thymocyte and NK cell development were examined. In a mouse strain termed εΔP, a neomycin cassette inserted within the CD3ε promoter abolishes CD3ε and δ expression and also abolishes CD3γ expression in all but a small minority (≤1%) of prothymocytes. These prothymocytes became deficient in CD3ε alone upon reconstitution of CD3δ expression and were severely, but not completely, arrested at the DN stage, as small numbers of double positive thymocytes were detected. In de facto CD3γδεζnull mice generated by crossing the εΔP mice with CD3ζ−/− mice, thymopoiesis were arrested at the CD44−CD25+ DN stage as observed in RAG−/− mice, DJ and VDJ recombination at the TCRβ locus was functional, and normal numbers of NK cells were detected. Together, the findings demonstrate that during thymocyte development, the CD3 complex collectively is not essential until the critical CD44−CD25+ DN stage in which pre-TCR begins to function, whereas CD3ε is critical for the assembly of pre-TCR. Moreover, CD3 proteins are dispensable for NK cell development.
Osteopontin Is Associated with T Cells in Sarcoid Granulomas and Has T Cell Adhesive and Cytokine-Like Properties In Vitro
Authors: O’Regan Anthony W. , Chupp Geoffrey L. , Lowry John A. , Goetschkes Margo , Mulligan Niall , Berman Jeffrey S.
Published: December 2022

Sarcoidosis is a systemic disease characterized by the accumulation of activated T cells and widespread granuloma formation. In addition, individual genetic predisposition appears to be important in this disease. Osteopontin, a noncollagenous matrix protein produced by macrophages and T lymphocytes, is expressed in the granulomas of tuberculosis, and is associated with genetic susceptibility to intracellular infection. The function of osteopontin in these T cell-mediated responses is unknown. We sought to elucidate the role of osteopontin in granulomatous inflammation by characterizing its expression in different stages of sarcoidosis and its effector function on T cells in vitro. Lymphocyte-associated expression of osteopontin in sarcoidosis was demonstrated by immunohistochemistry, and its expression correlated with granuloma maturity. In addition, osteopontin induced T cell chemotaxis, supported T cell adhesion (an effect enhanced by thrombin cleavage of osteopontin), and costimulated T cell proliferation. These results suggest a novel mechanism by which osteopontin and thrombin modulate T cell recruitment and activation in granulomatous inflammation.
The Mast Cell as Site of Tissue-Type Plasminogen Activator Expression and Fibrinolysis
Authors: Sillaber Christian , Baghestanian Mehrdad , Bevec Dorian , Willheim Martin , Agis Hermine , Kapiotis Stylianos , Füreder Wolfgang , Bankl Hans C. , Kiener Hans P. , Speiser Wolfgang , Binder Bernd R. , Lechner Klaus , Valent Peter
Published: December 2022

Recent data suggest that mast cells (MC) and their products (heparin, proteases) are involved in the regulation of coagulation and fibrino(geno)lysis. The key enzyme of fibrinolysis, plasmin, derives from its inactive progenitor, plasminogen, through catalytic action of plasminogen activators (PAs). In most cell systems, however, PAs are neutralized by plasminogen activator inhibitors (PAIs). We report that human tissue MC as well as the MC line HMC-1 constitutively produce, express, and release tissue-type plasminogen activator (tPA) without producing inhibitory PAIs. As assessed by Northern blotting, highly enriched lung MC (>98% pure) as well as HMC-1 expressed tPA mRNA, but did not express mRNA for PAI-1, PAI-2, or PAI-3. The tPA protein was detectable in MC-conditioned medium by Western blotting and immunoassay, and the MC agonist stem cell factor (c-Kit ligand) was found to promote the release of tPA from MC. In addition, MC-conditioned medium induced fibrin-independent plasmin generation as well as clot lysis in vitro. These observations raise the possibility that MC play an important role in endogenous fibrinolysis.
IL-12 Is a Heparin-Binding Cytokine
Authors: Hasan Maemunah , Najjam Saloua , Gordon Myrtle Y. , Gibbs Roslyn V. , Rider Christopher C.
Published: December 2022

