bluuebunny/crossref_metadata_2025 huggingface dataset:
Uptake of microparticle‐adsorbed protein antigen by bone marrow‐derived dendritic cells results in up‐regulation of interleukin‐1α and interleukin‐12 p40/p35 and triggers prolonged, efficient antigen presentation
Authors: Scheicher Christoph , Mehlig Maria , Dienes Hans‐Peter , Reske Konrad
Published: March 2007

Dendritic cells synthesize and express major histocompatibility complex (MHC) class II peptide‐binding elements constitutively and, therefore, belong to the category of professional antigen‐presenting cells. Unlike other cells that show constitutive class II expression, such as B cells and certain T cell clones, dendritic cells possess the unique capacity to activate naive T cells. Using dendritic cells generated in vitro by culture of mouse bone marrow in the presence of low doses of recombinant mouse granulocyte/macrophage colony‐stimulating factor, we found that discrete maturation stages of these cells can be distinguished which were correlated with defined functional capabilities. The striking observation was the presence of a progenitor dendritic cell expressing low levels of class II which, unlike its differentiated counterpart in vitro, possessed pronounced phagocytic activity. Adding protein antigen to dendritic cells in a particle‐adsorbed form, as compared to a soluble form, we demonstrate that phagocytosis of the particle‐adsorbed protein by progenitor dendritic cells involves an activation event. This is evidenced by the de novo synthesis of transcripts of interleukin‐1α and interleukin‐12 p40/p35 as well as transcripts of MHC class II. Most importantly, an augmented and prolonged antigen‐presentation capacity was observed when the antigen was given in particle‐adsorbed instead of soluble form. These findings indicate that progenitor dendritic cells are functionally more flexible and potent than fully differentiated dendritic cells and that they play a crucial role in antigen presentation. It is suggested that these findings will open up new possibilities to devise strategies for vaccine development.
A gut‐homing, oligoclonal CD4+ T cell population in severe‐combined immunodeficient mice expressing a rearranged, transgenic class I‐restricted αβ T cell receptor
Authors: Reimann Jörg , Rudolphi Angelika , Spieß Sibylle , Claesson Mogens H.
Published: March 2007

We studied the peripheral T cell compartment of H‐2b severe combined immunodeficient (scid) mice that express a transgenic (tg) αβ T cell receptor (TcR) specific for the H‐Y (male) epitope presented by the H‐2 class I Db molecule. Large populations of CD3+ NK1.1−TCRβT+ T cells were present in spleen, mesenteric lymph nodes, peritoneal cavity, lamina propria and epithelial layer of the small and large intestine of 6‐ to 10‐month‐old, male and female tg scid mice. Only low numbers of CD3+ T cells were recovered from inguinal, popliteal, or axillary lymph nodes. We studied CD4+ T cells in these tg scid mice. CD4+ T cells were found in the peritoneal cavity, in the mesenteric lymph nodes and in the intraepithelial layer and lamina propria of the gut. All CD4+ T cells were CD44+ (i.e. showed evidence of antigen‐driven differentiation) and expressed the tg Vβ8.2 TcR β‐chain (TcRβT+). Only few CD4+ T cells expressed the tg Vα3+ TcR α‐chain (TcRαT). cDNA was prepared from CD4+ T cells from spleen or mesenteric lymph nodes of individual male and female tg scid mice; sequence analyses of polymerase chain reaction‐amplified, endogenous TcR α‐chain (TcRαE) transcripts indicated that > 90% of the TcRαE‐chain transcripts were in‐frame, that the TcRαE repertoire in CD4+ T cell populations was oligoclonal, and that the TcRαE repertoire was different in individual tg scid mice. Hence, an oligoclonal, leaky CD4+ T cell population is selected in tg scid mice that apparently responds to gut‐derived antigens. No inflammatory bowel disease (IBD) was evident in the small or large intestine of 6‐ to 10‐month old tg scid mice. After adoptive transfer of purified CD4+ T cells (105 cells per mouse) from tg scid mice into non‐tg H‐2b scid mice, CD4+ TcRαT−βT+ cells were found in gut tissues of the immunodeficient host. Transplanted scid mice developed clinical and histological signs of IBD. An oligoclonal, gut‐homing, memory/effector CD4+ CD44+ TcRβT+ TcRαT− T cell subset from leaky tg scid mice thus has a pathogenic potential when released from the control of TcRβT+ TcRαT+ T cells.
Immunoglobulin variable gene analysis of human autoantibodies reveals antigen‐driven immune response to glutamate decarboxylase in type 1 diabetes mellitus
Authors: Richter Wiltrud , Jury Karin M. , Loeffler Daniela , Manfras Burkhard J. , Eiermann Thomas H. , Boehm Bernhard O.
Published: March 2007

