bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative analysis of the human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocyte (CTL) response at different stages of HIV-1 infection: differential CTL responses to HIV-1 and Epstein-Barr virus in late disease.
Authors: Carmichael A , Jin X , Sissons P , Borysiewicz L
Published: June 2004

Major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) are part of the cellular immune response to human persistent virus infections. Measurements of the frequency and specificity of human immunodeficiency virus type 1 (HIV-1)-specific CTL and their variation with time may indicate their relative importance in modulating the progression of HIV-1 infection. We have used limiting dilution analysis (LDA) to derive quantitative estimates of the frequency of HIV-1-specific CTL precursors in a cross-sectional study of 23 patients at different clinical stages of HIV-1 infection and to compare these with the frequency of CTL precursors specific for another persistent virus (Epstein-Barr virus [EBV]) in the same patients. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with autologous HIV-1-infected lymphoblasts and assayed for cytotoxicity in 51Cr release assays against autologous and MHC-mismatched lymphoblastoid B cells infected with recombinant vaccinia viruses expressing the three HIV-1 structural gene products. The frequency of MHC-restricted precursors was high in asymptomatic HIV-1-infected patients (env-specific CTL precursors up to 73/10(6) PBMC; gag-specific CTL precursors up to 488/10(6) PBMC), although the relative frequency against the different structural gene products varied from patient to patient. The HIV-1-specific CTL precursor frequency was reduced in patients who had more severe (< 400/microliters) CD4+ lymphocyte depletion, while in the majority of such patients the frequency of CTL precursors against EBV was maintained at levels observed in healthy controls. Direct CTL activity in unstimulated PBMC was observed in three of nine patients but no correlation was found between the presence of an activated CTL response and the magnitude of the CTL response detected after stimulation in LDA. Thus, CTL precursors were detected against all three HIV-1 structural gene products in patients with CD4+ lymphocyte counts > 400/microliters, at frequencies that are high compared with those reported for other persistent viruses. A CTL response directed against multiple protein antigens of HIV-1 may protect the patient against epitope variation. The fact that the EBV-specific CTL precursor frequencies were maintained in advanced HIV-1 infection suggests that there may be selective impairment of the HIV-1-specific CTL response associated with disease progression.
Pfs2400 can mediate antibody-dependent malaria transmission inhibition and may be the Plasmodium falciparum 11.1 gene product.
Authors: Feng Z , Hoffmann R N , Nussenzweig R S , Tsuji M , Fujioka H , Aikawa M , Lensen T H , Ponnudurai T , Pologe L G
Published: June 2004

Monoclonal antibodies (mAb) have been raised against Plasmodium falciparum gametocyte stage protein extracts, in an effort to identify novel parasite antigens that might mediate malaria transmission-blocking immunity. mAb 1A1 identified Pfs2400, a sexual stage-specific antigen of greater than 2 megadaltons, that is associated with the outer leaflet of the parasitophorous vacuole membrane in mature circulating gametocyte-infected red blood cells. Upon induction of gametogenesis, Pfs2400 partitions between the gamete plasmalemma and the degenerating erythrocyte membrane. The antigen is no longer detectable in the fully emerged gamete. mAb 1A1 dramatically reduces the number of oocysts formed in P. falciparum gametocyte-fed mosquitoes. The cognate antigen is probably the product of the Pf11.1 gene (Scherf et al. 1988. EMBO [Eur. Mol. Biol. Organ.]J. 7:1129) on the basis that a peptide composed of two copies of the degenerate nine amino acid repeat sequence in the Pf11.1 protein, can inhibit binding of mAb1A1 to the native antigen. The mechanism of transmission inhibition mediated by the Pfs2400 is discussed.
Localization of a site on bacterial superantigens that determines T cell receptor beta chain specificity.
Authors: Mollick J A , McMasters R L , Grossman D , Rich R R
Published: June 2004

A defining characteristic of superantigens is their ability to stimulate T cells based predominantly on the type of variable segment of the T cell receptor (TCR) beta chain (V beta). The V beta specificity of these toxins most likely results from direct contact between the toxin and the TCR, although the low affinity nature of this binding has prevented direct assessment of this interaction. To identify important functional sites on the toxin, we created chimeric enterotoxin genes between staphylococcal enterotoxins A and E (SEA and SEE) and tested the V beta specificity of the chimeric toxins. This approach allowed us to identify three amino acid residues in the extreme COOH terminus of these toxins that are largely responsible for their ability to stimulate either human V beta 5- or V beta 8-bearing T cells, or mouse V beta 3 or V beta 11. We also found that residues in the NH2 terminus were required for wild-type levels of V beta-specific T cell activation, suggesting that the NH2 and COOH ends of these superantigens may come together to form the full TCR V beta contact site. SEA and SEE also differ with respect to their class II binding characteristics. Using the same chimeric molecules, we demonstrate that the first third of the molecule controls the class II binding phenotype. These data lead us to propose that for SEA and SEE, and perhaps for all bacterial-derived superantigens, the COOH and NH2 termini together form the contact sites for the TCR and therefore largely determine the V beta specificity of the toxin, while the NH2 terminus alone binds major histocompatibility complex class II molecules. The predominant role of the COOH terminus of bacterial superantigens in determining V beta specificity resembles current models being proposed for virally encoded superantigens, suggesting that these molecules may demonstrate some structural relationship not seen at the amino acid level.
Cutaneous lymphoproliferation and lymphomas in interleukin 7 transgenic mice.
Authors: Rich B E , Campos-Torres J , Tepper R I , Moreadith R W , Leder P
Published: June 2004