Using an ELISA approach, we demonstrate that recombinant human IL-12 (rhIL-12) binds strongly to an immobilized heparin-BSA complex. This binding is completely displaceable with soluble heparin, IC50∼ 0.1 μg/ml, corresponding to ∼ 10 nM. By interpolation with our previous findings, this indicates an affinity for heparin greater than that of antithrombin III and comparable with that of FGF-2, two high-affinity heparin-binding proteins. Recombinant murine IL-12 also binds strongly to heparin. The binding of rhIL-12 to heparin shows specificity because chondroitin sulfates A and C fail to compete, whereas chondroitin B inhibits weakly. A highly sulfated heparan sulfate is a strong competitor, whereas other heparan sulfates show weak or no activity. Small heparin fragments inhibit binding, although activity decreases with size. An octasaccharide pool derived by cleavage of heparin with nitrous acid is a significantly stronger inhibitor than its heparinase I-derived counterpart, further indicating structural specificity in the interaction between rhIL-12 and heparin. The binding of recombinant p40 to heparin appears indistinguishable from that of the IL-12 heterodimer, implying that the heparin binding site is largely if not solely located in this subunit. These results show for the first time that IL-12 is a heparin-binding cytokine, a property common to the other Th1-response-inducing cytokines, IFN-γ and IL-2. Our findings strongly suggest that IL-12 will tend to be retained close to its sites of secretion in the tissues by binding to heparin-like glycosaminoglycans, thus favoring a paracrine role for IL-12.
High Affinity Very Late Antigen-4 Subsets Expressed on T Cells Are Mandatory for Spontaneous Adhesion Strengthening But Not for Rolling on VCAM-1 in Shear Flow
Authors: Chen Chun , Mobley James L. , Dwir Oren , Shimron Frida , Grabovsky Valentin , Lobb Roy R. , Shimizu Yoji , Alon Ronen
Published: December 2022

The very late Ag-4 (VLA-4) integrin supports both rolling and firm adhesion of leukocytes on VCAM-1 under shear flow. The molecular basis for the unique ability of a single adhesion molecule to mediate these versatile adhesive processes was investigated. VLA-4 occurs in multiple activation states, with different affinities to ligand. In this study we tested how these states regulate VLA-4 adhesiveness under shear flow in Jurkat T cells and PBL. VLA-4 on nonstimulated Jurkat cells supported rolling and spontaneous arrest on VCAM-1, whereas a Jurkat activation mutant with reduced VLA-4 affinity failed to spontaneously arrest after tethering to or during rolling on VCAM-1. The contribution of VLA-4 affinity for ligand to rolling and spontaneous arrests on immobilized VCAM-1 was dissected using soluble VLA-4 ligands, which selectively block high affinity states. VLA-4 saturation with ligand completely blocked spontaneous adhesion strengthening post-tethering to VCAM-1, but did not impair rolling on the endothelial ligand. High affinity VLA-4 was found to comprise a small subset of VLA-4 on resting Jurkat cells and PBL. This subset is essential for firm adhesion but not for tethering or rolling adhesions on VCAM-1. Interestingly, low and high affinity VLA-4 states were found to mediate similar initial tethering to ligand. High affinity VLA-4, constitutively expressed on circulating T cells, may control their early adhesion strengthening on VCAM-1-expressing endothelium before exposure to vascular chemokines and activation of additional integrins.
TGF-β Increases Leukotriene C4 Synthase Expression in the Monocyte-Like Cell Line, THP-1
Authors: Riddick Carl A. , Serio Kenneth J. , Hodulik Craig R. , Ring William L. , Regan Mark S. , Bigby Timothy D.
Published: December 2022

The goal of this study was to determine whether cytokines modulate leukotriene C4 (LTC4) synthase expression in mononuclear phagocytes. A panel of cytokines was surveyed for changes in LTC4 synthase mRNA in THP-1 cells. TGF-β1, -2, and -3 had significant stimulatory effects. The addition of TGF-β resulted in a time-dependent increase in LTC4 synthase mRNA at 6 h, which persisted through 48 h. Furthermore, this conditioning resulted in an increase in immunoreactive protein for LTC4 synthase through 7 days. TGF-β conditioning of cells resulted in a time- and dose-dependent increase in stimulated LTC4 synthase activity. Following transient transfection of THP-1 cells with a promoter-reporter construct containing 1.2 kb of the LTC4 synthase promoter, TGF-β treatment resulted in a 2-fold increase in reporter activity. Conditioning with TGF-β did not prolong the half-life of LTC4 synthase mRNA, as assessed by RNase protection assays in actinomycin D-treated cells. Cycloheximide exposure experiments revealed that new protein synthesis was not required for the observed stimulatory effect of TGF-β on LTC4 synthase mRNA. We conclude that LTC4 synthase expression is increased at a transcriptional level by TGF-β in mononuclear phagocytes.
One Active C1r Subunit Is Sufficient for the Activity of the Complement C1 Complex: Stabilization of C1r in the Zymogen Form by Point Mutations
Authors: Dobó József , Gál Péter , Szilágyi Katalin , Cseh Sándor , Lörincz Zsolt , Schumaker Verne N. , Závodszky Péter
Published: December 2022

