bluuebunny/crossref_metadata_2025 huggingface dataset:
A subclass of dendritic cells regulates the response of naive CD8 T cells by limiting their IL-2 production.
Authors: Kronin V , Winkel K , Süss G , Kelso A , Heath W , Kirberg J , von Boehmer H , Shortman K
Published: December 2022

Previous work indicated that a subclass of mouse spleen dendritic cells (DC), those bearing CD8alpha, expresses the Fas ligand and restricts peripheral CD4 T cell responses by initiating Fas-mediated apoptosis. To determine whether a similar regulation applies to CD8 T cells, they were purified from normal or from TCR-transgenic mice, and then cultured with purified splenic CD8+ DC or CD8- DC presenting either alloantigens or the specific Ag for the TCR transgene. In all systems studied, the proliferative response of CD8 T cells was markedly less on stimulation with CD8+ DC compared with conventional CD8- DC. However, the basis of this restricted proliferation in response to CD8+ DC was totally different for CD8 T cells than for CD4 T cells. The reduced proliferation of CD8 T cells occurred later in the response than with CD4 T cells. In contrast with CD4 T cells, the reduced proliferation of CD8 T cells occurred even with T cells from Fas-deficient Ipr mice, or with DC from Fas ligand-deficient gld mice, indicating that Fas-induced apoptosis was not involved. Also, in contrast with CD4 T cells, the reduced proliferation of CD8 T cells was completely reversed by the addition of exogenous IL-2. Furthermore, cultures of CD8 T cells with CD8+ DC were found to be deficient in IL-2 production. Accordingly, although CD8+ DC are very efficient at stimulating CD8 T cells into cell division, they are deficient at stimulating endogenous cytokine production. The implications of these different DC regulatory systems are discussed.
Intracellular IL-1beta is an inhibitor of Fas-mediated apoptosis.
Authors: Tatsuta T , Cheng J , Mountz J D
Published: December 2022

Fas-mediated apoptosis has been shown to be mediated by the IL-1beta converting enzyme (ICE) pathway. To determine the relationship between ICE and its substrate IL-1beta, we examined six human cell lines for susceptibility to Fas-mediated apoptosis and Fas induction of ICE-like activity. The human B lymphoblastoid cell line SKW6.4 and the human T lymphoma cell lines Jurkat, CEM-6, H-9, and MOLT4 were susceptible to Fas-mediated apoptosis, whereas the human promyelocytic leukemia cell line HL-60 was resistant to Fas-mediated apoptosis. ICE mRNA was highly expressed in SKW6.4, H-9, and HL-60 cells, and ICE-like activity increased during Fas-mediated apoptosis in SKW6.4 cells. In contrast, IL-1beta mRNA was highly expressed only in HL-60 cells. Acetyl-Tyr-Val-Ala-Asp-chloromethylketone, a tetrapeptidyl inhibitor of ICE, prevented Fas-mediated apoptosis strongly in SKW6.4 and H-9 cells but weakly or marginally in other cells. To examine whether intracellular IL-1beta is a proteolytic substrate or an endogenous competitive inhibitor against other substrates for Fas-ICE-mediated apoptosis in SKW6.4 cells, we established precursor IL-1beta transfectant clones using SKW6.4 cells. We demonstrated that stably transfected SKW6.4 cells expressing precursor IL-1beta, but not cells transfected with the empty vector, exhibited resistance to Fas-mediated apoptosis due to competitive inhibition of ICE-like activity, which was associated with increased cleavage of precursor IL-1beta to mature IL-1beta. These results suggest that Fas-mediated apoptosis is mediated by ICE cleavage of proteolytic substrates other than IL-1beta and that IL-1beta is an endogenous inhibitor of Fas-mediated apoptosis.
IL-12 enhances vaccine-induced immunity to schistosomes by augmenting both humoral and cell-mediated immune responses against the parasite.
Authors: Wynn T A , Reynolds A , James S , Cheever A W , Caspar P , Hieny S , Jankovic D , Strand M , Sher A
Published: December 2022

The production of Th1-type cytokines is associated with strong cell-mediated immunity, while Th2-type cytokines typically dominate humoral immune responses. In mice vaccinated a single time with attenuated cercariae of Schistosoma mansoni, the protection induced is associated with Th1 cytokine-dependent, cell-mediated immunity. In contrast, mice vaccinated multiple times display a more Th2-type dominant cytokine response and develop Ab-dependent resistance. We have previously shown that IL-12 enhances cell-mediated immunity in singly vaccinated mice. In the present study, we asked what effects administering IL-12 as an adjuvant would have on the development of a protective humoral response in multiply immunized animals. We found that multiply immunized/IL-12-treated mice displayed a marked increase in resistance to challenge infection, with some animals demonstrating complete protection. The IL-12-vaccinated mice developed strongly polarized Th1 responses but, importantly, also showed significant increases in parasite-specific Ab and, in particular, IgG2a, IgG2b, and IgG1 isotypes. Passive transfer demonstrated an enhanced ability of serum from these animals to protect naive recipients. In addition, animals vaccinated in the presence of IL-12 also developed macrophages with increased nitric oxide-dependent killing activity against the parasites. Together, these data demonstrate that IL-12, initially described as an adjuvant for cell-mediated immunity, may be used to simultaneously to promote both humoral and cell-mediated protective responses against infection.
Isolation of tyrosinase-specific CD8+ and CD4+ T cell clones from the peripheral blood of melanoma patients following in vitro stimulation with recombinant vaccinia virus.
Authors: Yee C , Gilbert M J , Riddell S R , Brichard V G , Fefer A , Thompson J A , Boon T , Greenberg P D
Published: December 2022

