bluuebunny/crossref_metadata_2025 huggingface dataset:
Disturbed MHC regulation in the IFN-gamma knockout mouse. Evidence for three states of MHC expression with distinct roles for IFN-gamma.
Authors: Goes N , Sims T , Urmson J , Vincent D , Ramassar V , Halloran P F
Published: December 2022

We compared the expression of MHC class I and II products in tissues of IFN-gamma knockout (GKO) vs normal (wild-type) BALB/c mice. We studied expression in the basal state, after local tissue injury, after stimuli that induce systemic MHC expression (allogeneic cells, oxazolone skin painting, or LPS), and after rIFN-gamma. Basal class II expression in interstitial cells was not reduced in GKO mice. However, GKO mice had less basal class I expression in kidney, liver, heart, and arterial endothelium than wild-type mice. Local renal ischemic injury increased class I and II expression in kidney tubules of both GKO and wild-type mice, but induction in GKO was less than in wild-type. Potent inflammatory stimuli increased systemic MHC class I and II markedly in kidney, liver, and heart of wild-type mice, but induced no increase in GKO mice. rIFN-gamma induced class I and II equally in GKO and wild-type mice. Thus, three states of MHC expression can be defined that differ in their dependencies on IFN-gamma: basal, locally induced, and systemically induced. Basal class II expression in interstitial cells is IFN-gamma independent, but basal class I expression, particularly in arterial endothelium, is partially dependent on IFN-gamma. The local increase in MHC class I and II in parenchymal cells in response to injury reflects both IFN-gamma and a non-IFN-gamma factor. Systemic MHC class I and II induction is almost exclusively due to IFN-gamma.
Regulation of IkB alpha phosphorylation by PKC- and Ca(2+)-dependent signal transduction pathways.
Authors: Steffan N M , Bren G D , Frantz B , Tocci M J , O'Neill E A , Paya C V
Published: December 2022

The Ca(2+)-dependent phosphatase calcineurin, a target of FK506 and CsA, synergizes with PKC-induced activation of nuclear factor (NF)-kappa B in T cell lines. We have investigated whether this synergy is present in other cell types and the mechanism(s) by which these two pathways lead to NF-kappa B activation. While this synergy is present in other cell types, in the monocytic cell line U937 calcineurin is also sufficient to activate NF-kappa B. Having previously shown that Ca(2+)- and PKC-dependent pathways synergize by accelerating the degradation of IkB alpha, we focused on the regulation of IkB alpha phosphorylation. While PKC-dependent pathways sequentially result in the phosphorylation and in an incomplete degradation of IkB alpha in T cell lines, co-activation of Ca(2+)-dependent pathways accelerates the rate of IkB alpha phosphorylation and results in its complete degradation. Activation of Ca(2+)-dependent pathways alone do not result in the phosphorylation and/or degradation of IkB alpha in Jurkat T or in U937 cells. Treatment of T cells with the selective PKC inhibitor GF109203X abrogates the PMA-induced IkB alpha phosphorylation/degradation irrespective of activation of Ca(2+)-dependent pathways, but not the phosphorylation and degradation of IkB alpha induced by TNF-alpha, a PKC-independent stimulus. Contrary to the interaction with PKC, Ca(2+)-dependent pathways synergize with TNF-alpha not at the level of IkB alpha phosphorylation, but at the level of its degradation. These results indicate that Ca(2+)-dependent pathways, including the phosphatase calcineurin, participate in the regulation of NF-kappa B in a cell specific fashion and synergize with PKC-dependent and -independent pathways at the level of IkB alpha phosphorylation and degradation.
Triggering of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) induces nuclear translocation of NF-kappa B (p50/p65) in human monocytes and enhances viral replication in HIV-infected monocytic cells.
Authors: Thieblemont N , Haeffner-Cavaillon N , Haeffner A , Cholley B , Weiss L , Kazatchkine M D
Published: December 2022

Monocyte/macrophages may harbor HIV in a nonproductive fashion for prolonged periods of time. Viral gene expression may be reactivated by stimulation of the cells with LPS or cytokines such as TNF-alpha in vitro. The effect of LPS and TNF-alpha is mediated by their ability to induce nuclear translocation of the DNA-binding heterodimer NF-kappa B (p50/p65), which binds to a specific sequence in the HIV-long terminal repeat. The present study demonstrates that triggering of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) enhances viral replication in HIV-infected human monocytic cells. Monocytic cell lines and normal peripheral blood monocytes were infected with HIV-1 in vitro and cultured in the presence or absence of F(ab')2 fragments of monoclonal anti-CR1 or anti-CR3 Abs or with C3 fragments. Stimulation of CR1 or CR3 induces a two- to fourfold increase in the amount of cell-associated and released p24 Ag in cell cultures that was equivalent to that observed in control cultures triggered with LPS. We further observed that stimulation of CR1 or CR3 induces the nuclear translocation of NF-kappa B p50/p65 in infected cells. Translocation of NF-kappa B p50/p65 was also observed following stimulation of CR1 or CR3 of uninfected peripheral blood monocytes from HIV-seronegative donors. The amount of protein translocated was similar to that observed when cells were stimulated with rhTNF-alpha. TNF-alpha did not mediate the translocation of NF-kappa B p50/p65 induced by triggering of complement receptors. Taken together, these observations suggest that HIV gene expression may be activated in infected monocytes through interaction of the cells with complement-opsonized particles and that enhanced viral replication is associated with C3 receptor-mediated nuclear translocation of the NF-kappa B complex.
A central role for IL-1 beta in the in vitro and in vivo regulation of hepatic inducible nitric oxide synthase. IL-1 beta induces hepatic nitric oxide synthesis.
Authors: Geller D A , de Vera M E , Russell D A , Shapiro R A , Nussler A K , Simmons R L , Billiar T R
Published: December 2022