The binding of C1 (the first component of complement) to immune complexes leads to the autoactivation of C1r through the cleavage of the Arg463-Ile464 bond in the catalytic domain. Spontaneous activation of C1r (and C1) also occurs in the fluid phase, preventing the characterization of the zymogen form of C1r. To overcome this difficulty, the zymogen form of human C1r was stabilized by mutating the Arg in the Arg463-Ile464 bond to Gln. This mutant was designated as mutant QI. Recombinant C1r (wild type (wt) or mutant) was expressed in insect cells using serum-free medium in functionally pure form; therefore, the cell culture supernatant was suitable to reconstruct C1 for the hemolytic assay. Mutant QI was a stable, nonactivable zymogen and showed no hemolytic activity in reconstituted C1. However, this stable zymogen C1r mutant could form an active mixed dimer with the wt C1r, indicating that one active C1r subunit in the C1 complex is sufficient for the full activity of the entire complex. Our experiments also showed that the exchange of C1r monomers between the C1r dimers is completed in less than 16 h even at pH 7 and 4°C. Two other mutants were also constructed by changing Arg463 to Lys, or Ile464 to Phe, and were designated as mutants KI and RF, respectively. Although these substitutions did increase the stability of the proenzyme in the cell culture supernatant, the mutant proteins retained their ability to autoactivate, and both had a wt-like hemolytic activity.
Varying Roles of E-Selectin and P-Selectin in Different Microvascular Beds in Response to Antigen
Authors: Hickey Michael J. , Kanwar Samina , McCafferty Donna-Marie , Granger D. Neil , Eppihimer Michael J. , Kubes Paul
Published: December 2022

Expression of E-selectin and P-selectin is critical in the effector phase of leukocyte recruitment in response to Ag. Whether their relative roles differ between tissues in response to the same Ag is unknown. In this study, a type I hypersensitivity response was elicited in C57BL/6 mice by systemic sensitization with OVA. Following local Ag challenge, endothelial selectin expression was examined in the skin and cremaster muscle microvasculature using a dual-radiolabeled mAb technique. Next, the dermal and muscle microcirculations were visualized using intravital microscopy to establish roles for P-selectin and/or E-selectin. In untreated mice, leukocyte recruitment in both skin and skeletal muscle was mediated entirely by P-selectin. Following Ag challenge, leukocyte rolling flux and adhesion were dramatically increased and leukocyte rolling velocity was unchanged in muscle. Only P-selectin expression increased in muscle, and leukocyte recruitment was entirely dependent upon this selectin. In contrast, in Ag-challenged skin, leukocyte rolling flux did not increase, but rolling velocity dropped profoundly. In skin, only E-selectin expression increased, and blockade of either E-selectin or P-selectin had minimal effect on either rolling flux or rolling velocity. Blockade of both selectins reduced rolling flux by 80% and increased rolling velocity sevenfold. These data highlight striking differences in expression of the endothelial selectins in separate microvascular beds in response to the same stimulus and demonstrate that these differences underlie very different patterns of leukocyte recruitment. The data underscore the importance of studying individual microvascular beds to understand tissue-specific leukocyte recruitment in vivo.
Antigen-Dependent and -Independent IFN-γ Modulation by Penicillins
Authors: Padovan Elisabetta , Greyerz Salome von , Pichler Werner J. , Weltzien Hans Ulrich
Published: December 2022

The activation of CD4+ T lymphocytes upon Ag stimulation plays a critical role in adverse immune responses including drug-specific hypersensitivity reactions. We examined the modulation of T cell phenotype induced by hapten-specific stimulation using the model of β-lactam antibiotics such as penicillin G (Pen G), Pen V, and ampicillin (Amp). When PBMC of donors suffering from hypersensitivity reactions against β-lactams were stimulated in vitro with different doses of Pen G, a preferential expansion of IL-4-producing TCRαβ+ cells was detected. A panel of T cell clones was then prepared from Pen G-specific lines after two cycles of restimulation with the hapten. For the majority of these clones, we found that high doses of Pen G induced optimal IL-4 secretion, whereas the amount of IFN-γ secreted was inversely correlated with the dose of Pen G, thus leading to a hapten-inducible shift of the fuctional phenotypes for some of the clones. Finally, Pen V and Amp were used to modulate different Ag-induced immune responses. We found that Amp had no influence on the cytokine pattern induced by specific Ag or mitogens. In contrast, Pen V inhibited the secretion of IFN-γ, but not IL-4, most likely by Ag-independent mechanisms. This last finding may open new applications for immune intervention in those diseases in which polarized Th1 responses are involved in the development of the pathology.
Increased Activation of Protein Kinase A Type I Contributes to the T Cell Deficiency in Common Variable Immunodeficiency
Authors: Aukrust Pål , Aandahl Einar Martin , Skålhegg Bjørn S. , Nordøy Ingvild , Hansson Vidar , Taskén Kjetil , Frøland Stig S. , Müller Fredrik
Published: December 2022