The identification of Ags expressed by tumor cells and recognized by autologous T cells has led to the prospect of treating cancer by adoptive transfer of tumor-reactive T cells selected for Ag specificity. Tyrosinase is an Ag expressed by normal melanocytes as well as melanoma cells for which responses by autologous T cells have been detected. To evaluate the frequency with which tyrosinase-specific T cells can be isolated from melanoma patients for potential use in therapy, a recombinant vaccinia virus expressing tyrosinase was constructed for infection of autologous APCs that could be used to stimulate T cells reactive with this protein. Eight patients were studied, with peripheral blood serving as the source of both responder T cells and autologous APCs. Tyrosinase-specific CD8+ CTL clones were isolated from five of the eight patients with melanoma. The tyrosinase-specific CTL generated in this manner recognized autologous tumor cells as well as targets expressing the recombinant virus vector. CTL clones from three of the individuals were restricted to HLA-A28, -B8, and -B60, which have not previously been identified as alleles that can present immunogenic tyrosinase peptides. Tyrosinase-specific CD4+ T cell clones were isolated from six of the eight patients by stimulation with autologous APCs infected with recombinant vaccinia virus, and all these CD4+ clones were capable of recognizing autologous tumor cells. These studies demonstrate a high prevalence of CD4+ and CD8+ tyrosinase-specific responses in peripheral blood and support the feasibility of using peripheral blood to generate T cells for tumor therapy without the requirement for isolating T cells that have infiltrated tumor sites.
In vitro and in vivo secretion of human ISG15, an IFN-induced immunomodulatory cytokine.
Authors: D'Cunha J , Ramanujam S , Wagner R J , Witt P L , Knight E , Borden E C
Published: December 2022

ISG15, a 15-kDa protein of unique primary amino acid sequence, functions intracellularly as a ubiquitin homologue and a cytokine that induces production of IFN-gamma and augments NK/lymphokine-activated killer cell proliferation and function. ISG15 is secreted from monocytes and lymphocytes, and in this study we have characterized in vitro and in vivo production of ISG15 in response to IFN-alphabeta. Low levels of ISG15 were present constitutively in PBMCs; dose-dependent ISG15 synthesis was observed in response to IFN-alpha or IFN-beta, but not IFN-gamma. High m.w. conjugates, present in PBMC extracts constitutively, were enhanced after IFN-alpha or IFN-beta treatment. Metabolic labeling experiments demonstrated that IFN-beta-induced ISG15 was released from primary cultures of peripheral blood CD3+ (including both CD4+ and CD8+ subpopulations). Furthermore, ISG15 was released from viable cell lines of monocyte, T lymphocyte, B lymphocyte, and epithelial origins. Since ISG15 was secreted in response to IFN treatment in vitro, its levels in the serum of healthy human volunteers treated with IFN-beta(ser) were quantitated by asymmetric sandwich ELISA. Both single and multiple doses of IFN-beta(ser) increased serum ISG15 levels significantly (p < 0.01) over baseline. A maximum 7.3-fold enhancement of serum ISG15 was obtained after multiple injections of 8 million units of IFN-beta(ser). Significant change was observed at 24 and 48 h of multiple 0.02-million-unit injections, yielding 1.2- and 1.7-fold increases over basal levels, respectively. These studies suggest that ISG15 is a novel member of the cytokine cascade that is synthesized and released in response to IFN-beta both in vitro and in vivo.
Escherichia coli hemolysin and Staphylococcus aureas alpha-toxin potently induce neutrophil adhesion to cultured human endothelial cells.
Authors: Krüll M , Dold C , Hippenstiel S , Rosseau S , Lohmeyer J , Suttorp N
Published: December 2022

Adhesion of polymorphonuclear leukocytes (PMN) to endothelial cells is an essential step in inflammatory reactions. We characterized the effects of two important bacterial exotoxins, Escherichia coli hemolysin (HlyA) and Staphylococcus aureus alpha-toxin (S. alpha-toxin) on PMN adhesion to cultured HUVEC. Both toxins increased adherence of human PMN to HUVEC in a dose- and time-dependent manner, peaking after 30 min at 0.01 hemolytic units/ml HlyA or 0.5 microg/ml S. alpha-toxin. Pretreatment of HUVEC with anti-P-selectin mAbs or of PMN with anti-CD11b/CD18 mAb reduced HlyA- and S. alpha-toxin-related cell adhesion significantly. Increased P-selectin expression on toxin-treated endothelial cells was demonstrated by cell surface ELISA. Compared with endotoxin, HlyA and S. alpha-toxin did not induce the expression of E-selectin, ICAM-1, or VCAM-1. FACS analysis showed increased CD11b/CD18 expression on HlyA-, but not on S. alpha-toxin-stimulated PMN. Platelet-activating factor, an important costimulatory factor for PMN adhesion and activation, was also active in the exotoxin-stimulated adhesion system, as evidenced by studies using the platelet-activating factor receptor antagonist BN50727. HPLC analysis of endothelial cell extracts confirmed enhanced toxin-mediated PAF synthesis. The capacity of exotoxins to stimulate PMN adhesion to endothelial cells may be relevant in patients with severe local or systemic bacterial infections.
TNF-alpha and IL-1beta selectively induce expression of 92-kDa gelatinase by human macrophages.
Authors: Sarén P , Welgus H G , Kovanen P T
Published: December 2022