We have previously demonstrated that high levels of inducible nitric oxide synthase (iNOS) expression in hepatocytes required a combination of LPS and TNF-alpha, IL-1 beta, and IFN-gamma. The need for such a complex regulatory system seemed unwarranted based on the importance of NO in the liver. Therefore, we investigated whether individual cytokines could induce NO synthesis in hepatocytes and characterized some of the mechanisms involved. Rat hepatocytes were stimulated in vitro with escalating doses of TNF-alpha, IL-1 beta, or IFN-gamma. Only IL-1 beta induced high levels of iNOS mRNA and corresponding NO2- + NO3- production, and dexamethasone and cycloheximide blocked a majority of this response. Nuclear run-on experiments revealed that IL-1 beta upregulated iNOS gene transcription. IL-1 receptor antagonist protein (IL-1ra) competitively inhibited IL-1 beta-stimulated NO synthesis, implying activation through a cell-specific receptor. Rats injected with both LPS and IL-1ra showed decreased hepatic iNOS mRNA and plasma NO2- + NO3- compared with rats given LPS alone, indicating that IL-1 beta plays a role in regulating iNOS expression within the liver in vivo during endotoxemia. The soluble TNF receptor antagonist, PEG-(rsTNF-RI)2, also suppressed hepatic iNOS mRNA levels and plasma NO2- + NO3- increases, supporting a role for this cytokine in LPS-induced iNOS expression. Finally, IL-1 beta at high doses also induced iNOS mRNA and significant NO2- + NO3- production in cultures of primary human hepatocytes. These data indicate an important role for IL-1 beta in the regulation of hepatic NO synthesis.
Construction and characterization of a humanized anti-gamma-Ig receptor type I (Fc gamma RI) monoclonal antibody.
Authors: Graziano R F , Tempest P R , White P , Keler T , Deo Y , Ghebremariam H , Coleman K , Pfefferkorn L C , Fanger M W , Guyre P M
Published: December 2022

The murine mAb 22 (M22) binds to the human high affinity receptor for the Fc portion of IgG (Fc gamma RI). This mAb recognizes an epitope on Fc gamma RI that is distinct from the natural ligand (Fc) binding site, and, therefore, can bind to Fc gamma RI in the presence of saturating levels of IgG found in vivo. Fc gamma RI is expressed only on cytotoxic effector cells and it acts as an efficient mediator (trigger molecule) of effector functions. Directing tumor cells or other pathogens to Fc gamma RI utilizing M22 conjugated to an anti-tumor- or to an anti-pathogen-specific mAb may represent an effective method to enhance their removal and destruction under physiologic conditions. Humanization of M22 may improve the therapeutic potential of the mAb since murine mAbs normally elicit a human anti-mouse Ab response. In this report we describe the humanization of M22 by CDR-grafting onto human V region frameworks based on KOL VH and REI V kappa attached to human IgG1 and kappa constant domains. This humanized mAb, H22, displays equivalent binding specificity and affinity as its murine counterpart. In addition, H22 is able to trigger superoxide release from Fc gamma RI-bearing cells, a finding that demonstrates that H22 triggers Fc gamma RI functions. The successful humanization of mAb 22 is an important step in the development of Fc gamma RI-directed immunotherapy.
Immobilized antibodies to CD45 induce rapid morphologic changes and increased tyrosine phosphorylation of p56lck-associated proteins in T cells.
Authors: Arendt C W , Hsi G , Ostergaard H L
Published: December 2022

CD45 is a transmembrane protein tyrosine phosphatase required for signal transduction through the Ag receptor complexes of T and B lymphocytes. Herein, we demonstrate that immobilized mAbs to the external domain of CD45 induce rapid and dramatic morphologic changes in a variety of T cell lines, including CD8+ cytotoxic clones. CD45-induced morphologic changes can be inhibited by the cytoskeletal inhibitors cytochalasin D and E and by the protein tyrosine kinase inhibitor herbimycin A. Consistent with the requirement for tyrosine kinase activity, tyrosine phosphorylation of proteins at about 60 to 75 kDa and 115 to 130 kDa is increased upon engagement with immobilized anti-CD45 mAb with kinetics paralleling the observed changes in morphology. The phosphorylation of these proteins is inhibited by tyrosine kinase inhibitors at concentrations that also inhibit changes in morphology. The phosphoproteins induced when cells are added to immobilized anti-CD45 are co-immunoprecipitated with p56lck, suggesting that this tyrosine kinase might play a role in the phosphorylation of these proteins. Consistent with this, there is no increase in the phosphorylation of these proteins in p56lck-deficient CTLL-2 cells in response to immobilized anti-CD45 mAb. An important role for p56lck in the morphologic pathway is further supported by the observation that p56lck-deficient human J.CAM1.6 cells, in contrast to the parental Jurkat line, cannot be induced to undergo morphologic changes. Taken together, these results suggest a possible role for CD45 in coordinating a cytoskeletal remodeling cascade that may be important in cell activation.
Oxygen radical production and thiol depletion are required for Ca(2+)-mediated endogenous endonuclease activation in apoptotic thymocytes.
Authors: Fernandez A , Kiefer J , Fosdick L , McConkey D J
Published: December 2022