The molecular mechanisms underlying the T cell dysfunction often present in common variable immunodeficiency (CVI) are not established. cAMP-dependent protein kinase A type I (PKAI) is an important inhibitor of T cell proliferation after Ag stimulation. We therefore investigated the possibility that activation of PKAI may be involved in the development of T cell dysfunction in CVI. An exogenously added PKAI-selective antagonist (Rp-8-Br-cAMPS) induced a significant increase in anti-CD3-stimulated PBMC proliferation in 20 CVI patients compared with no effect in 15 controls. Purified T cells from 7 CVI patients with strictly defined T cell deficiency had elevated endogenous cAMP levels compared with controls. Treatment of T cells from these CVI patients with Rp-8-bromo-cAMP-phosphorothioate markedly improved anti-CD3-stimulated proliferation (up to 3.7-fold), particularly in CD4+ lymphocytes, reaching proliferation levels comparable to control values. No effect of cAMP antagonist on T cell proliferation was seen in controls. In these CVI patients, cAMP antagonist also increased IL-2 production in anti-CD3-stimulated T cells. However, exogenously added IL-2 at concentrations comparable to the achieved increase in IL-2 levels after addition of cAMP antagonist had no effect on T cell proliferation. Furthermore, the stimulatory effects of exogenously added IL-2 at higher concentrations and cAMP antagonist on T cell proliferation were additive. Our findings indicate that increased PKAI activation may be an important molecular basis for the T cell defect in CVI and suggest that the cAMP/PKAI system may be a potential molecular target for immunomodulating therapy in these patients.
Specific Vβ T Cell Subsets Are Associated with Cat and Birch Pollen Allergy in Humans
Authors: Beyer Kirsten , Häusler Tom , Kircher Moritz , Nickel Renate , Wahn Ulrich , Renz Harald
Published: December 2022

Cognate interaction between TCRs and MHC class II molecules plays an important role in initiating the allergen-specific immune response. Therefore, we analyzed the TCR distribution of human PBLs of 56 atopic and nonatopic (NA) individuals, including 4 monozygotic twin pairs, from two extended and four nuclear families. The expression of 23 Vβ and 3 Vα elements was analyzed. The blood samples of symptomatic birch pollen-sensitized individuals that were taken ≤6 wk after the birch pollen season (n = 8) showed a significantly higher frequency of Vβ16.1+ and Vβ20.1+ T cells compared with the blood samples of birch pollen-sensitized individuals that were obtained out of allergen season (n = 10) or from NA individuals (p < 0.0005 and p < 0.0001, respectively). Allergen-specific lymphocyte proliferation was detected in the allergic individuals, and the distribution of Vβ16.1+ and Vβ20.1+ T cells returned to normal levels after the pollen season. The frequency of these Vβ-expressing T cells correlated with the levels of allergen-specific IgE Abs. In addition, cat-sensitized individuals (n = 8) showed a significantly higher frequency of Vβ17.1-expressing T cells than did NA individuals (p < 0.005). Our results indicate restricted TCR-Vβ gene usage in cat and birch pollen allergies; we suggest that both genetic and environmental factors contribute to TCR-Vβ gene expression and to the development of a specific T cell response.
Vasoactive Intestinal Peptide (VIP) and Pituitary Adenylate Cyclase-Activation Polypeptide (PACAP) Protect Mice from Lethal Endotoxemia Through the Inhibition of TNF-α and IL-6
Authors: Delgado Mario , Martinez Carmen , Pozo David , Calvo Juan R. , Leceta Javier , Ganea Doina , Gomariz Rosa P.
Published: December 2022

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) down-regulate cytokine production. Because human septic shock involves excessive cytokine production, the effect of VIP/PACAP was investigated in a high endotoxemia murine model. Both peptides protect against endotoxin-induced lethality and prevent septic shock-associated histopathological alterations. VIP/PACAP reduce serum and peritoneal TNF-α and IL-6, suggesting that the protective effect is exerted by inhibiting the production of endogenous TNF-α/IL-6. Consistent with this mechanism, VIP does not protect against septic shock induced by exogenous TNF-α. The immunomodulatory role of VIP in vivo is supported by the appearance of high levels of VIP in serum and peritoneal fluid following LPS administration. Thus, the neuropeptides VIP/PACAP protect from the lethal effect of high endotoxemia, presumably by down-regulating TNF-α and IL-6 production, and may offer an alternative in the treatment of human septic shock syndrome.
Cutting Edge: Induction of Enhanced CTL-Dependent Protective Immunity In Vivo by N-End Rule Targeting of a Model Tumor Antigen
Authors: Tobery Timothy , Siliciano Robert F.
Published: December 2022