Macrophages are present in inflammatory tissue sites where abnormal degradation of the extracellular matrix takes place. To evaluate the potential of macrophages to participate in such matrix destruction, we studied the effects of three cytokines present in inflammatory tissue sites, TNF-alpha, IL-1beta, and IFN-gamma, on the production of three matrix-degrading metalloproteinases, interstitial collagenase, stromelysin, and 92-kDa gelatinase, as well as their natural inhibitor, TIMP-1 (tissue inhibitor of metalloproteinases number 1), by human monocyte-derived macrophages differentiated in vitro. Spontaneous production of interstitial collagenase and stromelysin by these cells was minimal, and was not influenced by the cytokines. In contrast, the cells secreted substantial basal amounts of 92-kDa gelatinase, the secretion of which was stimulated (2- to 15-fold; on average 5-fold) by both TNF-alpha and IL-1beta, while the production of TIMP-1 was unaffected. IFN-gamma suppressed the production of the 92-kDa gelatinase induced by TNF-alpha- and IL-1beta. TNF-alpha and IL-1beta regulated the expression of 92-kDa gelatinase by monocyte-derived macrophages at the pretranslational level. The results show that expression of 92-kDa gelatinase, but not its natural inhibitor TIMP-1, by human tissue-type macrophages is selectively up-regulated by proinflammatory cytokines; which suggests that these cells, when actually present in an inflammatory environment, will actively participate in the destruction of the extracellular matrix.
Bronchoalveolar lavage neutrophilia is associated with obliterative bronchiolitis after lung transplantation: role of IL-8.
Authors: DiGiovine B , Lynch J P , Martinez F J , Flint A , Whyte R I , Iannettoni M D , Arenberg D A , Burdick M D , Glass M C , Wilke C A , Morris S B , Kunkel S L , Strieter R M
Published: December 2022

Obliterative bronchiolitis (OB) is a devastating complication in lung transplantation. We postulated that the pathogenesis of OB is mediated, in part, by neutrophils. We serially collected bronchoalveolar lavage (BAL) fluid from lung transplant recipients. Patients were divided into two groups depending on the presence or absence of OB. Samples from patients who never developed OB were further divided according to whether rejection was present. These samples were labeled healthy or rejection. Samples from patients who developed OB were divided according to whether the sample was obtained before (future OB) or at the time of diagnosis of OB (OB). The OB group, as compared with the healthy and rejection group, had significantly elevated neutrophil counts (3.9 x 10(5) +/- 1.8 x 10(5) vs 0.3 x 10(5) +/- 0.07 x 10(5) and 0.4 x 10(5) +/- 0.1 x 10(5), respectively, p < 0.01 for both) and levels of IL-8 (3131 +/- 1468 pg/ml vs 240 +/- 62 pg/ml and 172 +/- 47 pg/ml, p < 0.01 for both). Furthermore, we demonstrated immunolocalization of IL-8 associated with alpha smooth muscle actin-positive cells in the peribronchial region of OB. To confirm that the IL-8 present in BAL fluid from patients with OB was bioactive, we performed neutrophil chemotaxis experiments that showed that IL-8 accounted for a significant amount of the neutrophil chemotactic activity. We also found a trend toward higher levels of neutrophils and IL-8 in BALs from the future OB as compared with the healthy group (7.1 x 10(4) +/- 4.2 x 10(4) vs 3.4 x 10(4) +/- 0.7 x 10(4) and 500 +/- 306 pg/ml vs 240 +/- 62 pg/ml). In conclusion, we have provided the novel observation that in lung transplant recipients with OB, neutrophilia is present and highly correlated with the presence of IL-8.
Suppression of murine chronic relapsing experimental autoimmune encephalomyelitis by the oral administration of myelin basic protein.
Authors: Meyer A L , Benson J M , Gienapp I E , Cox K L , Whitacre C C
Published: December 2022

Chronic relapsing experimental autoimmune encephalomyelitis (EAE), induced in mice by the injection of myelin basic protein (MBP), is a T cell-mediated autoimmune disease characterized by periods of paralysis and remission. We have shown previously that the oral administration of MBP or MBP peptides renders Lewis rats refractory to EAE. This study was undertaken to examine the conditions necessary to produce oral tolerance in a chronic relapsing model of EAE in B10.PL mice. The optimal tolerizing regimen for the mouse was found to be a single feeding of 20 mg of MBP suspended in PBS. To determine the ability to suppress chronic disease, a range of doses (0.4-100 mg) was administered orally in a single dose before challenge. Larger oral doses (20 or 100 mg) of MBP provided the best protection from EAE, while 0.4 mg exacerbated the clinical course of disease. Secretion of the proinflammatory cytokines, IL-2 and IFN-gamma, were lowest in the group fed 20 mg. A single feeding of MBP before challenge or as late as the first day of clinical signs showed significant protection over the relapsing disease course. Once relapsing EAE was established, multiple oral doses of MBP were required to achieve suppression of clinical signs of disease. These findings suggest that vehicle, dosage, and timing are important considerations in the successful application of oral tolerance strategies for suppression of chronic disease processes.
Genetic bases of human complement C7 deficiency.
Authors: Nishizaka H , Horiuchi T , Zhu Z B , Fukumori Y , Volanakis J E
Published: December 2022