Glucocorticoid hormones stimulate apoptosis in thymocytes via a mechanism that involves changes in intracellular Ca2+, and exogenous Ca2+ can also directly promote the nuclear alterations of apoptosis (lamin degradation and chromatin cleavage) in isolated nuclei. Here we report that glucocorticoid treatment resulted in the production of reactive oxygen species and the depletion of reduced glutathione. Separation of apoptotic cells on Percoll gradients demonstrated that both effects selectively occurred in thymocytes undergoing apoptosis. Moreover, glucocorticoid-induced endonuclease activation was partially blocked by the antioxidant N-acetyl-L-cysteine. Although abrogation of methylprednisolone-induced Ca2+ increases using the intracellular Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid resulted in inhibition of endonuclease activation, it failed to prevent GSH depletion. However, N-acetyl-L-cysteine almost completely blocked methylprednisolone-induced elevations in cytosolic calcium levels, indicating that oxidative stress was playing a role in the Ca2+ response. Our results support the idea that oxidative stress is a key component of the apoptotic effector pathway in thymocytes, and that it interacts, at least in part, with the Ca2+ response.
Evidence for immunologic immaturity of cord blood T cells. Cord blood T cells are susceptible to tolerance induction to in vitro stimulation with a superantigen.
Authors: Takahashi N , Imanishi K , Nishida H , Uchiyama T
Published: December 2022

T cell blasts resulting from stimulating human cord blood (CB) mononuclear cells (MC) and adult peripheral blood (APB) MC with a bacterial superantigen, toxic shock syndrome toxin-1 (TSST-1), and then with human rIL-2 were investigated for their reactions to restimulation with TSST-1. Expression of TCR V beta 2, which determines the potential reactivity to TSST-1 and of CD45RO, was increased in both TSST-1-induced CB and APB T cell blasts. Most preparations of the TSST-1-induced CB T cell blasts exhibited low or no production of IL-2 and IL-4 in response to restimulation with TSST-1, while the APB T cell blasts showed high responses. TSST-1-induced T cell blasts derived from APB T cells depleted of both CD45RO+ T cells and HLA class II+ T cells showed high IL-2 production in response to restimulation with TSST-1. TSST-1-induced CB T cell blasts generated in the presence of DR+ L cells with high accessory cell activity still showed a low response to restimulation with TSST-1. These results indicate that CB T cells are inherently highly susceptible to tolerance induction by bacterial superantigens, suggesting the immunologic immaturity of CB T cells.
Human anti-porcine xenogeneic T cell response. Evidence for allelic specificity of mixed leukocyte reaction and for both direct and indirect pathways of recognition.
Authors: Yamada K , Sachs D H , DerSimonian H
Published: December 2022

Partially inbred, MHC homozygous miniature swine have been used to study the nature of the human xenogeneic cellular immune response to swine Ags in vitro. Human T cells responded to xeno-MHC Ags in MLR at least as well as they did to allo-MHC Ags and appeared to share similar requirements for APC of either stimulator (direct pathway) or responder (indirect pathway) derivation. In addition, mAb-blocking experiments indicated that the majority of the primary human anti-pig xeno-response was directed toward porcine MHC class II Ags and involved interaction with the human CD4 accessory molecule. Finally, the availability of intra-MHC recombinant haplotypes in our herds has made it possible to map genetically the antigenic specificities recognized in human anti-swine cellular responses. For this purpose, T cell clones were generated from human anti-swine MLR cultures and were tested for reactivity to stimulator cells from MHC homozygous and recombinant haplotypes. Clear evidence for antixenogeneic MHC Ags was observed. In all cases in which allelic differences between haplotypes were detected (the majority of clones), the reactivity could be mapped to the class II region of stimulator haplotypes. In addition, cross-reactivity between haplotypes was consistent with known sequence similarities between DR beta-chains. Our results indicate, therefore, that the human anti-porcine T cell response is similar in strength and specificity to an allogeneic response, and that the TCR repertoire, accessory molecule interactions, and cytokine production required for both direct and indirect pathways of recognition in the human anti-porcine MHC class II responses are functionally intact.
Mouse VH7183 recombination signal sequences mediate recombination more frequently than those of VHJ558.
Authors: Connor A M , Fanning L J , Celler J W , Hicks L K , Ramsden D A , Wu G E
Published: December 2022

Mouse VH gene segments are conventionally classified into 13 families on the basis of sequence similarity. The 7183 family lies close to the 3' end of the locus and is preferentially used in BALB/c mice; J558, the largest family, lies close to the 5' end of the VH stretch and is preferentially used in C57BL/6 mice. To investigate whether differential effectiveness of the RSSs in the two families might contribute to the overusage of 7183 in the primary repertoire of BALB/c, we constructed recombination substrates in which the recombination signal sequences (RSSs) of VH segments 7183 and J558 compete with each other for a single RSS, DFL16.1, after transfection into two transformed cell lines derived from C57BL/6 and two cell lines from BALB/c mice. In both strains, the 7183 RSS was found to be preferentially used (83%). Thus, the 7183 RSS mediates recombination more frequently than does that of J558, and this preference must thereby influence the primary repertoire, but the strain difference cannot be accounted for by a difference in the RSSs.
Cross-linking of alpha 4 beta 1 and alpha 5 beta 1 fibronectin receptors enhances natural killer cell cytotoxic activity.
Authors: Palmieri G , Serra A , De Maria R , Gismondi A , Milella M , Piccoli M , Frati L , Santoni A
Published: December 2022

