bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetics of Gag-specific cytotoxic T lymphocyte responses during the clinical course of HIV-1 infection: a longitudinal analysis of rapid progressors and long-term asymptomatics.
Authors: Klein M R , van Baalen C A , Holwerda A M , Kerkhof Garde S R , Bende R J , Keet I P , Eeftinck-Schattenkerk J K , Osterhaus A D , Schuitemaker H , Miedema F
Published: June 2004

To gain more insight into the role of HIV-1-specific cytotoxic T lymphocytes (CTL) in the pathogenesis of AIDS, we investigated temporal relations between HIV-1 Gag-specific precursor CTL (CTLp), HIV-1 viral load, CD4+ T cell counts, and T cell function. Six HIV-1-infected subjects, who were asymptomatic for more than 8 yr with CD4+ counts > 500 cells/mm3, were compared with six subjects who progressed to AIDS within 5 yr after HIV-1 seroconversion. In the long-term asymptomatics, persistent HIV-1 Gag-specific CTL responses and very low numbers of HIV-1-infected CD4+ T cells coincided with normal and stable CD4+ counts and preserved CD3 mAb-induced T cell reactivity for more than 8 yr. In five out of six rapid progressors Gag-specific CTLp were also detected. However, early in infection the number of circulating HIV-1-infected CD4+ T cells increased despite strong and mounting Gag-specific CTL responses. During subsequent clinical progression to AIDS, loss of Gag-specific CTLp coincided with precipitating CD4+ counts and severe deterioration of T cell function. The possible relationships of HIV-1 Gag-specific CTLp to disease progression are discussed.
Activated mast cells produce interleukin 13.
Authors: Burd P R , Thompson W C , Max E E , Mills F C
Published: June 2004

When mast cells are activated through their immunoglobulin (Ig)E receptors, release of low molecular weight mediators like histamine is followed by secretion of multiple cytokines, including interleukin (IL)-3, IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor. Here we report that stimulated mast cells also synthesize IL-13 mRNA and protein; secretion of this cytokine may be of particular importance because of its ability to stimulate IgE expression. IL-13 transcripts detected by a semiquantitative reverse transcriptase-mediated polymerase chain reaction assay were induced within 30 min after stimulation of mast cells by dinitrophenyl plus monoclonal IgE anti-dinitrophenyl, and peaked at about 1 h. Within 3 h of IgE stimulation, secreted IL-13 bioactivity, estimated by proliferation of an IL-13-dependent cell line, reached levels equivalent to 1-2 ng/ml of IL-13. When added to human B lymphocytes, the mast cell-derived IL-13 activity (like bone fide IL-13) induced Ig C epsilon transcripts, DNA recombination characteristic of the isotype switch to C epsilon, and the secretion of IgE protein. These results suggest a model of local positive feedback interactions between mast cells and B cells, which could play a role in the pathogenesis of atopy.
Urokinase plasminogen activator receptor, beta 2-integrins, and Src-kinases within a single receptor complex of human monocytes.
Authors: Bohuslav J , Horejsí V , Hansmann C , Stöckl J , Weidle U H , Majdic O , Bartke I , Knapp W , Stockinger H
Published: June 2004

The glycosylphosphatidylinositol (GPI)-anchored membrane protein urokinase plasminogen activator-receptor (uPA-R; CD87) is one of the key molecules involved in migration of leukocytes and tumor cells. uPA bound to uPA-R provides the cell proteolytic potential used for degradation of extracellular matrix. uPA-R is also involved in induction of cell adhesion and chemotaxis. Here, we provide a molecular explanation for these uPA-R-related cellular events. By size fractionation of monocyte lysate and affinity isolation on its natural ligand uPA, we demonstrate uPA-R as a component of a receptor complex of relatively large size. Reprecipitation and immunoblotting techniques allowed us to detect the protein tyrosine kinases (PTKs) p60fyn, p53/56lyn, p58/64hck, and p59fgr as components of this "uPA-R complex". Activation of monocytes even with enzymatically inactivated uPA resulted in induction of tyrosine phosphorylation, suggesting modulation of uPA-R-associated PTKs upon ligand binding. In spite of their presence in large complexes, we did not find the GPI-linked proteins CD14, CD58, and CD59 in the uPA-R complex, which indicates the presence of different receptor domains containing GPI-linked proteins in monocytes. However, we identified the leukocyte integrins LFA-1 and CR3 as components of the uPA-R complex as indicated by coisolation of these molecules, as well as by cocapping and comodulation of uPA-R and leukocyte integrins on the monocyte surface. The assemblage of uPA-R, PTKs and membrane spanning beta 2-integrins in one receptor complex indicates functional cooperation. In regard to the involvement of these molecules in pericellular proteolysis, signal transduction, as well as adhesion and chemotactic movement, we suggest uPA-R complex as a potential cellular device for cell migration.
Dual T cell receptor beta chain expression on human T lymphocytes.
Authors: Davodeau F , Peyrat M A , Romagné F , Necker A , Hallet M M , Vié H , Bonneville M
Published: June 2004

Allelic exclusion of lymphocyte antigen receptor chains has been hypothesized as a mechanism developed by the immune system to ensure efficient lymphocyte repertoire selection and tight control of lymphocyte specificity. It was effectively shown to be operative for both the immunoglobulin (Ig) and the T cell receptor (TCR) beta chain genes. Our present observations suggest that close to 1% of human T lymphocytes escape this allelic control, and express two surface TCR beta chains with distinct superantigenic reactivities. Since this high frequency of dual beta chain expressors did not result in any dramatic immune dysregulations, these results question the need for a mechanism ensuring clonal monospecificity through allelic exclusion.
Molecular characterization of major histocompatibility complex class II gene expression and demonstration of antigen-specific T cell response indicate a new phenotype in class II-deficient patients.
Authors: Hauber I , Gulle H , Wolf H M , Maris M , Eggenbauer H , Eibl M M
Published: June 2004