Complement C7 deficiency (C7D) is associated frequently with recurrent bacterial infections, especially meningitis caused by Neisseria meningitidis. We report in this work the molecular bases of C7D in two unrelated Japanese males. We used exon-specific PCR/single-strand conformation polymorphism analysis as a screening step for mutations. Subsequent direct sequencing of the target exons identified homozygous mutations in exon 16 of case 1 and in exon 15 of case 2. The mutation of case 1 was a homozygous T to A transversion at nucleotide 2250, the third nucleotide of the codon TGT for Cys728, leading to a stop codon TGA (C728X). In case 2, a homozygous 2-bp deletion (2137delTG/2138delGT/2139delTG) caused a frameshift, generating a premature termination codon 4 to 6 nucleotides downstream. Family study in case 1 confirmed the genetic nature of the defect. Moreover, we detected a novel polymorphism in intron 11 that presumably is linked to the mutation responsible for C7D in case 1. Our results indicate that the pathogenesis of C7D is heterogeneous like most of the other deficiencies of complement components.
Influence on CD8 of TCR/CD3-generated signals in CTL clones and CTL precursor cells.
Authors: Anel A , Martínez-Lorenzo M J , Schmitt-Verhulst A M , Boyer C
Published: December 2022

Alloreactive CTL clones and naive CTL precursor cells (CTLp) from TCR-transgenic mice were analyzed for their response in total and in TCR-associated kinase activation upon stimulation with the relevant class I allo-APCs. The responses were found to be stronger and more sustained for the CTL clone and CTLp expressing a TCR previously characterized as CD8 coreceptor independent than for the CTL clone and CTLp expressing a TCR characterized as CD8 dependent. Unexpectedly, it was found that also in response to CD3 engagement, total and TCR-associated kinase activation were stronger and more sustained in the CTL clone and CTLp expressing the CD8-independent TCR. In both types of CTL clones, p56(lck) was found associated with the TCR complex, and CD3 components were found associated with CD8 before CD3 engagement. Upon CD3 engagement, ZAP-70 was also found associated with the TCR complex and the kinase activity (p56(lck)) associated with CD8 increased. This increase was more pronounced for the CD8-independent than for the CD8-dependent clone. An increased association of CD3zeta with CD8 was also detected after CD3 engagement for each clone. These data indicate that signals resulting from exclusive CD3 engagement can influence CD8 molecular associations and activate CD8-bound p56(lck). They further suggest that clonal differences exist that influence the efficiency of signaling upon binding of the same CD3 ligand. The observation that this property was shared between independently derived CTL clone and CTLp expressing the same TCR suggests that it may be acquired during repertoire selection.
Oligonucleotide-mediated inhibition of CD28 expression induces human T cell hyporesponsiveness and manifests impaired contact hypersensitivity in mice.
Authors: Tam R C , Phan U T , Milovanovic T , Pai B , Lim C , Bard J , He L
Published: December 2022

Ligation of CD28 provides a costimulatory signal essential for Ag-mediated T cell activation via the TCR. Blocking CD28 ligation can inhibit cytokine expression and elicits a state of T cell hyporesponsiveness. In this study, we examined the effect of inhibiting CD28 expression on in vitro and in vivo T cell responses. To address this, we have synthesized a series of G-rich phosphorothioate oligonucleotides that inhibited activation-induced transcription and cell surface expression of CD28 on human T cells. CD28 blockade was selective, as expression of other activation-induced receptors was unaffected by oligonucleotide treatment. Using strategic changes to base composition, we identified a minimal 12-mer sequence, containing two sets of four contiguous guanosines separated by 3 to 5 bases, which conferred activity in vitro. Furthermore, inhibition of CD28 expression mediated by one representative active oligonucleotide, GR1, resulted in a concomitant dose-dependent diminution of anti-CD3/PMA-induced cytokine (IL-2, IFN-gamma, IL-8) production. Inhibition of IL-2 synthesis was dependent on CD28 expression, as GR1 failed to abrogate activated IL-2 production in a CD28-deficient T cell line, HUT 78. The inhibitory activity of GR1 reduced T cell proliferative responses in MLR and induced Ag-specific T cell hyporesponsiveness to alloantigens. Finally, s.c. administration of GR1 impaired in vivo contact hypersensitivity responses in mice and was associated with substantially decreased CD28 and IFN-gamma mRNA expression in lymph node cells. Collectively, our studies show the tolerogenic potential of oligonucleotide-mediated CD28 inhibition on T cell activation, in vitro and in vivo.
Mapping regions of herpes simplex virus type 1 glycoprotein I required for formation of the viral Fc receptor for monomeric IgG.
Authors: Basu S , Dubin G , Nagashunmugam T , Basu M , Goldstein L T , Wang L , Weeks B , Friedman H M
Published: December 2022

Glycoprotein E (gE) and glycoprotein I (gI) of herpes simplex virus type 1 (HSV-1) form a complex that binds the Fc domain of monomeric IgG. In this study, we used two approaches to map the regions of gI-1 required for formation of the HSV-1 Fc receptor for monomeric IgG. First, we constructed six plasmids encoding gD-1/gI-1 fusion proteins. Each fusion protein contains a large gI-1 peptide inserted into the ectodomain of gD-1. gD-1/gI-1 fusion proteins were coexpressed with gE-1 using a transfection-infection assay in which cells were transfected with individual fusion protein constructs and then infected with a gE+/gI- virus. Cells were then assayed for monomeric IgG binding using immunofluorescence microscopy. Transfection-infection with two of six fusion proteins conferred monomeric IgG binding activity to cells, whereas cells infected with gE+/gI- virus alone failed to bind IgG monomers. The smallest gI-1 peptide to confer monomeric IgG binding activity contained amino acids 43 to 192. To more precisely map the region of gI-1 required for monomeric IgG binding, we constructed a panel of 10 gI-1 linker insertion mutants. Transfection-infection studies identified two mutants containing linker insertions at gI-1 amino acids 128 and 145, which failed to bind monomeric IgG. The other eight mutants demonstrated wild-type IgG binding activity. Taken together, these results indicate that the region of gI-1 between amino acids 128 and 145 is required for formation of the HSV-1 Fc receptor for monomeric IgG.
Analysis of the contact sites on the CD4 molecule with class II MHC molecule: co-ligand versus co-receptor function.
Authors: Huang B , Yachou A , Fleury S , Hendrickson W A , Sekaly R P
Published: December 2022