There is much interest in vaccines that will enhance the induction of CTL. One mechanism to enhance Ag-specific CTL responses involves targeting Ag to undergo rapid cytoplasmic degradation by the N-end rule pathway. We have analyzed the ability of N-end rule targeting to confer protection in an immunization-challenge setting. Using the HIV-1 nef protein as a model tumor Ag, we found that in mice immunized with a vaccinia vector expressing a form of nef that is targeted for rapid cytoplasmic degradation, there was enhanced induction of nef-specific CTL and protection from a lethal challenge with the syngeneic CT26 tumor cells that had been transfected with nef. Protection from tumor challenge correlated with the magnitude of the CTL response. Thus, the targeting of tumor or viral Ags for rapid cytoplasmic degradation by the N-end rule pathway may represent a strategy for the induction of protective Ag-specific CTL responses in vivo.
The Role of the Antigen-Presenting Cell in Fas-Mediated Direct and Bystander Killing: Potential In Vivo Function of Fas in Experimental Allergic Encephalomyelitis
Authors: Thilenius Anja R. B. , Sabelko-Downes Kimberly A. , Russell John H.
Published: December 2022

Costimulatory molecules are critical in mediating Fas-dependent direct and bystander lysis. In direct lysis, the APC is the Fas-positive target. It presents Ag to the T cell, thereby activating the T cell. The activated T cell then up-regulates FasL, allowing it to kill the APC. In bystander lysis, the APC again induces FasL expression on the T cell, but the target is a third Fas-positive cell that may lack the appropriate MHC-restricting element to activate the T cell. This study shows that ICAM-1 and B7-1 can serve as important adhesion molecules in direct killing using CD4+ T cell effectors. In bystander killing, B7-1 appears to act as a signaling molecule as well. It has been demonstrated that lpr and gld mice are less susceptible to experimental allergic encephalomyelitis than their wild-type counterparts. In this study, we show that although microglia are poor targets of direct killing, they are capable of stimulating myelin basic protein-specific T cells to kill innocent Fas-positive targets. This presents a possible mechanism for the pathogenesis of experimental allergic encephalomyelitis.
Hepatitis C Virus Envelope Glycoprotein E1 Originates in the Endoplasmic Reticulum and Requires Cytoplasmic Processing for Presentation by Class I MHC Molecules
Authors: Selby Mark , Erickson Ann , Dong Christine , Cooper Stewart , Parham Peter , Houghton Michael , Walker Christopher M.
Published: December 2022

We investigated whether hepatitis C virus envelope glycoprotein E1 is transported from the endoplasmic reticulum (ER) to the cytoplasm of infected cells for class I MHC processing. Target cells expressing E1 were killed by CTL lines from a hepatitis C virus-infected chimpanzee, and synthetic peptides were used to define an epitope (amino acids 233-GNASRCWVA-241) presented by the Patr-B*1601 class I MHC molecule. An unusually high concentration (>100 nM) of this nonameric peptide was required for target cell lysis, but this could be reduced at least 1000-fold by replacing the asparagine at amino acid position 234 (Asn234) with aspartic acid (Asp), the anticipated anchor residue for NH2-terminal peptide binding to Patr-B*1601. Conspicuously, position 234 is part of an N-glycosylation motif (Asn-Xaa-Ser/Thr), suggesting that the Asn234 to Asp substitution might occur naturally within the cell due to deglycosylation/deamidation of this amino acid by the cytosolic enzyme peptide N-glycanase. In support of this model, we demonstrate that presentation of the epitope depended on 1) cotranslational synthesis of E1 in the ER, 2) glycosylation of the E1 molecule, and 3) a functional TAP transporter to shuttle peptide from the cytosolic to ER compartment. These results indicate for the first time that during infection of the host, viral envelope glycoproteins originating in the ER are processed in the cytoplasm for class I MHC presentation. That a posttranslational change in amino acid sequence from Asn to Asp alters the repertoire of peptides presented to CD8+ CTL has implications for the design of antiviral vaccines.
The Role of LY49 NK Cell Subsets in the Regulation of Murine Cytomegalovirus Infections
Authors: Tay Chin H. , Yu Lawrence Y. Y. , Kumar Vinay , Mason Llewelyn , Ortaldo John R. , Welsh Raymond M.
Published: December 2022

The distributions and functions of NK cell subsets, as defined by the expression of Ly49 NK cell receptors, were examined in murine CMV (MCMV)-infected mice. MCMV induced a reduction in NK1.1+ cell number in the spleen and an increase in the peritoneal exudate cells. Within the splenic NK1.1+ population, proportional increases in Ly49A+ and Ly49G2+ cells but decreases in Ly49C+ and Ly49D+ cells were observed 3 days post-MCMV infection, but within the peritoneal NK1.1+ cell populations there were proportional decreases in Ly49A+ cells and increases in Ly49C+, Ly49D+, and Ly49G2+ cells. Lymphocytic choriomeningitis virus did not elicit a comparable NK cell subset distribution. Lymphokine-activated killer cells were sorted into different Ly49 NK cell subsets and adoptively transferred into C57BL/6 suckling mice. Regulation of MCMV synthesis in these suckling mice was shown to be an IFN-γ-dependent, perforin- and Cmv-1-independent process, and each NK cell subset mediated anti-viral activity. In adult C57BL/6 mice, the control of MCMV in the spleen is mediated by a perforin-dependent mechanism, regulated in part by the Cmv-1 gene, which maps closely to the Ly49 family. In vivo depletions of either one or two of the Ly49 subsets in adult mice did not affect the ability of the residual NK cells to regulate MCMV synthesis. These data provide evidence of NK cell subset distribution and function in MCMV infection, but no individual subset was required for the Cmv-1-like regulation of MCMV synthesis.
Acquired Thymic Tolerance: Role of CTLA4 in the Initiation and Maintenance of Tolerance in a Clinically Relevant Autoimmune Disease Model
Authors: Issazadeh Shohreh , Zhang ManHua , Sayegh Mohamed H. , Khoury Samia J.
Published: December 2022

