bluuebunny/crossref_metadata_2025 huggingface dataset:
The integrin-triggered rescue of T lymphocyte apoptosis is blocked in HIV-1-infected individuals.
Authors: Ng T T , Kanner S B , Humphries M J , Wickremasinghe R G , Nye K E , Anderson J , Khoo S H , Morrow W J
Published: December 2022

HIV infection is associated with a disease status-dependent impairment of Ag-specific T cell responses, resulting in anergy or unchecked apoptotic cell death. beta1 integrins play an important role in the induction of T lymphocyte responses to antigenic challenge by providing a T cell costimulatory signal, and have been shown to rescue various cell types from undergoing apoptosis. We examined the integrin-triggered cell survival signal and associated pathways in CD3+ T cells derived from 69 HIV-1-infected individuals in comparison with healthy controls. We found beta1 integrin-mediated costimulation of TCR-induced T cell proliferation and protection from aberrant cell death to be absent in the majority of patients with AIDS, but intact in asymptomatic, infected individuals. The lack of integrin-mediated rescue may be partly due to an early impairment of TCR/integrin-costimulated secretion of IFN-gamma, a type 1 lymphokine that protects against TCR-induced apoptosis of T cells from HIV-seropositive donors, but not loss of integrin expression. The mechanism of integrin hyporesponsiveness appeared to correlate with a failure of the integrin-generated signal to induce pp125FAK mRNA and protein expression. Protein kinase C activation in CD3+ T cells following integrin stimulation was also impaired in HIV-infected individuals, mostly among the symptomatic/AIDS patients. Protein kinase C inactivation in T cells was shown to have a destabilizing effect in vitro on pp125FAK mRNA that contains an AUUUA motif in the 3'-untranslated region, a consensus sequence for the AU-rich elements responsible for mRNA destabilization. These aberrant changes in pp125FAK expression may have direct significance to the overall immunopathogenesis during infection with HIV-1.
Complement Fixation Reaction with Rabies and Herpes Viruses
Authors: Tang Fei-Fang , Castaneda M. Ruiz
Published: December 2022

In 1926 Takaki, Bonis and Koref (1) reported that they had been able to identify and differentiate the various forms of encephalitic viruses by means of the complement fixation reaction. Working on rabies, Kraus and Michalka (2) in the same year published very favorable results in the diagnosis of this disease by complement fixation. They used “cooked” and glycerin extract antigens. Their results were so definite that it was decided to repeat their experiments with rabies, and to use the same technic with herpes, in view of the fact that in herpes studies during the last two years we (3) have carried out a large number of complement fixation experiments with herpes virus, always with negative results.

Technic 1. Immunization of the rabbits Rabbit 1 was immunized against rabies by a prolonged treatment with Semple's vaccine (50 cc. in all) followed by three injections with living fixed virus (3.8 cc. in all).
Cutting Edge: Lyn-Mediated Down-Regulation of B Cell Antigen Receptor Signaling: Inhibition of Protein Kinase C Activation by Lyn in a Kinase-Independent Fashion
Authors: Katsuta Hitoshi , Tsuji Sachiyo , Niho Yoshiyuki , Kurosaki Tomohiro , Kitamura Daisuke
Published: December 2022

Stimulation of the B cell Ag receptor (BCR) induces activation of tyrosine kinases such as Lyn and Syk, phosphorylation and activation of multiple signaling components, and eventually, the expression of several genes including c-myc. Syk is required for activation of phospholipase C-γ2 and the subsequent phosphatidylinositol hydrolysis, leading to protein kinase C (PKC) activation and intracellular Ca2+ increase. In contrast, the function of Lyn remains obscure. Here, we report that BCR-mediated induction of c-myc promoter activity and of PKC activity, but not the expression level of functional PKC, was markedly augmented in Lyn-deficient chicken B cells. This enhancement was reversed to the level of wild-type cells by the expression of exogenous Lyn of kinase-inactive form. These results indicate that Lyn inhibits BCR-mediated activation of a large portion of PKC isozymes in a kinase-independent fashion. This finding reveals a novel role of Lyn in negative regulation of BCR signaling.
Cross-Linking CD21/CD35 or CD19 Increases Both B7-1 and B7-2 Expression on Murine Splenic B Cells
Authors: Kozono Yuko , Abe Ryo , Kozono Haruo , Kelly Robert G. , Azuma Takachika , Holers V. Michael
Published: December 2022

Activation of the complement cascade and ligation of complement C3 receptors on B cells represent an important bridge between innate and Ag-specific acquired immunity. We show here that cross-linking of mouse CD21 (complement receptor type 2, CR2, C3d receptor) and CD35 (complement receptor type 1, CR1, C3b/C4b receptor) or co-cross-linking of CD21/CD35 and surface IgM rapidly up-regulates both B7-1 and B7-2 expression on murine resting splenic B cells. CD21/CD35-mediated up-regulation of both B7-1 and B7-2 expression is observed within 14 h, while other stimuli up-regulate only B7-2 but not B7-1 at this early time point. Consistent with the increase in B7 levels, BALB/c B cells on which surface IgM and CD21/CD35 have been co-cross-linked stimulate C57BL/6 T cells more effectively than controls. This CD21/CD35-enhanced allogeneic MLR is blocked nearly completely by anti-B7-2 mAbs and partially by anti-B7-1 mAbs. In addition, cross-linking of CD19, which is physically associated with CD21/CD35, leads to increased B7-1 and B7-2 expression. These data suggest that CD21/CD35 ligation results in enhanced B cell Ag presentation using costimulatory mechanisms shared with other activators and thus works cooperatively in this process. Rapid up-regulation of B7-1 expression, a unique response to CD21/CD35 and CD19 cross-linking, may be a particularly important effect of C3-containing ligands. We propose that CD21/CD35- and CD19-mediated B7-1 and B7-2 up-regulation is an important mechanism by which complement activation links innate and acquired immunity.
IL-12 Down-Regulates Autoantibody Production in Mercury-Induced Autoimmunity
Authors: Bagenstose Lee M. , Salgame Padmini , Monestier Marc
Published: December 2022