Major histocompatibility complex (MHC) class II deficiency is an inherited autosomal recessive combined immunodeficiency. The disease is known as bare lymphocyte syndrome (BLS). BLS is characterized by a lack of constitutive MHC class II expression on macrophages and B cells as well as a lack of induced MHC class II expression on cells other than professional antigen-presenting cells (APCs) due to the absence of mRNA and protein of the human leukocyte antigen (HLA) class II molecules, designated HLA-DR, -DQ, and -DP. The defect in gene expression is located at the transcriptional level and affects all class II genes simultaneously. Here we have analyzed transcription and protein expression of class II antigens in Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines and mononuclear cells (MNCs) of twin brothers. Whereas flow cytometric analysis failed to detect class II antigens on the cell surface of the patients' EBV-B cells and MNCs, examination of the genes coding for HLA-DR, -DQ, -DP, and the invariant chain (Ii) by reverse transcriptase-polymerase chain reaction amplification resulted in an unusual mRNA pattern in the B cell lines of the patients (HLA-DR alpha +, -DR beta, -DQ alpha +, -DQ beta -, -DP alpha -; -DP beta +, Ii+). In accordance with these findings no HLA-DR beta-specific protein was detected by immunoblotting, whereas low levels of HLA-DR alpha and normal levels of Ii were present. In contrast to EBV-B cells, the MNCs of both patients displayed a residual HLA-DR beta, -DQ beta, and -DP alpha mRNA signal. Furthermore, HLA-DR beta-specific protein was found in addition to HLA-DR alpha by immunoblotting of cell lysates, even though it was clearly decreased as compared with controls. Our results indicate that the defect in class II antigen expression is not necessarily present to the same extent in B cells and cells of other lineages. mRNA levels of HLA-DR beta were found to be enriched in adherent cells within the MNC fraction. Further investigations indicated that the MHC class II expressed is functional in antigen presentation, as the two boys' CD4+ T cells became activated and expressed interleukin-2R after stimulation of peripheral blood mononuclear cell cultures with recall antigen (tetanus toxoid). Furthermore, T cells tested in one of the two patients responded to both MHC class I and II allostimulation, and this response was inhibited by monoclonal antibodies of the respective specificity.(ABSTRACT TRUNCATED AT 400 WORDS)
Regulation of JAK3 expression in human monocytes: phosphorylation in response to interleukins 2, 4, and 7.
Authors: Musso T , Johnston J A , Linnekin D , Varesio L , Rowe T K , O'Shea J J , McVicar D W
Published: June 2004

The Janus family of kinases (JAKs) has been shown to be involved in the signal transduction of a number of cytokine receptors. Recently, we have cloned a novel JAK family member, JAK3, that is expressed in natural killer and activated T cells and is coupled functionally and physically to the interleukin 2 (IL-2) receptor in these cells. Here we report that JAK3 was expressed at low but detectable levels in human monocytes. In contrast, JAK3 expression was strongly induced during activation by interferon gamma (IFN-gamma) or lipopolysaccharide. Moreover, JAK3 became tyrosine phosphorylated in response to IL-2, IL-4, and IL-7 but not response to IFN-gamma or granulocyte/macrophage colony-stimulating factor. Together, these findings suggest that JAK3 is functionally important in activated monocytes and cells of the myeloid lineage and is involved in signaling responses of cytokines that use the common gamma-chain of the IL-2 receptor.
Nonclassical behavior of the thymus leukemia antigen: peptide transporter-independent expression of a nonclassical class I molecule.
Authors: Holcombe H R , Castaño A R , Cheroutre H , Teitell M , Maher J K , Peterson P A , Kronenberg M
Published: June 2004

The thymus leukemia (TL) antigen is a major histocompatibility complex-encoded nonclassical class I molecule. Here we present data demonstrating that expression of the TL antigen, unlike other class I molecules, is completely independent of the function of the transporter associated with antigen processing (TAP). The TL antigen is expressed by transfected TAP-2-deficient RMA-S cells when these cells are grown at 37 degrees C. In transfected RMA cells, the kinetics of arrival of TL antigen on the cell surface are similar to those of a classical class I molecule. The kinetics are not altered in TAP-deficient RMA-S cells, demonstrating that surface TL expression in TAP-deficient cells is not due to the stable expression of a few molecules that leak out by a TAP-independent pathway. Soluble TL molecules produced by Drosophila melanogaster cells are highly resistant to thermal denaturation, unlike peptide-free classical class I molecules synthesized by these insect cells. In addition, these soluble TL molecules are devoid of detectable bound peptides. The results demonstrate that the TL antigen is capable of reaching the surface without bound peptide, although acquisition of peptide or some other ligand through a TAP-independent pathway cannot be formally excluded. We speculate that the ability of the TL antigen to reach the cell surface, under conditions in which other class I molecules do not, may be related to a specialized function of the TL molecule in the mucosal immune system, and possibly in the stimulation of intestinal gamma delta T cells.
Early human T cell development: analysis of the human thymus at the time of initial entry of hematopoietic stem cells into the fetal thymic microenvironment.
Authors: Haynes B F , Heinly C S
Published: June 2004

