bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective effect of neoglycoprotein-conjugated muramyl dipeptide against Leishmania donovani infection: the role of cytokines.
Authors: Sarkar K , Das P K
Published: December 2022

Active targeting of muramyl dipeptide (MDP) to macrophages was studied by conjugation with the neoglycoprotein, mannosyl human serum albumin (mannose-HSA) using visceral leishmaniasis as the model macrophage disease. Conjugation did not decrease the affinity of the neoglycoprotein for macrophage mannose receptor. Mannose-HSA-MDP was 50 times more efficient than free MDP in inhibiting the growth of Leishmania donovani inside peritoneal macrophages. Moreover, in a 60-day murine model of visceral leishmaniasis, 95% of the spleen parasite burden was reduced by mannose-HSA-MDP at a dose of 0.5 mg/kg/day given for 4 days. Free MDP at a similar dose had very little effect. In vitro exposure of MDP caused enhanced generation of O2- by macrophages, whereas generation of nitric oxide (NO) was not induced. The elevated antileishmanial activity of MDP-treated macrophages in culture was abrogated by O2- scavengers. In contrast, considerably enhanced amounts of NO and O2- were generated from macrophages of mannose-HSA-MDP-treated animals, and their splenocytes secreted soluble factors providing all the signals required for the induction of NO biosynthesis. The increase in NO production was paralleled by a concomitant increase in antileishmanial activity, which was reversed by NO synthesis inhibitors. Splenocyte supernatants treated with anti-IFN-gamma or anti-TNF-alpha Abs suppressed inducible NO generation by macrophages. Moreover, i.v. administration of anti-IFN-gamma and anti-TNF-alpha along with mannose-HSA-MDP greatly reduced protection against L. donovani infection. Neoglycoprotein-conjugated MDP, therefore, activated mouse macrophages in vivo to kill L. donovani, and this may depend on the physiologic generation of NO induced by IFN-gamma and TNF-alpha.
Intrinsic resistance to T cell infection with HIV type 1 induced by CD28 costimulation.
Authors: Riley J L , Carroll R G , Levine B L , Bernstein W , St Louis D C , Weislow O S , June C H
Published: December 2022

When HIV-infected leukocytes are activated by the CD28 costimulatory receptor, HIV-1 is rapidly cleared from cultures, suggesting that costimulation can render T cells resistant to HIV-1 infection. In this study we tested the hypothesis that enhanced secretion of cytokines or chemokines could account for CD28-induced antiviral effects. In an acute infection system, resistance to infection with macrophage-tropic strains of HIV-1 was shown to be comprised of both soluble and cell-associated components. Induction of HIV-1 resistance was specific for CD28 costimulation, in that a variety of other accessory receptors, such as CD2, CD4, CD5, and MHC class I, failed to confer the antiviral resistance. The soluble component was secreted by both CD4 and CD8 T cells, was not unique to CD28 costimulation, and could be neutralized by removal of C-C chemokines (RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory protein-1alpha and -1beta) from the culture supernatants of costimulated CD4 T cells. In contrast, CD28 stimulation of CD4 cells resulted in the specific induction of a pronounced intrinsic resistance to HIV-1 infection by macrophage tropic isolates of HIV-1.
A pronounced thymic B cell deficiency in the spontaneously diabetic BB rat.
Authors: Tullin S , Farris P , Petersen J S , Hornum L , Jackerott M , Markholst H
Published: December 2022

In an attempt to elucidate the origin of the T cell lymphopenia and/or the beta-cell-specific autoimmunity observed in diabetes-prone Bio-Breeding (DP-BB) rats, a thymic cDNA library was subjected to differential screening with thymic cDNA probes of DP-BB rats and nonlymphopenic nondiabetic controls. This approach resulted in the identification of a prominent lack of thymic B cells in DP-BB rats. This deficiency is distinct from a less pronounced peripheral B cell deficiency of different timing. The thymic B cell defect is linked to the lymphopenia trait on chromosome 4 and thereby with susceptibility to diabetes in crosses involving the DP-BB rat. In conclusion, our data suggest that the contribution of thymic B cells to the (negative) selection of thymocytes is inadequate in DP-BB rats, thus providing a plausible explanation for at least some of the spontaneous autoimmune phenomena in this animal model.
IL-2 induces Fas ligand/Fas (CD95L/CD95) cytotoxicity in CD8+ and CD4+ T lymphocyte clones.
Authors: Esser M T , Dinglasan R D , Krishnamurthy B , Gullo C A , Graham M B , Braciale V L
Published: December 2022

IL-2 is a T cell growth factor that has pleiotropic functions in T cell differentiation, induction of lymphokine-activated killer cells, and regulation of immune responses. In studying TCR triggering of perforin or Fas ligand (FasL)/Fas (CD95 ligand/CD95) cytotoxicity in our influenza-specific T cell clones, we found that IL-2 can also induce FasL/Fas cytotoxicity. IL-2 induces FasL/Fas cytotoxicity in our CD8+ and CD4+ Th1 clones, but not in our CD4+ Th2 clones. IL-2 induction of cytolytic activity occurs when the CD8+ T cells are refractory to IL-2-induced proliferation. This killing is Ag independent, MHC unrestricted, and blocked by Fas.Fc fusion protein. IL-2 induces FasL/Fas cytotoxicity in a dose-dependent manner, but does not induce high levels of FasL expression as detected by flow cytometry. TCR triggered FasL/Fas cytotoxicity is detectable in CD8+ and Th1 clones by 3 h and peaks at 6 h; high levels of killing are maintained for at least 24 h. Similarly, IL-2 induces FasL/Fas killing in CD8+ and Th1 clones within 3 h of stimulation and maintains high levels for at least 24 h. TCR-triggered FasL/Fas killing is inhibited by emetine and cyclosporin A, whereas IL-2-induced FasL/Fas killing is inhibited by emetine, but not by cyclosporin A. These results demonstrate a second mechanism to induce FasL/Fas cytotoxicity in CD8+ and Th1 clones and may explain IL-2 induction of Ag-independent MHC-unrestricted lymphokine-activated killer cell activity.
The TCR-delta repertoire in human intestine undergoes characteristic changes during fetal to adult development.
Authors: Holtmeier W , Witthöft T , Hennemann A , Winter H S , Kagnoff M F
Published: December 2022