In genetically susceptible H-2s mice, subtoxic doses of mercuric chloride (HgCl2) induce a complex autoimmune syndrome characterized by the production of anti-nucleolar IgG Abs, lymphoproliferation, increased serum levels of IgG1 and IgE Abs, and renal Ig deposits. Mercury-induced autoimmunity in H-2s mice provides a useful model for chemically related autoimmunity in humans. The increase in serum IgG1 and IgE, which are under IL-4 control, suggests a role for the Th2 subset in this syndrome. The IL-12 cytokine induces T cell proliferation and IFN-γ production and is necessary for differentiation of naive T cells into the Th1 subset. To gain an understanding of T cell control in this syndrome and, in particular, Th1/Th2 regulation, we assessed the effect of IL-12 administration in mercury-induced autoimmunity. Groups of A.SW mice (H-2s) received HgCl2 plus IL-12, HgCl2 alone, or IL-12 alone. IL-12 treatment resulted in a dramatic reduction of the anti-nucleolar Ab titers. IL-12 also inhibited the HgCl2-induced serum IgG1 increase, but, in contrast, did not significantly affect IgE induction in this model. This observation may be related to our unexpected finding that IL-12 further potentiated the HgCl2-triggered IL-4 induction in this model. The levels of renal Ig deposits were similar in mice receiving HgCl2 alone or HgCl2 plus IL-12. Our results indicate that IL-12 can down-regulate the autoimmune component of this experimental syndrome and that the various manifestations of mercury-induced autoimmunity are independently regulated.
Participation of the CD94 Receptor Complex in Costimulation of Human Natural Killer Cells
Authors: Voss Stephan D. , Daley John , Ritz Jerome , Robertson Michael J.
Published: December 2022

Optimal proliferation and expansion of human NK cells require mitogenic cytokines together with cell contact-dependent costimulation. Production of mAb that can modulate human NK cell proliferation yielded NKH3, which recognizes the CD94 Ag. NKH3 immunoprecipitates contain ∼70-kDa heterodimeric complexes consisting of a ∼25-kDa glycoprotein and ∼40- to 45-kDa molecules. Analysis by two-dimensional isoelectric focusing/SDS-PAGE suggests that several different 40- to 45-kDa species are present in the CD94 receptor complex in human NK cells. NKH3 reacted with essentially all resting NK cells, although CD94 is expressed at higher levels on the CD56bright (i.e., high level of CD56) CD16dim/neg (i.e., low level of or absent CD16) subpopulation than on the more abundant CD56dimCD16bright NK cell subset. Moreover, the Z199 mAb, which appears to recognize NKG2-A species that can form heterodimers with CD94, stained virtually all CD56bright NK cells, but only a subset of CD56dim NK cells. Ligation of CD94 augmented the proliferation of CD56bright NK cells in response to IL-2 or IL-15 by as much as 10-fold. Secretion of IFN-γ by CD56bright NK cells stimulated with IL-2 or IL-15 was also enhanced up to 10-fold after CD94 ligation. CD94 mAb did not consistently costimulate proliferation of or IFN-γ production by CD56dim NK cells cultured with IL-2 or IL-15. In contrast, irradiated K562 cells costimulated proliferation of both CD56bright and CD56dim NK cells. These results indicate that CD56bright and CD56dim NK cells can be costimulated through different receptors, which may allow these distinct NK cell subsets to be independently regulated in vivo.
Increased Apoptosis of T Cell Subsets in Aging Humans: Altered Expression of Fas (CD95), Fas Ligand, Bcl-2, and Bax
Authors: Aggarwal Sudeepta , Gupta Sudhir
Published: December 2022

Aging is associated with lymphopenia and progressive decline in T cell functions; however, the mechanisms underlying these defects are unclear. We analyzed the expression of genes promoting apoptosis (fas/fasL1 and bax) and those inhibiting apoptosis (bcl-2 and bcl-xL) in lymphocytes from aging and young subjects at the protein level, using flow cytometry/Western blotting, and at the mRNA level, using quantitative PCR. Susceptibility of T cell subsets to undergo anti-Fas-induced apoptosis was analyzed by propidium iodide staining, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay, DNA fragmentation assay, and staining with Hoechst 33342 dye. An increased expression of Fas and Fas ligand and a decreased expression of Bcl-2 were observed in both CD4+ and CD8+ T cells from aging as compared with young controls. Increased Fas and decreased Bcl-2 expression were also found in memory cells of both CD4+ and CD8+ T cell subsets from aging. Bax expression was increased in lymphocytes from aging at both the protein and mRNA level. No significant difference was observed in Bcl-xL expression between aging and young; however, the ratio of Bax:Bcl-xL was increased in aging. An increased proportion of CD4+ and CD8+ T cell subsets from aging underwent apoptosis following anti-Fas Ab treatment as compared with CD4+ and CD8+ T cell subsets from young controls. These data suggest that increased apoptosis may be one of the mechanisms responsible for lymphopenia and T cell deficiency associated with human aging.
The Cytokine Profile Expressed by Human Dendritic Cells Is Dependent on Cell Subtype and Mode of Activation
Authors: de Saint-Vis Blandine , Fugier-Vivier Isabelle , Massacrier Catherine , Gaillard Claude , Vanbervliet Béatrice , Aït-Yahia Smina , Banchereau Jacques , Liu Yong-Jun , Lebecque Serge , Caux Christophe
Published: December 2022