Injection of Ag into the thymus of adult animals induces specific systemic tolerance. The mechanisms of acquired thymic tolerance include anergy and the deletion of Ag-specific T cells. Here, we report that anergy to nominal Ag induced via acquired thymic tolerance requires CTL-associated Ag 4 (CTLA4) engagement. The role of CTLA4 in the induction and maintenance of tolerance was then investigated in the murine experimental autoimmune encephalomyelitis model. CTLA4 blockade abrogated the induction but not the maintenance phase of acquired thymic tolerance induced by intrathymic injection of myelin Ags. In addition, CTLA4 blockade had a restricted window of action after priming with Ag, which is consistent with the expression patterns of CTLA4 in vivo. We conclude that: 1) the induction of acquired thymic tolerance requires signaling through CTLA4 and 2) CTLA4 does not appear to be required for the maintenance of acquired thymic tolerance. This is the first report documenting the role of a CTLA4 negative signaling pathway in the induction of tolerance in an autoimmune disease model.
Functional Characteristics and Survival Requirements of Memory CD4+ T Lymphocytes In Vivo
Authors: London Cheryl A. , Perez Victor L. , Abbas Abul K.
Published: December 2022

The phenotypic and functional characteristics of Ag-specific memory CD4+ lymphocytes are poorly defined. To examine the properties and cytokine responsiveness of these cells, we have developed an adoptive transfer system using in vitro-activated T cells expressing the DO.11 transgenic TCR specific for OVA323–339+ I-Ad. In vitro-activated DO.11 CD4+ cells exhibit comparable survival patterns at 1, 6, and 10 wk after adoptive transfer, indicating that a stable population of memory cells has been generated. In the absence of Ag, previously activated T cells survive longer than their naive counterparts in vivo, rapidly revert to a partially naive phenotype, and maintain their effector cytokine profile. The DO.11 CD4+ memory cells are capable of proliferating in response to IL-2 and IL-4, while naive DO.11 CD4+ cells exhibit no such proliferative responses.
Role of Bcl-2 in αβ T Cell Development in Mice Deficient in the Common Cytokine Receptor γ-Chain: The Requirement for Bcl-2 Differs Depending on the TCR/MHC Affinity
Authors: Nakajima Hiroshi , Leonard Warren J.
Published: December 2022

Mice lacking the common cytokine receptor γ-chain (γc) exhibit severely compromised T cell development, with diminished Bcl-2 expression in mature (CD4+ or CD8+) thymocytes and peripheral T cells. Enforced expression of Bcl-2 in these mice partially rescued αβ T cell development but not γδ T cell development. Transgenic expression of the OVA-specific DO11.10 (DO10) TCR also could modestly increase thymocyte numbers, and T cells expressing the transgenic TCR (KJ1-26+ T cells) were found in the periphery. Interestingly, the presence of KJ1-26+ T cells was dependent on the MHC background and was seen in the moderate affinity H-2d/d background but not in the higher affinity H-2d/b background in γc-deficient mice. In contrast, KJ1-26+ T cells exist in the periphery in both the H-2d/d and H-2d/b backgrounds in DO10 transgenic γc wild-type mice. These results suggest that the importance of γc-dependent signals for T cell development differs depending on the affinity of TCR for MHC. Moreover, enforced expression of Bcl-2 had a much greater effect on the development of γc-deficient T cells expressing the DO10 TCR in the high affinity H-2d/b background than in the H-2d/d background, suggesting that γc-dependent Bcl-2 expression influences T cell development in a TCR/MHC-dependent manner.
The Mechanism of Chromosome 7 Inversion in Human Lymphocytes Expressing Chimeric γβ TCR
Authors: Retière Christelle , Halary Franck , Peyrat Marie-Alix , Le Deist Françoise , Bonneville Marc , Hallet Marie-Martine
Published: December 2022