To investigate the role of interleukin 7 (IL-7) in the development of the lymphoid system, we have generated two lines of transgenic mice carrying an IL-7 cDNA fused to an immunoglobulin heavy chain promoter and enhancer. This transgene is expressed in the bone marrow, lymph nodes, spleen, thymus, and skin provoking a perturbation of T cell development characterized by a marked reduction of CD4+ CD8+ (double-positive) thymocytes. Quite unexpectedly, however, both lines also develop a progressive cutaneous disorder involving a dermal lymphoid infiltrate that results in progressive alopecia, hyperkeratosis, and exfoliation. Although the infiltrate is primarily composed of T lineage cells, its development is not impeded in the athymic nu/nu background. Furthermore, the phenotype can be transmitted horizontally by transplanting lymphoid tissues or skin to syngeneic wild-type mice. Thus, the phenotype is conveyed by skin-homing, mobile cells (presumably the infiltrating lymphocytes) in a cell-autonomous fashion. In addition to the skin phenotype, this transgene also provokes the development of a lymphoproliferative disorder that induces B and T cell lymphomas within the first 4 mo of life. These findings suggest potential physiologic actions of IL-7 in T cell development and in cutaneous immunity. They also demonstrate that IL-7 can act as an oncogene in the living organism.
Superantigen-reactive CD4+ T cells are required to stimulate B cells after infection with mouse mammary tumor virus.
Authors: Held W , Shakhov A N , Izui S , Waanders G A , Scarpellino L , MacDonald H R , Acha-Orbea H
Published: June 2004

Superantigens are defined by their ability to stimulate a large fraction of T cells via interaction with the T cell receptor (TCR) V beta domain. Endogenous superantigens, classically termed minor lymphocyte-stimulating (Mls) antigens, were recently identified as products of open reading frames (ORF) in integrated proviral copies of mouse mammary tumor virus (MMTV). We have described an infectious MMTV homologue of the classical endogenous superantigen Mls-1a (Mtv-7). The ORF molecules of both the endogenous Mtv-7 and the infectious MMTV(SW) interact with T cells expressing the TCR V beta 6, 7, 8.1, and 9 domains. Furthermore, the COOH termini of their ORF molecules, thought to confer TCR specificity, are very similar. Since successful transport of MMTV from the site of infection in the gut to the mammary gland depends on a functional immune system, we were interested in determining the early events after and requirements for MMTV infection. We show that MMTV(SW) infection induces a massive response of V beta 6+ CDC4+ T cells, which interact with the viral ORF. Concomitantly, we observed a B cell response and differentiation that depends on both the presence and stimulation of the superantigen-reactive T cells. Furthermore, we show that B cells are the main target of the initial MMTV infection as judged by the presence of the reverse-transcribed viral genome and ORF transcripts. Thus, we suggest that MMTV infection of B cells leads to ORF-mediated B-T cell interaction, which maintains and possibly amplifies viral infection.
High-frequency representation of a single VH gene in the expressed human B cell repertoire.
Authors: Stewart A K , Huang C , Stollar B D , Schwartz R S
Published: June 2004

Idiotype (Id) 16/6 marks a variable (V) region structure that occurs frequently in the human immunoglobulin repertoire. The basis of the Id has been traced to a germline heavy chain gene segment, VH18/2 (VH26). To pursue the molecular basis for the frequency of Id 16/6, we have analyzed polymerase chain reaction-generated C mu, C gamma, and VH3 family V gene libraries derived from the circulating and tonsillar B cells of four normal individuals and from the B cells of two patients with active systemic lupus erythematosus (SLE). The frequency of VH18/2 in these libraries was compared with three control VH genes, VH56P1, VH21/28, and VHA57. Plaque lifts from C mu and C gamma VH cDNA libraries were screened with gene-specific oligonucleotide probes. The frequency of VH18/2 ranged from 4 to 10% of JH+ plaques (two of five times that of control VH genes). In four VH3 family-specific libraries derived from rearranged DNA, VH18/2 represented 19-33% of VH3+ plaques. Hybridizing VH18/2 plaques were 98-100% homologous to the germline VH gene; mutations when present were often in framework 3. Extensive variation was seen in the complementarity determining region 3 sequences of these rearranged V genes. The high frequency of VH18/2 expression in the B cell repertoire was confirmed by sequencing randomly picked JH+ plaques. In two patients with active SLE the frequency of use of VH18/2 was not greater than that observed in normal subjects. These results show that VH18/2 is overrepresented in the B cell repertoire of normal subjects and suggest that the immune repertoire may be dominated by relatively few V genes.
Characterization of a novel trans-sialidase of Trypanosoma brucei procyclic trypomastigotes and identification of procyclin as the main sialic acid acceptor.
Authors: Pontes de Carvalho L C , Tomlinson S , Vandekerckhove F , Bienen E J , Clarkson A B , Jiang M S , Hart G W , Nussenzweig V
Published: June 2004