The TCR-delta repertoire in adult human intestine is oligoclonal and unique in each individual. In the present study, changes in the junctional regions of TCR-delta transcripts in human intestine that occur during development from fetal to adult life were used to characterize fundamental changes in the TCR-delta repertoire in the human intestinal tract during ontogeny. At mid-gestation, the fetal repertoire was polyclonal, but limited, in its junctional diversity by the relative lack of N region nucleotide additions and by the frequent formation of coding region joins at regions of short sequence homology. In addition, identical TCRDV2 transcripts that resemble canonical TCR-delta sequences in mice were present in the intestine of different fetuses. In the early period after birth, the intestinal TCR-delta repertoire was polyclonal, and more diverse than the fetal repertoire, with junctional regions that contained extensive N nucleotide additions and frequently were as complex as those of adults. The intestinal TCR-delta repertoire showed increasing restriction with age and, by 14 to 17 yr, the repertoire was oligoclonal and resembled the repertoire of individuals in the sixth to seventh decade. Moreover, the adult TCR-delta repertoire was almost identical at multiple sites throughout the intestine, suggesting a model in which gammadelta T cell clones, selected by ligands in the intestinal tract, undergo expansion and recirculation before lodging throughout the small intestine or colon.
The regulation of p27Kip1 expression following the polyclonal activation of murine G0 T cells.
Authors: Kwon T K , Buchholz M A , Ponsalle P , Chrest F J , Nordin A A
Published: December 2022

Polyclonal activation of murine G0 T cells with immobilized anti-CD3 induces entry into the cell cycle as well as the subsequent cytokine-dependent proliferative response. G0 T cells express high levels of p27Kip1 protein and specific mRNA, which decline rapidly following activation. The decline in the expression of p27Kip1 and sequestering of the inhibitory protein by cdk4 and cdk6 correlated with the increase in cdk2 kinase activity during the G1 phase. Anti-CD3 activation of G0 T cells in the presence of cyclosporin A or rapamycin inhibited the down-regulation of p27Kip1, the cellular levels of the inhibitor remained high, and the cells remained in the G1 phase. PBu2 activation of G0 T cells also did not result in the down-regulation of p27Kip1 and the cells remained in G1. In each instance IL-2 restored the down-regulation of p27Kip1, resulting in a significant reduction in the level of the inhibitor, and stimulated the cells to progress through the cell cycle. Jurkat cells transfected with the p27GL-988 plasmid containing +1 to -988 nt of the p27Kip1 promoter region and subsequently exposed to rIL-2 resulted in a significant reduction in the activity of the p27Kip1 promoter. These findings suggest that in addition to providing the signals required for activated T cells to traverse G1/S, IL-2 also influences the promoter function of p27Kip1, which effectively induces transcriptional down-regulation of the gene.
Overexpression of the heat shock protein 70 enhances the TCR/CD3- and Fas/Apo-1/CD95-mediated apoptotic cell death in Jurkat T cells.
Authors: Liossis S N , Ding X Z , Kiang J G , Tsokos G C
Published: December 2022

To investigate whether the protective effects of the 70-kDa heat shock protein (hsp70) extend to the apoptotic mode of cell death, we transfected Jurkat T cells with the gene for the human hsp70 and challenged the cells with an anti-Fas mAb or with two different murine anti-CD3 mAbs. The anti-Fas mAb-triggered apoptotic cell death and the anti-CD3 mAb-mediated activation-induced cell death were significantly enhanced in the gene-transfected Jurkat cells overexpressing hsp70 compared with the unmanipulated and the vector-transfected cells. On the other hand, the well-established protective effect that this protein offers to the cells was unaffected, as determined by enhanced viability of gene-transfected cells exposed to a lethal heat shock. To investigate the mechanisms that are responsible for the increased susceptibility of the gene-transfected cells to apoptotic death, we studied the TCR/CD3-initiated events that showed a significant down-regulation of the protein tyrosine phosphorylation levels and the cytoplasmic free Ca2+ responses. As for the Fas/Apo-1/CD95-mediated early events, the activity of protein serine/threonine phosphatases was markedly increased in the cells overexpressing hsp70. Our study demonstrates that hsp70 overexpression offers thermoprotection but enhances TCR/CD3- and the Fas-induced apoptotic cell death. This phenomenon is associated with a down-regulation of the Ag receptor-initiated early signal transduction pathways and with an up-regulation of Fas-mediated early metabolic events.
Functional in vivo MHC class II loading by endogenously synthesized glycoprotein during viral infection.
Authors: Oxenius A , Bachmann M F , Mathis D , Benoist C , Zinkernagel R M , Hengartner H
Published: December 2022