In the present study, we have analyzed the pattern of cytokines expressed by two independent dendritic cell (DC) subpopulations generated in vitro from human cord blood CD34+ progenitors cultured with granulocyte-macrophage CSF and TNF-α. Molecularly, we confirmed the phenotypic differences discriminating the two subsets: E-cadherin mRNA was only detected in CD1a+-derived DC, whereas CD68 and factor XIIIa mRNAs were observed exclusively in CD14+-derived DC. Semiquantitative reverse-transcriptase PCR analysis revealed that both DC subpopulations spontaneously expressed IL-1α, IL-1β, IL-6, IL-7, IL-12 (p35 and p40), IL-15, IL-18, TNF-α, TGF-β, macrophage CSF, and granulocyte-macrophage CSF, but not IL-2, IL-3, IL-4, IL-5, IL-9, and IFN-γ transcripts. Both subpopulations were shown to secrete IL-12 after CD40 triggering. Interestingly, only the CD14+-derived DC secreted IL-10 after CD40 activation, strengthening the notion that the two DC subpopulations indeed represent two independent pathways of DC development. Furthermore, both DC subpopulations expressed IL-13 mRNA and protein following activation with PMA-ionomycin, but not with CD40 ligand, in contrast to IL-12 and IL-10, revealing the existence of different pathways for DC activation. Finally, we confirmed the expression of IL-7, IL-10, and IL-13 mRNA by CD4+CD11c+CD3− DC isolated ex vivo from tonsillar germinal centers. Thus, CD14+-derived DC expressing IL-10 and factor XIIIa seemed more closely related to germinal center dendritic cellsGCDC than to Langerhans cells.
Dendritic Cells and Resting B Cells Form Clusters In Vitro and In Vivo: T Cell Independence, Partial LFA-1 Dependence, and Regulation by Cross-Linking Surface Molecules
Authors: Kushnir Natalyia , Liu LiMing , MacPherson G. Gordon
Published: December 2022

Initiation of an Ab response requires interaction between dendritic cells (DC), T cells, and B cells in a T cell area. We demonstrate that rat DC and B cells form T cell-independent clusters in vitro and in vivo. In vitro clusters form within 1 h and dissociate within 24 to 48 h. Clustering is restricted to resting B cells, is energy, cytoskeleton, and protein kinase C dependent, and is inhibited by anti-LFA-1 but not anti-ICAM-1 mAbs. Spleen and lymph node B cells cluster more strongly than those from lymph or blood, suggesting up-regulation of adhesiveness during transendothelial migration. Bone marrow B cells do not form clusters. DC from spleen and lymph nodes show the most clustering, lymph-borne DC are intermediate, and DC from lamina propria, Peyer’s patches, and those grown from bone marrow form the fewest clusters. Clustering is stimulated by cross-linking MHC class II (whole mAb or F(ab′)2) on DC or B cells or Thy-1 on DC, but not MHC class I, CD45, or CD44. Stimulation by mAb is energy, cytoskeletal, and protein kinase C dependent, but is not inhibited by anti-LFA-1 mAbs, suggesting involvement of other, unidentified adhesion molecules. We suggest that interactions between DC and B cells will occur regularly during B cell recirculation. Cross-linking of MHC class II-peptide molecules on DC by specific T cells would increase binding avidity, causing retention of Ag-specific B cells on DC long enough for the B cells to process Ag, thereby facilitating cognate interactions between T and B cells.
The Vasoactive Peptide Maxadilan from Sand Fly Saliva Inhibits TNF-α and Induces IL-6 by Mouse Macrophages Through Interaction with the Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Receptor
Authors: Soares Milena B. P. , Titus Richard G. , Shoemaker Charles B. , David John R. , Bozza Marcelo
Published: December 2022

Maxadilan is a vasodilatory peptide encoded by a gene cloned from Lutzomyia longipalpis salivary glands. In this study we investigated the effects of maxadilan on macrophage functions. Maxadilan treatment of LPS-stimulated BALB/c macrophages inhibited TNF-α release but increased IL-6. Further, it also induced IL-6 release in a dose-dependent manner from unstimulated macrophages. Maxadilan increased production of PGE2, and the inhibition of TNF-α was completely abrogated by indomethacin. Others have recently shown that maxadilan is a selective agonist of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor. Treatment with the receptor antagonist PACAP 6–38 blocked maxadilan activities on macrophages. The natural endogenous ligand, PACAP 38, had the same effects as maxadilan on TNF-α and IL-6 production. Finally, in a dose- and time-dependent fashion, maxadilan induced the intracellular accumulation of cAMP in macrophages. Taken together, the results presented here indicate a modulatory effect of ligands of PACAP type I receptor on cytokine production by macrophages and suggest that activation of this receptor, with the subsequent elevation of intracellular cAMP in macrophages, could participate in a negative-feedback mechanism that controls certain inflammatory responses.
Cutting Edge: Virus-Activated CD8 T Cells and Lymphokine-Activated NK Cells Express the Mast Cell Function-Associated Antigen, An Inhibitory C-Type Lectin
Authors: Blaser Claudine , Kaufmann Martina , Pircher Hanspeter
Published: December 2022

The mast cell function-associated Ag (MAFA) is an inhibitory C-type lectin that was originally identified on the cell surface of a rat mucosal mast cell line, RBL-2H3. We have cloned the mouse homologue of the rat MAFA gene, and Northern blot analysis revealed that mouse MAFA (mMAFA) gene expression was strongly induced in effector CD8 T cells and lymphokine-activated NK cells but not in effector CD4 T cells and in mouse mast cells. Moreover, mMAFA gene expression was only found in effector CD8 T cells that had been primed in vivo with live virus because in vitro activated CD8 T cells did not express mMAFA. Primary sequence comparison revealed a high degree of conservation (89% similarity) between rat MAFA and mMAFA. Thus, the MAFA molecule in the mouse is a putative inhibitory receptor on anti-viral CD8 T cells induced in vivo and on NK cells.
Cutting Edge: Nuclear Factor of Activated T Cells and AP-1 Are Insufficient for IL-2 Promoter Activation: Requirement for CD28 Up-Regulation of RE/AP
Authors: Shapiro Virginia Smith , Mollenauer Marianne Newton , Weiss Arthur
Published: December 2022