To determine events that transpire during the earliest stages of human T cell development, we have studied fetal tissues before (7 wk), during (8.2 wk), and after (9.5 wk to birth) colonization of the fetal thymic rudiment with hematopoietic stem cells. Calculation of the approximate volumes of the 7- and 8.2-wk thymuses revealed a 35-fold increase in thymic volumes during this time, with 7-wk thymus height of 160 microM and volume of 0.008 mm3, and 8.2-wk thymus height of 1044 microM and volume of 0.296 mm3. Human thymocytes in the 8.2-wk thymus were CD4+ CD8 alpha+ and cytoplasmic CD3 epsilon+ cCD3 delta+ CD8 beta- and CD3 zetta-. Only 5% of 8-wk thymocytes were T cell receptor (TCR)-beta+, < 0.1% were TCR-gamma+, and none reacted with monoclonal antibodies against TCR-delta. During the first 16 wk of gestation, we observed developmentally regulated expression of CD2 and CD8 beta (appearing at 9.5 wk), CD1a,b, and c molecules (CD1b, then CD1c, then CD1a), TCR molecules (TCR-beta, then TCR-delta), CD45RA and CD45RO isoforms, CD28 (10 wk), CD3 zeta (12-13 wk), and CD6 (12,75 wk). Whereas CD2 was not expressed at the time of initiation of thymic lymphopoiesis, a second CD58 ligand, CD48, was expressed at 8.2 wk, suggesting a role for CD48 early in thymic development. Taken together, these data define sequential phenotypic and morphologic changes that occur in human thymus coincident with thymus colonization by hematopoietic stem cells and provide insight into the molecules that are involved in the earliest stages of human T cell development.
Coordinate regulation of the human TAP1 and LMP2 genes from a shared bidirectional promoter.
Authors: Wright K L , White L C , Kelly A , Beck S , Trowsdale J , Ting J P
Published: June 2004

Recently, four genes (TAP1, TAP2, LMP2, LMP7) involved or potentially involved in the processing and transport of major histocompatibility complex class I-associated antigen to the endoplasmic reticulum have been identified. We now report the initial characterization of the bidirectional promoter for the human transporter associated with antigen processing 1 (TAP1) and low molecular mass polypeptide 2 (LMP2) genes. These genes are divergently transcribed from a central promoter region of only 593 bp. Functional analysis using a bidirectional reporter system demonstrates the minimal 593-bp promoter is sufficient for concurrent expression in both directions. There is no TATA box homology at either end but there is a prevalence of GC boxes. Transcription is initiated at multiple sites for each gene without any of the TAP1 transcripts overlapping with the LMP2 transcripts. The region proximal to the TAP1 gene is required for maximal basal level expression of not only TAP1 but also LMP2. Furthermore, this region is necessary for tumor necrosis factor alpha (TNF-alpha) induction of both genes. Site-specific mutations of an NF-kappa B element in the TAP1 proximal region blocked induction by TNF-alpha in both the TAP1 and LMP2 directions. An adjacent GC box was required for basal expression of both genes as well as augmenting the TNF-alpha induction of the distal LMP2 gene. In vivo genomic foot-printing of this region revealed strong protein/DNA interactions at the NF-kappa B and GC box consensus sequences. In vitro binding studies confirmed the capacity of the NF-kappa B site to bind p50/p65 and p52/p65 heterodimers and of the GC box to bind Sp1. Thus, the promoter elements proximal to the TAP1 gene play a significant role in regulating basal and induced expression of both TAP1 and LMP2. The findings presented in this report clearly link LMP2 expression with TAP1 expression and provide additional suggestive evidence linking LMP2 to class I antigen presentation.
CD11c/CD18, a transmembrane signaling receptor for lipopolysaccharide.
Authors: Ingalls R R , Golenbock D T
Published: June 2004

CD11c/CD18 is a member of the leukocyte integrin family, heterodimeric adhesion molecules that interact with a diverse repertoire of ligands, including bacterial lipopolysaccharide (LPS). Their role as signal transducing receptors remains uncertain. We used a heterologous expression system to determine if CD11c/CD18 was capable of initiating signal transduction in response to LPS-binding, as assessed by the induced translocation of nuclear factor-kappa B. We have previously reported that Chinese hamster ovary (CHO)-K1 fibroblasts, normally unresponsive to LPS, acquire serum-dependent macrophage-like responses to LPS when transfected with CD14 (Golenbock, D.T., Y. Liu, F. Millham, M. Freeman, and R. Zoeller. 1993. J. Biol. Chem. 268:22055-22059), a known LPS receptor. In contrast, CHO cells acquired serum-independent responses to Gram-negative bacteria and LPS when transfected with CD11c/CD18 (CHO/CD11c). In comparison to CHO cells transfected with CD14 (CHO/CD14), responses in CHO/CD11c cells were slower, required higher endotoxin concentrations for maximal response, and were not inhibited by the presence of antibodies to CD14. CD11c/CD18 is, thus, the second phagocyte receptor, in addition to CD14, which has been shown to have the capacity to activate cells after binding to LPS. The function of this receptor in normal phagocytes may be limited to the recognition of LPS in infected tissues, where LPS-CD14 interactions are not favored because of the absence of serum proteins.
Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine.
Authors: von Freeden-Jeffry U , Vieira P , Lucian L A , McNeil T , Burdach S E , Murray R
Published: June 2004

