bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular analysis of the cytoplasmic domain of CD4: overlapping but noncompetitive requirement for lck association and down-regulation by Nef.
Authors: Gratton S , Yao X J , Venkatesan S , Cohen E A , Sékaly R P
Published: January 2023

Expression of the HIV Nef protein results in the down-regulation of cell surface expression of CD4, with a di-leucine motif in the cytoplasmic domain of CD4 being required for this effect. However, our results indicate that this motif is not sufficient to confer sensitivity to down-regulation by Nef. Using site-directed mutagenesis and a transient expression system, we demonstrate that an alpha-helical stretch of amino acids of the cytoplasmic tail of CD4 is also required for the down-regulation of CD4 induced by Nef. Some CD4 mutations allowed the discrimination between PMA- and Nef-induced down-regulation, suggesting the existence of multiple pathways. In addition, our results demonstrate that this motif is involved in the association of CD4 with the tyrosine kinase p56lck, thus defining a multifunctional domain of CD4. Although there is overlap between the sequence requirement for lck association and susceptibility to Nef, we fail to detect any preferential decrease in lck association with CD4 when Nef is expressed during acute HIV infection. Altogether, these results demonstrate that there is an overlapping, but noncompetitive, sequence requirement in the cytoplasmic domain of CD4 for lck association and down-regulation by Nef.
Isolation from human placenta of the IgG transporter, FcRn, and localization to the syncytiotrophoblast: implications for maternal-fetal antibody transport.
Authors: Leach J L , Sedmak D D , Osborne J M , Rahill B , Lairmore M D , Anderson C L
Published: January 2023

The IgG transporter responsible for ferrying maternal IgG across the human placenta to fetal circulation has not been identified, although the human homologue of the neonatal rat Fc receptor (FcRn), a heterodimer with pH-dependent IgG affinity, structurally similar to MHC Class I molecules, was recently proposed as a candidate. Affirming this hypothesis, we describe herein the specific copurification from human placenta of 46- and 14-kDa proteins by IgG affinity at acid pH. The larger protein, characterized by its amino acid sequence and by immunoblot, is the alpha-chain of human FcRn (hFcRn). The smaller is beta2-microglobulin. Their coisolation by ligand affinity suggests that they comprise the hFcRn heterodimer. Placenta sections stained immunohistochemically with anti-hFcRn alpha-chain peptide Abs show extensive expression of hFcRn in the syncytiotrophoblast and traces in the endothelium and other unidentified cells of the villus stroma. We find alpha-chain mRNA by Northern analysis in human placenta and in human trophoblast-like cell lines (JEG-3, ED27) but not in a human myelocytic cell line (HL60). We suggest that the placental hFcRn heterodimer may transport IgG to the fetus by a mechanism in which maternal IgG is pinocytosed nonspecifically and then carried to fetal tissues by a pH gradient from acidic endosomes to the pH-neutral basolateral surface of the syncytiotrophoblast. Furthermore, the known characteristics of FcRn suggest a wider function, that it is the receptor postulated by Brambell in the 1960s to regulate tissue and serum IgG concentrations by controlling IgG transport and catabolism.
Involvement of peptide antigens in the cytotoxicity between 70-kDa heat shock cognate protein-like molecule and CD3+, CD4-, CD8-, TCR-alpha beta- killer T cells.
Authors: Takashima S , Sato N , Kishi A , Tamura Y , Hirai I , Torigoe T , Yagihashi A , Takahashi S , Sagae S , Kudo R , Kikuchi K
Published: January 2023

We previously reported that the 70-kDa heat shock cognate protein-like molecule (hsc70) is expressed on the cell surface along with the neoplastic transformation of rat fibroblast and that this molecule is recognized by CD3+, CD4-, CD8-, NKR-P1-, and TCR-alphabeta- T (DNT) killer cells in an MHC class I-unrestricted fashion. We investigated the mechanism of interaction between hsc70 and DNT cells. H-ras oncogene-transformed rat fibrosarcoma W31 cells expressed hsc70 on the cell surface in almost the same density when the cells were growing in a conventional 5% FCS (5% W31) as when the cell growth was inhibited in the cultivation with 1% FCS (1% W31). However, DNT cells lysed only 5% W31, but not 1% W31. Since these observations suggest that certain peptide Ags of the fast growing W31 cells may play a role in the interaction between hsc70 and DNT cells, we pulsed 1% W31 cells with trifluoroacetic acid (TFA)-extracted fast growing W31 tumor Ags of less than 3000 Da in molecular size. We also pulsed 1% W31 with TFA-extracted Ags from moderate growing W14 tumors and whole fetus tissues. Our data indicated that DNT cells were clearly cytotoxic to 1% W31 pulsed only with TFA-extracted Ags from W31 tumors. Anti-rat hsc70 mAb completely blocked this cytotoxicity. In addition, pronase K treatment of Ags clearly inhibited the cytotoxicity by DNT cells. Taken together, these data suggest that the complex of peptide Ag and hsc70 is involved in the cytotoxic mechanism between hsc70 and DNT cells.
Kinetics of generation and persistence on membrane class II molecules of a viral peptide expressed on foreign and self proteins.
Authors: Bot A , Bot S , Antohi S , Karjalainen K , Bona C
Published: January 2023