MHC class II presentation of antigenic peptides derived from soluble proteins is usually preceded by antigenic uptake via (nonreceptor-mediated) endocytosis by professional APCs, followed by processing in endosomal compartments. Although in vitro alternative pathways for MHC class II loading have been described for certain intracellularly synthesized proteins, the importance of these pathways has not been assessed in vivo. We have shown previously that endogenously produced membrane-associated glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), a noncytopathic virus, can be presented in vitro on MHC class II molecules in the absence of the invariant chain (Ii), whereas the cytosolic LCMV nucleoprotein (LCMV-NP) failed to be presented under the same conditions. Taking advantage of this system, we analyzed presentation of LCMV-GP and LCMV-NP in vivo in Ii-deficient mice and followed the induced Th cell and B cell responses. At early time points after LCMV infection of li-deficient mice, we found in vivo MHC class II loading exclusively by the endogenously synthesized LCMV-GP, whereas no MHC class II loading by LCMV-NP could be detected. As a direct consequence, LCMV-specific Th cells exhibited initially only LCMV-GP specificity. In contrast, both LCMV-GP- and LCMV-NP-derived epitopes were presented in comparable amounts on APCs upon LCMV infection of normal mice, and LCMV-GP- as well as LCMV-NP-specific Th cells were comparably induced in vivo. Thus, cell internal MHC class II loading pathways are functional in vivo and may become dominant if the usual Ag presentation pathways are hampered.
TCR-gamma genes are rearranged but not transcribed in IL-7R alpha-deficient mice.
Authors: Perumal N B , Kenniston T W , Tweardy D J , Dyer K F , Hoffman R , Peschon J , Appasamy P M
Published: December 2022

IL-7, a cytokine produced by bone marrow and thymic stroma, is a growth factor for B and T lymphocytes very early in their development. The IL-7R is a heterodimer of an alpha-chain that specifically binds IL-7 and the common gamma-chain, gamma(c), which is also a component of the receptors for IL-2, IL-4, IL-9, and IL-15. IL-7 has also been hypothesized to play a role in the differentiation of gammadelta T cells, which is supported by the recent findings that mice deficient in the alpha-chain of the IL-7R (IL-7R alpha -/-) or IL-7 (IL-7 -/-) have a complete absence of gammadelta T cells, but not alphabeta T cells. We show in this work that Vgamma4 and Vgamma6 TCR genes are rearranged, and sterile Vgamma4 and Vgamma6 TCR-gamma transcripts are expressed in IL-7R alpha -/- thymocytes, but these TCR-gamma genes, and Vgamma5, are not transcribed in thymocytes from IL-7R alpha -/- mice. RAG-1 and RAG-2 genes are transcriptionally active in fetal and adult IL-7R alpha -/- thymocytes. The IL-7-inducible transcription factor, STAT5, is not active in the fetal thymus of IL-7R alpha -/- compared with IL-7R alpha +/+ mice. These data point to a specific role for IL-7/IL-7R signaling in regulating the transcriptional activity, possibly mediated by STAT5, of the rearranged TCR-gamma complex during development of gammadelta T cells, and point to mechanistic differences in the regulation of rearrangement of Vgamma4 and Vgamma6 genes vs Vgamma5.
Fas ligand-mediated bystander lysis of syngeneic cells in response to an allogeneic stimulus.
Authors: Smyth M J
Published: December 2022

Using a mouse allospecific lymph node (LN) CTL (H-2b) response to transplanted cells (H-2k), effector cells were shown to lyse C3H/HeJ or L929-Fas (H-2k) target cells in either a perforin-dependent or a Fas ligand (FasL)-dependent manner. C3H/HeJ.lpr targets or L929 targets lacking Fas were not sensitive to these effectors generated in perforin-deficient (Po) mice. By contrast, C3H/HeJ.lpr target cells and L929 were as responsive as C3H/HeJ and L929-Fas targets to the effectors generated in perforin wild-type (P+/+) mice. To measure potential bystander lysis in allogeneic responses, these same effector LN from either C57BL/6 P+/+ or P(o) mice were examined for lysis of C3H/HeJ (or C3H/HeJ.lpr) or L929-Fas (or L929) cocultured with 51Cr-labeled C57BL/6 (H-2b) target cells. Bystander lysis of C57BL/6 targets was observed only in those allogeneic responses where FasL was the defined mechanism of lysis. By contrast, perforin-mediated lysis was restricted by allo-recognition. Syngeneic (H-2b) targets that were not sensitive to Fas-mediated lysis were not lysed in a bystander fashion. Depletion of subsets of LN Po effectors (b anti-k) demonstrated that CD4+ T cells were primarily responsible for FasL-mediated direct and bystander lysis. FasL-mediated bystander lysis was also observed in H-2k anti-b reactions, except when the effectors were FasL mutant (gld). These data suggest that CTL responses to foreign Ag may evoke a certain amount of collateral damage to local host Fas-sensitive cells.
Contribution of the complement control protein modules of C2 in C4b binding assessed by analysis of C2/factor B chimeras.
Authors: Xu Y , Volanakis J E
Published: December 2022

To identify the complement control protein (CCP) module(s) of C2 that are required for C4b recognition, we constructed a panel of C2/factor B chimeras by substituting intact or partial factor B CCP modules for the corresponding ones of C2. Epitope mapping indicated that the anti-C2b mAb 3A3.3, which inhibits binding of C2 to C4b, reacts with the second CCP of C2 and similarly the anti-Ba mAb HA4-1A, which inhibits binding of factor B to C3b, reacts with the second CCP of factor B. The hemolytic activity of the chimeras CP1, CP2, and CP3a containing CCP1, CCP2, and a fragment of CCP3 of factor B, respectively, was substantially decreased compared with that of wild-type C2. The CP3 and CP1-3 chimeras, in which CCP3 and all three CCP modules of factor B, respectively, were substituted, had no hemolytic activity. Loss of activity could be attributed to the resistance of these two chimeras to C1s cleavage, which was probably due to conformational changes of the cleavage site. The combined results indicate that all three CCP modules of C2 contribute structural elements to the C4b-binding site of C2b. This site has been shown previously to be necessary for the initial binding of C2 to C4b which leads to the formation of the classical pathway C3 convertase.
IL-15 enhances immune functions during HIV infection.
Authors: Chehimi J , Marshall J D , Salvucci O , Frank I , Chehimi S , Kawecki S , Bacheller D , Rifat S , Chouaib S
Published: December 2022