Insulin‐dependent (type 1) diabetes mellitus is an organ‐specific autoimmune disease frequently associated with an islet‐specific humoral autoimmune response. The role of islet cell autoantibodies in the disease process is unclear; in particular, it is not known whether they are a non‐specific side effect of islet cell destruction or play a role in the autoimmune network leading to type 1 diabetes. Here we report the immunoglobulin gene usage and somatic mutation rates of a panel of seven human monoclonal islet cell autoantibodies (MICA 1–7) directed towards the major islet cell autoantigen glutamate decarboxylase (GAD). These autoantibodies were produced from cells from two patients with newly diagnosed type 1 diabetes. VH1, VH4 and Vλ2 gene segments were frequently used in the MICA, but no correlation between V gene usage and epitope recognition was found. The nonrandom ratio of replacement versus silent mutations in the variable gene region, an accumulation of replacement mutations in the complementarity determining regions, which confer antigen binding, and the high relative avidity for GAD observed for MICA 1, 3, 4, and 6, suggested that the immune response to GAD is driven by the antigen. In contrast, MICA 2, 5, and 7, revealing a lower affinity for antigen, have accumulated a large number of silent mutations. These latter antibodies may, therefore, be characteristic for later stages of the chronic autoimmune disease. Our results argue in favor of an antigen‐driven autoantibody response to islets in human type 1 diabetes. They suggest that GAD is an important target of autoimmunity associated with type 1 diabetes.
Repression of the immunoglobulin heavy chain 3′ enhancer by helix‐loop‐helix protein Id3 via a functionally important E47/E12 binding site: implications for developmental control of enhancer function
Authors: Meyer Kerstin B. , Skogberg Michaela , Margenfeld Cecilia , Ireland John , Pettersson Sven
Published: March 2007

The activity of the immunoglobulin 3′ enhancer is restricted to the late stages of B lymphoid development. Here we further examine the molecular basis for the temporally restricted activity of the B‐lymphoid IgH 3′ enhancer. We demonstrate that a binding site (E5 site) for the E47 and/or E12 proteins is functionally important for enhancer activity. The multimerized E5 site acts as a B cell‐specific enhancer and, when assayed in COS cells, can be transactivated by E47/E12 proteins. This transactivation in COS cells, as well as the activity of the full length 3′ enhancer in plasma cells, can be repressed by overexpression of the dominant negative nuclear regulator Id3. When examining the tissue distribution of Id3 in murine cell lines, we find that Id3 is expressed throughout the pre‐B and B cell stages, but is down‐regulated at the plasma cell stage. Thus, Id3 may contribute to the temporal regulation of the IgH 3′ enhancer.
The type I interleukin‐1 receptor acts in series with tumor necrosis factor (TNF) to induce arthritis in TNF‐transgenic mice
Authors: Probert Lesley , Plows David , Kontogeorgos George , Kollias George
Published: March 2007

The pro‐inflammatory cytokines tumor necrosis factor (TNF) and interleukin‐1 (IL‐1) have been shown to be primary mediators in the pathogenesis of chronic inflammatory joint diseases. However, the relative contributions of these molecules to the development and progression of disease is not known. In the present study, we have investigated the involvement of the type I IL‐1 receptor in the development and progression of chronic arthritis in a previously described TNF‐transgenic mouse model of this disease. A neutralizing monoclonal antibody to the murine type I IL‐1 receptor was injected intraperitoneally three times a week from birth to 9 weeks of age. In the positive control group of untreated transgenic mice the incidence of arthritis was 100% after 9 weeks of age. The injection of normal hamster IgG did not influence the incidence or development of arthritis. In contrast, the injection of antibody to the type I IL‐1 receptor completely prevented the development of arthritis. Clinical evaluation of disease was confirmed histologically, anti‐receptor antibody‐treated mice showed no sign of arthritic change. Moreover, the analysis of sera taken at the end of the study period showed that the neutralization of arthritis by IL‐1 receptor antibody treatment was accompanied by significantly lower levels of circulating human TNF compared to the control groups and to untreated transgenic mice prior to disease onset. Taken together, these results show that IL‐1 signaling blockade exerts a direct negative feedback effect on TNF production. Our results show that in TNF‐transgenic mice the IL‐1 receptor accepts the whole pathogenic load of TNF, thereby acting as a potent downstream mediator in the pathogenesis of chronic arthritis.
Induction of the increased Fyn kinase activity in anergic T helper type 1 clones requires calcium and protein synthesis and is sensitive to cyclosporin A
Authors: Gajewski Thomas F. , Fields Patrick , Fitch Frank W.
Published: March 2007