Cell/extracellular matrix interactions mediated by integrins regulate differentiation, migration, and effector functions of immune system components. Human NK cells express alpha 4 beta 1 and alpha 5 beta 1 integrins, which mediate their binding to fibronectin (FN). We have investigated the ability of FN and its beta 1 integrin receptors to modulate NK cytotoxicity. Our data show that the presence of immobilized FN significantly augments the spontaneous cytotoxic activity of in vitro cultured human NK cells against several NK-susceptible, but not NK-resistant, target cells; Ab-dependent cytotoxicity against Ab-coated P815 target cells and the redirected lysis of anti-CD16 hybridomas are also enhanced in the presence of FN. Solid-phase-bound anti-human beta 1, or its F(ab')2 fragment, anti-alpha 4 and anti-alpha 5 mAbs, all consistently enhance Ab-dependent cytotoxicity against Ab-coated murine target cells. The 120-kDa (alpha 5 beta 1-binding), but not the 40-kDa (alpha 4 beta 1-binding), FN fragment fully reproduced the enhancing effect observed with the entire molecule. Our data also demonstrate that alpha 4 beta 1 and alpha 5 beta 1 cross-linking on NK cells induces an increase of intracellular Ca2+ concentration that is abrogated by EGTA, thus suggesting that the capacity to mobilize Ca2+ is involved in the coactivating role of alpha 4 beta 1 and alpha 5 beta 1 FN receptors in the cytotoxic functions of NK cells.
CD14-independent responses to LPS require a serum factor that is absent from neonates.
Authors: Cohen L , Haziot A , Shen D R , Lin X Y , Sia C , Harper R , Silver J , Goyert S M
Published: December 2022

Monocytes/macrophages and neutrophils can respond to endotoxin via a high-affinity receptor (CD14), requiring low levels of LPS (< 1 ng/ml) as well as through another pathway(s) that requires high levels of LPS (> 10 ng/ml). Both pathways result in the secretion of high levels of cytokines, such as TNF-alpha, and the up-regulation of various other effector molecules. To further define the activation of cells by LPS via a pathway that does not involve CD14, we have used an experimental model that distinguishes CD14-dependent from CD14-independent responses using saturating amounts of an anti-CD14 Ab to block the CD14-dependent response. Analysis of the ability of various individuals to respond to LPS via via both the CD14-dependent and CD14-independent pathways shows that adults can respond via both pathways; furthermore, in the presence of 100 ng of LPS/ml, the primary response is CD14 independent. In contrast to the response by adults, neonates can only respond via the CD14-dependent pathway. Further analysis has shown that the CD14-independent pathway requires a non-CD14 plasma protein present in adult plasma that is either missing or nonfunctional in neonate (cord) plasma.
Prevention of autoimmune disease by retroviral-mediated gene therapy.
Authors: Ally B A , Hawley T S , McKall-Faienza K J , Kündig T M , Oehen S U , Pircher H , Hawley R G , Ohashi P S
Published: December 2022

T lymphocytes have been implicated in a variety of autoimmune diseases, and therefore one potential therapeutic approach would be to tolerize the pathogenic self-reactive T cells. In this study, we examined whether retroviral gene therapy could be used to induce tolerance and prevent autoimmunity using a transgenic mouse model for experimentally induced diabetes. In this model, the lymphocytic choriomeningitis virus (LCMV) glycoprotein (gp) is expressed on the beta-islet cells of the pancreas under the control of the rat insulin promoter (RIP). Previous work showed that the T cells specific for the gp remain unaware of the transgenic gp Ag expressed by the iselt cells, and infection with LCMV leads to immune-mediated diabetes. To tolerize the gp-specific pathogenic T cells, a retroviral vector (RV) expressing the LCMV gp was constructed, RV-gp. Replication-defective recombinant retroviruses were used to transduce bone marrow cells, which were subsequently infused into host RIP-gp transgenic animals. Unlike control animals, RV-gp chimeric animals did not possess T cells specific for the gp Ag as measured by proliferation and cytotoxic function, and further analysis suggested that tolerance of the gp-specific self-reactive T cells occurred by clonal deletion. Further experiments demonstrated that chimeric RIP-gp transgenic animals generated using bone marrow transduced with RV-gp did not develop experimentally induced diabetes. Our animal model demonstrates that retroviral gene therapy may cure immune-mediated diabetes by providing long lasting Ag-specific tolerance.
Human anti-mouse xenorecognition. Provision of noncognate interactions reveals plasticity of T cell repertoire.
Authors: Batten P , Heaton T , Fuller-Espie S , Lechler R I
Published: December 2022

DAP.3 transfectants expressing native H-2E molecules with or without human LFA-3 and ICAM-1 (intercellular adhesion molecule-1) failed to induce proliferation by human peripheral blood T cells. Introduction of sequence from the DR beta 2 domain into the H-2E molecule led to the induction of detectable proliferation, which was substantially augmented by co-expression of human LFA-3 and ICAM-1 to levels comparable to those induced by DAP.3 cells co-expressing wild-type DR alloantigens with human LFA-3/ICAM-1. In marked contrast, cells expressing native H-2A molecules together with human accessory molecules provoked strong primary proliferative responses. The results of Ab inhibition experiments confirmed that this was caused by direct xenorecognition. In limiting dilution assays the frequency of anti-H-2A, IL-2-secreting, CD4+ human T cells was only fivefold lower than that measured against a DR alloantigen expressed on the same background. No measurable frequency was recorded against H-2E-expressing cells. Evidence to suggest that this difference was a result of isotype-specific differences in the interaction with CD4 was provided using transfectants expressing DR alloantigens with either the H-2E or H-2A beta 2 domain. DR molecules with the H-2A beta 2 domain stimulated a substantially stronger response than those with the H-2E beta 2 domain. These results challenge the view that xenogeneic T cell responses between evolutionarily distant species are weak; further emphasize the influence of the interaction between the T cell co-receptor molecule CD4, with its MHC class II molecular ligand on the strength of primary xenoresponses; and suggest that MHC class II isotypes may differ substantially in their interaction with CD4.
Ex vivo coating of islet cell allografts with murine CTLA4/Fc promotes graft tolerance.
Authors: Steurer W , Nickerson P W , Steele A W , Steiger J , Zheng X X , Strom T B
Published: December 2022