IL-2 gene transcription in T cells requires both TCR and costimulatory signals. IL-2 promoter activation in Jurkat T cells stimulated with superantigen presented by Raji B cells requires CD28 activation. The addition of rCTLA4Ig, which blocks CD28 binding to its ligand, to the cultures decreased IL-2 promoter activation by >80%. Interestingly, CTLA4Ig did not significantly inhibit the activation of either NF of activated T cells (NFAT) or AP-1 reporters. Therefore, activation of NFAT and AP-1 is insufficient for IL-2 promoter activation. In contrast, an RE/AP reporter was blocked by CTLA4Ig by >90%. Thus, the requirement for CD28 in IL-2 promoter activation appears to be due to RE/AP and not the NFAT or AP-1 sites. In addition, these data suggest that transcriptional activation of RE/AP is not mediated by NFAT, because activation of a NFAT reporter is not affected by the addition of CTLA4Ig.
Cutting Edge: TCR Signaling Induces Selective Exclusion of CD43 from the T Cell-Antigen-Presenting Cell Contact Site
Authors: Sperling Anne I. , Sedy John R. , Manjunath N. , Kupfer Abraham , Ardman Blair , Burkhardt Janis K.
Published: December 2022

CD43, a large highly glycosylated molecule, is arguably the most abundant molecule on the surface of T cells. Nevertheless, the function of CD43 remains unclear. Utilizing fluorescence microscopy, we find that CD43 is excluded from the T cell-APC contact site. This exclusion is Ag dependent since optimal CD43 exclusion requires Ag-pulsed APC, and since signaling through CD3, in the absence of any other receptor ligand interactions, can induce the modulation of CD43. These data suggest that CD43 may function as a barrier to nonspecific T cell-APC interactions that is removed as a result of T cell activation. Exclusion from the interaction site is a unique feature of CD43 and not universally found for all large highly glycosylated molecules since CD45 is not excluded. Thus, CD43 may represent a novel regulatory molecule on the T cell surface that can direct T cell interactions by changing its location on the cell surface.
Increased Activity of Oleate-Dependent Type Phospholipase D During Actinomycin D-Induced Apoptosis in Jurkat T Cells
Authors: Kasai Takeshi , Ohguchi Kenji , Nakashima Shigeru , Ito Yuzuru , Naganawa Takashi , Kondo Naomi , Nozawa Yoshinori
Published: December 2022

Apoptosis is an active form of cell death that can be induced by a wide variety of agents and conditions. In response to actinomycin D, hydrogen peroxide (H2O2), or TNF-α, Jurkat T cells underwent typical apoptosis. Phospholipase D (PLD) activity in intact cells determined by phosphatidylbutanol generation was up-regulated by these agents. The PLD activation was in a time-dependent manner during apoptosis. It was also shown that the PLD activity measured by using exogenous substrate in the lysate from apoptotic cells was higher than that in the lysate from control untreated cells. The PLD activity in lysate from control untreated cells was stimulated by unsaturated fatty acids (UFA), but not by guanosine 5′-O-(3-thiotriphosphate). However, the PLD activity in the apoptotic cell lysate was no longer enhanced by the addition of oleate, suggesting that the increased PLD activity during apoptosis was attributed to the PLD of UFA-dependent type, but not the small G protein-dependent one. In fact, the release of free UFA was increased during apoptosis. The caspase inhibitors, z-DEVD and z-VAD, effectively suppressed PLD activation and apoptosis, but UFA release was unaffected. These results suggest the possibility that UFA-dependent type PLD may be implicated in apoptotic process in Jurkat T cells. This is the first demonstration that the PLD of UFA-dependent type would be involved in cellular responses.
CD27/CD70 Interaction Augments IgE Secretion by Promoting the Differentiation of Memory B Cells into Plasma Cells
Authors: Nagumo Haruo , Agematsu Kazunaga , Shinozaki Koji , Hokibara Sho , Ito Susumu , Takamoto Masaya , Nikaido Toshio , Yasui Kozo , Uehara Yoshio , Yachie Akihiro , Komiyama Atsushi
Published: December 2022

The induction of IgE switching in B cells requires several signals given by cytokines and cell contact-delivered signals. Here, we investigated the role of CD27/CD70 interaction in B cell IgE synthesis. The addition of CD27 ligand (CD70) transfectants to B cell cultures increased the IgE synthesis synergistically in the presence of IL-4 plus anti-CD40 mAb (anti-CD40). The effect of CD70 transfectants was dose dependent and was completely blocked by anti-CD70 mAb. CD27+ B cells had the ability to produce IgE, which was increased by contact with CD70 transfectants, whereas CD27− B cells did not produce IgE. CD27/CD70 interaction enhanced B cell proliferation in the presence of IL-4 or IL-4 plus anti-CD40. The augmentation of B cell proliferation by CD70 transfectants was apparent in CD27+ B cells, but was mild in CD27− B cells. The helper activity for IgE synthesis by the CD27/CD70 interaction did not contribute to the enhancement of germline ε transcripts. Flow cytometric and morphological analyses demonstrated that the addition of CD70 transfectants to B cell cultures remarkably promoted differentiation into plasma cells in the presence of IL-4 and CD40 signaling. Finally, CD27 cross-linking resulted in the up-regulation of positive regulatory domain I-binding factor-1. Taken together, our findings indicate that signaling via CD27 on B cells induces IgE synthesis, in cooperation with IL-4 and CD40 signaling, by promoting the generation of plasma cells through up-regulation of positive regulatory domain I-binding factor-1.
Stimulation of Stat5 by Granulocyte Colony-Stimulating Factor (G-CSF) Is Modulated by Two Distinct Cytoplasmic Regions of the G-CSF Receptor
Authors: Dong Fan , Liu Xiuwen , de Koning John P. , Touw Ivo P. , Henninghausen Lothar , Larner Andrew , Grimley Philip M.
Published: December 2022