Interleukin (IL)-7 is a potent stimulus for immature T and B cells and, to a lesser extent, mature T cells. We have inactivated the IL-7 gene in the mouse germline by using gene-targeting techniques to further understand the biology of IL-7. Mutant mice were highly lymphopenic in the peripheral blood and lymphoid organs. Bone marrow B lymphopoiesis was blocked at the transition from pro-B to pre-B cells. Thymic cellularity was reduced 20-fold, but retained normal distribution of CD4 and CD8. Splenic T cellularity was reduced 10-fold. Splenic B cells, also reduced in number, showed an abnormal population of immature B cells in adult animals. The remaining splenic populations of lymphocytes showed normal responsiveness to mitogenic stimuli. These data show that proper T and B cell development is dependent on IL-7. The IL-7-deficient mice are the first example of single cytokine-deficient mice that exhibit severe lymphoid abnormalities.
A recombinant Leishmania antigen that stimulates human peripheral blood mononuclear cells to express a Th1-type cytokine profile and to produce interleukin 12.
Authors: Skeiky Y A , Guderian J A , Benson D R , Bacelar O , Carvalho E M , Kubin M , Badaro R , Trinchieri G , Reed S G
Published: June 2004

Leishmania braziliensis causes cutaneous and mucosal leishmaniasis in humans. Most patients with cutaneous leishmaniasis heal spontaneously and may therefore have developed protective immunity. There appears to be a mixed cytokine profile associated with active cutaneous or mucosal disease, and a dominant T helper (Th)1-type response associated with healing. Leishmanial antigens that elicit these potent proliferative and cytokine responses from peripheral blood mononuclear cells (PBMC) are now being identified. Herein, we report on the cloning and expression of a L. braziliensis gene homologous to the eukaryotic ribosomal protein eIF4A (LeIF) and patient PBMC responses to rLeIF. Patients with mucosal and self-healing cutaneous disease had significantly higher proliferative responses than those with cutaneous lesions. Whereas the parasite lysate stimulated patient PBMC to produce a mixed Th1/Th2-type cytokine profile, LeIF stimulated the production of interferon gamma (IFN-gamma), interleukin 2 (IL-2), and tumor necrosis factor alpha but not IL-4 or IL-10. Recombinant LeIF (rLeIF) downregulated both IL-10 mRNA in the "resting" PBMC of leishmaniasis patients and LPS-induced IL-10 production by patient PBMC. rLeIF also stimulated the production of IL-12 in cultured PBMC from both patients and uninfected individuals. The production of IFN-gamma by patient PBMC stimulated with either rLeIF or parasite lysate was IL-12 dependent, whereas anti-IFN-gamma monoclonal antibody only partially blocked the LeIF-induced production of IL-12. In vitro production of both IFN-gamma and IL-12 was abrogated by exogenous human recombinant IL-10. Therefore, we have identified a recombinant leishmanial antigen that elicits IL-12 production and Th1-type responses in patients as well as IL-12 production in normal human PBMC.
Tumor dormancy and cell signaling. II. Antibody as an agonist in inducing dormancy of a B cell lymphoma in SCID mice.
Authors: Racila E , Scheuermann R H , Picker L J , Yefenof E , Tucker T , Chang W , Marches R , Street N E , Vitetta E S , Uhr J W
Published: June 2004

Tumor dormancy can be induced in a murine B cell lymphoma (BCL1) by immunizing BALB/c mice with the tumor immunoglobulin (Ig) before tumor cell challenge. In this report, we have investigated the immunological and cellular mechanisms underlying the induction of dormancy. BCL1 tumor cells were injected into SCID mice passively immunized with antibody against different epitopes on IgM or IgD with or without idiotype (Id)-immune T lymphocytes. Results indicate that antibody to IgM is sufficient to induce a state of dormancy. Antibodies against other cell surface molecules including IgD and CD44 (Pgp1) had no effect on tumor growth. Id-immune T cells by themselves also had no effect on tumor growth in SCID mice. However, simultaneous transfer of anti-Id and Id-immune T cells enhanced both the induction and duration of the dormant state. In vitro studies indicated that antibody to IgM induced apoptosis within several hours and cell cycle arrest by 24 h. Hyper cross-linking increased apoptosis. The Fc gamma RII receptor played little or no role in the negative signaling. Antibodies that did not negatively signal in vitro did not induce dormancy in vivo. The results suggest that anti-IgM plays a decisive role in inducing tumor dormancy to BCL1 by acting as an agonist of IgM-mediated signal transduction pathways.
Protection against HIV-1 gp120-induced brain damage by neuronal expression of human amyloid precursor protein.
Authors: Mucke L , Abraham C R , Ruppe M D , Rockenstein E M , Toggas S M , Mallory M , Alford M , Masliah E
Published: June 2004

Expression of the HIV-1 envelope protein gp120 in brains of transgenic (tg) mice induces extensive neurodegeneration (Toggas, S. M., E. Masliah, E. M. Rockenstein, G. F. Rall, C. R. Abraham, and L. Mucke. 1994. Nature [Lond.]. 367:188-193.). To further analyze the pathogenesis of gp120-induced neurotoxicity and to assess the neuroprotective potential of human amyloid precursor proteins (hAPPs) in vivo, different hAPP isoforms were expressed in neurons of gp120/hAPP-bigenic mice: hAPP751, which contains a Kunitz-type protease inhibitor domain, or hAPP695, which lacks this domain. Bigenic mice overexpressing hAPP751 at moderate levels showed significantly less neuronal loss, synapto-dendritic degeneration, and gliosis than singly tg mice expressing gp120 alone. In contrast, higher levels of hAPP695 expression in bigenic mice failed to prevent gp120-induced brain damage. These data indicate that hAPP can exert important neuroprotective functions in vivo and that the efficiency of this protection may depend on the hAPP isoform expressed and/or on the level of neuronal hAPP expression. Hence, molecules that mimic beneficial APP activities may be useful in the prevention/treatment of HIV-1-associated nervous system damage and, perhaps, also of other types of neural injury.
Novel packages of viral and self-antigens are generated during apoptosis.
Authors: Rosen A , Casciola-Rosen L , Ahearn J
Published: June 2004