IL-15, a new cytokine primarily produced by macrophages, has been shown to exhibit several functional properties shared with IL-2. Treatment of PBMC from HIV-infected patients with IL-15 resulted in an increase in NK cell cytotoxicity to levels similar to those of untreated PBMC from healthy donors. This effect is independent of several well-characterized regulatory cytokines, as it is not prevented by Abs that neutralize IFNs, TNF-alpha, IL-2, or IL-12. Enhanced cytotoxicity was accompanied by a significant increase in expression of cytotoxic granules. IL-15 enhanced the proliferative ability in both controls and HIV-seropositive in response to mitogen and recall Ags. Although the addition of IL-15 has a preventive effect on the appearance of spontaneous cell death, this effect was not seen during mitogen-induced apoptosis. The production of IL-15 by PBMC from patients in response to Staphylococcus aureus Cowan strain 1 appeared heterogeneous and was not negatively regulated by cytokines that inhibited IL-12 production. No correlation was found between in vitro HIV infection and IL-15 production, as viral infection had no effect on the ability of monocytes to produce IL-15 in response to S. aureus. Interestingly IL-15 restored the deficient production of IL-12 by PBMC from HIV+ people and had no major effect on modulating viral expression in latently infected cell lines or PBMC from naturally infected people. Taken together, these results suggest a potent immunoregulatory role of IL-15 during HIV infection.
Diminished HIV-specific CTL activity is associated with lower type 1 and enhanced type 2 responses to HIV-specific peptides during perinatal HIV infection.
Authors: Wasik T J , Jagodzinski P P , Hyjek E M , Wustner J , Trinchieri G , Lischner H W , Kozbor D
Published: December 2022

The early development of symptoms and the rapid progression of disease in some vertically infected infants are thought to reflect in part the immaturity of their immune systems. We examined the relationship between HIV-specific CTL activity and the profile of cytokine production induced by mAb to CD3 and HIV envelope (env) peptides P18 and T1 in PBMC derived from 0.6- to 3.6-yr-old children with perinatal HIV infection. Cellular immunity against HIV was demonstrated only during early stages of disease, whereas the responses were either undetectable or at background levels in HIV-infected children with rapidly progressing disease and in uninfected children of HIV+ and HIV- mothers. Levels of IL-2 mRNA in anti-CD3 mAb- and env peptide-induced PBMC varied and were increased in the infected children with high frequencies of HIV-specific CTL precursors. Analysis of IFN-gamma and IL-4 production by CD4+ T cell clones obtained from cultures stimulated with anti-CD3 mAb or the env peptides showed an increased proportion of Th2 and Th0 clones in HIV-infected children with lower HIV-specific CTL activity, whereas children with high CTL activity had increased numbers of Th1 clones. The results of these studies suggest that decreases in CTL activity to the virus might be associated with the induction of a type 2 cytokine response. These findings underline the role of cytokines in the generation of HIV-specific CTL responses and may be important for the development of immunomodulatory and vaccine strategies to interrupt vertical transmission of HIV.
Identification through bioinformatics of two new macrophage proinflammatory human chemokines: MIP-3alpha and MIP-3beta.
Authors: Rossi D L , Vicari A P , Franz-Bacon K , McClanahan T K , Zlotnik A
Published: December 2022

An increasing number of proinflammatory peptides, known as chemokines, are constantly being described and characterized. Because of their proven biologic functions in allergy, AIDS and, in general, inflammatory processes, these proteins have recently gained more attention. In this study we report the identification through bioinformatics of two new human chemokines: MIP-3alpha and MIP-3beta. Both of them belong to the beta- or CC chemokine family. Expression studies indicate that MIP-3alpha is predominantly expressed in lymph nodes, appendix, PBL, fetal liver, fetal lung and several cell lines. However, MIP-3beta expression is restricted to lymph nodes, thymus and appendix. Interestingly enough, both chemokines manifested a pattern of expression strongly regulated by IL-10. In contrast with other CC chemokines, MIP-3beta maps to chromosome 9. Here we show the importance of bioinformatics to discover new molecules with possible therapeutic effects and regulatory functions.
CD8 alphabeta+ TCR alphabeta(intermediate) lymphocytes expressing skewed TCRVbeta repertoire in the liver of aged athymic nu/nu mice.
Authors: Emoto M , Emoto Y , Kaufmann S H
Published: December 2022

Principally, TCRalphabeta cells differentiate and mature in the thymus. However, evidence has accumulated to suggest that some TCRalphabeta cells develop extrathymically. Such cells have been identified in athymic nu/nu mice that are devoid of a functional thymus. They appear relatively late in ontogeny and randomly increase in number as a function of age. Recently, the liver has been suggested as a candidate site for thymus-independent development of T cells. Here we show that CD8alphabeta+ T cells that express TCRalphabeta at intermediate intensity (TCRalpha(beta)int) are preferentially localized in the liver of nu/nu mice. This population encompassed cells expressing lectin cell adhesion molecule-1 and/or IL-2Rbeta and cells lacking either of these surface molecules. The CD8alphabeta+ TCRalpha(beta)int liver lymphocytes in nu/nu mice also differed in their LFA-1 expression marginally, whereas in C57BL/6 mice all CD8alphabeta+ TCRalpha(beta)int liver lymphocytes expressed LFA-1 at low intensity. Although the TCRVbeta repertoire markedly differed among individual animals, the CD8alphabeta+ TCRalpha(beta)int liver lymphocytes in nu/nu mice preferentially used TCRVbeta5 and/or TCRVbeta8. These findings show that CD8alphabeta+ TCRalpha(beta)int cells develop in the liver of nu/nu mice and that they arise randomly.
G(alpha)i2-deficient mice with colitis exhibit a local increase in memory CD4+ T cells and proinflammatory Th1-type cytokines.
Authors: Hörnquist C E , Lu X , Rogers-Fani P M , Rudolph U , Shappell S , Birnbaumer L , Harriman G R
Published: December 2022