Several alterations in T cell receptor‐associated signal transduction have been observed following induction of anergy of T helper type 1 (Th1) clones, including a modified intracellular free calcium ([Ca2+]i) response and increased kinase activity associated with the protein tyrosine kinase p59fyn. In the current study, we demonstrate that, although the kinetics of acquisition of both of these signaling alterations correlated with the generation of anergy, a normal calcium response returned within 48 h after removal from the anergizing stimulus, whereas the increased p59fyn activity persisted and the cells remained hyporesponsive. Generation of both the anergic state and the increased p59fyn activity was prevented in the presence of calcium‐free medium, cycloheximide (CHX), or cyclosporin A (CsA), and could be mimicked by the calcium ionophore ionomycin. In contrast, the altered calcium response was inhibited by stimulation in the presence of calcium‐free medium or CsA, but not CHX. Thus, surprisingly, these data suggest that a chronic elevation of [Ca2+]i is proximal to and necessary for the increase in p59fyn‐associated kinase activity observed in anergic Th1 clones. Increased p59fyn activity, but not the altered calcium response, correlates with maintenance of the anergic state.
Apoptosis of ileal Peyer's patch B cells is increased by glucocorticoids or anti‐immunoglobulin antibodies
Authors: Motyka Bruce , Bhogal Hardeep S. , Reynolds John D.
Published: March 2007

The ileal Peyer's patch (PP) in sheep plays a central role in the development and production of B cells. Associated with a tremendous amount of B cell proliferation in this site is the extensive diversification of the Ig repertoire by somatic hypermutation. Very few (<5%) of the B cells produced in the ileal PP differentiate and emigrate; instead, the vast majority of these cells soon die, and we have previously shown that death is associated with apoptosis. When placed in culture, ileal PP B cells die rapidly by apoptosis, such that after 24h, 60 ± 1 % of DNA is fragmented. Here, we show that the extent of this spontaneous B cell apoptosis in culture, as quantitated by DNA fragmentation, was significantly increased in a dose‐dependent manner by the glucocorticoids hydrocortisone or dexamethasone. Furthermore, treatment of lambs with 2–2.5 mg/kg of dexamethasone resulted in a marked increase in the number of apoptotic cells in the ileal PP and an increase in ileal PP B cell DNA fragmentation to 20 ± 6%, compared with 2.4 ± 0.1 % in untreated lambs. Anti‐immunoglobulin (Ig) antibodies also increased the extent of DNA fragmentation in cultured ileal PP B cells. After 24 or 48 h of culture with anti‐Ig (PIg47A), DNA fragmentation was 74 ± 2 % and 75 ± 3 %, respectively. Ileal PP B cells are rescued from apoptosis by agents that activate protein kinase C and increase cytosolic Ca2+, and here we show that this treatment also results in apoptotic rescue in the presence of dexamethasone or anti‐Ig. We speculate that the apoptosis of ileal PP B cells in situ may be modulated by glucocorticoids and by the cross‐linking of surface Ig. Apoptosis, induced by a signal through surface Ig, may be an important mechanism in the deletion of self‐reactive B cells during the expansion of the Ig repertoire in the ileal PP.
Hyaluronan binding by CD44 is regulated by a phosphorylation‐independent mechanism
Authors: Uff Caroline R. , Neame Stephen J. , Isacke Clare M.
Published: March 2007

CD44 is an adhesion receptor for which the major characterized ligand is the extracellular matrix glycosaminoglycan, hyaluronan. This interaction underlies CD44‐mediated cell attachment, cell migration, and matrix remodelling during development and wound healing. Truncation of the CD44 cytoplasmic domain does not prevent cell surface expression of this hyaluronan receptor but it dramatically impairs ligand binding. In this study we have examined the role of phosphorylation in regulating this function by mutating the target serine residues to either neutral amino acids with the aim of creating a phosphorylation‐incompetent molecule, or to acidic residues to mimic a fully phosphorylated CD44. In transfected AKR1 cells the behavior of both the neutral and acidic mutants was indistinguishable from wild‐type CD44, indicating that there is a phosphorylation‐independent mechanism involved in regulating hyaluronan binding.
Comparative analysis of B7‐1 and B7‐2 expression in Langerhans cells: differential regulation by T helper type 1 and T helper type 2 cytokines
Authors: Kawamura Tatsuyoshi , Furue Masutaka
Published: March 2007