We had previously shown that a genetically engineered Ig expressing an immunodominant CD4+ T epitope corresponding to the 110-120 amino acids of influenza virus hemagglutinin (HA), activated T cells more efficiently than the synthetic peptide. Taking advantage of a T cell hybridoma specific for HA 110-120 and transfected with a reporter gene under the IL-2 promoter, we studied the kinetics of generation and persistence on surface MHC-II of the HA 110-120 peptide derived from various carriers. Our results show that the generation rate of immunogenic MHC II-peptide complex is dependent on the nature of the carrier. Abs specific for HA 110-120 peptide or for other epitopes on HA affect through various mechanisms the presentation of HA 110-120 peptide to T cells. The persistence of immunogenic MHC II-peptide complex on the surface of APC is not dependent on the nature of the carrier and correlates with the half-life of class II molecules, suggesting an irreversible binding of peptides to MHC-II molecules in 2PK3 murine B lymphoma cells.
Molecular interactions between transfected human TCR, immunodominant myelin basic protein peptide 152-165, and HLA-DR13.
Authors: Hastings A E , Hurley C K , Robinson E D , Salerno K , Hernandez E , Richert J R
Published: January 2023

Chimeric TCR transfectants expressing human extracellular sequences and murine intracellular/transmembrane sequences were generated to analyze the trimolecular interaction between myelin basic protein (MBP) autoantigen, HLA, and a TCR isolated from a patient with multiple sclerosis. Chimeric transfectants responded to TCR activation by CD3- and TCRBV22S1-specific mAbs and by superantigen. Additionally, chimeric transfectants responded to autoantigen-specific activation with MBP 152-165 when presented by DR(alpha,beta1*1301) independent of the CD4 adhesion molecule. Transfectants did not respond to Ag presented by other HLA-DR molecules, including the closely related DR(alpha,beta1*1302). In peptide-binding studies with a panel of serial alanine-substituted MBP peptides, HLA contact residues necessary for anchoring MBP 152-165 to DR(alpha,beta1*1301) were also defined: 154 (F), 159 (R), and 162 (R). The chimeric TCR transfectant's differential response to a similar panel of MBP analogues defined residues that interact with the TCR: 153 (I), 155 (K), 156 (L), 160 (D), and 161 (S). Analysis of molecular interactions, such as those described in this work, may be central to developing new strategies for suppressing Ag-specific responses in human autoimmune disease.
Detection of Borrelia burgdorferi-specific CD8+ cytotoxic T cells in patients with Lyme arthritis.
Authors: Busch D H , Jassoy C , Brinckmann U , Girschick H , Huppertz H I
Published: January 2023

T cells are believed to play an important role in the pathogenesis of Lyme arthritis (LA), an inflammatory joint disease caused by the spirochete Borrelia burgdorferi (Bb). The presence or absence of certain Bb-specific CD4+ T helper cells has been associated with prognosis. Since recent observations suggested the activation of CD8+ T cells during infection with Bb, we searched for CD8+ cytotoxic T lymphocytes in patients with LA. CD8+ T cell lines were generated from peripheral blood and synovial fluid of five patients with LA. In addition, CD8+ T cells were expanded by Ag-specific stimulation in bulk cultures. A cytotoxicity assay was established using target cells infected with recombinant vaccinia viruses expressing the borrelial proteins outer surface protein (Osp) A, OspB, or flagellin. We found Bb-specific CTL lines derived from the peripheral blood of three patients with LA with specificity for flagellin, OspA, and OspB. All Bb-specific CTL lines were CD3+, CD8+, and TCRalphabeta, and cytotoxic activity was HLA class I restricted. Moreover, CD8+ T cells expanded by Ag-specific stimulation in vitro demonstrated Bb-specific and HLA class I-restricted lysis toward individual borrelial proteins. Interestingly, Bb-specific lytic activity was only detected in patient samples obtained after the disappearance of arthritis. We report the detection of Bb-specific cytotoxic CD8+ T cells in patients with LA. The induction of specific CD8+ T cells may play an important role in disease control and may have important bearings for the development of effective vaccines against Lyme borreliosis.
Natural resistance to intracellular infections: Nramp1 encodes a membrane phosphoglycoprotein absent in macrophages from susceptible (Nramp1 D169) mouse strains.
Authors: Vidal S M , Pinner E , Lepage P , Gauthier S , Gros P
Published: January 2023

The mouse Nramp1 gene (Bcg/Ity/Lsh) controls innate defense to infection with intracellular parasites such as Mycobacterium, Salmonella, and Leishmania. Sequence analysis of Nramp1 predicts a hydrophobic, membrane-associated protein expressed exclusively in monocyte/macrophage lineages. A single G169D substitution within the fourth predicted transmembrane domain of Nramp1 is associated with susceptibility to infection (Bcg) in inbred mouse strains. To initiate the biochemical characterization of the Nramp1 protein and to analyze the molecular basis of susceptibility associated with the Nramp1(D169) allele, oligopeptides derived from Nramp1 and two fusion proteins containing the first 54 and the last 35 residues of Nramp1 were used to raise specific anti-Nramp1 antisera. In addition, a c-Myc epitope (EQKLISEEDL) was introduced in-frame at the C terminus of Nramp1 to follow its expression in a yeast heterologous system. Western analysis of crude membrane fractions from yeast demonstrated that Nramp1 is indeed an integral membrane protein (resistant to urea extraction). In resident Bcg(r) (129sv, Nramp1(G169)) macrophages, one of the anti-Nramp1 antisera identified by immunoprecipitation a protein of 90,000 to 100,000 apparent m.w. that was absent in macrophages from knockout mice bearing a null mutation at Nramp1. The Nramp1 protein could be metabolically labeled with orthophosphate and was sensitive to glycosylase treatment, verifying that Nramp1 is an integral membrane phosphoglycoprotein. Parallel analysis of macrophage extracts from Bcg(s) mouse strains (C57BL6/J and BALB/c, Nramp1(D169)) failed to detect mature Nrampl protein in these cells, suggesting that the G169D mutation at Nramp1 prevents proper maturation or membrane integration of the protein, resulting in its rapid degradation.
Expression of type II nitric oxide synthase in primary human astrocytes and microglia: role of IL-1beta and IL-1 receptor antagonist.
Authors: Liu J , Zhao M L , Brosnan C F , Lee S C
Published: January 2023