Mice with targeted deletion of the G protein G(alpha)i2 develop an inflammatory bowel disease closely resembling ulcerative colitis. To better define disease pathogenesis, the mucosal immune system in G(alpha)i2-deficient mice was studied. Phenotypic analysis of large intestine lamina propria lymphocytes revealed a large increase in memory CD4+ T cells (CD44high, CD45RBlow, CD62Llow). Furthermore, expression of the mucosal homing receptor integrin beta7 was increased on mucosal, but not systemic, CD4+ T cells. Analysis of cytokine production revealed a marked increase in proinflammatory Th1-type cytokines in inflamed colons, as compared with wild-type mice or G(alpha)i2-deficient mice without colitis. Thus, IFN-gamma and IL-1beta levels were increased 13-fold and 30-fold, respectively, with more modest increases in IL-6 levels (5-fold) and TNF levels (2-fold). Inflamed colons of G(alpha)i2-deficient mice also demonstrated increased IL-12 p40 mRNA levels. No increase in IL-2, IL-4, IL-5, and IL-10 was seen. Large intestinal epithelial cells in G(alpha)i2-deficient mice with colitis were found by immunohistochemistry to express increased levels of both MHC class I and class II Ags. Colitis was associated with increased IgG levels (60-fold increase), predominantly IgG2a (135-fold increase), in large but not small intestinal secretions. This was shown by ELISPOT analysis to result from local production within the lamina propria.
Mouse CD38 is down-regulated on germinal center B cells and mature plasma cells.
Authors: Oliver A M , Martin F , Kearney J F
Published: December 2022

Germinal center formation is the result of antigenic stimulation of B cells in a T cell-rich area. B cells cycle through the germinal centers, and a small percentage survive to become plasma cells or memory B cells. The transformation from a mature B cell into a germinal center B cell and finally into a terminally differentiated B cell is not well understood. Human CD38 is highly expressed on both germinal center B cells and plasma cells, and is useful in delineating these B cell subsets and in understanding the signaling events involved in the development of these B cells. To determine whether CD38 expression on activated germinal center B cells and postgerminal center B cells influences germinal center differentiation, we studied the expression of CD38 in the mouse. CD38 is expressed on follicular B cells in the Peyer's patches but is down-regulated on germinal center B cells located within the Peyer's patches. CD38dim/-B220+ germinal center B cells are also found in the spleens of immunized but not control mice, suggesting that Ag-stimulated germinal center formation is involved in the production of CD38dim/-B220+ B cells. Furthermore, mature plasma cells isolated from in vitro LPS cultures do not express CD38, but do contain high levels of cytoplasmic Ig. These results are in contrast to studies in humans in which CD38 is not found on follicular B cells but is highly expressed on germinal center B cells and plasma cells.
Preferential rearrangements of the T cell receptor-delta-deleting elements in human T cells.
Authors: Verschuren M C , Wolvers-Tettero I L , Breit T M , Noordzij J , van Wering E R , van Dongen J J
Published: December 2022

The major part of the TCR-delta locus is flanked by the so-called TCR-delta-deleting elements deltaRec and psi(J)alpha, which preferentially rearrange to each other in human thymocytes. On the basis of our combined Southern blot and PCR analyses, we also identified the prominent deltaRec-Jalpha58 rearrangement and three other preferential deltaRec rearrangements. The latter rearrangements concern deltaRec rearrangements to the Ddelta3, Jdelta1, and Jdelta3 gene segments. These deltaRec rearrangements do not delete the complete TCR-delta locus and are homologous to Vdelta-Jdelta rearrangements, because the majority of their junctional regions contain Ddelta gene segments. In contrast, the prominent deltaRec-psi(J)alpha and deltaRec-Jalpha58 rearrangements, representing approximately 68 and approximately 23% of all deltaRec rearrangements, respectively, are homologous to Valpha-Jalpha rearrangements, because Ddelta gene segments are absent in their junctional regions. Additional PCR analysis of Vdelta-psi(J)alpha and Vdelta-Jalpha58 coding and signal joints revealed also that these rearrangements resemble Valpha-Jalpha rearrangements, except for Vdelta2-psi(J)alpha and Vdelta2-Jalpha58 rearrangements, which resemble Vdelta-Jdelta rearrangements. Our data show that virtually all TCR-delta-deleting rearrangements are Valpha-like, and the high frequency of these rearrangements in the human thymus suggests that most thymocytes use these rearrangements to further differentiate into the TCR-alphabeta lineage. Based on the very low frequency of deltaRec and psi(J)alpha rearrangements in 4% of the T cell acute lymphoblastic leukemia patients (n = 151) and 6% of the T cell lines (n = 26), we hypothesize that rearranged TCR-delta-deleting elements exist for only an extremely short period during thymocyte differentiation, probably due to rapid subsequent Valpha-Jalpha rearrangements.
An anti-inflammatory role for gamma delta T lymphocytes in acquired immunity to Mycobacterium tuberculosis.
Authors: D'Souza C D , Cooper A M , Frank A A , Mazzaccaro R J , Bloom B R , Orme I M
Published: December 2022