Here we report the presence of a trans-sialidase on the surface of Trypanosoma brucei culture-derived procyclic trypomastigotes. The enzyme is not detected in lysates of bloodstream trypomastigotes enriched for either stumpy or slender forms. The trans-sialidase catalyzes the transfer of alpha(2-3)-linked sialic acid residues to lactose. beta-galactopyranosyl residues are at least 100 times better acceptors for sialic acid than alpha-galactopyranosyl residues. In the absence of efficient acceptors, the purified enzyme transfers sialic acid to water, i.e., it acts as a sialidase. Although the T. cruzi and T. brucei trans-sialidases have very similar donor and acceptor specificities, they are antigenically distinct. Sodium dodecyl sulfate-polyacramide gel electrophoresis under nonreducing conditions and silver staining of the purified trans-sialidase reveals a single band of 63 kD. When the surface membrane of live procyclic trypomastigotes is trans-sialylated, using radioactive sialyllactose as the donor substrate, it appears that the only sialylated surface molecule is procyclin. Pronase treatment of live parasites removes only part of the surface sialic acid, in agreement with recent data showing that the glycosylphosphatidylinositol anchor of procyclin is sialylated (Ferguson, M. A. J., M. Murray, H. Rutherford, and M. J. McConville. 1993. Biochem. J. In press).
Deficient biosynthesis of N-acetylglucosaminyl-phosphatidylinositol, the first intermediate of glycosyl phosphatidylinositol anchor biosynthesis, in cell lines established from patients with paroxysmal nocturnal hemoglobinuria.
Authors: Takahashi M , Takeda J , Hirose S , Hyman R , Inoue N , Miyata T , Ueda E , Kitani T , Medof M E , Kinoshita T
Published: June 2004

Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic disorder caused by a deficiency of biosynthesis of the glycosyl phosphatidylinositol (GPI) anchor, but the biochemical defect is not completely understood. In the present study, we have analyzed affected cell lines established recently from two Japanese patients with PNH. Two lines of evidence indicate that these cells do not synthesize N-acetylglucosaminyl-phosphatidylinositol, the first intermediate in the GPI anchor biosynthesis. First, somatic cell hybridization analysis using Thy-1-deficient murine thymoma cell lines with known biochemical defects as fusion partners showed that the PNH cell lines belong to complementation class A, which is known not to synthesize N-acetylglucosaminyl-phosphatidylinositol. Second, analysis of in vitro glycolipid biosynthesis demonstrated that cell lysates of these PNH cell lines in fact did not support biosynthesis of N-acetylglucosaminyl-phosphatidylinositol. Thus, we have characterized for the first time the exact biochemical defect leading to PNH.
Interferon gamma inhibits interleukin 10 production by monocytes.
Authors: Chomarat P , Rissoan M C , Banchereau J , Miossec P
Published: June 2004

Interleukin 10 (IL-10) was first described for its ability to inhibit interferon gamma (IFN-gamma) production. Herein, we studied the balance between IFN-gamma and IL-10 production by human peripheral blood mononuclear cells (PBMC) in response to Staphylococcus aureus Cowan (SAC) or lipopolysaccharide (LPS). Monocyte depletion reduced IL-10 production by 90% and resulted in an increased IFN-gamma production. Addition of anti-IL-10 antibody to PBMC cultures also strongly increased IFN-gamma production. In contrast, among various cytokines, only IFN-gamma strongly reduced IL-10 synthesis by SAC- or LPS-activated PBMC and monocytes. Thus, IFN-gamma has proinflammatory effects through the combination of two mechanisms: (a) induction of early tumor necrosis factor alpha (TNF-alpha) and IL-1 beta synthesis; and (b) inhibition of the delayed production of IL-10, an inhibitor of TNF-alpha and IL-1 beta synthesis. Taken together, the present data indicate that IFN-gamma and IL-10 antagonize each other's production and function.
Tyrosine phosphorylation is required for Fc receptor-mediated phagocytosis in mouse macrophages.
Authors: Greenberg S , Chang P , Silverstein S C
Published: June 2004

Although Fc receptor-mediated phagocytosis is accompanied by a variety of transmembrane signaling events, not all signaling events are required for particle ingestion. For example, Fc receptor-mediated phagocytosis in mouse inflammatory macrophages (Di Virgilio, F., B. C. Meyer, S. Greenberg, and S. C. Silverstein. 1988. J. Cell Biol. 106:657; Greenberg, S., J. El Khoury, F. Di Virgilio, and S. C. Silverstein. 1991. J. Cell Biol. 113:757) and neutrophils (Della Bianca, V., M. Grzeskowiak, and F. Rossi. 1990. J. Immunol. 144:1411) occurs in the absence of cytosolic calcium transients. We sought to identify transmembrane signaling events that are essential for phagocytosis. Here we show that tyrosine phosphorylation is an early event after Fc receptor ligation in mouse inflammatory macrophages, and that the formation of tyrosine phosphoproteins coincides temporally with the appearance of F-actin beneath phagocytic cups. The distribution of tyrosine phosphoproteins that accumulated beneath phagocytic cups was punctate and corresponded to areas of high ligand density on the surface of the antibody-coated red blood cells, which provided the phagocytic stimulus. A tyrosine kinase inhibitor, genistein, but not several inhibitors of protein kinase C, blocked the appearance of tyrosine phosphoproteins as assessed by immunofluorescence, the focal accumulation of F-actin beneath immunoglobulin G-opsonized particles, and the ingestion of these particles as well. We suggest that tyrosine phosphorylation is a critical signaling event that underlies Fc receptor-mediated phagocytosis in mouse macrophages, and is necessary for the engulfment per se.
Glutamic acid decarboxylase 67-reactive T cells: a marker of insulin-dependent diabetes.
Authors: Honeyman M C , Cram D S , Harrison L C
Published: June 2004