Epidermal Langerhans cells (LC) are Ia‐bearing potent antigen‐presenting cells (APC) of dendritic cell lineage that play a crucial role in primary and secondary T cell‐dependent immune responses. LC express several costimulatory molecules such as B7, which has been implicated as one of the important determinants of professional APC. Recently, B7 antigens have been shown to include three distinct molecules termed B7‐1, B7‐2, and B7‐3, and the expression of B7‐1 and B7‐2 in LC has been already confirmed. However, little is known of the regulation of B7‐1 and B7‐2 expression in LC. We demonstrated that LC do not express B7‐1 and B7‐2 in situ; however, the expression of both molecules is rapidly induced during the first 3 days of culture, and high levels of expression are maintained at least until day 6. We show that the expression of B7‐2 in LC is much higher than that of B7‐1 in each experiment, and that B7‐1 and B7‐2 expression is reproducibly augmented by interleukin (IL)‐4 in a dose‐dependent manner; however, IL‐2 affected expression very little. Finally, B7‐1 expression is significantly and dose‐dependently down‐regulated by interferon (IFN)‐γ or IL‐10, and B7‐2 expression is consistently inhibited by IL‐10, but not by IFN‐γ. The effects of these cytokines are active only in the induction phase (during first 3 days of culture) of B7 expression: the modulatory effects of cytokines are hardly detected in the plateau phase (days 4 to 6 of culture) of B7 expression in LC. These findings suggest that B7‐1 and B7‐2 expression are indeed selectively and differentially regulated by these T cell‐derived cytokines, and that the cytokines may modulate the synthesis of B7 molecules rather than the degradation of already‐expressed B7 molecules.
Spontaneous apoptosis of dendritic cells is efficiently inhibited by TRAP (CD40‐ligand) and TNF‐α, but strongly enhanced by interleukin‐10
Authors: Ludewig Burkhard , Graf Daniel , Gelderblom Hans R. , Becker Yechiel , Kroczek Richard A. , Pauli Georg
Published: March 2007

In the lymphoid tissues, adaptive immune responses are initiated by the interaction of interdigitating dendritic cells (IDC) with naive T cells. To understand this interplay better, we used mature Langerhans cells (mLC), migrating from human epidermis, as the correlate of IDC ex vivo to evaluate the different effects of tumor necrosis factor (TNF)‐α, TNF‐related activation protein (TRAP; CD40‐ligand) and interleukin‐10 (IL‐10) on induction or prevention of apoptotic cell death in these cells. Spontaneous decrease of mLC viability in culture was due to apoptosis, as determined by the appearance of typical morphological changes such as dilatation of the endoplasmic reticulum (ER), chromatin condensation and membrane blebbing. IL‐10 strongly reduced mLC viability, whereas TRAP and TNF‐α facilitated the survival of mLC. Spontaneous DNA fragmentation was detectable after 24 h in culture. IL‐10 led to an earlier onset of DNA fragmentation, whereas TRAP and TNF‐α delayed internucleosomal DNA cleavage. We found that IL‐10‐treated mLC were readily ingested and removed by macrophages. TNF‐α and TRAP, in contrast, reduced engulfment of mLC by macrophages. Interestingly, IL‐10, even at low concentrations, reverted the effects of TNF‐α and TRAP in inhibiting mLC apoptosis. Furthermore, IL‐10 led to the down‐regulation of various surface antigens, especially of CD86 and CD54, whereas TNF‐α and TRAP enhanced the expression of MHC class I and II antigens and of the accessory molecules CD40, CD54, CD80 and CD86. Taken together, these results show that mLC spontaneously undergo apoptosis in culture and that the progression of mLC to apoptosis is inhibited by TRAP and TNF‐α, but accelerated by IL‐10.
Early appearance of T cell receptor αβ+ CD4− CD8− T cells with a skewed variable region repertoire after infection with Listeria monocytogenes
Authors: Matsuzaki Goro , Li Xiao‐Yan , Kadena Tsutomu , Song Fei , Hiromatsu Kenji , Yoshida Hiroki , Nomoto Kikuo
Published: March 2007