To test the hypothesis that blockade of B7-triggered costimulation by donor cells could preclude allograft rejection, we coated crude islet allograft preparations in vitro for 1 h with a murine CTLA4/Fc fusion protein. Murine CTLA4/Fc blocks the proliferative response in primary mixed lymphocyte cultures (MLC) and Con A-stimulated murine spleen cell cultures by 85 to 95%. Responder cells from a primary MLC containing mCTLA4/Fc were hyporesponsive upon restimulation to the same stimulator cells in a secondary MLC lacking mCTLA4/Fc. Because of mutations in the Fc gamma RI and C'1q binding sites of the Fc portion of the murine CTLA4/Fc fusion protein, the molecule binds to, but does not target, cells for Ab-dependent cellular cytotoxicity or complement-directed cytolysis. Although systemic immunosuppression was not applied, 42% (10 of 24) of B6AF1 recipients of islet allografts pretreated with CTLA4/Fc were permanently engrafted. Further, 50% of hosts bearing functioning islet allografts more than 150 days post-transplant were formally proved to be tolerant to donor tissues. A persistent CD4+ and CD8+ T cell infiltrate surrounding, but not invading, islet grafts in tolerant hosts was discerned. In control experiments, 89% (8 of 9) of islet allografts coated with mIgG3, and 100% (n = 10) pretreated with media alone were rejected. Thus, we conclude that 1) B7-triggered costimulation by donor APCs is an important element of rejection, and 2) blockade of the B7 pathway by in vitro allograft manipulation is able to induce tolerance.
Kinetic analysis of the interactions of recombinant human VpreB and Ig V domains.
Authors: Hirabayashi Y , Lecerf J M , Dong Z , Stollar B D
Published: December 2022

The surrogate light chain, composed of VpreB and lambda 5/14.1 proteins, is selectively expressed on B cell precursors, and is important for B cell development. The surrogate light chain associates with cell surface mu-chains on preB cells, but little is known about the ligand specificity and affinity of VpreB binding. To analyze its interactions with Igs, we made recombinant human VpreB protein and measured its affinity for H and L chain V domains using surface plasmon resonance. The recombinant VpreB protein existed as a homodimer in solution. VpreB chains associated with each other with an apparent Kd = 5 x 10(-7) M, and bound to a human VH domain, a mouse VH domain, and a human VL domain with a similar affinity. VpreB protein also bound to human Fab fragments of IgG with an apparent Kd = 6 x 10(-7) M, but showed a very low affinity for human Fc fragments of IgG. VpreB-Fab complex formation was reproduced by the formation of a trimolecular VpreB-VH-VL complex. Thus, VpreB proteins can associate with each other and also form complexes with Ig at sites different from those involved in VH-VL interaction. By flow cytometry, biotinylated VpreB protein bound to surface Ig-positive B cells but not T cells. Receptors that contain VpreB could be cross-linked by either Ig or by self-association.
Human anti-Gal heavy chain genes. Preferential use of VH3 and the presence of somatic mutations.
Authors: Wang L , Radic M Z , Galili U
Published: December 2022

Anti-Gal is the most abundant natural Ab known in humans. It interacts specifically with the carbohydrate structure Gal alpha 1-3 Gal beta 1-4GlcNAc-R (termed the alpha-galactosyl epitope), constitutes approximately 1% of circulating Ig, and is found in all three isotypes in the serum. Anti-Gal is produced in Old World monkeys, apes, and humans, and in no other mammalian species. The objective of this study was to determine the VH genes involved in the synthesis of anti-Gal. B lymphocytes from various individuals were transformed by EBV, the clones producing anti-Gal were isolated, the specificity and affinity of the Abs were determined, and the VH genes were sequenced. The affinity of anti-Gal clones for the free radiolabeled alpha-galactosyl epitope ranged between 1.1 x 10(6) M-1 and 5 x 10(8) M-1. Eight of the nine clones studied used VH3 family genes and one clone used a VH1 family gene. Four of the five VH3 genes used were found to form a cluster of related sequences, suggesting that functional constraints may lead to the use of VH3 genes with structural motifs that are suited for specific interactions with the alpha-galactosyl epitope. Comparison with known germline sequences for all of the clones studied and analysis of autologous germ-line genes in two of the clones indicate that anti-Gal VH genes undergo somatic mutations. These somatic mutations may provide a pool of variants that are available for affinity maturation.
Identification of a tyrosinase epitope recognized by HLA-A24-restricted, tumor-infiltrating lymphocytes.
Authors: Kang X , Kawakami Y , el-Gamil M , Wang R , Sakaguchi K , Yannelli J R , Appella E , Rosenberg S A , Robbins P F
Published: December 2022

A number of Ags recognized by class I-restricted, melanoma-specific T cells have recently been identified. In this report we demonstrated that tumor-infiltrating lymphocytes (TIL) from melanoma patient 1413 recognize a tumor Ag, tyrosinase, in the context of HLA-A24. This Ag had previously been shown to be recognized by an HLA-A24-restricted TIL, TIL 888, as well as HLA-A2-restricted, melanoma-specific T cells isolated from two additional patients. The peptide epitope recognized by TIL 1413 was then identified through the use of sequential deletions of the tyrosinase cDNA, as well as through prediction of HLA-A24 binding peptides based on a previously identified motif. Two peptides, a 9-amino acid peptide (AFLPWHRLF) and an overlapping 10-amino acid peptide (AFLPWHRLFL) containing an additional leucine at the carboxyl terminus, were both recognized by TIL 1413. Anti-peptide-specific CTL could be induced by repeated stimulation of peripheral blood lymphocytes from melanoma patient 1413, and this CTL line specifically recognized both HLA-A24+ B cell lines pulsed with the peptide and HLA-A24+ tyrosinase+ melanoma cells. This peptide thus represents a reagent that may be used to generate melanoma-specific T cells for adoptive immunotherapy, as well as in peptide vaccines for HLA-A24+ melanoma patients.
Variation of LPS-binding capacity, epitope expression, and shedding of membrane-bound CD14 during differentiation of human monocytes.
Authors: Pedron T , Girard R , Chaby R
Published: December 2022