In this work, we studied the expression of type II nitric oxide synthase (NOS) in primary cultures of human astrocytes and microglia. Cytokine-activated human fetal astrocytes expressed a 4.5-kb type II NOS mRNA that was first evident at 8 h, steadily increased through 48 h, and persisted through 72 h. The inducing signals for astrocyte NOS II mRNA expression were in the order IL-1beta + IFN-gamma > IL-1beta + TNF-alpha > IL-1beta. SDS-PAGE analysis of cytokine-stimulated astrocyte cultures revealed an approximately 130-kDa single NOS II band that was expressed strongly at 48 and 72 h (72 h > 48 h). Specific NOS II immunoreactivity was detected in cytokine-treated astrocytes, both in the cytosol and in a discrete paranuclear region, which corresponded to Golgi-like membranes on immunoelectron microscopy. In human microglia, cytokines and LPS failed to induce NOS II expression, while the same stimuli readily induced TNF-alpha expression. In cytokine-treated human astrocytes, neither NOS II mRNA/protein expression nor nitrite production was inhibited by TGF-beta, IL-4, or IL-10. In contrast, IL-1 receptor antagonist exerted near complete inhibition of NOS II mRNA and nitrite induction. Monocyte chemoattractant peptide-1 mRNA was induced in TGF-beta-treated astrocytes, demonstrating the presence of receptors for TGF-beta in astrocytes. These results confirm that in humans, cytokines stimulate astrocytes, but not microglia, to express NOS II belonging to the high output nitric oxide system similar to that found in rodent macrophages. They also show that the regulation of type II NOS expression in human glia differs significantly from that in rodent glia. A crucial role for the IL-1 pathway in the regulation of human astrocyte NOS II is shown, suggesting a potential role for IL-1 as a regulator of astrocyte activation in vivo.
Recombinant human IL-11 attenuates the inflammatory response through down-regulation of proinflammatory cytokine release and nitric oxide production.
Authors: Trepicchio W L , Bozza M , Pedneault G , Dorner A J
Published: January 2023

To elucidate the molecular mechanisms regulating the anti-inflammatory activities of recombinant human (rh)IL-11, the ability of rhIL-11 to reduce serum levels of inflammatory mediators such as TNF-alpha, IL-1beta, IL-12, and IFN-gamma in LPS-treated mice and to down-regulate macrophage function in culture was investigated. In a mouse model of endotoxemia, pretreatment with rhIL-11 blocked LPS-induced elevation of TNF-alpha, IL-1beta, and IFN-gamma serum levels, but had no effect on IL-12 p40, IL-6, or IL-10 serum levels. The effects of rhIL-11 on the production of inflammatory mediators in vivo may occur in part through direct interactions with macrophages. rhIL-11 pretreatment of thioglycollate-elicited peritoneal macrophages resulted in greater than 60% inhibition of LPS-induced production of TNF-alpha, IL-1beta, IL-12 p40, and nitric oxide. The activity of rhIL-11 was not mediated through induction of IL-10, IL-6, or TGF-beta1. These results indicate that the ability of rhIL-11 to modulate the inflammatory response is not dependent on known anti-inflammatory cytokines and substantiate a role for this cytokine in the attenuation of inflammatory conditions.
Env-specific CTL predominate in cats protected from feline immunodeficiency virus infection by vaccination.
Authors: Flynn J N , Keating P , Hosie M J , Mackett M , Stephens E B , Beatty J A , Neil J C , Jarrett O
Published: January 2023

Animal models of HIV-1 have a key role to play in elucidation of the cellular mechanisms responsible for protective immunity. Vaccination of domestic cats with whole inactivated feline immunodeficiency virus (FIV) elicits virus-neutralizing Abs and virus-specific CTL in the peripheral blood and lymphoid organs and affords protection from homologous virus challenge. In the present study we confirm the induction of virus-specific CTL following immunization with whole inactivated FIV vaccine and demonstrate that cats are protected for up to 1 yr following vaccination. Long term protection in vaccinated cats correlates with both higher levels of FIV Env-specific CTL in the peripheral blood following vaccination and the presence of FIV Env-specific memory CD8+ CTL in the lymph nodes, which persist for up to 1 yr following challenge in the absence of detectable virus. The CTL responses observed in vaccinated protected cats differ qualitatively from those in FIV-infected cats. The latter cats either do not generate a memory CTL response or exhibit a Gag-specific memory CTL response. These results show that the protective immunity observed in whole inactivated virus-vaccinated cats is associated with the induction of high levels of Env-specific CTL activity.
Molecular characterization of properdin deficiency type III: dysfunction produced by a single point mutation in exon 9 of the structural gene causing a tyrosine to aspartic acid interchange.
Authors: Fredrikson G N , Westberg J , Kuijper E J , Tijssen C C , Sjöholm A G , Uhlén M , Truedsson L
Published: January 2023

Inherited properdin deficiency is an X-linked recessive disorder clinically manifested by susceptibility to meningococcal disease. Deficiency of properdin is characterized by complete absence (type I), very low level presence (type II), or the presence of a dysfunctional properdin protein in serum as found in one Dutch family (type III). To better understand the dysfunctional protein on the molecular level, samples from three members of the Dutch family were analyzed by direct genomic sequencing. The sequence of the complete gene, including 10 exons and 9 introns, covering about 6500 bases was determined. The dysfunctional properdin was found to be caused by a single T to G mutation in exon 9, which gives rise to a substitution of a tyrosine by an aspartic acid residue at position 387. This change to a hydrophilic amino acid affects the function of the properdin molecule, although the oligomerization of dysfunctional properdin molecules was similar to that of normal properdin. In binding studies with C3b and properdin in serum, no properdin deposition was detected with the type III deficient serum. Inhibition studies with different decapeptides revealed distinct inhibitory sequences, and indicated also that the part of properdin containing the type III mutation was not directly involved in the binding to C3b. The mutation most likely causes conformational changes that make the properdin molecule dysfunctional by affecting its binding to C3b.
Ex vivo treatment of antigen-presenting cells with CTLA4Ig and encephalitogenic peptide prevents experimental autoimmune encephalomyelitis in the Lewis rat.
Authors: Khoury S J , Gallon L , Verburg R R , Chandraker A , Peach R , Linsley P S , Turka L A , Hancock W W , Sayegh M H
Published: January 2023