We found that the number of T cell receptor (TCR)αβ+ CD4− CD8− T cells increased in the peritoneal cavity on day 5 after an intraperitoneal infection with Listeria monocytogenes strain EGD together with TCRγδ+ CD4− CD8− T cells. Thereafter, the TCRαβ+ CD4− CD8− T cells decreased to a normal level by day 14. The TCRαβ+ CD4− CD8− T cells showed an activated T cell phenotype (L‐selectin‐CD44+) and expressed CD45/B220 and interleukin‐2 receptor β, but did not express heat stable antigen, which is expressed by the immature CD4− CD8− thymocytes. Furthermore, 20–30% of the TCRαβ+ CD4− CD8− T cells expressed the NK1.1 natural killer cell marker. Analyses of the TCR V region repertoire of the TCRαβ+ CD4− CD8− T cells induced by L. monocytogenes infection showed that more than 80% of the TCRαβ+ CD4− CD8− T cells expressed TCR Vβ8 detected by anti‐TCR Vβ8.1 and 8.2 mAb, and a reverse transcription‐polymerase chain reaction analysis of Vα14 relative to Vα11 expression revealed that the TCRαβ+ CD4− CD8− T cells expressed a higher level of Vα14, which was reported to be preferentially expressed by TCRαβ+ CD4− CD8− thymocytes rather than conventional CD4+ T cells. The TCRαβ+ CD4− CD8− T cells showed a proliferative response to anti‐TCRαβ mAb stimulation. In contrast, they showed no response to stimulation with either Listeria antigen or 65‐kDa heat shock protein of Mycobacterium bovis, which do stimulate the Listeria‐specific TCRαβ+ CD4+ CD8− T cells and the Listeria‐induced TCR γδ+ T cells, respectively. These results suggest that the TCRαβ+ CD4− CD8− T cells may recognize a restricted set of self antigens induced by L. monocytogenes infection, and that they contribute to host protection at an early stage of the infection.
Cassette baculovirus vectors for the production of chimeric, humanized, or human antibodies in insect cells
Authors: Poul Marie‐Alix , Cerutti Martine , Chaabihi Hassan , Ticchioni Michel , Deramoudt François‐Xavier , Bernard Alain , Devauchelle Gérard , Kaczorek Michel , Lefranc Marie‐Paule
Published: March 2007

Plasmid cassette‐transfer vectors pBHuCϰ and pBHuCy1 have been designed which enable the construction of recombinant baculoviruses directing the co‐expression of complete immunoglobulin in insect cells. We describe the application of these vectors for the expression of a human/mouse chimeric monoclonal antibody of potential immunosuppressive clinical value derived from a mouse anti‐human CD29 monoclonal antibody (Mu‐K20). The chimeric K20 light and heavy chains produced in sf9 insect cells were correctly processed and assembled into a normal immunoglobulin which is secreted into the culture medium of infected cells. The chimeric mAb Ch‐K20‐sf9 reproduces in vitro the functional properties of the parental mouse K20, including affinity and inhibition of lymphocyte proliferation. These results demonstrate that the baculovirus/insect cell expression system is suitable for the expression of fully active monoclonal antibodies of therapeutic value. Our generic cassette approach makes this system a very flexible and convenient one for the rapid production of either chimeric, humanized or human mAb with heavy and light chains of any isotype.
Cell cycle regulation of immunoglobulin class switch recombination and germ‐line transcription: potential role of Ets family members
Authors: Lundgren Mats , Ström Lena , Bergquist Lars‐Olof , Skog Sven , Heiden Thomas , Stavnezer Janet , Severinson Eva
Published: March 2007

Previous studies have indicated that transcription of germ‐line (GL) CH genes is necessary to obtain immunoglobulin (Ig) class switching. We report here a correlation between proliferation, switching and GL transcripts. Sμ‐Sγ1 switch recombination in lipopolysaccharide (LPS) + interleukin‐4 (IL‐4)‐activated mouse B cells was assayed by a digestion‐circularization polymerase chain reaction. Switching to γ1 is reduced upon inhibition of DNA synthesis with hydroxyurea (HU) or aphidicholin (AC). Incubation of activated B cells with HU severely reduces steady‐state levels of GL γ1 and ϵ RNA. By utilizing elutriation to synchronize B cell blasts in different phases of the cell cycle, it was found that GL γ1 transcripts are mainly expressed in G1 and S phases, but not in G0. Using the electrophoretic mobility shift assay, we characterized two major LPS‐induced complexes, which bind to the GL γ1 promoter and are expressed at levels which correlate with the amount of LPS‐induced DNA synthesis. Furthermore, the intensity of the complexes is reduced when cells are arrested with the DNA synthesis inhibitors HU or AC. Elutriation experiments revealed that the complexes are expressed in G1 and S, but not in G0. They bind to an Ets consensus element near the major initiation sites used in proliferating cells. The possible implications of these findings for Ig isotype switching are discussed.
B7.2 provides co‐stimulatory functions in vivo in response to staphylococcal enterotoxin B
Authors: Muraille Eric , de Smedt Thibaut , Urbain Jacques , Moser Muriel , Leo Oberdan
Published: March 2007