The CD4 molecule interacts with the alpha2 and beta2 domains of the MHC class II molecules. However, the class II contact sites on CD4 are less clear. Involvement of different regions throughout D1, D2, and D3 domains have been suggested. To further delineate the class II MHC contact sites on CD4, a crystal structure-informed mutagenesis was performed. Alanine scan mutants were generated for exposed residues located throughout D1 and the FG loop of D2, and in the "hinge-like" region, a short and flexible region between D2 and D3. Mutants were tested in a co-ligand (Dd stimulation) and a co-receptor (staphylococcal enterotoxin B stimulation) assay. In the co-ligand assay, TCR and CD4 interact with two distinct ligands (Dd or HLA-DR), while in the co-receptor assay both molecules interact with the same ligand, namely HLA-DR. Results show that residues from both lateral faces of D1 and the FG loop of D2 are implicated in interaction with class II, although a bigger surface of CD4 was involved in co-receptor compared with co-ligand function. The potential involvement of residues on both the top and two side faces of CD4 is consistent with a bivalent model, which involves the interaction between a single CD4 and two class II molecules. Alternatively, our results can be interpreted with a model of a specifically organized CD4 and/or class II oligomerization event. Finally, results from the hinge region mutants revealed a very important role in maintaining the overall structural integrity of CD4, its topology, and function.
Regulation of IL-15 secretion via the leader peptide of two IL-15 isoforms.
Authors: Onu A , Pohl T , Krause H , Bulfone-Paus S
Published: December 2022

The secretion of IL-15, a potent modulator of T, B, and NK lymphocyte functions, is likely to be tightly controlled. Here, we show that human T lymphoblasts transcribe the IL-15 gene and generate an alternative splicing product that codes for the same amino acid composition as the mature IL-15 protein, but produces an IL-15 precursor protein with a shorter signal peptide. Both alternative splicing products are transcribed by non-IL-15-secreting lymphocytes, suggesting that IL-15 secretion is not primarily controlled at the level of transcription. We generated an in vitro system for correlating the expression, translation, and secretion of IL-15 or IL-15-IgG1 fusion protein. This revealed that the two isoforms of IL-15 or a truncated IL-15 variant, both alone and fused to human IgG1, are all transcribed and translated, but not efficiently secreted. After replacing the IL-15 leader peptide with a foreign one, translation and secretion clearly increase. These results suggest that IL-15 is mainly controlled at the level of translation and secretion.
Antigen-driven but not lipopolysaccharide-driven IL-12 production in macrophages requires triggering of CD40.
Authors: DeKruyff R H , Gieni R S , Umetsu D T
Published: December 2022

We demonstrated that two distinct pathways exist for the induction of IL-12 in APC. The first pathway for IL-12 production occurred during responses to T cell-dependent Ags such as OVA and required triggering of CD40 molecules on the APC. IL-12 production in this T cell-dependent system increased in direct proportion to Ag concentration and required TCR ligation but not CD28 costimulation. The second pathway occurred when bacterial products such as LPS or heat-killed Listeria monocytogenes were used to activate macrophages to produce IL-12 in the complete absence of T cells. In this second pathway, IL-12 production was completely independent of CD40 triggering. In both pathways, the presence of IFN-gamma was not required for induction of IL-12 synthesis when splenic adherent cells (SAC) from normal mice were used. However, addition of IFN-gamma to cultures of Th2 T cells and SAC increased IL-12 production two- to fivefold, and addition of rTNF-alpha with IFN-gamma further enhanced IL-12 production. The addition of TNF-alpha in the absence of IFN-gamma, however, had no effect on IL-12 production in the T cell-dependent pathway. Similarly, addition of TNF-alpha in the presence or the absence of IFN-gamma to cultures of LPS or heat-killed Listeria and SAC did not increase IL-12 production, but addition of IFN-gamma alone greatly enhanced IL-12 production, consistent with the idea that bacterial stimuli induce significant quantities of endogenous TNF-alpha production. These results indicate that the requirements for the induction of IL-12 production in T cell-dependent and T cell-independent responses differs mainly with regard to CD40 triggering. Furthermore, these results suggest that IL-12 production can be induced by bacterial products in patients with hyper-IgM syndrome who lack CD40 ligand expression and in those treated with soluble gp39 to interrupt CD40-CD40 ligand interactions.
Neutrophil CD18-dependent arrest on intercellular adhesion molecule 1 (ICAM-1) in shear flow can be activated through L-selectin.
Authors: Gopalan P K , Smith C W , Lu H , Berg E L , McIntire L V , Simon S I
Published: December 2022