Glutamic acid decarboxylase (GAD) has been shown to be a target of autoantibodies in insulin-dependent diabetes (IDD). Two forms of GAD, with molecular weights of 67,000 and 65,000, have been cloned from separate genes. As pancreatic islet beta cell destruction DD is an autoimmune process mediated by T cells, we sought to determine if recombinant GAD67 was recognized by T cells in IDD subjects and particularly their first-degree relatives with islet cell antibodies known to be at risk for IDD. The central regions of human islet and brain GAD67 (amino acids 208-404) were cloned as fusion proteins with glutathione-S-transferase (GST). Proliferation of peripheral blood T cells in the presence of recombinant GAD67 was significantly higher in both at-risk relatives and recent-onset IDD subjects than in other autoimmune disease subjects and human histocompatibility leukocyte antigen (HLA)-matched healthy controls. Thus, 12 of 29 (41%) at-risk relatives and 11 of 29 (38%) recent-onset IDD subjects responded to GAD67, compared with 1 of 7 (14%) other autoimmune disease subjects and 1 of 23 (4%) HLA-matched controls. T cell responses to GST alone or to tetanus toxoid were not different between the groups. These findings demonstrate that GAD67 is a target autoantigen of T cells in IDD and suggest the possibility that GAD-reactive T cells may delineate asymptomatic subjects at increased risk for IDD.
T cell receptor ligation induces interleukin (IL) 2R beta chain expression in rat CD4,8 double positive thymocytes, initiating an IL-2-dependent differentiation pathway of CD8 alpha+/beta- T cells.
Authors: Park J H , Mitnacht R , Torres-Nagel N , Hünig T
Published: June 2004

The role of interleukin (IL)2 in intrathymic T cell development is highly controversial, and nothing is known about IL-2R expression on thymocytes of the T cell receptor (TCR) alpha/beta lineage undergoing TCR-driven differentiation events. We analyze here IL-2R alpha and beta mRNA expression in an in vitro system where newly generated rat CD4,8 double positive (DP) thymocytes respond to TCR ligation plus IL-2 (but not to either stimulus alone) with rapid differentiation to functional CD8 single positive T cells (Hünig, T., and R. Mitnacht. 1991. J. Exp. Med. 173:561). TCR ligation induced expression of IL-2R beta (but not alpha) chain mRNA in DP thymocytes. Addition of IL-2 then lead to functional maturation and expression of the IL-2R alpha chain. To investigate if the CD8 T cells generated via this IL-2R beta-driven pathway in vitro correspond to the bulk of CD8 T cells seeding peripheral lymphoid organs in vivo, we compared their phenotype to that of lymph node CD8 T cells. Surprisingly, analysis of CD8 cell surface expression using a novel anti-CD8 monoclonal antibody specific for the alpha/beta heterodimeric isoform, and of CD8 alpha and beta chain mRNA revealed that T cells generated by TCR ligation plus IL-2 resemble thymus-independent rather than thymus-derived CD8 cells in that they express CD8 alpha without beta chains. These findings demonstrate that TCR crosslinking induces functional IL-2R on immature DP rat thymocytes. In addition, they show that at least in vitro, CD8 alpha/alpha T cells are generated from TCR-stimulated DP thymocytes (which express the CD8 alpha/beta in the heterodimeric isoform) along an IL-2-driven pathway of T cell differentiation.
Interferon gamma regulates antigen-induced eosinophil recruitment into the mouse airways by inhibiting the infiltration of CD4+ T cells.
Authors: Iwamoto I , Nakajima H , Endo H , Yoshida S
Published: June 2004

We have previously shown that antigen-induced eosinophil recruitment into the tissue of sensitized mice is mediated by CD4+ T cells and interleukin 5. To determine whether interferon gamma (IFN-gamma) regulates antigen-induced eosinophil recruitment into the tissue, we studied the effect of recombinant (r) murine IFN-gamma and of anti-IFN-gamma monoclonal antibody (mAb) on the eosinophil infiltration of the trachea induced by antigen inhalation in mice. The intraperitoneal administration of rIFN-gamma prevented antigen-induced eosinophil infiltration in the trachea of sensitized mice. The administration of rIFN-gamma also decreased antigen-induced CD4+ T cell but not CD8+ T cell infiltration in the trachea. On the other hand, pretreatment with anti-IFN-gamma mAb enhanced antigen-induced eosinophil and CD4+ T cell infiltration in the trachea. These results indicate that IFN-gamma regulates antigen-induced eosinophil recruitment into the tissue by inhibiting CD4+ T cell infiltration.
Human peripheral blood basophils primed by interleukin 3 (IL-3) produce IL-4 in response to immunoglobulin E receptor stimulation.
Authors: Brunner T , Heusser C H , Dahinden C A
Published: June 2004

In contrast to most cytokines, interleukin 4 (IL-4) expression is restricted to T lymphocytes, with the exception of mast cell lines and mast cells, as more recently demonstrated in rodents. Little is known, however, about the capacity of human nonlymphoid cells to produce IL-4. In this study we show that mature human basophils are capable of expressing IL-4 and examine the regulation of IL-4 production in comparison with the lipid mediator leukotriene C4. IL-4 was produced upon immunoglobulin E receptor (IgER) activation of basophils cultured with IL-3, a cytokine previously shown to prime these cells for enhanced release of inflammatory mediators. In some experiments, IL-3 or IgER activation alone also induced IL-4 production close to the detection limit. The effect of IL-3 on IgER-dependent IL-4 expression was dose and time dependent: maximal IL-4 production occurred between 18 and 48 h preexposure of basophils to 3-10 ng/ml IL-3. IgER-induced IL-4 synthesis and release by basophils cultured with IL-3 was rapid and complete after 6 h. In contrast to IL-3, other cytokines (IL-5, granulocyte/macrophage colony-stimulating factor, and nerve growth factor) that also prime basophils for enhanced histamine and leukotriene C4 release did not promote IgER-induced IL-4 synthesis. Basophils appear to secrete a "TH2-like" cytokine profile since no detectable IL-2 or interferon gamma was produced upon IgER activation. Mononuclear cells (depleted of basophils), cultured in parallel, did not release IL-4 in response to IL-3 and/or IgER activation, and produced approximately ten times less IL-4 than basophils upon nonspecific activation by phorbol ester and calcium ionophore. Thus, human basophils are an important cellular source of IL-4, and may, therefore, in addition to their inflammatory effector functions, also regulate the differentiation of T helper cells and B cells, in particular in allergic diseases.
Delayed progression of a murine retrovirus-induced acquired immunodeficiency syndrome in X-linked immunodeficient mice.
Authors: Hitoshi Y , Okada Y , Sonoda E , Tominaga A , Makino M , Suzuki K , Kinoshita J , Komuro K , Mizuochi T , Takatsu K
Published: June 2004