Although a role for gammadelta receptor-bearing T cells in the acquired immune response to infection with Mycobacterium tuberculosis is suggested by several lines of evidence, the only data indicating a possible role in specific protective immunity have been provided by very high dose i.v. infection models. In the current study, more modest low dose inocula delivered by the aerosol route grew identically in wild-type controls and in mutant mice in which the Cdelta gene of the gammadelta TCR has been disrupted by homologous recombination. This situation did not change if the inoculum size was increased or if an aerosol challenge with an M. tuberculosis strain of higher virulence was given. However, while the control and containment of these infections was similar, the mutant mice exhibited a substantial pyogenic form of the granulomatous response compared with the lymphocytic response seen in control animals, a finding that may well explain mortality in the former group if high i.v. doses are given. These data indicate that gammadelta T cells do not directly contribute to protection against tuberculosis or that they do so only when bacterial loads are very high. Instead, the data suggest that gammadelta T cells perhaps play an important role by influencing local cellular traffic, promoting the influx of lymphocytes and monocytes, and limiting the access of inflammatory cells that do not contribute to protection but may cause tissue damage.
Mechanisms of heterosubtypic immunity to lethal influenza A virus infection in fully immunocompetent, T cell-depleted, beta2-microglobulin-deficient, and J chain-deficient mice.
Authors: Epstein S L , Lo C Y , Misplon J A , Lawson C M , Hendrickson B A , Max E E , Subbarao K
Published: December 2022

Immunity that is cross-protective between different influenza A virus subtypes (termed heterosubtypic immunity) can be demonstrated readily in some animals but only rarely in humans. Induction of heterosubtypic immunity in humans by vaccines would provide public health benefit, perhaps offering some protection against pandemics or other new influenza A strains. Therefore, we studied mechanisms mediating heterosubtypic immunity in mice. Immunization with either A/H1N1 or A/H3N2 virus protected mice against mortality following heterosubtypic challenge while providing modest reductions in lung virus titers. No cross-protection was seen with distantly related type B influenza virus. Depletion of CD4+ or CD8+ T cells or both around the time of challenge had no significant effect on survival, indicating that these cells are not required at the effector stage. beta2-microglobulin knockout mice could be protected readily against heterosubtypic challenge, confirming that class I-restricted T cells are not required. In beta2-microglobulin -/- mice, depletion of CD4+ T cells partially abrogated heterosubtypic immunity, showing that they play a role in these mice. Passive transfer of Abs to naive recipients protected against subsequent challenge with homologous but not heterosubtypic virus. Because a role for secretory Abs has been suggested, we studied dependence on the J chain, which is required for polymeric Ig receptor-mediated IgA transport. J chain knockout mice were readily protected by heterosubtypic immunity, indicating that polymeric Ig receptor-mediated transport is not required. Better understanding of heterosubtypic immunity should be valuable in analyzing new vaccines, including peptide and DNA vaccines, intended to induce broadly cross-reactive immunity.
Enhancement of cellular and humoral immune responses to hepatitis C virus core protein using DNA-based vaccines augmented with cytokine-expressing plasmids.
Authors: Geissler M , Gesien A , Tokushige K , Wands J R
Published: December 2022

Development of a broad based cellular and humoral immune response to hepatitis C virus (HCV) structural proteins may be important for irradication of infection. DNA-based immunization is a promising approach to generate HCV-specific immune responses. Previous studies of DNA-based immunizations in mice using an HCV core DNA expression plasmid (pHCV2-2) demonstrated an efficient CTL response against HCV core epitopes; however, the humoral and Th cell proliferative responses were found to be weak. To enhance the immunogenicity of this nonsecreted viral structural protein at the B and T cell level, we coimmunized mice with pHCV2-2 and DNA expression constructs encoding for mouse IL-2, IL-4, and granulocyte-macrophage CSF proteins. Under these experimental conditions, a seroconversion frequency to anti-HCV core increased from 40 to 80% in immunized mice. The CD4+ inflammatory T cell proliferative responses as well as CD8+ CTL activity to HCV core protein were enhanced substantially after coimmunization with the IL-2 and granulocyte-macrophage CSF DNA expression constructs. In contrast, coimmunization with an IL-4-producing construct induced differentiation of Th cells toward a Th0 subtype and suppressed HCV core-specific CTL activity. Taken together, these studies emphasize that generation of antiviral immune responses using DNA-based immunization may be modified by local cytokine production at the site of Ag presentation.
IFN regulatory factor-1 gene transfer into an aggressive, nonimmunogenic sarcoma suppresses the malignant phenotype and enhances immunogenicity in syngeneic mice.
Authors: Yim J H , Wu S J , Casey M J , Norton J A , Doherty G M
Published: December 2022

IFN-gamma has a direct antitumor effect on many tumor cell lines mediated through the IFN-gammaR. One effect of IFN-gamma is to induce the nuclear transcription factor IFN regulatory factor-1 (IRF-1), which may function as a tumor suppressor. In this study, mouse IRF-1 cDNA under a high constitutive expression promoter was transfected into the highly aggressive, nonimmunogenic MCA 101 murine sarcoma. Clones were obtained by G418 selection and screened for IRF-1 mRNA expression by reverse transcriptase-PCR (RT-PCR). High expression clones had high levels of two MHC class I proteins (H-2Kb and H-2Db) on the cell surface that correlated with increased levels of class I mRNA by RT-PCR. Furthermore, these clones also had increased levels of MHC class II protein (I-Ab), which correlated with increased levels of one subunit of class II mRNA by RT-PCR. IRF-1-expressing clones had markedly diminished cell growth in vitro and decreased anchorage-independent growth in a soft agar assay. These clones also demonstrated markedly prolonged tumor latency and slowed growth in syngeneic C57BL/6 mice. IRF-1 gene-transfected cells had shortened tumor latency and formed faster growing tumors in gamma-irradiated immunodeficient mice compared with results in immunocompetent mice. Mice immunized with IRF-1-transfected cells were protected against subsequent challenge with IRF-1 transfected cells and also demonstrated greater tumor latency and slower tumor growth against subsequent challenge with untransfected cells compared with mice immunized with empty vector-transfected cells. These studies demonstrate a tumor suppressor effect of IRF-1, which acts in vivo through both partial reversion of the malignant phenotype and enhanced immune recognition and may play a role in the antitumor effects of IFN-gamma.
Angiogenesis induced in vivo by hepatocyte growth factor is mediated by platelet-activating factor synthesis from macrophages.
Authors: Camussi G , Montrucchio G , Lupia E , Soldi R , Comoglio P M , Bussolino F
Published: December 2022