We used a novel approach to study the role of CD28-B7 T cell costimulatory blockade in experimental autoimmune encephalomyelitis (EAE) in the Lewis rat model. APCs were incubated in vitro with CTLA4Ig and the encephalitogenic peptide p71-90 of myelin basic protein. Systemic injection of APCs treated ex vivo with p71-90 and CTLA4Ig before immunization protected animals from clinical EAE. Systemic injection of APCs treated with CTLA4Ig alone, CTLA4Ig and control peptide, peptide alone, or peptide and control Ig was not protective. Injection of APCs treated ex vivo with CTLA4Ig and p71-90 on the day of immunization was also protective, but delaying the injection till day 7 after immunization impaired the protective effect. Immunohistologically, protected animals had decreased inflammatory responses, with inhibition of Th1 and sparing of Th2 cytokines in the brain. Preincubation of APCs with p71-90 and a mutant form of CTLA4Ig that binds only B7-1 also protected animals from developing EAE. These results suggest that ex vivo blockade of CD28-B7-1 leads to the generation of regulatory cells, presumably Th2, which inhibit the generation or priming of encephalitogenic T cells and suppress the autoimmune response to the specific Ag in vivo. These observations have therapeutic implications for autoimmune diseases and transplantation.
A CD4-CDR3 peptide analog inhibits both primary and secondary autoreactive CD4+ T cell responses in experimental allergic encephalomyelitis.
Authors: Marini J C , Jameson B A , Lublin F D , Korngold R
Published: January 2023

A structure-based design approach was used to develop a cyclized peptide analog of the murine CD4-CDR3-like region as a potential inhibitor of autoimmune CD4+ T cells responsible for the pathogenesis of experimental allergic encephalomyelitis (EAE). Our results indicate that this peptide, referred to as rD-mPGPtide, is able to significantly inhibit the clinical and pathologic symptoms of EAE in the SJL mouse model when administered on day 12 of induction. The optimum effective dosage range for the peptide, injected i.v., was between 0.125 and 0.5 mg and dosages of as high as 5 mg had no observable toxic effects. Treated mice had normal levels of lymphocytes less than 2 wk later and exhibited normal in vitro primary responses to alloantigen and secondary responses to keyhole limpet hemocyanin Ag. The specificity of the rD-mPGPtide treatment for autoreactive T cells was demonstrated by inhibiting proteolipid protein (p139-151)-induced EAE and finding that the lymph node T cells from these mice had suppressed responses to this Ag, but normal responses to alloantigen or other nominal Ag. Importantly, rD-mPGPtide was found to be effective on secondary T cell responses in an EAE rechallenge situation and was able to establish conditions for long-term resistance to further Ag exposure. Analysis of the cytokine profile of responding T cells during late effector stages of disease revealed that the levels of IFN-gamma and IL-4 are significantly reduced in rD-mPGPtide-treated mice. These results strongly suggest that the administration of a CD4-CDR3 peptide analog is an effective therapeutic approach for the inhibition of the CD4+ T cell-mediated autoimmune response in EAE.
Concurrent or sequential delta and beta TCR gene rearrangement during thymocyte development: individual thymi follow distinct pathways.
Authors: Margolis D , Yassai M , Hletko A , McOlash L , Gorski J
Published: January 2023

Analysis of TCR rearrangement profiles of well-defined thymocyte populations in a number of individual thymi provides evidence for a new pathway of lineage commitment. In all of the thymi analyzed, alphabeta thymocytes have rearrangements in the delta locus that are enhanced for out-of-frame rearrangements. Thus, not only did alphabeta thymocytes pass through a stage in differentiation that included delta rearrangement, but they also constitute a population that was relatively unsuccessful at these rearrangements. Interestingly, in some thymi, gammadelta thymocytes have out-of-frame beta rearrangements. This represents a novel pathway in which delta and beta rearrangement happens concurrently. In this pathway, selection favors whichever gene is in frame, thus improving the chances of generating useful T cells. In other thymi, gammadelta cells showed no obvious beta gene rearrangement, indicating a sequential rearrangement. Thus, alphabeta/gammadelta lineage commitment can follow at least two distinct pathways in different individuals.
The generation of MHC class I-associated peptides is only partially inhibited by proteasome inhibitors: involvement of nonproteasomal cytosolic proteases in antigen processing?
Authors: Vinitsky A , Antón L C , Snyder H L , Orlowski M , Bennink J R , Yewdell J W
Published: January 2023

The proteasome is believed to participate in the generation of a large percentage of peptide ligands for MHC class I molecules. This conclusion is based largely on the activities of peptidyl aldehydes that block proteasome activity. We tested the ability of a panel of proteasome inhibitors to affect the generation of MHC class I binding peptides in mouse L929 cells. Included in the panel are peptidyl aldehydes and a microbial product, lactacystin, that blocks proteasome activity in a distinct and more specific manner. Contrary to expectations, proteasome inhibitors failed to block the generation of a large portion of high affinity peptides as inferred by measuring cell surface expression of newly synthesized MHC class I molecules. These findings were confirmed by examining the effects of the inhibitors on the presentation of individual antigenic determinants from endogenously synthesized or exogenously delivered influenza virus proteins. Presentation of peptides derived from exogenous basic polymerase 1, endogenous basic polymerase 1, and nonstructural-1 proteins was decreased by inhibitors in a manner consistent with proteasomal involvement. Presentation of peptides derived from endogenous nucleoprotein was not significantly affected by the proteasome inhibitors, while presentation of exogenous hemagglutinin and nucleoprotein was enhanced by the proteasome inhibitors. These data are consistent with the involvement of both proteasomes and nonproteasomal cytosolic proteases in the generation of a significant portion of MHC class I binding peptides.
Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes.
Authors: Vremec D , Shortman K
Published: January 2023