The murine acquired immunodeficiency syndrome (MAIDS) caused by defective LP-BM5 murine leukemia virus (MuLV) is a disease that shows severe immunodeficiency with abnormal lymphoproliferation, and hypergammaglobulinemia in susceptible C57BL/6 (B6) mice. To examine the cellular mechanisms of development of MAIDS, we injected LP-BM5 MuLV intraperitoneally into B6 mice bearing the X chromosome-linked immunodeficiency (xid). xid mice lack functionally mature B cells including Ly-1 B cells (also known as B-1 cells). All B6 mice died by 20 wk after LP-BM5 MuLV inoculation. In marked contrast, xid mice have continued to survive without any sign of MAIDS-related symptoms till at least 20 wk after the inoculation. The delayed progression of MAIDS in xid mice appears to depend on xid mutation, according to our experiments using both sexes of (B6.xid x B6)F1 and (B6 x B6.xid)F1 mice. Furthermore, Ly-1 B cells, enriched by a FACS, were shown to integrate the defective genome and appeared to be a major virus-infected B cell population. Our data corroborate that Ly-1 B cells play an important role in the induction and progression of MAIDS.
Voltage-gated potassium channels regulate calcium-dependent pathways involved in human T lymphocyte activation.
Authors: Lin C S , Boltz R C , Blake J T , Nguyen M , Talento A , Fischer P A , Springer M S , Sigal N H , Slaughter R S , Garcia M L
Published: June 2004

The role that potassium channels play in human T lymphocyte activation has been investigated by using specific potassium channel probes. Charybdotoxin (ChTX), a blocker of small conductance Ca(2+)-activated potassium channels (PK,Ca) and voltage-gated potassium channels (PK,V) that are present in human T cells, inhibits the activation of these cells. ChTX blocks T cell activation induced by signals (e.g., anti-CD2, anti-CD3, ionomycin) that elicit a rise in intracellular calcium ([Ca2+]i) by preventing the elevation of [Ca2+]i in a dose-dependent manner. However, ChTX has no effect on the activation pathways (e.g., anti-CD28, interleukin 2 [IL-2]) that are independent of a rise in [Ca2+]i. In the former case, both proliferative response and lymphokine production (IL-2 and interferon gamma) are inhibited by ChTX. The inhibitory effect of ChTX can be demonstrated when added simultaneously, or up to 4 h after the addition of the stimulants. Since ChTX inhibits both PK,Ca and PK,V, we investigated which channel is responsible for these immunosuppressive effects with the use of two other peptides, noxiustoxin (NxTX) and margatoxin (MgTX), which are specific for PK,V. These studies demonstrate that, similar to ChTX, both NxTX and MgTX inhibit lymphokine production and the rise in [Ca2+]i. Taken together, these data provide evidence that blockade of PK,V affects the Ca(2+)-dependent pathways involved in T lymphocyte proliferation and lymphokine production by diminishing the rise in [Ca2+]i that occurs upon T cell activation.
B lymphocytes in vivo fail to prime naive T cells but can stimulate antigen-experienced T lymphocytes.
Authors: Ronchese F , Hausmann B
Published: June 2004

The ability of B cells or macrophages and dendritic cells (DC) to elicit class II-restricted T cell responses in vivo was compared using a mouse chimera model. Severe combined immunodeficient (SCID) mice (H-2d), reconstituted either with T or T+B lymphocytes from (H-2d x H-2b) donors, were immunized subcutaneously with protein antigen (Ag) to induce a class II-restricted T cell response. The frequency and major histocompatibility complex restriction of the resulting Ag-specific T cells were analyzed to establish whether B cells were necessary for the induction of class II-restricted T cell responses, and to determine the cell type on which priming had occurred. The results indicated that: (a) B cells are not necessary for the induction of a class II-restricted T cell response in vivo, as the frequencies of interleukin 2 (IL-2)- or IL-3-secreting T cells induced in the presence or absence of B cells were comparable. (b) Activation of naive T cells requires presentation of Ag on DC; Ag presented only on B cells is not sufficient to elicit a response. No H-2b-restricted, IL-3-secreting cells could in fact be detected in SCID mice reconstituted with naive (H-2d x H-2b) T cells and nonimmune or antigen-primed (H-2d x H-2b) B cells. (c) Previously primed T cells are able to be stimulated by Ag presented by both B cells and DC. H-2b-restricted, IL-3-secreting cells could in fact be readily demonstrated in SCID mice reconstituted with antigen-primed (H-2d x H-2b) T and B cells. Irrespective of whether the T cells were naive or previously activated, B cells were able to respond with an Ag-specific immunoglobulin G response, indicating that B cells were functional and able to present Ag in order to receive specific T cell help. Therefore, it appears that B cells are not necessary and do not participate in the initial priming of T cells; however, Ag presented by B cells can reactivate previously primed T cells. Taken together, these data indicate that during the course of an immune response Ag is first presented to naive T cells via DC, and only subsequently primed T cells can be stimulated by Ag presented by B cells.
Repression of major histocompatibility complex IA expression by glucocorticoids: the glucocorticoid receptor inhibits the DNA binding of the X box DNA binding protein.
Authors: Celada A , McKercher S , Maki R A
Published: June 2004