The myeloid differentiation Ag CD14 is considered to play a critical role in the binding of LPS to monocytes. To determine if differences in LPS-binding capacities of cells could reflect a variability of CD14 molecules, we analyzed the interactions of various reagents with these molecules in human blood monocytes and in promyelocytic (HL60) and monocytic (THP-1) cell lines. The expression of CD14 epitopes was analyzed with the fluorescent anti-CD14 mAbs My4 and LeuM3. Expression of LPS-binding sites (LPS+ molecules) was detected with LPS-FITC. THP-1 cells stimulated with calcitriol (VitD3), as well as the majority of blood monocytes (50-90%) were My4+/LPS+. However, untreated THP-1 cells, and a substantial population (10-50%) of human monocytes from healthy donors, were My4+/LPS-, thus suggesting the existence of CD14 isoforms with different LPS-binding capacities. In line with this assumption, monocytes stimulated with PMA selectively shed LeuM3+ molecules, but almost no My4+ and LPS+ constituents. Analysis of monocytes after treatment with phosphatidylinositol-specific phospholipase C indicated that among CD14 molecules with LPS-binding capacity, some are susceptible and others are resistant to the enzyme, each type being mainly expressed by a different monocyte subset. Studies of uninduced and chemically induced THP-1 cells showed that wheat-germ agglutinin blocked the binding of My4 to constitutive, but not to chemically inducible CD14. The overall results suggest the existence of at least three different forms of CD14, which may reflect different stages of cell maturation.
Activation of p42mapk/erk2 following engagement of tumor necrosis factor receptor CD120a (p55) in mouse macrophages.
Authors: Winston B W , Riches D W
Published: December 2022

Cross-linking of the dichotomous cell surface receptors for TNF-alpha (CD120a (p55) and CD120b (p75)) induces the activation of a variety of macrophage responses that mediate the role of this cell in inflammation and host defense. Although significant progress has been made in understanding the role that lipid second messengers play in mediating the action(s) of this multifunctional cytokine, less is known about the role of specific kinases in TNF-alpha-initiated signaling. We show that exposure of mouse macrophages to TNF-alpha stimulates a rapid and transient tyrosine phosphorylation and activation of p42mapk/erk2. By contrast, p44mapk/erk1 was found to be constitutively phosphorylated, with minimal further response to TNF-alpha. To investigate the use of CD120a (p55) and CD120b (p75) in the activation of p42mapk/erk2 in mouse macrophages, we determined the effects of blocking Ab on the activation of p42mapk/erk2 in response to TNF-alpha. In addition, we independently cross-linked each receptor with specific agonistic Ab, which have previously been shown to mimic the effects of TNF-alpha. Collectively, the results from these experiments indicate that cross-linking of CD120a (p55), but not that of CD120b (p75), was both necessary and sufficient for the activation of p42mapk/erk2 in mouse macrophages.
The antirheumatic drug disodium aurothiomalate inhibits CD4+ T cell recognition of peptides containing two or more cysteine residues.
Authors: Griem P , Takahashi K , Kalbacher H , Gleichmann E
Published: December 2022

The mechanism of action of antirheumatic gold drugs, such as disodium aurothiomalate (Au(I)TM), has not been clearly identified. Gold drugs inhibit T cell activation induced by mitogen and anti-CD3 mAb in vitro at relatively high concentrations. However, since gold drugs fail to induce immunosuppression in vivo, the pharmacologic relevance of this finding is doubtful. In this study, we asked whether Au(I)TM interferes with processing and presentation of defined Ags to T cells. Using a panel of murine CD4+ T cell hybridomas, we found that low concentrations of Au(I)TM (< or = 10 microM) led to a markedly reduced IL-2 release of T cell hybridoma clones that recognized peptides containing two or more cysteine (Cys) residues, such as bovine insulin A1-14. Since disodium thiomalate alone had no effect, the inhibition was due to Au(I). IL-2 production induced by anti-CD3 mAb stimulation was not affected by the low concentration of Au(I)TM used. Au(I)TM had no effect on the presentation of peptides containing no or only one Cys residue(s). In contrast to the unmodified insulin peptide A1-14, Au(I) could not inhibit recognition of an insulin peptide in which Cys residues in positions 6 and 11 were replaced by serine. Most likely, the observed inhibition is mediated by formation of chelate complexes between Au(I) and two Cys thiol groups of the affected antigenic peptides. The peptide-specific inhibitory effect of Au(I) on Ag presentation described here might contribute to the therapeutic effect of Au(I) compounds in rheumatoid arthritis.
Autoimmunity induction by human T cell leukemia virus type 1 in transgenic mice that develop chronic inflammatory arthropathy resembling rheumatoid arthritis in humans.
Authors: Iwakura Y , Saijo S , Kioka Y , Nakayama-Yamada J , Itagaki K , Tosu M , Asano M , Kanai Y , Kakimoto K
Published: December 2022