Immune context is an essential determinant of the host response to potential autoantigens. The clustering of the autoantigens targeted in systemic lupus erythematosus within surface blebs of apoptotic cells generates high concentrations of autoantigen within discrete subcellular packages. We demonstrate here that when apoptosis is induced by Sindbis virus infection, viral antigens and autoantigens cocluster exclusively in small surface blebs of apoptotic cells. The surface of these blebs is rich in viral glycoproteins, and virions can be seen blebbing from their surface. We propose that these blebs of mixed foreign and self-origin define a novel immune context that may challenge self-tolerance.
Identification and characterization of a 100-kD ligand for CD6 on human thymic epithelial cells.
Authors: Patel D D , Wee S F , Whichard L P , Bowen M A , Pesando J M , Aruffo A , Haynes B F
Published: June 2004

CD6 is a 130-kD glycoprotein expressed on the surface of thymocytes and peripheral blood T cells that is involved in TCR-mediated T cell activation. In thymus, CD6 mediates interactions between thymocytes and thymic epithelial (TE) cells. In indirect immunofluorescence assays, a recombinant CD6-immunoglobulin fusion protein (CD6-Rg) bound to cultured human TE cells and to thymic fibroblasts. CD6-Rg binding to TF and TE cells was trypsin sensitive, and 54 +/- 4% of binding was divalent cation dependent. By screening the blind panel of 479 monoclonal antibodies (mAbs) from the 5th International Workshop on Human Leukocyte Differentiation Antigens for expression on human TE cells and for the ability to block CD6-Rg binding to TE cells, we found one mAb (J4-81) that significantly inhibited the binding of CD6-Rg to TE cells (60 +/- 7% inhibition). A second mAb to the surface antigen identified by mAb J4-81, J3-119, enhanced the binding of CD6-Rg to TE cells by 48 +/- 5%. Using covalent cross-linking and trypsin digestion, we found that mAb J4-81 and CD6-Rg both bound to the same 100-kD glycoprotein (CD6L-100) on the surface of TE cells. These data demonstrate that a 100-kD glycoprotein on TE cells detected by mAb J4-81 is a ligand for CD6.
Altered peptide ligands can control CD4 T lymphocyte differentiation in vivo.
Authors: Pfeiffer C , Stein J , Southwood S , Ketelaar H , Sette A , Bottomly K
Published: June 2004

Antigen priming of naive CD4 T cells can generate effector CD4 T cells that produce interleukin 4 (T helper [Th]2-like) or interferon-gamma (Th1-like). Using a system in which priming leads to responses dominated by one or the other of these cell types, we show that varying either the antigenic peptide or the major histocompatibility complex class II molecule can determine whether Th1-like or Th2-like responses are obtained. Our results show that peptide/major histocompatibility complex class II complexes that interact strongly with the T cell receptor favor generation of Th1-like cells, while those that bind weakly favor priming of Th2-like T cells. Thus, signals from the T cell receptor can influence the differentiation of CD4 T cells into specific types of effector cells.
Human T lymphocyte virus 1 from a leukemogenic cell line mediates in vivo and in vitro lymphocyte apoptosis.
Authors: Leno M , Simpson R M , Bowers F S , Kindt T J
Published: June 2004

HTLV-1 is implicated in the development of diverse diseases. However, most HTLV-1-infected individuals remain asymptomatic. How HTLV-1 infection leads to disparate consequences remains a mystery, despite extensive investigation of HTLV-1 isolates from infected individuals. As in human infection, experimental HTLV-1 infection in rabbits is generally benign, although HTLV-1-infected rabbit T cell lines that mediate lethal leukemia-like disease have been reported. We report here that thymuses from mature outbred rabbits inoculated with a lethal leukemia-like disease have been reported. We report here that thymuses from mature outbred rabbits inoculated with a lethal HTLV-1 T cell line (RH/K34) showed morphological and biochemical evidence of apoptosis, whereas thymuses from rabbits inoculated with nonlethal HTLV-1 T cell lines showed no signs of apoptosis. Exposure of rabbit or human lymphocytes to purified virus from RH/K34 caused rapid induction of apoptosis, providing an in vitro correlate to the pathogenic effects. By contrast, virus isolated from a nonlethal cell line mediated dose-dependent lymphocyte proliferation. These data implicate lymphocyte apoptosis as a potential mechanism by which the lethal HTLV-1 cell line causes fulminant disease and provide a means to identify factors contributing to HTLV-1 disease. Results from this HTLV-1 infection model can provide insight into variations in HTLV-1 pathogenicity in human infection.
Identification of the ligand-binding domains of CD22, a member of the immunoglobulin superfamily that uniquely binds a sialic acid-dependent ligand.
Authors: Engel P , Wagner N , Miller A S , Tedder T F
Published: June 2004

CD22 is a B cell-restricted member of the immunoglobulin (Ig) superfamily that functions as an adhesion receptor for leukocytes and erythrocytes. CD22 is unique among members of the Ig superfamily in that it has been suggested to bind a series of sialic acid-dependent ligands, potentially through different functional domains expressed by different splice variants of CD22. In this study, the epitopes identified by a large panel of function-blocking and non-function-blocking CD22 monoclonal antibodies were localized to specific Ig-like domains, revealing that all function-blocking monoclonal antibodies bound to the first and/or second Ig-like domains. Consistent with a single ligand-binding region, the two amino-terminal domains were the functional unit that mediated CD22 adhesion with lymphocytes, neutrophils, monocytes, and erythrocytes. The predominant cell surface species of CD22 was a full length 140,000 relative molecular mass seven Ig-like domain glycoprotein and a minor 130,000 relative molecular mass form lacking the fourth domain. While the two amino-terminal Ig-like domains of CD22 are structurally similar to those found in other members of the Ig superfamily involved in cell adhesion and containing an amino acid sequence motif associated with integrin recognition, site-directed mutagenesis of critical residues surrounding this motif did not disrupt CD22-mediated adhesion. These results demonstrate that the unique ligand-binding properties of CD22 are distinct from those of other members of the Ig superfamily involved in integrin-mediated cell adhesion.
Stochastic coreceptor shut-off is restricted to the CD4 lineage maturation pathway.
Authors: Lucas B , Vasseur F , Penit C
Published: June 2004