Glucocorticoids are effective repressors of major histocompatibility complex (MHC) class II gene expression. The repression occurs in B cells, which constitutively express MHC class II, as well as in macrophages, which only express MHC class II after the cells are treated with interferon gamma. For the MHC class II gene IA beta, this negative regulation has been linked to the X box DNA sequence, located with the IA beta promoter. The addition of the glucocorticoid receptor was shown to inhibit the DNA binding of the X box DNA binding protein to the X box. The DNA binding of two other DNA binding proteins that recognize elements within this promoter was unaffected by the addition of glucocorticoid receptor. It is likely that the repression of IA beta gene expression by glucocorticoids occurs because the X box DNA binding protein is prevented from binding to the DNA and activating transcription.
Regulation of thymocyte development through CD3. I. Timepoint of ligation of CD3 epsilon determines clonal deletion or induction of developmental program.
Authors: Levelt C N , Ehrfeld A , Eichmann K
Published: June 2004

Several recent observations suggest that successful rearrangement of the T cell receptor (TCR) beta locus induces several important events in thymocyte maturation. Allelic exclusion is achieved by interruption of further rearrangement of the beta locus, and CD4-8- interleukin (IL)-2R+ cells enter the CD4+8+IL-2R- stage. The actual molecular events regulating this important control point are unknown, but may be related to the expression of the TCR-beta locus in immature CD4-8- thymocytes. It is not clear whether maturation is induced by intracellular appearance of TCR-beta chain or by signal transduction through an immature TCR complex on the thymocyte membrane, possibly involving TCR-beta chain homodimers and CD3. Here we show that early addition of anti-CD3 mAb to fetal thymic organ cultures induces all known events associated with the acquisition of the CD4+8+ stage. Expression of CD4 and CD8 is accelerated, IL-2R alpha is downregulated, and the cells fail to produce TCR-beta, possibly based on premature cessation of beta gene rearrangement. Upon stimulation with anti-CD3 antibodies, we see calcium mobilization in 15% of all CD4-8- thymocytes with no detectable surface TCR expression. These results suggest that functional CD3 is expressed on immature thymocytes at very low concentrations before the appearance of a complete TCR-beta chain. Ligation of CD3 at this stage may mimic the maturation signal normally generated by the immature TCR-beta homodimer-CD3 complex. The results are consistent with the notion that acquisition of the CD4+8+ stage involves signal transduction through an immature TCR complex. Later in thymocyte development, ligation of CD3 results in deletion of CD4+8+ cells. Thus, signal transduction through CD3 may result in entirely different cellular responses, depending on the stage of thymocyte differentiation. These results suggest an involvement of CD3 as a link in signal transduction for at least two different decision points in the development of a thymocyte.
Lymphotoxin is an autocrine growth factor for Epstein-Barr virus-infected B cell lines.
Authors: Estrov Z , Kurzrock R , Pocsik E , Pathak S , Kantarjian H M , Zipf T F , Harris D , Talpaz M , Aggarwal B B
Published: June 2004

Because human lymphotoxin (LT) was originally isolated from a lymphoblastoid cell line, we investigated the role of this molecule in three newly established Epstein-Barr virus (EBV)-infected human B cell lines. These lines were derived from acute lymphoblastic leukemia (Z-6), myelodysplastic syndrome (Z-43), and acute myelogenous leukemia (Z-55) patients who had a prior EBV infection. Each lymphoblastoid cell line had a karyotype that was different from that of the original parent leukemic cells, and all expressed B cell, but not T cell or myeloid surface markers. In all three lines, rearranged immunoglobulin heavy chain joining region (JH) bands were found, and the presence of EBV DNA was confirmed by Southern blotting. Z-6, Z-43, and Z-55 cell lines constitutively produced 192, 48, and 78 U/ml LT, respectively, as assessed by a cytotoxicity assay and antibody neutralization. Levels of tumor necrosis factor (TNF) were undetectable. Scatchard analysis revealed that all the cell lines expressed high-affinity TNF/LT receptors with receptor densities of 4197, 1258, and 1209 sites/cell on Z-6, Z-43, and Z-55, respectively. Furthermore, labeled TNF binding could be reversed by both unlabeled TNF, as well as by LT. Studies with p60 and p80 receptor-specific antibodies revealed that the three lines expressed primarily the p80 form of the TNF receptor. When studied in a clonogenic assay, exogenous LT stimulated proliferation of all three cell lines in a dose-dependent fashion at concentrations ranging from 25 to 500 U/ml. Similar results were obtained with [3H]TdR incorporation. Monoclonal anti-LT neutralizing antibodies at concentrations of 25-500 U/ml inhibited cellular multiplication in a dose-dependent manner. It is interesting that in spite of a common receptor, TNF (1,000 U/ml) had no direct effect on Z-55 cell growth, whereas it partially reversed the stimulatory effect of exogenous LT. In addition, TNF inhibited Z-6 and Z-43 cell proliferation, and its suppressive effect was reversed by exogenous LT. Both p80 and p60 forms of soluble TNF receptors suppressed the lymphoblastoid cell line proliferation and their inhibitory effect was partially reversed by LT. Our data suggest that (a) LT is an autocrine growth factor for EBV-transformed lymphoblastoid B cell lines; and (b) anti-LT antibodies, soluble TNF/LT receptors, and TNF itself can suppress the growth of lymphoblastoid cells, probably by modulating or competing with LT.(ABSTRACT TRUNCATED AT 400 WORDS)
Interleukin (IL) 4 differentially regulates monocyte IL-1 family gene expression and synthesis in vitro and in vivo.
Authors: Wong H L , Costa G L , Lotze M T , Wahl S M
Published: June 2004