Freshly isolated, mature dendritic cells (DC) from mouse lymphoid organs were analyzed by immunofluorescent labeling and flow cytometry to determine the number of discrete subpopulations and to assess possible lineage markers. The permanence of surface markers was then determined by overnight culture of the DC. Three DC subtypes were discerned, CD8alpha- DEC-205-, CD8alpha+ DEC-205+, and CD8alpha- DEC-205+, with different tissue distributions. The majority of DC expressed high levels of class II MHC, expressed CD11c, and expressed the costimulator molecules CD80, CD86, and CD40; CD80 and CD40 were further up-regulated on culture. DC also expressed low levels of L-selectin that were up-regulated on culture. Thymus contained predominantly CD8alpha+ DEC205+ CD11b- DC, resembling a major subpopulation of DC in other tissues but unique in expressing BP-1. Spleen contained predominantly two DC populations in equal proportions: one CD8alpha+ DEC-205+ CD11b- as in the thymus, and the other CD8alpha- DEC-205- CD11b+. Lymph nodes contained the same two DC populations as in spleen, but in addition a third population of CD8alpha- DEC-205+ CD11b- DC. The CD8alpha expression of splenic DC subpopulations did not change on culture. Although DEC-205 was up-regulated on culture so all DC became positive, the difference in the level between subpopulations was maintained. However, CD11b was up-regulated on culture, so all subpopulations became positive and finally expressed equivalent levels. Some aspects of this complex, but discrete, pattern of surface marker expression can be correlated with differences in lineage origin and functional activity of the DC.
Induction of tolerance to human gamma-globulin in FcR gamma- and Fc gammaRII-deficient mice.
Authors: Whitmer K J , Romball C G , Weigle W O
Published: January 2023

FcR gamma-deficient mice were used to examine the role of Fc gamma receptors in the induction of peripheral tolerance to human gamma-globulin (HGG). FcR gamma-deficient mice injected with HGG in adjuvant demonstrated a CD4+ T cell response to in vitro challenge with HGG, as assayed by proliferation, cytokine secretion, and Ag-specific help for B cell Ab production. In vitro kinetics of Ag-specific proliferation were similar in both conventional and knockout mice. Peripheral tolerance could be established in these mice with a single dose of deaggregated protein, despite the lack of functional Fc gammaRI, the high affinity receptor for monomeric IgG. Establishment of unresponsiveness was observed at both the T and B cell levels. T cell tolerance was manifested in the reduction of T cell helper function and Ag-induced release of Th1- and Th2-like cytokines, as well as decreased proliferation to Ag-specific stimulation. B cell tolerance was demonstrated in knockout and normal mice by failure to detect HGG-specific Ab production using an immunization protocol for Ab production that bypasses the need for Ag-specific T cells. These results demonstrate that induction of tolerance in CD4+ cells to HGG does not require transduction of a signal through Fc gammaRI. Furthermore, the ability to induce tolerance to HGG in B cells in Fc gammaRII-deficient mice suggests that down-regulation of Ag-specific B cells through Fc gammaRII is not the mechanism by which B cell tolerance is induced. However, Fc gammaRII plays a role in regulating the immune response since the Ab response to immunogenic HGG in Fc gammaRII-deficient mice is markedly enhanced.
Increased lymphocyte apoptosis in Fas ligand transgenic mice.
Authors: Cheng J , Liu C , Yang P , Zhou T , Mountz J D
Published: January 2023

Fas ligand (fasL) transgenic (Tg) mice were produced by introducing a murine fasL cDNA under the control of a TCR beta-chain enhancer minigene. Higher levels of fasL expression with increased biologic activity were observed in the Tg mice compared with non-Tg mice. Numbers of CD4+ CD8+ T cells in the thymus and T cells in the lymph node and spleen were lower in the fasL Tg mice compared with the non-Tg mice. This is consistent with a reduction in the size of the T cell areas in fasL Tg mice compared with non-Tg mice. Conversely, in fasL Tg mice, there was an increase in the number and size of apoptotic foci associated with phagocytic cells, as determined by in vivo TUNEL (TdT-mediated dUTP nick-end labeling) staining. Stimulation of non-Tg mice in vivo with anti-CD3 Ab for 3 days resulted in greatly increased apoptosis of CD4+ CD8+ thymocytes and lymph node T cells. Surviving thymocytes and T cells of lymph node and spleen expressed Fas at low levels. After similar stimulation of fasL Tg mice, however, a discreet population of surviving cells expressed high levels of Fas, indicating that a novel population of Fas apoptosis-resistant cells develops in these mice. These results indicate that high levels of fasL can result in both increased Fas-mediated apoptosis and the development of T cells that express high levels of Fas, but are resistant to Fas-mediated apoptosis.
Evolution of proteasome subunits delta and LMP2: complementary DNA cloning and linkage analysis with MHC in lower vertebrates.
Authors: Nonaka M , Namikawa-Yamada C , Sasaki M , Salter-Cid L , Flajnik M F
Published: January 2023