Kinetics of mature T cell generation in the thymus of normal or major histocompatibility complex (MHC) class I- or II-deficient mice were studied by the bromodeoxyuridine pulse labeling method. As previously described, the early activation and final maturation phases were found to be synchronous for the two T cell lineages, but CD4+8- cells were generated faster than CD4-8+ cells in MHC class I- and II-deficient mice, respectively. CD8 downregulation started on day 2 after cell proliferation even in the absence of MHC class II expression. CD8 downregulation thus appears to be stochastic at its beginning. By contrast, CD4 shut-off was found totally instructive, as the generation of CD4lo8+ cells with a high TCR density was not observed in class I-deficient mice. The analysis of the V beta 14 TCR frequencies in CD4/8 subsets in normal and MHC-deficient mice confirmed that CD4 and CD8 generation pathways are not symmetrical. These findings show that commitment towards the CD4+8- or CD4-8+ phenotype is controlled at the CD8lo step for the former and at the CD4+8+ double-positive stage for the latter.
Intermediate steps in positive selection: differentiation of CD4+8int TCRint thymocytes into CD4-8+TCRhi thymocytes.
Authors: Lundberg K , Heath W , Köntgen F , Carbone F R , Shortman K
Published: June 2004

The differentiation potential of putative intermediates between CD4+8+ thymocytes and mature T cells has been examined. Such intermediate populations were sorted, in parallel with CD4+8+ thymocytes, from three types of C57BL/6 mice: major histocompatibility complex (MHC) class II-deficient mice, mice transgenic for an alpha/beta T cell receptor (TCR) restricted by class I MHC and normal mice. The sorted populations were then transferred into the thymus of nonirradiated C57BL/Ka mice differing in Thy 1 allotype, and the progeny of the transferred cells were analyzed 2 d later. Surprisingly, with all three types of donor mice, a major proportion of the CD4+8intTCRint-derived progeny were found to be CD4-8+TCRhi cells, thus delineating a new alternative pathway for development of the CD8 lineage. In contrast, the transfer of CD4int8+TCRint thymocytes produced CD4-8+TCRhi cells but no significant proportion of CD4+8-TCRhi cells, suggesting that there is no equivalent alternative pathway for the CD4 lineage. The results negate some of the evidence for a stochastic/selective model of lineage commitment, and point to an asymmetry in the steps leading to CD4-8+ versus CD4+8- T cells.
Signals through T cell receptor-zeta chain alone are insufficient to prime resting T lymphocytes.
Authors: Brocker T , Karjalainen K
Published: June 2004

Activation studies performed with transfected T cell hybridomas and tumors revealed that chimeric molecules containing the CD3 epsilon or zeta chain intracytoplasmic portions can induce the complete effector functions normally seen only when the complete T cell receptor (TCR)/CD3 complexes of T lymphocytes are triggered. Therefore, the zeta chain, with its three antigen recognition activation motives, is thought to connect the antigen-binding Ti chains with the intracellular signaling machinery of the T cell. Here we demonstrate that the cytoplasmic portion of the TCR-zeta chain is not sufficient to activate resting T lymphocytes when cells from transgenic mice expressing a chimeric zeta receptor are used. However, after (in vivo and in vitro) activation through their endogenous TCR/CD3 complexes, the preactivated T lymphocytes could be triggered through the zeta chimera to the same extent as when they were activated through their endogenous TCR/CD3 complexes. They were able to proliferate and elicit cytotoxic functions when triggered through their zeta chimeras. These results suggest that the triggering requirements for effector functions seem to be different in resting than in activated T cells.
Fas and activation-induced Fas ligand mediate apoptosis of T cell hybridomas: inhibition of Fas ligand expression by retinoic acid and glucocorticoids.
Authors: Yang Y , Merćep M , Ware C F , Ashwell J D
Published: June 2004

Activation of T cell hybridomas induces a G1/S cell cycle block and apoptosis. We isolated a variant of the 2B4.11 T cell hybridoma that, when activated via the TCR, produced IL-2 and underwent growth inhibition but did not die. Analysis of a variety of cell surface molecules revealed that the variant cell line, termed VD1, expressed very low levels of Fas compared to the wild type cells. Unlike 2B4.11 cells, VD1 cells were not killed by Fas ligand (FasL)-bearing effector cells. To determine if Fas is involved in activation-induced apoptosis, two different reagents that specifically bind Fas without killing the T cell hybridomas, a monoclonal antibody and a soluble Fas:Fc chimeric molecule, were added to activated T cell hybridomas. Both treatments prevented activation-induced apoptosis in a dose-dependent manner, but had no effect on IL-2 production or growth inhibition. Northern blot analysis revealed that unactivated 2B4.11 cells expressed negligible levels of FasL mRNA, but transcripts were detectable as early as 2 h after activation and continued to increase up to 4-6 h after activation. Anti-TCR induced activation of 2B4.11 cells in the presence of a TCR- 2B4.11 variant resulted in death of the unactivated "bystander" cells, which was inhibited by anti-Fas antibodies. Finally, treatment of T hybridoma cells with 9-cis retinoic acid or glucocorticoids, which are known to prevent activation-induced T cell apoptosis, inhibited the up-regulation of FasL. We conclude that up-regulated expression of FasL and its subsequent interaction with Fas accounts for the apoptotic response of T cell hybridomas to activation, and that retinoic acid and corticosteroids inhibit activation-induced apoptosis by preventing up-regulation of FasL.
Intracellular targeting of antigens internalized by membrane immunoglobulin in B lymphocytes.
Authors: Mitchell R N , Barnes K A , Grupp S A , Sanchez M , Misulovin Z , Nussenzweig M C , Abbas A K
Published: June 2004