Interleukin (IL) 4 is a multifunctional T cell-derived cytokine that inhibits cytokine production and certain effector functions in human monocytes, while enhancing others. We show that IL-4 may contribute to the downregulation and resolution of an inflammatory response by selectively promoting expression of the IL-1 receptor antagonist (IL-1ra) that blocks the action of IL-1. IL-1ra specifically binds to the IL-1 receptor without initiating signal transduction. Peripheral blood monocytes obtained from cancer patients, before and immediately after a regimen of IL-4 immunotherapy, were examined for IL-1ra gene expression. After IL-4 therapy, monocytes from the patients showed a marked increase in IL-1ra mRNA. This selective induction of IL-1ra mRNA in circulating monocytes was reflected by significantly enhanced serum levels of IL-1ra (p < 0.01) during IL-4 therapy, which declined after IL-4 treatment. In vitro analysis of IL-4 regulation of monocytes from normal individuals revealed a dose-dependent induction of IL-1ra mRNA within 2-4 h after stimulation without a concomitant effect on the expression of IL-1 mRNA. Increased IL-1ra mRNA was not due to RNA stabilization, but occurred at the level of transcription. In the presence of LPS, IL-4 not only augmented IL-1ra levels, but markedly inhibited LPS-induced IL-1 mRNA expression. The selective upregulation of IL-1ra by resting or activated monocytes, coupled with inhibition of IL-1 production by activated monocytes, as we demonstrate both in vitro and in vivo, suggests that IL-4 may prove clinically useful as a systemic antiinflammatory agent.
Regulation of leukocyte rolling and adhesion to high endothelial venules through the cytoplasmic domain of L-selectin.
Authors: Kansas G S , Ley K , Munro J M , Tedder T F
Published: June 2004

L-selectin (leukocyte adhesion molecule 1/MEL-14), a member of the selectin family of cell adhesion molecules, mediates leukocyte rolling and leukocyte adhesion to endothelium at sites of inflammation. In addition, L-selectin mediates the binding of lymphocytes to high endothelial venules (HEV) of peripheral lymph nodes. The strong amino acid sequence conservation of the cytoplasmic domain of L-selectin between humans and mice suggests an important role for this region. Deletion of the COOH-terminal 11 amino acids from the approximately 17 amino acid cytoplasmic domain of L-selectin eliminated binding of lymphocytes to HEV in the in vitro frozen section assay, and also abolished leukocyte rolling in vivo in exteriorized rat mesenteric venules, but did not alter the lectin activity of L-selectin. Pretreatment of cells with cytochalasin B, which disrupts actin microfilaments, also abolished adhesion without affecting carbohydrate recognition. Therefore, the cytoplasmic domain of L-selectin regulates leukocyte adhesion to endothelium independent of ligand recognition, by controlling cytoskeletal interactions and/or receptor avidity.
Receptor editing in self-reactive bone marrow B cells.
Authors: Tiegs S L , Russell D M , Nemazee D
Published: June 2004

A central paradigm of immunology is clonal selection: lymphocytes displaying clonally distributed antigen receptors are generated and subsequently selected by antigen for growth or elimination. Here we show that in mice transgenic for anti-H-2Kk,b antibody genes, in which a homogeneous clone of developing B cells can be analyzed for the outcome of autoantigen encounter, surface immunoglobulin M+/idiotype+ immature B cells binding to self-antigens in the bone marrow are induced to alter the specificity of their antigen receptors. Transgenic bone marrow B cells encountering membrane-bound Kb or Kk proteins modify their receptors by expressing the V(D)J recombinase activator genes and assembling endogenously encoded immunoglobulin light chain variable genes. This (auto)antigen-directed change in the specificity of newly generated lymphocytes is termed receptor editing.
A marker for neoplastic progression of human melanocytes is a cell surface ectopeptidase.
Authors: Morrison M E , Vijayasaradhi S , Engelstein D , Albino A P , Houghton A N
Published: June 2004

Adenosine deaminase binding protein (ADAbp) is a cell surface glycoprotein that is expressed by normal melanocytes but not by melanoma, the malignant counterpart. ADAbp is specifically downregulated during malignant transformation of melanocytes. Recently, we have developed a system that progressively transforms melanocytes in vitro in defined steps. Transduction with v-Ha-ras oncogene followed by long-term culture leads to a cell phenotype and genotype that specifically mimics human melanoma. Loss of ADAbp expression occurred concomitantly with the emergence of growth factor independence and appearance of specific chromosomal abnormalities. The cellular function of ADAbp has not been defined. To characterize ADAbp, the mature 110-kD form was purified from human kidney. Five tryptic peptides from purified human ADAbp revealed 100% homology to a serine protease, human dipeptidyl peptidase IV (DPP IV), also known as CD26. DPP IV activity was detected in lysates from human melanocytes and renal carcinoma cells but not melanoma cells, and DPP IV activity could be specifically isolated from melanocytes by binding to ADA or to S27 monoclonal antibody against ADAbp. These findings show that ADAbp is a cell surface ectopeptidase that is tightly regulated during neoplastic transformation of melanocytes.
Thymus epithelium induces tissue-specific tolerance.
Authors: Bonomo A , Matzinger P
Published: June 2004