In a manner similar to many other cytokines, treatment of cells with granulocyte CSF (G-CSF) has been shown to induce the tyrosine phosphorylation of the STAT proteins. Activation of Stat1 and Stat5 by G-CSF requires the membrane-proximal cytoplasmic domain of the receptor, including box1 and box2, while G-CSF-stimulated tyrosine phosphorylation of Stat3 also requires a region distal to box 2. In this study, we show that although the membrane-proximal 55 amino acids of the G-CSF receptor are sufficient for activation of Stat5, the maximal rate of Stat5 activation requires an additional 30 amino acids of the cytoplasmic domain. In contrast, the distal carboxyl-terminal region of the receptor appears to down-regulate Stat5 activation in that deletion of this carboxyl terminus results in increased amplitude and prolonged duration of Stat5 activation by G-CSF. Significantly, expression of a truncated dominant-negative Stat5 protein in hemopoietic cells not only inhibits G-CSF-dependent cell proliferation, but also suppresses cell survival upon G-CSF withdrawal. We further show that a potential protein tyrosine phosphatase may play a critical role in the down-regulation of G-CSF-stimulated Stat5 activation. These results demonstrate that two distinct cytoplasmic regions of the G-CSF receptor are involved in the regulation of the intensity and duration of Stat5 activation, and that Stat5 may be an important player in G-CSF-mediated cell proliferation and survival.
Cytotoxic T Cell Responses to DNA Vaccination: Dependence on Antigen Presentation via Class II MHC
Authors: Maecker Holden T. , Umetsu Dale T. , DeKruyff Rosemarie H. , Levy Shoshana
Published: December 2022

This study was designed to test whether cytotoxic T cell (CTL) responses to DNA vaccination are dependent upon MHC class II-restricted priming of CD4+ T cells. Because DNA vaccination may directly transfect dendritic cells, and dendritic cells may be capable of directly stimulating CD8+ T cell responses, such priming might be unnecessary. To test this hypothesis, C57BL/6 mice were immunized intramuscularly or intradermally with DNA encoding either whole OVA, a class I (Kb)-restricted peptide epitope of OVA (amino acids 257–264, SIINFEKL), or this class I-restricted epitope plus the adjacent class II (I-Ab)-restricted epitope of OVA (amino acids 265–280, TEWTSSNVMEERKIKV). Very low to negligible CTL responses were observed in mice vaccinated with the SIINFEKL construct, whereas mice vaccinated with the SIINFEKLTEWTSSNVMEERKIKV or with the complete OVA construct made equally robust CTL responses. These responses were sensitive to blocking by anti-CD8 mAb and were shown to be SIINFEKL-specific by using SIINFEKL peptide-pulsed EL-4 cells as targets. To ensure that the generation of these CTL responses was indeed dependent upon CD4+ T cell help, mice were depleted of either CD4+ or CD8+ cells before immunization. Depletion of CD4+ cells completely abrogated the CTL response to OVA DNA, as did depletion of CD8+ cells. Thus, we conclude that the CTL response to both intramuscular and intradermal DNA vaccination is highly dependent upon the generation of CD4+ T cell help via a class II MHC-dependent pathway. These results will be relevant for the construction of minimal-epitope vaccines for DNA immunization.
Regulation of NK1.1 Expression During Lineage Commitment of Progenitor Thymocytes
Authors: Carlyle James R. , Zúñiga-Pflücker Juan Carlos
Published: December 2022

We recently identified a stage in fetal ontogeny (NK1.1+/CD117+) that defines committed progenitors for T and NK lymphocytes. These cells are found in the fetal thymus as early as day 13 of gestation, but are absent in the fetal liver. Nonetheless, multipotent precursors derived from both the fetal thymus and fetal liver are capable of rapidly differentiating to the NK1.1+ stage upon transfer into fetal thymic organ culture (FTOC). This suggests that expression of NK1.1 marks a thymus-induced lineage commitment event. We now report that a subset of the most immature fetal thymocytes (NK1.1−/CD117+) is capable of up-regulating NK1.1 expression spontaneously upon short-term in vitro culture. Interestingly, fetal liver-derived CD117+ precursors remain NK1.1− upon similar culture. Spontaneous up-regulation of NK1.1 surface expression is minimally affected by transcriptional blockade, mitogen-induced activation, or exposure of these cells to exogenous cytokines or stromal cells. These data suggest that induction of NK1.1 expression on cultured thymocytes may be predetermined by exposure to the thymic microenvironment in vivo. Importantly, multipotent CD117+ thymocytes subdivided on the basis of NK1.1 expression after short-term in vitro culture show distinct precursor potential in lymphocyte lineage reconstitution assays. This demonstrates that even the earliest precursor thymocyte population, although phenotypically homogeneous, contains a functionally heterogeneous subset of lineage-committed progenitors. These findings characterize a thymus-induced pathway in the control of lymphocyte lineage commitment to the T and NK cell fates.
The Role of Stat4 in Species-Specific Regulation of Th Cell Development by Type I IFNs
Authors: Rogge Lars , D’Ambrosio Daniele , Biffi Mauro , Penna Giuseppe , Minetti Lisa J. , Presky David H. , Adorini Luciano , Sinigaglia Francesco
Published: December 2022

Type I IFNs (IFN-α/β), in addition to IL-12, have been shown to play an important role in the differentiation of human, but not mouse, Th cells. We show here that IFN-α/β act directly on human T cells to drive Th1 development, bypassing the need for IL-12-induced signaling, whereas IFN-α cannot substitute IL-12 for mouse Th1 development. The molecular basis for this species specificity is that IFN-α/β activate Stat4 in differentiating human, but not mouse, Th cells. Unlike IL-12, which acts only on Th1 cells, IFN-α/β can activate Stat4 not only in human Th1, but also in Th2 cells. However, restimulation of human Th2 lines and clones in the presence of IFN-α does not induce the production of IFN-γ. These results suggest that activation of Stat4, which is necessary for the differentiation of naive T cells into polarized Th1 cells, is not sufficient to induce phenotype reversal of human Th2 cells.
The Development of Autoimmune Inflammatory Arthropathy in Mice Transgenic for the Human T Cell Leukemia Virus Type-1env-pXRegion Is Not Dependent on H-2 Haplotypes and Modified by the Expression Levels of Fas Antigen
Authors: Iwakura Yoichiro , Itagaki Keiko , Ishitsuka Chiho , Yamasaki Yoshiki , Matsuzawa Akio , Yonehara Shin , Karasawa Shigeru , Ueda Susumu , Saijo Shinobu
Published: December 2022