Neutrophil emigration through endothelial cells under shear flow involves several adhesion processes including cell rolling, arrest, and transmigration. Rolling is mediated by selectins, while arrest and transmigration both require activated CD18 integrins. One mode of CD18 activation is via selectins expressed on neutrophils and endothelial cells. We have recently reported that cross-linking of L-selectin (CD62L) resulted in the rapid activation of CD18-dependent adhesion. In the current study, we examine whether binding of E-selectin (CD62E) and L-selectin can activate neutrophil CD18-dependent adhesion under shear flow. Human ICAM-1 (CD54) and E-selectin were co-transfected into L cells. Neutrophil capture, rolling, and arrest on these monolayers were quantitated in a parallel plate flow chamber at a wall shear stress of 2.0 dyne/cm2. Under these conditions, E-selectin supported cell capture and rolling on the monolayer, but did not trigger CD18-mediated cell arrest within 200 microm of rolling. However, when neutrophils were treated with anti-L-selectin mAb and cross-linked with a secondary mAb, approximately 50% of the cells arrested within 54 microm. Cell arrest was also observed in response to IL-8 stimulation. A subthreshold level of IL-8 in combination with L-selectin cross-linking potentiated the level of cell arrest due to either stimulus alone. The transition to cell arrest involved both LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18). Blocking either subunit alone failed to reduce arrest, while blocking both molecules with mAbs reduced the number to baseline levels. These data support the conclusion that L-selectin, but not E-selectin, can signal the transition from neutrophil rolling to cell arrest under shear flow.
Ig N-glycan orientation can influence interactions with the complement system.
Authors: White K D , Cummings R D , Waxman F J
Published: December 2022

This study was prompted by the paradoxical observation that a pair of dinitrophenyl-specific murine monoclonal IgG2a Abs had similar monosaccharide content and yet differed in their binding to lectins. The differential lectin-binding properties were lost when the Abs were denatured, suggesting that variations in lectin binding reflected the conformational accessibility of the N-glycans rather than intrinsic differences in the lectin binding capacity of the glycans themselves. This hypothesis was supported by experiments indicating that the degree to which the N-glycans on the Abs were reactive with beta-1,4-galactosyltransferase or susceptible to peptide N-glycosidase F corresponded directly to their relative accessibility to lectins. Moreover, the relative susceptibility to these enzymes and accessibility to lectins was inversely related to the capacity of the Abs to activate the classical pathway, suggesting that the orientation of the more accessible N-glycan might inhibit C1q binding. This hypothesis was supported by evidence that enzymatic cleavage of the more accessible N-glycan resulted in enhanced Clq, C4b, and C3b deposition. Conversely, removal of the less accessible N-glycan expressed by the other Ab inhibited C1q, C4b, and C3b deposition. The respective increase or decrease in C3b deposition on the two deglycosylated Abs was magnified when complement activation was performed in factor B-depleted serum, suggesting that N-glycan conformation primarily affects the classical pathway. Collectively, these data suggest that the orientation of the N-glycan expressed on Igs can profoundly influence interaction with the complement system.
Highly restricted TCR-alpha beta usage by autoreactive human T cell clones specific for DNA topoisomerase I: recognition of an immunodominant epitope.
Authors: Kuwana M , Medsger T A , Wright T M
Published: December 2022

Autoantibody responses to DNA topoisomerase I (Topo I) are highly specific to patients with systemic sclerosis (SSc). We recently demonstrated that Topo I-specific T cells are components of the T cell repertoire of patients with SSc and healthy individuals. These autoreactive T cells were essential for the Ag-specific activation of B cells resulting in anti-Topo I Ab production in vitro and therefore are believed to play a central role in autoantibody production. To characterize the Topo I-specific T cell, 15 T cell clones reactive with Topo I were generated from two patients with SSc and three healthy donors, all of whom shared the MHC class II allele DR11. All clones expressed a CD3+CD4+CD8- phenotype and were restricted by HLA-DR. When eight rTopo I fragments were tested individually as Ags, all clones responded to F5, which encodes amino acids 209 through 386 of Topo I, but not to F10, which encodes amino acids 209 through 276, indicating that one or more immunodominant epitopes on Topo I is located between amino acids 276 and 386. Analysis of TCR gene usage showed that the predominant V(alpha) segment of the functionally rearranged TCR-alpha gene was Vdelta5, which was used by seven clones. Most strikingly, all except one T cell clone had functional rearrangements of TCR beta-chain genes using the Vbeta120.la and Jbeta1.1 gene segments. Comparison of the CDR3 sequences of the TCRs revealed limited diversity, and, of note, all clones contained the amino acid motif PGGN (or minor variations) in the CDR3 of their TCR beta-chains. Furthermore, identical beta-chain CDR3 amino acid sequences were encoded by cDNAs generated from T cell clones derived from multiple individuals, including patients with SSc and healthy donors.
Nuclear import of HIV-1 DNA in resting CD4+ T cells requires a cyclosporin A-sensitive pathway.
Authors: Sun Y , Pinchuk L M , Agy M B , Clark E A
Published: December 2022