Functional chimeric TCR chains, encoded by VγJγCβ or VγJβCβ hybrid gene TCR, are expressed at the surface of a small fraction of αβ T lymphocytes in healthy individuals. Their frequency is dramatically increased in patients with ataxia-telangiectasia, a syndrome associated with inherited genomic instability. As the TCR γ and β loci are in an inverted orientation on chromosome 7, the generation of such hybrid genes requires at least an inversion event. Until now, neither the sequences involved in this genetic mechanism nor the number of recombinations leading to the formation of functional transcriptional units have been characterized. In this manuscript, we demonstrate that at least two rearrangements, involving classical recombination signal sequence and the V(D)J recombinase complex, lead to the formation of productive hybrid genes. A primary inversion 7 event between Dβ and Jγ genic segments generates CγVβ and CβVγ hybrid loci. Within the CγVβ locus, secondary rearrangements between Vγ and Jγ or Vγ and Jβ elements generate functional genes. Besides, our results suggest that secondary rearrangements were blocked in the CβVγ locus of normal but not ataxia-telangiectasia T lymphocytes. We also provide formal evidence that the same Dβ-3′ recombination signal sequence can be used in successive rearrangements with Jγ and Jβ genic segments, thus showing that a signal joint has been involved in a secondary recombination event.
A Role forRAD51in the Generation of Immunoglobulin Gene Diversity in Rabbits
Authors: Barrington Robert A. , Fasullo Michael , Knight Katherine L.
Published: December 2022

Ig VDJ genes in rabbit somatically diversify by both hyperpointmutation and gene conversion. To elucidate the mechanism of gene conversion of IgH genes, we cloned a rabbit homologue of RAD51, a gene involved in gene conversion in Saccharomyces cerevisiae (yeast), and tested whether it could complement a yeast rad51 mutant deficient in recombination repair. We found that rabbit RAD51 partially complemented the defect in switching mating types by gene conversion as well as in DNA double-strand break repair after γ-irradiation. Further, by Western blot analysis, we found that levels of Rad51 were higher in appendix-derived B lymphocytes of 6-wk-old rabbits, a time at which IgH genes diversify by somatic gene conversion. We suggest that Rad51 is involved in somatic gene conversion of rabbit Ig genes.
Identification of Quantitative Trait Loci Governing Arthritis Severity and Humoral Responses in the Murine Model of Lyme Disease
Authors: Weis Janis J. , McCracken Barbara A. , Ma Ying , Fairbairn Daryl , Roper Randall J. , Morrison Tom B. , Weis John H. , Zachary James F. , Doerge R. W. , Teuscher Cory
Published: December 2022

A spectrum of disease severity has been observed in patients with Lyme disease, with ∼60% of untreated individuals developing arthritis. The murine model of Lyme disease has provided strong evidence that the genetic composition of the host influences the severity of arthritis following infection with Borrelia burgdorferi: infected C3H mice develop severe arthritis while infected C57BL/6N mice develop mild arthritis. Regions of the mouse genome controlling arthritis severity and humoral responses during B. burgdorferi infection were identified in the F2 intercross generation of C3H/HeNCr and C57BL/6NCr mice. Rear ankle swelling measurements identified quantitative trait loci (QTL) on chromosomes 4 and 5, while histopathological scoring identified QTL on a unique region of chromosome 5 and on chromosome 11. The identification of QTL unique for ankle swelling or histopathological severity suggests that processes under distinct genetic control are responsible for these two manifestations of Lyme arthritis. Additional QTL that control the levels of circulating Igs induced by B. burgdorferi infection were identified on chromosomes 6, 9, 11, 12, and 17. Interestingly, the magnitude of the humoral response was not correlated with the severity of arthritis in infected F2 mice. This work defines several genetic loci that regulate either the severity of arthritis or the magnitude of humoral responses to B. burgdorferi infection in mice, with implications toward understanding the host-pathogen interactions involved in disease development.
Anti-TGF-β Treatment Promotes Rapid Healing ofLeishmania majorInfection in Mice by Enhancing In Vivo Nitric Oxide Production
Authors: Li Jian , Hunter Christopher A. , Farrell Jay P.
Published: December 2022

CB6F1 mice display intermediate susceptibility to Leishmania major infection compared with the highly susceptible BALB/c and resistant C57BL/6 parental strains. During early weeks of infection, these mice develop dominant Th2 type responses to L. major, although they eventually exhibit a Th2 to Th1 switch and spontaneously resolve their infections. In this study, we have examined the effects of either IL-12 or anti-TGF-β therapy on the immune response and course of disease in chronically infected CB6F1 mice. Local treatment with IL-12 inoculated into the parasitized lesion at 4 wk of infection induced a marked increase in IFN-γ production but did not result in a significant reduction in numbers of parasite or promote more rapid healing. However, local treatment with an Ab to TGF-β led to both a decrease in parasite numbers and more rapid healing, despite the fact that such treatment did not significantly alter the pattern of IL-4 and IFN-γ production. Immunohistochemical studies showed that anti-TGF-β treatment resulted in increased nitric oxide production within parasitized lesions. Our results suggest that TGF-β may play an important regulatory role during chronic stages of a L. major infection by suppressing macrophage production of nitric oxide and that, in the absence of TGF-β, even the relatively low levels of IFN-γ observed in mice with dominant Th2-type responses are sufficient to activate macrophages to destroy amastigotes within parasitized lesions.
Cutting Edge: MHC Class I Triggering by a Novel Cell Surface Ligand Costimulates Proliferation of Activated Human T Cells
Authors: Agrawal Samir , Marquet Jeanine , Freeman Gordon J. , Tawab Abdul , Bouteiller Philippe Le , Roth Patricia , Bolton Wade , Ogg Graham , Boumsell Laurence , Bensussan Armand
Published: December 2022