This study shows that the neoangiogenesis induced by hepatocyte growth factor (HGF) was associated with a local synthesis of platelet-activating factor (PAF) and was inhibited by the specific PAF receptor antagonist WEB 2170 in a murine model in which matrigel was injected s.c. as a substratum for angiogenesis. The synthesis of PAF was concomitant with the early migration of endothelial cells and infiltration of MAC-1-positive macrophages. Infiltration of lymphocytes and polymorphonuclear leukocytes was never observed. In vitro studies demonstrated that mouse peritoneal macrophages, but not two murine microvascular endothelial cell lines or human and bovine endothelial cells from large vessels, synthesized PAF after stimulation with HGF. Furthermore, macrophages expressed the transcript of HGF receptor encoded by the MET proto-oncogene and migrated after HGF challenge. The binding of HGF to its receptor was followed by the activation of the receptor tyrosine kinase domain and phosphorylation of the beta subunit. Leukocyte depletion with 5-fluorouracil and anti-MAC-1 Ab added to matrigel prevented the infiltration of macrophages, the synthesis of PAF and the angiogenesis induced by HGF. PAF extracted and purified from mice challenged with HGF induced a rapid angiogenic response, inhibited by WEB 2170. These results suggest that the angiogenic effect of HGF in vivo is mediated, at least in part, by PAF synthesized from macrophages infiltrating the matrigel plug.
Lipopolysaccharide-induced biphasic inositol 1,4,5-trisphosphate response and tyrosine phosphorylation of 140-kilodalton protein in mouse peritoneal macrophages.
Authors: Shinji H , Akagawa K S , Tsuji M , Maeda M , Yamada R , Matsuura K , Yamamoto S , Yoshida T
Published: December 2022

We previously showed that a relatively high dose of LPS induced the selective translocation of protein kinase C-beta (PKC-beta) in LPS-responsive mouse macrophages. This result suggested that phosphatidylinositol-specific phospholipase C (PLC) might be activated in the upstream of PKC-beta. Stimulation of C3H/HeN mouse macrophages by LPS induced the characteristic phosphatidylinositol-1,4,5-trisphosphate (IP3) response, that is, a biphasic response consisting of a rapid increase occurring within the first 1 min, and another increase beginning at around 1 min after stimulation. Only the first response was disappeared when cells were treated with a platelet-activating factor receptor antagonist. LPS-inducible TNF-alpha gene activation, however, was not suppressed by the same antagonist, but suppressed by PKC inhibitors. LPS-stimulated macrophage lysates showed tyrosine phosphorylation of some proteins, and the strongest phosphorylation was observed at molecular mass of 140 kDa. The phosphorylation of this protein started at 40 s after LPS stimulation and continued to increase. Anti-PLC-gamma2 Ab seemed to recognize the same protein as the tyrosine-phosphorylated 140-kDa protein. A low dose of LPS (1 ng/ml) could not induce the tyrosine phosphorylation of this protein. Furthermore, LPS induced only the first phase change, but not the second phase increase in LPS-hyporesponsive C3H/HeJ mouse macrophages. These results indicate that the first phase rapid IP3 change, which is also seen in HeJ macrophages, is mediated via a platelet-activating factor receptor, and is not responsible for TNF-alpha production, while the second phase change mediated by a molecule other than CD14 is responsible for PKC-beta translocation and TNF-alpha production. The results also suggest that the later IP3 change is considered to be mediated through a gamma2 type of phosphatidylinositol-specific PLC.
Age-dependent intestinal lymphoproliferative disorder due to stem cell factor receptor deficiency: parameters in small and large intestine.
Authors: Laky K , Lefrançois L , Puddington L
Published: December 2022

Signaling through c-Kit/stem cell factor (SCF) is crucial for normal development of erythroid and myeloid hematopoietic precursors and of melanocytes and germ cells. While peripheral lymphoid populations of W/Wv and SI/SId mice appear normal, we demonstrated that the intraepithelial lymphocyte (IEL) populations of small (SI) and large (LI) intestine were significantly affected. IEL populations of young W/Wv animals were indistinguishable from those of their control littermates, but an age-dependent decrease in SI and LI TCRgamma delta IEL occurred in c-Kit mutant mice. In SI, but not in LI, this diminution was accompanied by gross expansion of TCRalpha beta IEL that resulted in significantly increased IEL:epithelial cell ratios in c-Kit mutant mice. Bromodeoxyuridine labeling studies revealed that the increase in cell numbers was due to lymphoproliferation that occurred in situ. Interestingly, TCRgamma delta IEL expressed cell surface c-Kit, while the expanding population of TCRalpha beta IEL did not. Analysis of radiation bone marrow chimeras demonstrated that the dysregulation required either disruption of stromal cell SCF or IEL c-Kit and showed that the effect on IEL or their precursors was not due to other changes in the intestinal microenvironment. Lamina propria T cell populations in these mice were unaffected, reinforcing the idea that the developmental requirements of these gut-resident lymphocyte populations are distinct. Overall, the results demonstrated that the development of intestinal TCRgamma delta IEL, regardless of location, shares common requirements for SCF, while SI and LI TCRalpha beta IEL may develop along distinct pathways. Possible mechanisms for the loss of proliferative regulation in gut T cells in c-Kit/SCF deficiency are discussed.
Signaling by adhesion in human neutrophils: activation of the p72syk tyrosine kinase and formation of protein complexes containing p72syk and Src family kinases in neutrophils spreading over fibrinogen.
Authors: Yan S R , Huang M , Berton G
Published: December 2022