Using PCR to monitor early (LTR/LTR (long terminal repeat)) and late (LTR/gag) products of reverse transcription and the formation of HIV-1 LTR circles (indicating nuclear import), we explored the relationship between T cell activation signals and early events in the life cycle of HIV-1 infection. The combination of TCR ligation with either CD28 cross-linking or exogenous IL-2 was required for HIV-1 LTR circle formation in resting CD4+ T cells. Ligation of the TCR or CD28 receptors or addition of IL-2 alone did not induce this process. However, cross-linking the TCR, or IL-2 alone, unlike CD28 ligation, could induce the completion of viral reverse transcription. In contrast, the initiation of HIV-1 reverse transcription could occur in resting CD4+ T cells without any stimulation. Cyclosporin A (CsA), an inhibitor of T cell activation, completely blocked HIV-1 DNA nuclear import in activated CD4+ T cells. The completion of HIV-1 reverse transcription was blocked by CsA in infected CD4+ T cells activated by TCR ligation and IL-2, but not in cells stimulated by TCR and CD28 ligation. The costimulation of CD3 and CD28 mAbs in the presence of IL-2 could not overcome the CsA inhibitory effect on nuclear import of viral DNA. Therefore, the factor(s) involved in a CsA-sensitive pathway plays a critical role in HIV-1 DNA transport from the cytoplasm into the nucleus. Production and movement of HIV-1 DNA in resting CD4+ T cells require two signals: one signal from the TCR, which normally regulates the G0 to G1 transition, induces completion of viral reverse transcription; the other signal through CD28 or an IL-2R-dependent process is sensitive to CsA treatment and regulates viral DNA entry into the nucleus.
RAG1 and RAG2 in developing rabbit appendix subpopulations.
Authors: Fuschiotti P , Fitts M G , Pospisil R , Weinstein P D , Mage R G
Published: December 2022

The appendix of young rabbits is a site of primary heavy chain variable region-gene diversification and B cell selection. Appendix cells from 6- to 9-wk-old rabbits were stained and sorted for surface CD43 and IgM. We found that the CD43+IgM- and double-negative CD43-IgM- cells contained RAG1 transcripts and RAG2 protein. The presence of RAG gene products in appendix raised the possibility that pro-/pre-B cells were present in young rabbit appendix. Although an early suggestion that RAG2 plays a role in variable region-gene diversification by gene conversion in chicken bursa was not supported by studies of RAG2 protein in this tissue, we produced anti-rabbit RAG2 Abs to determine whether RAG2 protein was present in rabbit appendix, where cells that recently underwent gene conversion are found. We detected RAG2 protein in the four subpopulations of rabbit appendix lymphocytes, distinguished by surface CD43 and IgM markers. The appearance of RAG gene products during different stages of B cell maturation may reflect the function of the young rabbit appendix as a site of both B cell development and diversification.
Triggering of motile behavior in T lymphocytes via cross-linking of alpha 4 beta 1 and alpha L beta 2.
Authors: Hauzenberger D , Klominek J , Holgersson J , Bergström S E , Sundqvist K G
Published: December 2022

The mechanisms by which T lymphocytes are transformed from passively transported cells during circulation in the vascular system to actively migrating cells during extravasation are unknown. Therefore, the possibility that lymphocyte receptors are capable of inducing motility was investigated using a modified Boyden chamber assay. Cross-linking of alphaL beta2 and alpha4 beta1 on human T lymphocytes (T cell line and peripheral blood T cells) with immobilized mAbs induced motile behavior on fibronectin, laminin, collagen type IV, and poly-L-lysine. This induction of T cell migration was very potent and in most cases more efficient than pretreatment of the cells with phorbol esters. In contrast, control Abs to several other integrin- and non-integrin molecules present on T lymphocytes did not induce T cell migration. Anti-CD3 Abs themselves did not trigger motile behavior. However, anti-CD3 promoted T cell migration in the Boyden chamber system if present simultaneously with 40-kDa alpha4 beta1 binding fibronectin fragments or alphaL beta2 binding intercellular adhesion molecule-1/hIgG1Fc fusion proteins on the upper side of the filter. Abs to other surface components on T cells did not trigger motility when presented together with the 40-kDa fibronectin fragments or the intercellular adhesion molecule-1/hIgG1Fc fusion proteins. The induction of motile behavior could be blocked if the T cells were pretreated with Genistein and Calphostin C, indicating the involvement of a protein tyrosine kinase and protein kinase C-dependent signaling pathway in triggering of T cell motility via integrins. These results indicate that alphaL beta2 and alpha4 beta1 on T lymphocytes can selectively trigger motile behavior when cross-linked by their endothelial or extracellular matrix ligands. Furthermore, these data indicate that cross-linking of CD3 facilitates ligand binding and subsequent triggering of a motile phenotype by alphaL beta2 and alpha4 beta1.
CD4 ligands inhibit the formation of multifunctional transduction complexes involved in T cell activation.
Authors: Jabado N , Pallier A , Le Deist F , Bernard F , Fischer A , Hivroz C
Published: December 2022

Ligands binding to the CD4 molecule can inhibit TCR-mediated T cell activation. We have previously reported that transcription factors regulating the expression of the IL-2 gene, NF-AT, NF-kappaB, and AP-1, are targets of this inhibitory effect in an in vitro model using peripheral human CD4+ T cells activated by a CD3 mAb. Two T cell activation pathways involved in the regulation of these transcription factors, calcium flux and the p21ras pathway, were investigated as potential targets. Binding of HIV envelope glycoprotein gp160/gp120 or a CD4 mAb to the CD4+ T cells, prior to TCR/CD3 activation, inhibited the intracellular calcium elevation. This event strongly suggested an inhibition of PLCgamma1 activity. Tyrosine phosphorylation of PLCgamma1, induced by CD3 activation, was not affected, but its association with tyrosine-phosphorylated proteins, including a 62-kDa protein, was disrupted. This PLCgamma1-associated p62 was found to be immunoreactive to p62-Sam68 Abs. The activation-induced phosphorylation of two p21ras effectors, Raf-1 and Erk2, was inhibited by the CD4 ligands, indirectly pointing to inhibition of the p21ras activation pathway. In addition, we demonstrate that TCR activation of normal CD4+ T cells induced the formation of p120GAP and PLCgamma1-containing complexes. These complexes also contain other unidentified proteins. CD4 ligand binding induced a defective formation of these transduction complexes. This may result in inefficient signaling, partially accounting for the inhibitory effects of the CD4 ligands on both p21ras and calcium-activation pathways.
Class I- and class II-reactive TCRs coexpressed on CD4+ T cells both trigger CD4/CD8-shared and CD4-unique functions.
Authors: Asnagli H , Schmitt-Verhulst A M , Guimezanes A
Published: December 2022