The class II region of the mammalian MHC harbors two proteasome subunit genes, LMP2 and LMP7. These genes are induced by IFN-gamma, and their products are incorporated into proteasomes substituting for their closest relatives, the delta and X subunits, respectively. This substitution is believed to change the proteolytic specificity of proteasomes, making it more suitable for generation of peptides to be presented by class I molecules. To elucidate the phylogenetic origin of LMP2 and the linkage of its gene with the MHC, reverse transcriptase-PCR amplification of Xenopus laevis and lamprey liver mRNA was performed with primers designed to amplify both the mammalian LMP2 and delta sequences. Both LMP2 and delta were amplified from X. laevis, whereas only delta was amplified from lamprey, suggesting that delta/LMP2 gene duplication occurred after divergence of cyclostomes but before divergence of amphibians. The linkage between the LMP2 gene and the MHC was observed in a diploid Xenopus species, Xenopus tropicalis, but not in a tetraploid species, X. laevis, indicating that this linkage was established before the divergence of amphibian from higher vertebrates, but that this linkage was lost in X. laevis, probably by a gene reorganization accompanying the tetraploidization. The X. laevis LMP2 and LMP7 mRNA showed a similar tissue distribution, indicating that the genetic linkage is not required for apparently coordinated tissue-specific expression of these genes. Sequence and linkage analyses suggest that LMP2 may not play as vital a role as LMP7 in Ag presentation.
Deviated overexpression of TCR-beta, TCR-gamma, CD4, and CD8 on thymic lymphomas induced by 1-propyl-1-nitrosourea: destruction of the allelic exclusion of TCR-beta and expression of functional TCR-betagamma heterodimer on a lymphoma, cFTL53.
Authors: Kinebuchi M , Matsuura A , Ogiu T , Kikuchi K
Published: January 2023

Thymic lymphomas (FTLs) induced by the chemical carcinogen 1-propyl-1-nitrosourea (PNU) in F344 rats showed deviated overexpression of TCR-beta, TCR-gamma, CD4, and CD8. Even though most FTLs were in the CD4+ CD8+ stage, all FTLs expressed TCR-beta mRNA with TCR-gamma mRNA, but without TCR-alpha mRNA or TCR-delta mRNA. One of the FTLs, cFTL53, expressed two kinds of TCR-beta mRNA and two kinds of TCR-gamma mRNA, but did not express any mRNA of TCR-alpha or TCR-delta. Both alleles of TCR-beta loci were rearranged on cFTL53. cDNA cloning and sequencing analysis showed that one TCR-gamma mRNA, Vgamma4-Jgamma1-Cgamma1, and both TCR-beta mRNA, Vbeta2-Dbeta2-Jbeta2.1 and Vbeta19-Dbeta2-Jbeta2.1-Cbeta2, on cFTL53 were in the productive form, while the other TCR-gamma mRNA, Vgamma1-Jgamma4-Cgamma4L, was not. Both TCR beta-chains and a TCR gamma-chain were expressed on cFTL53, making a novel set of TCR-betagamma heterodimer. Cross-linking of TCR-betagamma heterodimer on cFTL53 resulted in a calcium flux, indicating that TCR-betagamma works as a signal transduction receptor. Thus, there are four strange phenomena on FTLs; CD4 and CD8 are expressed without TCR-alphabeta or TCR-gammadelta, TCR-beta mRNA and TCR-gamma mRNA were expressed simultaneously without TCR-alpha and TCR-delta mRNA on FTLs, the allelic exclusion of TCR-beta was destroyed in cFTL53, and a novel set of functional TCR-betagamma heterodimer was expressed on cFTL53.
Human colorectal cancer (CRC) antigen CO17-1A/GA733 encoded by adenovirus inhibits growth of established CRC cells in mice.
Authors: Li W , Berencsi K , Basak S , Somasundaram R , Ricciardi R P , Gönczöl E , Zaloudik J , Linnenbach A , Maruyama H , Miniou P , Herlyn D
Published: January 2023

The human colorectal carcinoma (CRC)-associated Ag CO17-1A/GA733, originally defined by mAbs CO17-1A and GA733, has been a useful target in passive immunotherapy of CRC patients with mAb and in active immunotherapy with anti-idiotypic Abs mimicking the CO17-1A or GA733 epitope. Both approaches have targeted single epitopes. We investigated the capacity of full-length CO17-1A/GA733 Ag expressing multiple potentially immunogenic epitopes and encoded by recombinant adenovirus 5 (Ad5 GA733-2) to induce humoral, cellular, and/or protective immunity in mice. Ad5 GA733-2 induced Ag-specific Abs that reacted predominantly to CO17-1A- and GA733-unrelated epitopes on the Ag and lysed Ag-positive CRC targets in conjunction with effector cells. Ad5 GA733-2-immune mice developed Ag-specific, proliferative lymphocytes of Th1 type and cytolytic lymphocytes. The use of Ad5 GA733-2 to immunize mice bearing established syngeneic CRC cells transfected with the human Ag induced significant and specific tumor regression. Cured mice resisted rechallenge with human CO17-1A/GA733 Ag-negative parental CRC cells, suggesting that targeting the human Ag on the murine transfectants induced protective immunity to other Ag expressed by the parental tumor. These results may explain the high potency of the recombinant vaccine. Thus, rAd5 GA733-2 may have potential as a vaccine for CRC patients.
IL-4 protects against TNF-alpha-mediated cachexia and death during acute schistosomiasis.
Authors: Brunet L R , Finkelman F D , Cheever A W , Kopf M A , Pearce E J
Published: January 2023

To examine the role of the Th2-type response during schistosomiasis mansoni we compared disease progression in wild type (wt), and Th2-response deficient IL-4(-/-) mice. Whereas wt C57BL/6 mice tolerate infection and develop chronic disease, IL-4(-/-) C57BL/6 animals are highly susceptible, exhibiting severe acute cachexia followed by death. Data point toward morbidity in the IL-4(-/-) C57BL/6 mice being mediated by TNF-alpha, possibly through the uncontrolled production of nitric oxide in target organs such as the ileum. We propose that IL-4 prevents severe disease during schistosomiasis by regulating macrophage activation.
Activation of phosphatidylinositol 3'-kinase by insulin-like growth factor-I rescues promyeloid cells from apoptosis and permits their differentiation into granulocytes.
Authors: Liu Q , Schacher D , Hurth C , Freund G G , Dantzer R , Kelley K W
Published: January 2023