Src family kinases are implicated in signaling by integrins in polymorphonuclear neutrophils (PMN). To identify proteins present in complexes with Src family kinases, we subjected p58(c-fgr) or p53/56(lyn) immunoprecipitates from Triton X-100 lysates of PMN incubated on fibrinogen-coated surfaces to in vitro kinase assays. Assays on p58(c-fgr) or p53/56(lyn) immunoprecipitates from Triton lysates of PMN induced to spread over fibrinogen in response to TNF-alpha showed that several proteins form complexes with Src family kinases and can be phosphorylated in vitro. Immunoblot analysis showed that the p72(syk) tyrosine kinase is one of these proteins. Formation of protein complexes containing Src family kinases and p72(syk) required PMN spreading because p72(syk) was not detected in p58(c-fgr) or p53/56(lyn) immunoprecipitates from PMN, which were stimulated with TNF-alpha, in suspension. In addition, induction of PMN spreading with Mn2+ in the absence of TNF-alpha promoted the formation of protein complexes containing Src family kinases and p72(syk). We also found that p72(syk)-autophosphorylating kinase activity was enhanced, and a fraction of p72(syk) was translocated to a Triton-insoluble cytoskeletal fraction in PMN induced to spread over fibrinogen by TNF-alpha or Mn2+. In vitro kinase assays on CD18 (beta 2 integrin subunit) immunoprecipitates from Triton lysates of spread PMN showed that several proteins formed complexes with CD18 and could be phosphorylated in vitro. Immunoblot analysis of CD18 immunoprecipitates allowed us to identify p72(syk) as one of these proteins. These findings show that PMN spreading is accompanied by activation of p72(syk) and formation of multimolecular complexes in which Src family kinases and p72(syk) colocalize.
Biochemical analysis of p120/130: a protein-tyrosine kinase substrate restricted to T and myeloid cells.
Authors: da Silva A J , Rosenfield J M , Mueller I , Bouton A , Hirai H , Rudd C E
Published: December 2022

T cell activation is mediated by a cascade of intracellular events involving protein-tyrosine kinases and their substrates. p56(lck) and p59(fyn) are protein-tyrosine kinases that associate with CD4/CD8 and the TCRzeta/CD3 complex, respectively. We previously reported the appearance of a protein doublet at 120 and 130 kDa that preferentially associates with p59(fyn) and undergoes tyrosine phosphorylation upon receptor ligation. In this paper, we demonstrate that p120/130 is a novel protein that is restricted in expression to T cells, thymocytes and myeloid cells. Internal peptide sequencing and immunoblotting using an anti-p120/130 antisera showed that p120/130 is a unique protein that is distinct from p130(cas) and p125(cbl). By contrast, p120 and p130 shared similar peptide patterns and are structurally related. Alkaline phosphatase digestion of precipitates showed that they are not related due to phosphorylation. p120/130 was found to associate constitutively with a 55-kDa protein of unknown identity, but which is distinct from p56(lck) and Shc. p120/130 also undergoes a unique kinetics of phosphorylation and associates with the Ag receptor in response to TCR ligation. In keeping with the association with p59(fyn), T cells from p59(fyn)-negative mice exhibit reduced phosphorylation of the protein. p120/130 therefore represents a novel TCR associated intracellular molecule with potential to play a role in T cell signaling.
B7-CD28/CTLA-4 costimulatory pathways are required for the development of T helper cell 2-mediated allergic airway responses to inhaled antigens.
Authors: Keane-Myers A , Gause W C , Linsley P S , Chen S J , Wills-Karp M
Published: December 2022

We have previously demonstrated that the development of allergen-induced airway hyperresponsiveness in a murine model is CD4+ T cell dependent. In the present study, we examined the role of the B7/CD28-CTLA4 costimulatory T cell activation pathway in the pathogenesis of allergen-induced airway hyperresponsiveness in this murine model. Sensitized A/J mice develop significant increases in airway responsiveness, bronchoalveolar lavage eosinophils, serum IgE levels, and Th2-associated cytokine production following aspiration challenge with OVA. Administration of CTLA4-Ig either before Ag sensitization or before pulmonary Ag challenge abolished Ag-induced airway hyperresponsiveness and pulmonary eosinophilia. Examination of cytokine protein levels in the bronchoalveolar lavage showed a significant decrease in the level of the Th2 cytokine, IL-4, after CTLA4-Ig treatment either before sensitization or before challenge, with no significant change in the concentration of the Th1 cytokine, IFN-gamma. Further, the Ag-specific Ab isotypes IgG1 and IgE were significantly decreased in animals treated with CTLA4-Ig before challenge, while there was no significant change in the IgG2a Ab isotype. These data demonstrate that administration of CTLA4-Ig is effective in ablating allergen-induced airway dysfunction concomitant with a significant reduction in the Th2 response. We conclude that B7/CD28-CTLA-4 costimulation is required for the development of many of the immunologic and physiologic features of asthma, possibly by promoting a pathologic type 2-associated response.