An important function of membrane immunoglobulin (mIg), the B cell antigen receptor, is to endocytose limiting quantities of antigen for efficient presentation to class II-restricted T cells. We have used a panel of mIg mutants to analyze the mechanism of mIg-mediated antigen presentation, and specifically to explore the ability of mIg to target internalized antigen to intracellular processing compartments. Transfected mIgs carrying substitutions for the transmembrane Tyr587 residue fail to efficiently present specifically bound antigen. However, these mutants internalize antigen normally, and their defect cannot be attributed to a lack of mIg-associated Ig alpha/Ig beta molecules. A novel functional assay for detecting antigenic peptides in subcellular fractions shows that wild-type mIg transfectants generate class II-peptide complexes intracellularly, whereas only free antigenic peptides are detectable in the mutant mIg transfectants. Furthermore, an antigen competition assay reveals that antigen internalized by the mutant mIgs fails to enter the intracellular processing compartment accessed by wild-type mIg. Therefore, mIg specifically targets bound and endocytosed antigen to the intracellular compartment where processed peptides associate with class II molecules, and the transmembrane Tyr587 residue plays an obligatory role in this process. Targeting of internalized antigen may be mediated by receptor-associated chaperones, and may be a general mechanism for optimizing the presentation of specifically bound and endocytosed antigens in b lymphocytes and other antigen-presenting cells.
Detection of functional class II-associated antigen: role of a low density endosomal compartment in antigen processing.
Authors: Barnes K A , Mitchell R N
Published: June 2004

We have developed a functional assay to identify processed antigen in subcellular fractions from antigen-presenting cells; stimulatory activity in this assay may be caused by either free peptide fragments or by complexes of peptide fragments and class II molecules present on organellar membrane sheets and vesicles. In addition, we have developed a functional assay to identify proteolytic activity in subcellular fractions capable of generating antigenic peptides from intact proteins. These techniques permit the direct identification of intracellular sites of antigen processing and class II association. Using a murine B cell line stably transfected with a phosphorylcholine (PC)-specific membrane-bound immunoglobulin (Ig), we show that PC-conjugated antigens are rapidly internalized and efficiently degraded to generate processed antigen within an early low density compartment. Proteolytic activity capable of generating antigenic peptide fragments from intact proteins is found within low density endosomes and a dense compartment consistent with lysosomes. However, neither processed peptide nor peptide-class II complexes are detected in lysosomes from antigen-pulsed cells. Furthermore, blocking the intracellular transport of internalized antigen from the low density endosome to lysosomes does not inhibit the generation of processed antigen. Therefore, antigens internalized in association with membrane Ig on B cells can be efficiently processed in low density endosomal compartments without the contribution of proteases present within denser organelles.
Fibrin regulates neutrophil migration in response to interleukin 8, leukotriene B4, tumor necrosis factor, and formyl-methionyl-leucyl-phenylalanine.
Authors: Loike J D , el Khoury J , Cao L , Richards C P , Rascoff H , Mandeville J T , Maxfield F R , Silverstein S C
Published: June 2004

We have examined the capacity of four different chemoattractants/cytokines to promote directed migration of polymorphonuclear leukocytes (PMN) through three-dimensional gels composed of extracellular matrix proteins. About 20% of PMN migrated through fibrin gels and plasma clots in response to a gradient of interleukin 8 (IL-8) or leukotriene B4 (LTB4). In contrast, < 0.3% of PMN migrated through fibrin gels in response to a gradient of tumor necrosis factor alpha (TNF) or formyl-methionyl-leucyl-phenylalanine (FMLP). All four chemoattractants stimulated PMN to migrate through gels composed of collagen IV or of basement membrane proteins (Matrigel), or through filters to which fibronectin or fibrinogen had been adsorbed. PMN stimulated with TNF or FMLP adhered and formed zones of close apposition to fibrin, as measured by the exclusion of a 10-kD rhodamine-polyethylene glycol probe from the contact zones between PMN and the underlying fibrin gel. By this measure, IL-8- or LTB4-treated PMN adhered loosely to fibrin, since 10 kD rhodamine-polyethylene glycol permeated into the contact zones between these cells and the underlying fibrin gel. PMN stimulated with FMLP and IL-8, or FMLP and LTB4, exhibited very little migration through fibrin gels, and three times as many of these cells excluded 10 kD rhodamine-polyethylene glycol from their zones of contact with fibrin as PMN stimulated with IL-8 or LTB4 alone. These results show that PMN chemotaxis is regulated by both the nature of the chemoattractant and the composition of the extracellular matrix; they suggest that certain combinations of chemoattractants and matrix proteins may limit leukocyte movements and promote their localization in specific tissues in vivo.
Differential ability of isolated H-2 Kb subsets to serve as TCR ligands for allo-specific CTL clones: potential role for N-linked glycosylation.
Authors: Shen L , Kane K P
Published: June 2004