Insulin-like growth factor-I (IGF-I) promotes cell division and prevents programmed cell death in hemopoietic progenitors. Human HL-60 promyeloid cells differentiate toward the granulocytic lineage when stimulated with retinoic acid (RA) in serum-containing medium. When deprived of serum, however, we found that these cells differentiate poorly in the presence of RA, as assessed by expression of the alpha subunit of the beta2 integrin heterodimer, CD11b/CD18. However, when IGF-I is added to RA-treated cells, the proportion of CD11b-positive cells increases to a level similar to that in RA-treated cells cultured in serum-containing medium. Cells treated with RA alone not only differentiate poorly but also undergo apoptosis, as assessed by flow cytometry using propidium iodide and HO33342. In serum-free medium, one-third of RA-treated cells become apoptotic compared with only 5% apoptotic cells in the absence of RA. However, addition of IGF-I to RA-treated cells prevents the appearance of this apoptotic population and increases phosphatidylinositol 3'-kinase (PI 3-kinase) activity by fivefold. Wortmannin, a PI 3-kinase inhibitor, potently decreases this IGF-I-induced lipid kinase activity, blocks the ability of IGF-I to prevent apoptosis, and inhibits IGF-I-enhanced CD11b expression. These data demonstrate that IGF-I acts on RA-treated progenitors to promote their differentiation along the granulocytic lineage. IGF-I acts by rescuing these cells from apoptotic cell death via a downstream pathway that is dependent upon PI 3-kinase.
Cytokine-induced neutrophil-mediated injury of human endothelial cells.
Authors: Bratt J , Palmblad J
Published: January 2023

To understand the pathogenesis of vasculitides, we analyzed how cytokine stimulation of HUVEC in vitro activates the cytotoxic capacity of polymorphonuclear (PMN) granulocytes. IL-1beta, IFN-gamma, or TNF-alpha caused highly significant dose and time-dependent HUVEC injury. TNF-alpha-treated HUVEC activated the PMN by means of phospholipase C-related event, since coincubations conferred PMN to react with a rise of cytosolic calcium concentrations, [Ca2+]i. Ab blockade of ICAM-1 on HUVEC inhibited 50 to 70% of the injury induced by these cytokines, whereas a mAb to E-selectin reduced 45 to 65% of IL-1beta- and TNF-alpha-, but not IFN-gamma-induced cytotoxicity. The role of nitric oxide (NO) was of significance since injury induced by each cytokine was reduced by 60 to 87% by specific NO-synthase inhibitors, as well as by scavenging extracellular NO by oxyhemoglobin. In contrast, injury induced by TNF-alpha was inhibited by neither superoxide dismutase or catalase, alpha1-antitrypsin, alpha2-macroglobulin, nor the platelet-activating factor receptor antagonist WEB-2086. Moreover, PMN from a patient with chronic granulomatous disease were fully capable of mediating cytotoxicity. The possibility that IL-8, produced by HUVEC in response to TNF-alpha, mediated activation of PMN was not corroborated since addition of an IL-8-blocking mAb did not modify HUVEC injury. Nonetheless, the IL-8 mAb (but not WEB-2086) blocked the rise of [Ca2+]i. Thus, in this in vitro model of vasculitis, the effect of IL-1beta, IFN-gamma, and TNF-alpha as promotors of cytokine-mediated neutrophil-dependent injury to HUVEC is a process dependent on expression of adhesion molecules and probably associated with NO produced in the system.
Roles of CD9 molecules in survival and activation of human eosinophils.
Authors: Kim J T , Gleich G J , Kita H
Published: January 2023

CD9 is a cell surface glycoprotein belonging to the transmembrane 4 superfamily. In this report, we demonstrate that cross-linking CD9 with different forms of mAb activates distinct functions of human eosinophils. Anti-CD9 mAb (clone ALB6) immobilized onto tissue culture plates induced eosinophil degranulation. This effect of anti-CD9 mAb (clone ALB6) was unique because other immobilized clones of anti-CD9 mAb (clones FMC56 and ML13) or anti-HLA Class I mAb failed to induce degranulation. In addition, neutrophil degranulation was not provoked by the immobilized clone ALB6, consistent with a lack of expression of CD9 on neutrophils. Eosinophil degranulation induced by anti-CD9 mAb (clone ALB6) was abolished by pretreatment of eosinophils with anti-CD18 mAb, suggesting that beta2 integrins are involved in the reaction. In contrast to the results with immobilized clones, all clones of anti-CD9 mAb in soluble form enhanced eosinophil survival. This enhanced survival was inhibited by anti-granulocyte-macrophage-CSF (GM-CSF) mAb, suggesting that autocrine production of GM-CSF by eosinophils mediated the enhanced survival. In fact, by ELISA and RT-PCR, GM-CSF protein and mRNA were detected in supernatants and cell lysates of eosinophils stimulated by soluble anti-CD9 mAb, but not by immobilized mAb. These results indicate that CD9 serves as a molecule that delivers stimulation signals on eosinophils. The eosinophil's cellular responses may differ and be dependent on the manner of receptor ligation.
Endotoxin shock in antibody-deficient mice: unraveling the role of natural antibody and complement in the clearance of lipopolysaccharide.
Authors: Reid R R , Prodeus A P , Khan W , Hsu T , Rosen F S , Carroll M C
Published: January 2023