Previously, we reported that human T cell leukemia virus type-1 env-pX region-introduced transgenic (pX-Tg) mice develop an inflammatory polyarthropathy. Although autoimmune pathogenesis was suggested, the detailed mechanisms remain to be elucidated. In this report, we examined effects of the MHC and fas genes on the development of the disease. When pX-Tg mice were backcrossed with different inbred strains, the incidence of arthritis differed among strains; 64% and 72% in BALB/cAn (H-2d), 25% and 46% in C3H/HeN (H-2k), and 0% and 2% in C57BL/6J (H-2b) background at 3 and 6 months of age, respectively. Rheumatoid factor levels in the serum correlated with the susceptibility to the disease, whereas IL-1β and MHC gene expression were similarly elevated in all of these strains, suggesting involvement of immune regulatory genes in this strain difference. However, introduction of the H-2d locus into C57BL/6J pX-Tg mice did not increase the incidence of arthritis, and substitution of the BALB/cAn H-2 locus with the H-2b did not decrease it. The results indicate that the H-2 locus is not the major determinant of the disease. Then, since previous study indicated a defect in Fas-mediated apoptosis of transgenic T cells, the effects of fas gene modification on the disease were examined. The incidence increased when these pX-Tg mice were crossed with lpr/lpr mice, while it decreased when crossed with fas-transgenic mice. These observations suggest that aberration of Fas-mediated apoptosis of peripheral lymphocytes, rather than negative selection in the thymus, is involved in the development of autoimmune arthropathy in pX-Tg mice.
Dependence of Both Spontaneous and Antibody-Dependent, Granule Exocytosis-Mediated NK Cell Cytotoxicity on Extracellular Signal-Regulated Kinases
Authors: Trotta Rossana , Puorro Kristin A. , Paroli Marino , Azzoni Livio , Abebe Bekele , Eisenlohr Laurence C. , Perussia Bice
Published: December 2022

Extracellular signal-regulated kinases (ERK, also known as mitogen-activated protein kinases) are serine-threonine kinases transducing signals elicited upon ligand binding to several tyrosine kinase-associated receptors. We have reported that ERK2 phosphorylation and activation follows engagement of the low affinity receptor for the Fc portion of IgG (CD16) on NK cells, and is necessary for CD16-induced TNF-α mRNA expression. Here, we analyzed the involvement of ERK in NK cell-mediated cytotoxicity and IFN-γ expression induced upon stimulation with targets cells, coated or not with Abs. Our data indicate that, as with immune complexes, ERK2 phosphorylation occurs in human primary NK cells upon interaction with target cells sensitive to granule exocytosis-mediated spontaneous cytotoxicity, and that this regulates both target cell- and immune complex-induced cytotoxicity and IFN-γ mRNA expression. A specific inhibitor of mitogen-activated protein kinase kinase reduced both spontaneous and Ab-dependent cytotoxicity in a dose-dependent manner involving, at least in part, inhibition of granule exocytosis without affecting effector/target cell interaction and rearrangement of the cytoskeleton proteins actin and tubulin. Involvement of ERK in the regulation of Ca2+-dependent cell-mediated cytotoxicity was confirmed, using a genetic approach, in primary NK cells infected with a recombinant vaccinia virus encoding an ERK inactive mutant. These data indicate that the biochemical pathways elicited in NK cells upon engagement of receptors responsible for either spontaneous or Ab-dependent recognition of target cells, although distinct, utilize ERK as one of their downstream molecules to regulate effector functions.
A Region of Conformational Variability Outside the Peptide-Binding Site of a Class I MHC Molecule
Authors: Kuhns Scott T. , Pease Larry R.
Published: December 2022

Peptide binding is known to influence the conformation of the surface of class I molecules as detected with mAbs and TCR. A new conformationally sensitive epitope on the mouse class I molecule Kb is defined by mAb AF6-88.5. The recognized structure is affected by amino acid substitutions in any of the three external domains of the class I heavy chain and, in addition, is influenced by the substitution of human for mouse β2-microglobulin. Interestingly, the epitope for this Ab is not affected by mutations within the peptide-binding cleft or by the nature of the peptide bound. These findings indicate that the effect of a change in one domain of class I can radiate to other parts of the molecule. Furthermore, the existence of conformationally sensitive structures outside of the peptide-binding site suggests the possibility that class I molecules may change their structure in response to binding by receptors and ligands such as the TCR and the coligand CD8. Such structural changes may represent signals that can influence cellular activation events.
Antitumor Response Elicited by a Superantigen- Transmembrane Sequence Fusion Protein Anchored onto Tumor Cells
Authors: Wahlsten Jennifer L. , Mills Charles D. , Ramakrishnan S.
Published: December 2022