Most current models of T cell development include a positive selection step in the thymus that occurs when T cells interact with thymic epithelium and a negative selection step after encounters with bone marrow-derived cells. We show here that developing T cells are tolerized when they recognize antigens expressed by thymic epithelium, that the tolerance is tissue specific, and that it can occur by deletion of the reactive T cells.
Mercury-induced autoreactive anti-class II T cell line protects from experimental autoimmune encephalomyelitis by the bias of CD8+ antiergotypic cells in Lewis rats.
Authors: Castedo M , Pelletier L , Rossert J , Pasquier R , Villarroya H , Druet P
Published: June 2004

Brown-Norway (BN) rats injected with HgCl2 develop a systemic autoimmune disease associated with a polyclonal B cell activation, due to autoreactive T cells specific for self-class II molecules, while Lewis (LEW) rats injected with HgCl2 do not exhibit autoimmunity and develop a non-antigen-specific, CD8-mediated immunosuppression assessed by a depression of T cell functions, and a protection against experimental autoimmune encephalomyelitis (EAE). Resistance to HgCl2-induced autoimmunity is not due to these suppressor cells since treatment with an anti-CD8 monoclonal antibody (mAb) did not allow autoimmunity to appear. The absence of autoimmunity in this strain could result from the absence of autoreactive T cells, or from quantitative or qualitative differences of these cells between susceptible and resistant strains. In the present study, we show that CD4+ anti-class II T cells are present in HgCl2-injected LEW rats and are as frequent as in BN rats when assessed by limiting dilution analysis. LEW CD4+ autoreactive T cell lines were derived. They proliferated in the presence of normal class II-bearing cells, secreted interleukin 2, and did not induce B cells to produce immunoglobulins. Transfer of one of these lines, LEW Hg A, into normal LEW rats led to the appearance of CD8+ cells responsible for a non-antigen-specific immunosuppression that induced complete protection from EAE. Immunosuppression was abrogated after treatment with an anti-CD8 mAb. In vitro, CD8+ cells from rats injected with the LEW Hg A T cell line proliferated in the presence of activated T cells whatever their origin. We conclude that HgCl2 induces CD4+ autoreactive T cells that proliferate in the presence of class II+ cells in susceptible BN as well as in resistant LEW rats. But while these cells collaborate with B cells to produce autoantibodies in BN rats, they initiate in LEW rats a suppressor circuit involving antiergotypic CD8+ suppressor cells.
Activated human lymphocytes and aggressive non-Hodgkin's lymphomas express a homologue of the rat metastasis-associated variant of CD44.
Authors: Koopman G , Heider K H , Horst E , Adolf G R , van den Berg F , Ponta H , Herrlich P , Pals S T
Published: June 2004

A recently described splice variant of CD44 expressed in metastasizing cell lines of rat tumors, has been shown to confer metastatic potential to nonmetastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies raised against a bacterial fusion protein encoded by variant CD44 sequences, we have explored the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and on non-Hodgkin's lymphomas. Normal lymphohematopoietic cells express barely detectable low levels of variant CD44 glycoproteins, whereas T lymphocytes, upon activation by mitogen or antigen, transiently upregulate expression of specific CD44 variant glycoproteins. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that in the rat confers metastatic capability. It is interesting that overexpression of v6 was also found in several aggressive, but not low-grade, non-Hodgkin's lymphomas.
A rat CD4 mutant containing the gp120-binding site mediates human immunodeficiency virus type 1 infection.
Authors: Simon J H , Somoza C , Schockmel G A , Collin M , Davis S J , Williams A F , James W
Published: June 2004

CD4 is the primary receptor for the human immunodeficiency virus type 1 (HIV-1). Early mutational studies implicated a number of residues of CD4, centered in the region 41-59, in binding to gp120. However, further mutational analyses, together with studies using inhibitory antibodies or CD4-derived peptides, have suggested that other regions of CD4 are also involved in binding or postbinding events during infection. To resolve these ambiguities, we used rat CD4 mutants in which particular regions were replaced with the corresponding sequence of human CD4. We have previously shown that some of these are able to bind HIV-1 gp120, and here we test their ability to act as functional receptors. We find that the presence of human CD4 residues 33-62 is enough to confer efficient receptor function to rat CD4, and we conclude that it is unlikely that regions of CD4 outside this sequence are involved in specific interactions with HIV-1 during either infection or syncytium formation.
Receptor editing: an approach by autoreactive B cells to escape tolerance.
Authors: Gay D , Saunders T , Camper S , Weigert M
Published: June 2004

To determine the fate of anti-DNA antibody-bearing B cells in normal mice, we generated transgenic mice bearing the heavy (H) and light (L) chain genes of a well-characterized anti-double-stranded DNA antibody. This antibody was originally isolated from a diseased MRL/lpr mouse and has characteristics common to spontaneously arising anti-DNA antibodies. Results show that the H/L transgene (tg) immunoglobulin receptor is not expressed by animals bearing both tgs, although single tg animals (H or L) express their transgenes. Young H/L tg animals express few B cells, whereas adult H/L tg animals maintain almost normal B cell numbers. Analysis of the immunoglobulin receptors used by adult B cells shows that all contain the tg H chain in association with endogenous L chains. These B cells transcribe the L tg as well as the rearranged endogenous L chain gene, and loss of endogenous L chain gene transcription results in resurrection of the 3H9 H/L tg product. Examination of the endogenous L chains used by these cells shows that they represent a highly restricted subset of V genes. Taken together, these data suggest that autoreactive transgenic B cells can rearrange endogenous L chain genes to alter surface receptors. Those L chains that compete successfully with the L tg for H chain binding, and that create a nonautoreactive receptor, allow the B cell to escape deletion. We suggest that this receptor editing is a mechanism used by immature autoreactive B cells to escape tolerance.