It is not known whether all forms of cell surface peptide-class I complexes, when bound with relevant peptide antigen, are recognized by T cells. We demonstrate herein that two distinct subsets of the murine H-2 Kb molecule can be separately isolated from H-2b-expressing cell lines using Y3 mAb immunoaffinity chromatography. Although both isolated Kb subsets were found to be strongly reactive with Y3 mAb by ELISA, one Kb subset is S19.8 mAb reactive (Ly-m11+Kb subset) and exhibits low reactivity with the M1/42 antibody, while the other subset is negative for the Ly-m11 epitope and highly reactive with the M1/42 antibody (M1/42high Kb subset). More importantly, whereas the M1/42high Kb subset is a very effective ligand for both TCR and CD8, the Ly-m11+ Kb subset could only function as a CD8 ligand, as determined in allo-specific CD8+ CTL clone adhesion and degranulation assays. Peptides acid-eluted from both Kb subsets sensitized Kb-transfected T2 cells expressing "peptide empty" Kb for lysis to a similar extent by allo-CTL clones, indicating that relevant endogenous peptide antigens are not limiting in the Ly-m11+ Kb subset. The major distinction identified between the two Kb subsets is that they differ substantially in their degree of N-linked glycosylation, with the Ly-m11+ subset containing Kb molecules with larger and more complex carbohydrate modifications than the M1/42high subset. The differences in glycosylation may explain the functional differences observed between the two Kb subsets. It is therefore possible that some forms of glycosylation on class I molecules interfere with TCR recognition and may limit CD8+ T cell responses, perhaps under circumstances where peptide antigen is limiting.
Anti-DNA antibodies bind to DNase I.
Authors: Puccetti A , Madaio M P , Bellese G , Migliorini P
Published: June 2004

Polyspecificity is a well-known property of the anti-DNA antibodies produced by autoimmune animals. In our search for antigen targets of anti-DNA antibodies within tissue extracts, we identified a 32-kD polypeptide that was recognized by a large panel of anti-DNA antibodies. Direct sequencing of this protein disclosed its identity with DNase I. 22 monoclonal anti-DNA antibodies bound to DNase I in direct and competitive immunoassays; out of 15 autoantibodies that did not bind DNA, none had the ability to bind DNase I. The ability of anti-DNA antibodies to interfere with DNase I enzymatic activity was evaluated in an assay based on the enzyme digestion of phage double strand DNA. Six monoclonal anti-single strand DNA antibodies that did not bind double strand DNA were tested in this assay. Three out of six inhibited DNase I-mediated digestion of phage DNA. The interaction of anti-DNA antibodies with DNase I was further investigated by testing their ability to bind a synthetic peptide that corresponds to the catalytic site of the molecule. 4 out of 22 anti-DNA antibodies bound the active site peptide; two of these had been shown to inhibit DNase I enzymatic activity. This report show that anti-DNA antibodies recognize both DNA and its natural ligand DNase I. Some anti-DNA antibodies inhibit DNase I enzymatic activity, thus displaying the potential to modulate DNA catabolism. The dual specificity of anti-DNA antibodies offers a clue for understanding the mechanisms that lead to anti-DNA antibody production in autoimmune animals.
Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins.
Authors: Lévesque J P , Leavesley D I , Niutta S , Vadas M , Simmons P J
Published: June 2004

Cytokines are known to be important regulators of normal hemopoiesis, acting in concert with components of the bone marrow microenvironment. Interactions with this microenvironment are known to regulate the proliferation, differentiation, and homing of hemopoietic progenitor (CD34+) cells. Adhesive interactions with the extracellular matrix retain CD34+ cells in close proximity to cytokines, but may also provide important costimulatory signals. Thus, the functional states of adhesion receptors are critical properties of CD34+ cells, but the physiological mechanisms responsible for regulating functional properties of cell adhesion receptors on primitive hemopoietic cells are still unknown. We confirm that the integrins very late antigen (VLA)-4 and VLA-5 are expressed on the CD34+ cell lines MO7e, TF1, and on normal bone marrow CD34+ progenitor cells, but in a low affinity state, conferring on them a weak adhesive phenotype on fibronectin (Fn). Herein, we show that the cytokines interleukin (IL)-3, granulocyte-macrophage CSF (GM-CSF), and KIT ligand (KL) are physiological activators of VLA-4 and VLA-5 expressed by MO7e, TF1, and normal bone marrow CD34+ progenitor cells. Cytokine-stimulated adhesion on Fn is dose dependent and transient, reaching a maximum between 15 and 30 min and returning to basal levels after 2 h. This cytokine-dependent activation is specific for VLA-4 and VLA-5, since activation of other beta 1 integrins was not observed. The addition of second messenger antagonists staurosporine and W7 abolished all cytokine-stimulated adhesion to Fn. In contrast, genistein inhibited KL-stimulated adhesion, but failed to inhibit GM-CSF- and IL-3-stimulated adhesion. Our data suggest that cytokines GM-CSF and IL-3 specifically stimulate beta 1 integrin function via an "inside-out" mechanism involving protein kinase activity, while KL stimulates integrin activity through a similar, but initially distinct, pathway via the KIT tyrosine-kinase. Thus, in addition to promoting the survival, proliferation, and development of hemopoietic progenitors, cytokines also regulate adhesive interactions between progenitor cells and the bone marrow microenvironment by modifying the functional states of specific integrins. These data are of importance in understanding the fundamental processes of beta 1 integrin activation and cellular response to mitogenic cytokines as well as on the clinical setting where cytokines induce therapeutic mobilization of hematopoietic progenitors.