CD4+ and CD8+ T cells emerge from thymic selection expressing a TCR restricted by MHC class II (TCRII) and MHC class I (TCRI), and upon Ag stimulation develop respectively into Th and CTL effector cells. The influence of thymic differentiation and antigenic stimulation on the determination of T cell functions was studied, with CD4+ T cells expressing a transgenic TCRI that reacts with the class I alloantigen H-2K(b) in a CD8-independent fashion. Such T cells additionally express a TCR, probably TCRII, in which the transgenic TCR beta-chain is associated with endogenously rearranged TCR alpha-chains. Upon in vitro stimulation with H-2K(b)-expressing cells, both CD8+ and CD4+ transgenic TCR+ T cells developed into CTL capable of killing Ag-expressing target cells through a perforin-dependent mechanism, and secreted IL-2 and IFN-gamma. Fas ligand-dependent killing could also be induced in both CD8+ and CD4+ in vitro stimulated T cells. The capacity to secrete IL-4 was restricted to the CD4+ T cells, however, suggesting that both CD8/CD4-shared and CD4-unique programs can be elicited by stimulation of CD4 T cells through a TCRI. Acquisition of CTL function was also induced upon class II alloantigen stimulation through the endogenously rearranged TCRII, which represents a polyclonal set of TCRs. IL-2, IFN-gamma, and after restimulation, IL-4, were also produced. Thus: 1) events associated with intrathymic selection influence the gene program activated in response to the same TCRI/APC interaction; and 2) CD4+ T cells expressing a TCRI and a TCRII can activate the same gene program after engagement of either one of these TCRs.
gamma1 heavy chain transgenes are responsive to IFN-gamma repression and CD40 ligation.
Authors: Elenich L A , Ford C S , Collins J T , Dunnick W A
Published: December 2022

The cis-acting elements that regulate production of germ-line transcripts from Ig heavy chain genes and subsequent switch recombination to those genes are poorly defined. We reported that a 17-kb transgene that includes Igamma1, Sgamma1, and Cgamma1 is regulated for germ-line transcription like the endogenous gamma1 gene. Transcripts from such transgenes are expressed only in B cells treated with both LPS and IL-4, and not in B cells treated with LPS alone or in thymocytes or nonlymphoid tissues. We have now found that transcripts from these transgenes are induced by treatment of transgenic B cells by IL-4 alone. As reported by others, IFN-gamma acts to inhibit the IL-4-mediated induction of germ-line transcripts of the endogenous gamma1 gene. We have found that LPS-plus IL-4-induced germ-line transcription of gamma1 transgenes is likewise inhibited by treatment of B cells with IFN-gamma, so the gamma1 gene must include the cis-acting element(s) that confers this inhibition. It is also known that CD40 ligation induces a modest amount of germ-line transcripts from the endogenous gamma1 gene and synergizes with IL-4 to induce large amounts of germ-line transcripts. The gamma1 transgenes are likewise induced by CD40 ligation, suggesting that the response element(s) for CD40 ligation can be found in the gamma1 gene. The promoter region for the germ-line transcripts and the I exon are likely to include some of these cis-acting elements. A series of transgenic mice with the promoter/Igamma1 region conferred low level, lymphoid-specific, RNA expression to a reporter gene, including significant expression in thymocytes. However, the promoter/Igamma1 transgenes were not regulated like the endogenous gamma1 gene, in that transcription in splenic B cells was not increased by LPS plus IL-4.
Costimulation of T cell proliferation by a chimeric B7-2 antibody fusion protein specifically targeted to cells expressing the erbB2 proto-oncogene.
Authors: Gerstmayer B , Altenschmidt U , Hoffmann M , Wels W
Published: December 2022

T cells require at least two signals for activation and clonal expansion. The first signal conferring specificity is initiated by interaction of the T cell receptor with antigenic peptides in the context of MHC molecules. The second, costimulatory signal can be provided by cell surface molecules on APCs such as B7-1 (CD80) and B7-2 (CD86), which interact with their counter-receptors on T cells. The absence of costimulatory signals presents one possible mechanism for tumor cells to escape immune surveillance. In experimental models transfection of B7 genes into tumor cells can result in T cell-dependent tumor rejection. We have developed a novel approach to direct the costimulatory B7-2 molecule to the surface of target cells. Our approach is based on a chimeric fusion protein that consists of the extracellular domain of human B7-2 fused to a single-chain Ab domain (scFv) specific for the ErbB2 protein, a type I growth factor receptor overexpressed in a high percentage of human adenocarcinomas. This B7-2(225)-scFv(FRP5) molecule expressed in the yeast Pichia pastoris and purified from culture supernatants is functionally active and binds to B7 counter-receptors and to ErbB2. B7-2(225)-scFv(FRP5) localizes specifically to the surface of ErbB2-expressing target cells, thereby providing a costimulatory signal that results in enhanced proliferation of syngeneic T cells. Our results suggest that effective tumor vaccines for cancer immunotherapy could be created by targeting such chimeric ligands to the surface of tumor cells.