Superantigens stimulate T cells bearing certain TCR β-chain variable regions when bound to MHC II molecules. We investigated whether the superantigen toxic shock syndrome toxin-1 (TSST1) could induce an antitumor immune response when anchored onto MHC II-negative tumor cells. Our approach was to facilitate association of TSST1 with cell membranes by fusing its coding region to the transmembrane region (TM) sequence of the proto-oncogene c-erb-B-2. TSST1-TM was expressed in bacteria with an N-terminal histidine tag and purified using nickel-agarose affinity chromatography. Purified TSST1-TM added to cultures of several different MHC II-negative tumor cells spontaneously associated with cell membranes, as detected by flow cytometry. Because superantigens can direct cell-mediated cytotoxicity against MHC II-positive cells, a TM fusion protein lacking the TSST1 MHC II binding domain (TSST88–194-TM) was also constructed. Tumor cells precoated with TSST1-TM or TSST88–194-TM stimulated proliferation of human peripheral blood lymphocytes in vitro whereas uncoated tumor cells did not. Mice preimmunized with TSST1-TM- or TSST88–194-TM-coated tumor cells mounted a systemic response that resulted in significant antitumor immunity as measured by regression of a parental tumor challenge. TSST1-TM and TSST88–194-TM fusion proteins represent a useful new strategy for attaching superantigens or potentially other proteins onto tumor cell surfaces without genetic manipulation.
Expression of the Third Component of Complement, C3, in Regenerating Limb Blastema Cells of Urodeles
Authors: Del Rio-Tsonis Katia , Tsonis Panagiotis A. , Zarkadis Ioannis K. , Tsagas Andreas G. , Lambris John D.
Published: December 2022

In this study we have shown that complement component C3 is expressed in the regenerating tissue during urodele limb regeneration. C3 was expressed in the dedifferentiated regeneration blastema and in the redifferentiated limb tissues in the axolotl, Amblystoma mexicanum, and in Notophthalmus viridescens. This expression was verified by immunofluorescent staining using an Ab against axolotl C3 and by in situ hybridization with an axolotl C3 cDNA probe. In the early stages of regeneration C3 appeared to be equally present in all mesenchymal cells and in the wound epithelium, whereas in the later stages it was mainly expressed in the differentiating muscle cells. Since no expression was seen in the developing limb, it appears that the C3 expression was specific to the regeneration process. We then demonstrated by hybridization experiments that a blastema cell line of myogenic origin expresses C3. All these findings implicate C3 in the dedifferentiation process and may indicate a new role for this molecule in muscle differentiation.
The Type II IL-1 Receptor Interacts with the IL-1 Receptor Accessory Protein: A Novel Mechanism of Regulation of IL-1 Responsiveness
Authors: Lang Detlef , Knop Johannes , Wesche Holger , Raffetseder Ute , Kurrle Roland , Boraschi Diana , Martin Michael U.
Published: December 2022

IL-1 binds to two types of receptors on the cell membrane, of which only type I (IL-1RI) transduces signals in concert with the coreceptor IL-1 receptor accessory protein (IL-1RAcP) while type II (IL-1RII) allegedly functions solely as ligand sink and decoy receptor without participating in IL-1 signaling. To investigate the regulatory role of IL-1RII on IL-1 responsiveness, a chimeric receptor encompassing the extracellular and transmembrane portions of IL-1RII and the cytoplasmic signal-transducing domain of IL-1RI was transfected into two murine EL-4-derived sublines that do or do not express IL-1RAcP, respectively. The chimeric receptor was able to transduce the IL-1 signal and induce IL-2 production only in the cell line which expressed IL-1RAcP, suggesting effective interaction between the extracellular domains of IL-1RII and IL-1RAcP in the presence of IL-1. The physical association of ligated IL-1RII with IL-1RAcP was proven by crosslinking experiments with radio-iodinated IL-1 and subsequent immunoprecipitations in normal human B cells and in EL-4 D6/76 cells transiently cotransfected with IL-1RII and IL-1RAcP, respectively. Based on these findings, it is proposed that upon IL-1 binding IL-1RII can recruit IL-1RAcP into a nonfunctional trimeric complex and thus modulate IL-1 signaling by subtracting the coreceptor molecule from the signaling IL-1RI. In this novel mechanism of coreceptor competition, the ratio between IL-1RII and IL-1RI becomes the central factor in determining the IL-1 responsiveness of a cell and the availability of IL-1RAcP becomes limiting for effective IL-1 signaling.
Successful Induction of Adjuvant Arthritis in Mice by Treatment with a Monoclonal Antibody Against IL-4
Authors: Yoshino Shin , Murata Yuzo , Ohsawa Motoyasu
Published: December 2022

Adjuvant arthritis (AA) is an experimental model of autoimmune disease in rats induced by immunization with Mycobacterium tuberculosis (MT). Induction of AA in other species, including mice, has been shown to be difficult. In the present study, we found that AA could be induced in mice if the animals were treated with a mAb (11B11 mAb) against IL-4. Histologically, the joints exhibited synovial edema with infiltration of many neutrophils in the early phase of inflammation. In its late phase, there were proliferation of synovium, cell infiltrate in which mononuclear cells predominated, and destruction of cartilage and subchondral bone. The joint inflammation was passively transferred to normal syngeneic recipient mice with lymphoid cells but not with sera from mice immunized with MT followed by treatment with the anti-IL-4 Ab. Delayed-type hypersensitivity (DTH) and proliferative responses of lymphoid cells to purified protein derivative were markedly augmented in 11B11 mAb-treated mice. Furthermore, the induction of arthritis was associated with a marked decrease in IL-4 secretion but a significant increase in IFN-γ and IL-2 production. Thus, the neutralization of IL-4 by an anti-IL-4 Ab appears to be required for the induction of AA in mice.
IL-4 Inhibits Mouse Mast Cell FcεRI Expression Through a STAT6-Dependent Mechanism
Authors: Ryan John J. , DeSimone Shirley , Klisch Gregory , Shelburne Christopher , McReynolds Lisa J. , Han Kathy , Kovacs Reka , Mirmonsef Paria , Huff Thomas F.
Published: December 2022