We recently reported on an inflammatory arthropathy resembling rheumatoid arthritis that develops in high incidence among transgenic mice that carry the env-pX region of the human T cell leukemia virus type 1 genome. In an effort to elucidate the pathogenesis of this disease, we found that genes for inflammatory cytokines, including IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, transforming growth factor-beta 1, IFN-gamma, and IL-2, as well as MHC genes were activated in transgenic joints. Serum levels of IL-1 beta and IL-6 were also elevated. Interestingly, these mice produced Ab against IgG, type II collagen (IIC), and heat shock proteins accompanied by IgG hypergammaglobulinemia. The cellular immune response to IIC as well as that to heat shock proteins were activated. Moreover, these mice became immunologically responsive to exogenously administered IIC and developed arthritis, in contrast to their nontransgenic littermates, which showed little response to IIC. Taken together, the results suggest that human T cell leukemia virus type 1 can cause immune system hyperreactivity and induce autoimmunity. The possibility that elevated cytokine and/or MHC gene expression are involved in the development of autoimmunity and arthropathy are discussed.
Characterization of intercellular adhesion molecule-1 (ICAM-1)-augmented degranulation by cytotoxic T cells. ICAM-1 and anti-CD3 must be co-localized for optimal adhesion and stimulation.
Authors: Berg N N , Ostergaard H L
Published: December 2022

Purified intercellular adhesion molecule-1 (ICAM-1), a ligand of the LFA-1, was used to analyze the contribution of ICAM-1 to the activation of CTL. ICAM-1 facilitates degranulation when co-immobilized with substimulatory amounts of anti-CD3. This facilitated response is most likely mediated through LFA-1, since Abs to this molecule significantly inhibit the response, Interestingly, when ICAM-1 and anti-CD3 are immobilized on separate beads and presented to the CTL, no ICAM-1-enhanced degranulation is observed. The ICAM-1 and anti-CD3 must be immobilized on the same surface to augment the response, suggesting that ICAM-1 either does not transmit signals into the cell or it transmits a very localized signal, since the ICAM-1 and anti-CD3 must be juxtaposed. Consistent with this finding, we demonstrate that ICAM-1 does not induce tyrosine phosphorylation or a Ca(2+)-flux in the CTL clone, but does potentiate these responses when co-immobilized with substimulatory anti-CD3. Finally ICAM-1 and anti-CD3 must be immobilized on the same bead for stable adhesion of CTL to ICAM-1. When ICAM-1 and anti-CD3 are immobilized on separate beads, there is only a transient, low level of adhesion to the ICAM-1 beads. Taken together, these results suggest that LFA-1 is acting principally as an adhesion molecule, with respect to ICAM-1, in CTL and that this adhesion is regulated through the TCR complex.
Lysosome-associated membrane protein-1-mediated targeting of the HIV-1 envelope protein to an endosomal/lysosomal compartment enhances its presentation to MHC class II-restricted T cells.
Authors: Rowell J F , Ruff A L , Guarnieri F G , Staveley-O'Carroll K , Lin X , Tang J , August J T , Siliciano R F
Published: December 2022

A subset of endogenously synthesized Ags can be processed for class II-restricted presentation, probably through multiple mechanisms. Processing of exogenous Ags for class II-restricted presentation appears to occur in unique endosomal processing compartments with lysosomal characteristics including the presence of the lysosomal membrane protein LAMP-1. Therefore, we attempted to enhance the efficiency of class II-restricted presentation of an endogenous Ag, the HIV-1 envelope (env) protein, by specifically targeting the Ag to class II processing compartments through the pathway followed by LAMP-1. Because the env protein associates tightly with CD4 shortly after synthesis, we first targeted the env protein using a chimeric CD4 protein consisting of the extracellular domain of CD4 and the transmembrane and cytoplasmic domains of LAMP-1. When co-expressed with this chimeric protein, the env protein was efficiently localized to lysosome-like compartments. Enhanced stimulation of env-specific CD4+ T cell clones by APC expressing the env protein and the CD4-LAMP-1 chimera was readily demonstrated in both cytotoxicity assays and proliferation assays. We also targeted the env protein directly as a chimeric protein consisting of the extracellular domain of the env protein and the transmembrane and cytoplasmic domains of LAMP-1. The proliferative response of env-specific CD4+ T cell clones to the env-LAMP-1 chimera was greatly enhanced compared with wild-type env protein, especially when limiting numbers of stimulator cells were used. The enhanced stimulatory capacity of APC expressing LAMP-1-targeted Ags has important implications for vaccine design.
Differential regulation of alternative 3' splicing of epsilon messenger RNA variants.
Authors: Diaz-Sanchez D , Zhang K , Nutman T B , Saxon A
Published: December 2022

Alternative 3' splicing of the one active human epsilon heavy chain gene results in variants of epsilon mRNA encoding distinct IgE proteins. The same relative amounts of these epsilon mRNA variants were produced by non-atopic donor B cells when driven in a variety of T-dependent or T-independent systems. The most abundant variants were those for classic secreted epsilon a novel secreted form (CH4-M2"). In contrast, cells from subjects with high levels of serum IgE secondary to parasitic infection or atopy spontaneously produced higher relative levels of the CH4-M2' epsilon mRNA variant, lower relative amounts of both the membrane and CH4-M2" secreted variants, and very low levels of the CH4'-CH5 variant. The existence of and corresponding changes in levels of the CH4-M2'-encoded secreted protein were demonstrated. IL-10 induced this same differential expression of epsilon splice variants in vitro when used to costimulate IL-4 plus CD40-driven B cells and could differentially enhance the production of CH4-M2' protein by established IgE-secreting cell lines. Inhibition of IgE by cross-linking the low affinity IgE receptor (CD23) decreased the levels of epsilon mRNA and resulted in a distinct pattern of epsilon mRNA characterized by a dramatic decrease in CH4-M2' splice variant. IL-6, IL-2, or IFN-gamma did not change the epsilon mRNA pattern. Overall, the absolute and relative amounts of the different epsilon mRNA splice variants produced appear to be controlled in a differentiation-related fashion.
Expression cloning and chromosomal mapping of the leukocyte activation antigen CD97, a new seven-span transmembrane molecule of the secretion receptor superfamily with an unusual extracellular domain.
Authors: Hamann J , Eichler W , Hamann D , Kerstens H M , Poddighe P J , Hoovers J M , Hartmann E , Strauss M , van Lier R A
Published: December 2022