BY55 is a human cell surface molecule whose expression is restricted to NK cells, a subset of circulating CD8+ T lymphocytes, and all intestinal intraepithelial T lymphocytes. Here, we report that BY55 is a novel NK receptor showing broad specificity for both classical and nonclassical MHC class I molecules, and that optimal binding requires a prior aggregation of MHC class I complexes. Using BY55 transfectants, we have identified functional consequences of MHC class I/ligand interactions for the class I-bearing cell. The triggering of MHC class I molecules on human T cell clones by BY55 delivered a potent proliferative signal in the presence of soluble CD3 mAb. The costimulatory signal provided by MHC class I ligation was only seen in activated, and not resting, peripheral blood T cells. This observation represents an additional and/or alternative pathway to CD28 costimulation and may be of particular relevance in memory T cells lacking CD28, such as intestinal intraepithelial T lymphocytes, which are CD28− but BY55+.
Antigen-Induced Coreceptor Down-Regulation on Thymocytes Is Not a Result of Apoptosis
Authors: McGargill Maureen A. , Hogquist Kristin A.
Published: December 2022

The various stages of T cell development are typically characterized by the expression level of the two coreceptors, CD4 and CD8. During the CD4+CD8+ (double-positive, DP) stage of development, thymocytes that perceive a low avidity signal through the TCR go on to differentiate (positive selection), and ultimately down-regulate one coreceptor to express either CD4 or CD8. Alternatively, thymocytes that perceive a high avidity signal down-regulate both coreceptors and are induced to die via apoptosis (negative selection). However, it has recently been suggested that positively selected thymocytes may also partially down-regulate both coreceptors before up-regulating the one coreceptor that is ultimately expressed. This would imply that coreceptor down-regulation (dulling) is not a consequence of commitment to the death pathway. To explore this possibility, we have utilized an in vitro assay to demonstrate that dulling occurred in response to both positive and negative selecting ligands in vitro, was not a result of nonspecific membrane perturbation, was not dependent on the type of APC, and occurred before death in vitro. Furthermore, when thymocyte apoptosis was blocked, CD4 and CD8 were down-regulated in response to TCR stimulation. These data suggest that dulling in response to TCR ligation is distinct from death, and support a model in which DP dulling occurs during both positive and negative selection. The biological implications of this phenomenon are discussed.
IL-17 Is Produced by Some Proinflammatory Th1/Th0 Cells But Not by Th2 Cells
Authors: Aarvak Tanja , Chabaud Martine , Miossec Pierre , Natvig Jacob B.
Published: December 2022

IL-17 is defined as a proinflammatory cytokine and produced by activated CD4+ T cells. In rheumatoid arthritis synovial tissue, high levels of IL-17 contribute to IL-6 production by synoviocytes. The present study was performed to see whether Th cells that produce IL-17 are associated with the Th1, Th2, or Th0 subset. Thirty-three CD4+, αβ+ T cell clones were developed from synovial membranes and synovial fluid of rheumatoid arthritis patients. Thirteen clones were defined as Th1 since they produced IFN-γ but not IL-4, and four clones were defined as Th0 type that produced both IL-4 and IFN-γ. Sixteen clones were defined as Th2 since they produced high levels of IL-4 and/or IL-10 but not IFN-γ. IL-17 was measured in a bioassay, where IL-6 production from synoviocytes was a measurement for IL-17 activity in the presence and absence of blocking anti-IL-17 mAb. Three Th1 clones and two Th0 clones produced IL-17. In contrast, none of the sixteen Th2 clones analyzed produced IL-17. In addition, six Th2 clones were further cultured in conditions that induced a switch to Th1 type. Induction of this Th1 phenotype also led to production of IL-17 in two of these clones. The results demonstrate that some cells of the Th1/Th0 phenotype produce IL-17 but not cells of the Th2 phenotype. Thus, IL-17 may define a new subset of T cells, and IL-17 production appears to be a mechanism for Th1/Th0 cells, the most frequent Th subtype present in the rheumatoid synovium, to contribute to the local inflammatory reactions.