Mast cell activation by IgE-mediated stimuli is a central event in atopic disease. The regulation of the mast cell high affinity receptor, FcεRI, is poorly understood. We show that IL-4 can inhibit FcεRI expression on mouse bone marrow-derived mast cells and fetal liver-derived mast cell progenitors. This effect could be observed at 2.5 ng/ml IL-4 and was dose dependent. IL-4-mediated inhibition of cultured BMMC required 4 days of stimulation and was sustained at maximum levels for at least 21 days. The inhibition of FcεRI expression resulted in decreased sensitivity to IgE-mediated stimulation, as measured by serotonin release, and the induction of mRNA for IL-4, IL-5, IL-6, and IL-13. Additionally, IL-4 could abrogate the IgE-mediated increase in FcεRI expression. Lastly, IL-4-mediated inhibition was dependent upon expression of the STAT6 transcription factor, as STAT6-deficient bone marrow-derived mast cells did not decrease FcεRI levels in response to IL-4. These data argue for a homeostatic role of IL-4 in the regulation of FcεRI expression, a role that could be critical to understanding atopic disease.
Activation of Phosphatidylinositol 3-Kinase, Protein Kinase B, and p70 S6 Kinases in Lipopolysaccharide-Stimulated Raw 264.7 Cells: Differential Effects of Rapamycin, Ly294002, and Wortmannin on Nitric Oxide Production
Authors: Salh B. , Wagey R. , Marotta A. , Tao J. S. , Pelech S.
Published: December 2022

Phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B are critical players in cell proliferation and survival. Their downstream effector protein kinase, p70 S6 kinase, has an established role in protein translation. The mechanism by which bacterial LPS induces production of nitric oxide (NO) in murine macrophages is incompletely understood, and a role for PI 3-kinase/p70 S6 kinase pathway had not been previously investigated. In this study we demonstrate that LPS induced a fivefold activation of p70 S6 kinase and a twofold stimulation of PI 3-kinase. Pretreatment of Raw 264.7 cells with either rapamycin or Ly290042 completely blocked LPS-induced activation of p70 S6 kinase. Protein kinase B was also activated (twofold) by LPS and was only minimally affected by these inhibitors. PI 3-kinase activity was inhibited by both Ly294002 and wortmannin. The effects on NO production by these agents were strikingly different. While both rapamycin and Ly294002 resulted in almost complete inhibition of NO production, wortmannin was ineffective. Surprisingly, none of the inhibitors reduced the production of the inducible nitric oxide synthase protein (iNOS) as determined by immunoprecipitation. In vivo labeling studies revealed that the iNOS protein was phosphorylated in concordance with the production of NO. We conclude that LPS-mediated NO production occurs via a PI 3-kinase-independent, but FKBP12-rapamycin-associated protein-dependent, pathway in RAW cells by a mechanism probably involving phosphorylation of iNOS.
Environmental Antigen-Induced IL-13 Responses Are Elevated Among Subjects with Allergic Rhinitis, Are Independent of IL-4, and Are Inhibited by Endogenous IFN-γ Synthesis
Authors: Li Yan , Simons F. Estelle R. , HayGlass Kent T.
Published: December 2022

Human immediate hypersensitivity diseases represent the most common example of chronic excessive Th2-like activation in developed nations. While IL-13 shares many functional properties with IL-4, the intensity and regulation of environmental Ag-stimulated IL-13 synthesis by allergic vs nonallergic individuals remain ill defined. Here, we examine the intensity of polyclonally and Ag-stimulated IL-13 production by PBMC of 20 subjects with seasonal allergic rhinitis and 20 healthy controls. Polyclonally driven IL-13 responses did not differ significantly (Mann-Whitney U test, p = 0.68). In contrast, the median CD4-dependent IL-13 response among atopics was markedly stronger than nonatopics in Ag-stimulated primary culture (p = 0.0031) and exhibited a strong correlation with IL-5 (r = 0.76, p = 0.0009), but not IL-4 (r = 0.14, p > 0.05), responses. IL-13 production was unaffected by blocking endogenous IL-4 or IL-5 activity or by addition of rIL-4 or rIL-5. In contrast, it was inhibited by low levels of rIFN-γ and strongly enhanced upon addition of neutralizing anti-IFN-γ mAb. Collectively, the data are consistent with a negative regulatory role for endogenous IFN-γ synthesis in controlling the intensity of systemic IL-13 responses evoked in both atopic and nonatopic populations following exposure to common Ags. They also suggest that the elevated levels of IL-4 and IL-5 characteristic of type 2-dominated responses in vivo are without detectable impact on the maintenance of recall Ag-stimulated IL-13 production.
Terminal Deoxynucleotidyl Transferase Deficiency Reduces the Incidence of Autoimmune Nephritis in (New Zealand Black × New Zealand White)F1 Mice
Authors: Conde Carmen , Weller Sandra , Gilfillan Susan , Marcellin Luc , Martin Thierry , Pasquali Jean-Louis
Published: December 2022

Terminal deoxynucleotidyl transferase (TdT) enzyme activity in lymphocytes generates diversity in the Ag receptor repertoires by adding template-independent N nucleotides and disrupting homology-directed rearrangements. The importance of this diversity in vivo and the significance of the suppression of TdT during fetal life remain uncertain. Previous studies have shown that in TdT knockout mice (TdT°) 1) the T cell repertoire is less peptide oriented; and 2) natural autoantibody, particularly anti-DNA autoantibodies, are less polyreactive, and their mean affinities are reduced. Consequently, the suppression of TdT during early T/B cell ontogeny may participate in controlling autoimmunity. To study the impact of TdT suppression in autoimmune-prone mice, we introduced the TdT null mutation into the (NZB × NZW)F1 (B/W) mouse strain. We show that TdT deficiency significantly reduces the incidence of autoimmune nephritis and prolongs survival compared with those in control mice. Surprisingly, the long-term survivor TdT° mice produced amounts of anti-ADN and anti-histone autoantibodies similar to those of their TdT+ littermates. However, these TdT° mice showed no evidence of renal inflammation, and the immune deposits were restricted to the mesangium, whereas basal membrane deposits were clearly correlated with overt renal disease. The present study supports the idea that the absence of TdT enzyme activity in lymphocytes protects mice against autoimmunity and could offer a therapeutic approach to autoimmune diseases. Moreover, our results may help to unravel the mechanisms of lupus nephritis.