While the significance of natural Ab is not entirely clear, one proposed role is clearance of bacterial Ags. To determine whether natural Ab was involved in clearance of endotoxin, we have examined novel strains of mice with either a total or selective deficiency in Ig. Recombinase-activating gene-2 (RAG-2(-/-))-deficient mice, which have no serum Ig due to arrested development of B cells at the pro-B stage, demonstrate increased sensitivity to endotoxin that correlates with an impaired clearance. When RAG-2(-/-) mice are reconstituted with pooled sera from normal mice, both survival and clearance of circulating endotoxin are enhanced. To further define the nature of the protective Ab, Bruton's tyrosine kinase (Btk)-deficient mice were characterized in the high dose LPS model. Like RAG-2(-/-) mice, they are highly sensitive to endotoxin and have an impaired clearance of LPS. Reconstitution of Btk(-/-) mice, which have reduced levels of IgG3 and IgM, with purified normal mouse IgM dramatically enhances their ability to clear endotoxin compared with mock (saline)-reconstituted littermates. The cellular source of natural anti-LPS IgM was identified as the peritoneal-residing B-1 cell by enzyme-linked immunospot (ELISPOT) assay. Taken together, these studies demonstrate the important role of natural Ab and complement in the clearance of pathogenic substances from the circulation.
Effect of TGF-beta1 on cell cycle regulatory proteins in LPS-stimulated normal mouse B lymphocytes.
Authors: Bouchard C , Fridman W H , Sautès C
Published: January 2023

The cell cycle events accompanying TGF-beta1-induced growth arrest of normal mouse resting B lymphocytes stimulated by LPS were investigated. We showed that TGF-beta1 prevents the retinoblastoma protein (pRb) phosphorylation and induces growth arrest in mid- to late G1. To explore the molecular basis of the effect of TGF-beta1, we analyzed the in vitro kinase activities of cyclin/cyclin-dependent kinase (cdk) complexes involved in the progression through G1 phase and in the G1/S transition, by using the glutathione S-transferase-pRb fusion protein as a substrate. Cdk2-associated kinase activity was strongly induced in mitogen-treated B cells. It was dramatically inhibited by TGF-beta1 as were the cyclin E- and cyclin A-dependent kinase activities. TGF-beta1 treatment had no significant effect on the expression of two G1/S phase proteins, cyclin E and cdk2. In contrast, the appearance of cyclin A, occuring in late G1 phase, was almost totally inhibited by TGF-beta1. We also showed that expression of the cdk inhibitor protein p27Kip1 decreased as cells progressed through the G1 phase. An accumulation of p27 was found in TGF-beta1-treated cells, showing that TGF-beta1 prevented LPS-induced decline of p27. Finally we found that the lack of kinase activity associated with cyclin E/cdk2 complexes was correlated with increased amounts of cdk2- and cyclin E-bound p27. Overall, these results suggest that both cyclin A and cdk2 may be active participants in the TGF-beta1-induced cell cycle arrest in normal mouse B cells and indicate the involvement of p27 in this mechanism.
Altered peptide ligands trigger perforin- rather than Fas-dependent cell lysis.
Authors: Bachmann M F , Ohteki T , Faienza K M , Zakarian A , Kägi D , Speiser D E , Ohashi P S
Published: January 2023

CTLs lyse Fas-expressing target cells by the concomitant action of a perforin- and a Fas-dependent mechanism. This study analyzed whether target cells pulsed with T cell antagonists and other altered peptide ligands (APLs) were susceptible selectively to only one of these two mechanisms. In vivo and in vitro activated T cells from transgenic mice expressing a TCR specific for lymphocytic choriomeningitis virus were used as effector cells. To distinguish between perforin- and Fas-dependent cytotoxicity, T cells from normal or perforin-deficient mice were used to lyse peptide-pulsed Fas-positive or Fas-negative target cells. In contrast to previous reports that have shown that APLs selectively induce the Fas-dependent pathway of cytotoxicity, our results demonstrate that target cells pulsed with T cell antagonists and other APLs are lysed predominantly by the perforin-dependent pathway. The contribution of Fas-mediated cytotoxicity was similar for the full agonist and the APLs. Thus, full agonists, partial agonists, and antagonists trigger similar and not distinct pathways of cytotoxicity.
Differential tyrosine phosphorylation of inhibitory versus activating Ly-49 receptor proteins and their recruitment of SHP-1 phosphatase.
Authors: Mason L H , Gosselin P , Anderson S K , Fogler W E , Ortaldo J R , McVicar D W
Published: January 2023

Killer cell inhibitory receptors represent a family of p58/70-Ig-like proteins expressed on the surface of human NK cells. Engagement of class I MHC by killer cell inhibitory receptors turns off the lytic machinery of NK cells. This receptor/ligand interaction results in phosphorylation of intracellular tyrosine residues of p58/70 proteins. Murine NK cells express surface receptors of an unrelated family of type II lectin-like proteins, Ly-49, that have similar functions. Ly-49A, -C, and -G2 represent murine inhibitory receptors. However, Ly-49D functions as an activation receptor on the surface of NK cells. This dichotomy of function between Ly-49 family members suggested different signaling events upon receptor/ligand interaction. Here we demonstrate that: 1) in transfected Cos7 and murine NK cells, Ly-49A, -C, and -G2 are phosphorylated following pervanadate stimulation, whereas Ly-49D is not; 2) mAb-induced receptor ligation mediates tyrosine phosphorylation of Ly-49A and -G2, but not Ly-49D; 3) SHP-1 coprecipitates with Ly-49A and -G2 following receptor phosphorylation; and 4) tyrosine phosphorylation of Ly-49 inhibitory receptors depends on tyrosine residues restricted to the immunoreceptor tyrosine-based inhibitory motif. Our data further support the involvement of immunoreceptor tyrosine-based inhibitory motifs as crucial sequences regulating receptor-mediated inhibitory functions in NK cells.