CD97 is a monomeric glycoprotein of 75 to 85 kDa that is induced rapidly on the surface of most leukocytes upon activation. We herein report the isolation of a cDNA encoding human CD97 by expression cloning in COS cells. The 3-kb cDNA clone encodes a mature polypeptide chain of 722 amino acids with a predicted molecular mass of 79 kDa. Within the C-terminal part of the protein, a region with seven hydrophobic segments was identified, suggesting that CD97 is a seven-span transmembrane molecule. Sequence comparison indicates that CD97 is the first leukocyte Ag in a recently described superfamily that includes the receptors for secretin, calcitonin, and other mammalian and insect peptide hormones. Different from these receptors, CD97 has an extended extracellular region of 433 amino acids that possesses three N-terminal epidermal growth factor-like domains, two of them with a calcium-binding site, and a single Arg-Gly-Asp (RGD) motif. The existence of structural elements characteristic for extracellular matrix proteins in a seven-span transmembrane molecule makes CD97 a receptor potentially involved in both adhesion and signaling processes early after leukocyte activation. The gene encoding CD97 is localized on chromosome 19 (19p13.12-13.2).
Activation of IL-2 receptor alpha-chain gene by individual members of the rel oncogene family in association with serum response factor.
Authors: Pierce J W , Jamieson C A , Ross J L , Sen R
Published: December 2022

Expression of the IL-2R alpha gene is regulated by members of the c-Rel/NF-kappa B family of transcription factors binding to the kappa B site in the promoter. Previous work has not defined the role of individual members of the c-Rel family in the activation of the IL-2R alpha gene. Using the COS cell system, we were able to reconstitute the regulation of the IL-2R alpha promoter by expressing cloned Rel family members with serum response factor (SRF). We found that c-rel alone activated the IL-2R alpha promoter only weakly but worked with the p50 subunit of NF-kappa B (NFKB1) to give a higher level of expression. We showed that c-rel heterodimerizes with p50 and the amount of this heterodimer correlated with the level of IL-2R alpha gene expression. Our results provide evidence that c-rel/p50 heterodimers activate gene expression in the context of a cellular promoter. We show that c-rel or p65 can cooperate with SRF in the activation of this promoter and the transactivation by c-rel with SRF was enhanced by p50. Synergistic activation required both kappa B and CArG sites, and binding studies show that these adjacent sites can be occupied simultaneously. The transactivation observed with cloned transcription factors mimics the physiologic induction of the IL-2R alpha gene since multiple sequence elements cooperate to give gene activation. The data support the model that c-rel/p50 or p65 can cooperate with SRF to specifically target the expression of the IL-2R alpha gene in activated T cells.
Differences in the shedding of soluble TNF receptors between endotoxin-sensitive and endotoxin-resistant mice in response to lipopolysaccharide or live bacterial challenge.
Authors: Carpenter A , Evans T J , Buurman W A , Bemelmans M H , Moyes D , Cohen J
Published: December 2022

TNF-alpha plays a pivotal role in the pathogenesis of septic shock. It exerts its effects by binding two cell surface receptors, designated TNF-R I and II, also referred to as the p55 and p75 receptors, respectively. TNF-Rs are transmembrane proteins, which on cleavage of their extracellular domains, result in the release of soluble fragments (sTNF-R). sTNF-R levels increase markedly during infection, and may serve to modulate TNF-alpha bioactivity. The mechanisms regulating this process are uncertain. To investigate this, we measured sTNF-R release in endotoxin-sensitive C3H/HeN and endotoxin-resistant C3H/HeJ mice given LPS or live Gram-negative bacteria. In C3H/HeN mice, there was a rapid early response during the first 4 h, and a second peak at 8 h, particularly noticeable in the case of the p75 receptor. Prior administration of neutralizing Abs to TNF-alpha or IFN-gamma had no effect on receptor shedding. Surprisingly, C3H/HeJ mice also responded to both bacterial challenge and to LPS by shedding sTNF-R; the magnitude and duration of the early response was not substantially different from C3H/HeN mice, although the second peak was absent. Peritoneal macrophages from C3H/HeN mice responded promptly (5 h) when stimulated with LPS in vitro, and by 22 h levels had increased five- to 10-fold. In contrast, cells from C3H/HeJ mice demonstrated only a very modest response at 22 h following maximal stimulation. The data suggest that there may be at least two separately regulated pathways that control sTNF-R shedding in these mice.
Defective ceramide response in C3H/HeJ (Lpsd) macrophages.
Authors: Barber S A , Perera P Y , Vogel S N
Published: December 2022

Lipid second messengers are gaining recognition as important mediators of extracellular signals. One such lipid, ceramide, generated from membrane sphingomyelin following stimulation with TNF-alpha, IL-1 beta, or IFN-gamma, activates ceramide-activated kinase (CAK). A recent study demonstrated that LPS activated CAK without generating ceramide, suggesting that the LPS stimulation of cells mimics the second messenger function of ceramide. To compare ceramide to LPS signaling, we assessed the ability of LPS-responsive (Lpsn) and LPS-hyporesponsive (Lpsd) macrophages to respond directly to ceramide for enhanced expression of LPS-inducible genes. In contrast to macrophages from C3H/Ouj (Lpsn) mice, C3H/Hej (Lpsd) macrophages failed to respond to cellpermeable analogues of ceramide (C2,C6,C16) or sphingomyelinase. These results suggest that a common critical molecule, encoded by the Lps gene, regulates both ceramide and LPS signaling pathways.