Excessive T cell activation induced by bacterial superantigens plays an important role in the pathology associated with Gram‐positive bacteremia. To gain insight into the early phases of T cell activation by bacterial enterotoxins in vivo, we investigated the ability of antibodies to well‐defined co‐stimulatory molecules to inhibit T cell activation and the subsequent toxic shock syndrome induced in BALB/c mice following the injection of staphylococcal enterotoxin B (SEB). We demonstrate here that a single dose of anti‐B7.2 antibodies, but not anti‐B7.1 antibodies, significantly inhibits T cell activation, as judged by lower systemic IL‐2 release, blastogenesis and IL‐2 receptor expression, and reduces the lethal effect of SEB in D‐galactosamine‐sensitized mice. These results demonstrate that co‐stimulation through the B7.2 molecule plays an important role in the activation of T cells in response to SEB in vivo and suggest alternative therapies for septic shock caused by bacterial enterotoxins based on blocking antibodies to co‐stimulatory molecules.
Regulation of murine B cell growth and differentiation by CD30 ligand
Authors: Shanebeck Kurt D. , Maliszewski Charles R. , Kennedy Mary K. , Picha Kathleen S. , Smith Craig A. , Goodwin Ray G. , Grabstein Kenneth H.
Published: March 2007

A ligand for CD30 has been recently cloned, and has been shown to have sequence homology with the tumor necrosis factor family of cytokines. CD30 ligand (CD30L) was found to be induced on helper T cell clones, and its receptor was expressed on freshly isolated and activated murine B cells. Recombinant murine CD30L was found to share many functional properties with CD40 ligand (CD40L) in the regulation of murine B cell growth and differentiation in vitro. CD30L stimulated B cell proliferation, antigen‐specific antibody production, and polyclonal immunoglobulin secretion in a cytokine‐dependent manner. In particular, the stimulation of B cell proliferation by CD30L required interleukin (IL)‐4 and IL‐5, induction of anti‐sheep red blood cell antibody‐secreting B cells by CD30L required IL‐2 and IL‐5, and optimal induction of polyclonal immunoglobulin secretion required IL‐4 and IL‐5. Under these conditions, the polyclonal secretion of IgGl, IgA, IgG3 and IgE was induced. The induction of immunoglobulin secretion by CD30L was independent of CD40L, as B cells from CD40L deficient‐mice responded normally to CD30L treatment. We conclude that CD30L is a potent mediator of B cell growth and differentiation in vitro and may play a role in cognate T cell‐B cell interactions.
Antigen receptor‐initiated growth inhibition is blocked in CD45‐loss variants of a mature B lymphoma, with limited effects on apoptosis
Authors: Ogimoto Mami , Katagiri Tatsuo , Mashima Keisuke , Hasegawa Kiminori , Mizuno Kazuya , Yakura Hidetaka
Published: March 2007

Role of CD45 in B cell antigen receptor (BcR)‐mediated signaling events in mature B cells was examined using BAL‐17 and its CD45‐negative clones. In the CD45‐negative clones, BcR stimulation induced tyrosine phosphorylation almost identical to the parental cells, with a few exceptions of reduced phosphorylation, especially of a protein of about 60 kDa. BcR‐induced calcium responses were reduced in the CD45‐negative clones, but the kinetics were similar to the parent. BcR stimulation led to growth inhibition in the parental cells, but signals for growth inhibition were completely blocked in the CD45‐negative clones. Interestingly, the same stimulation induced low, but significant levels of apoptosis both in the parent and in the CD45‐negative clones. Thus, in mature BAL‐17 cells, CD45 subtly mediates early signaling events (tyrosine phosphorylation and Ca2+ mobilization), and is absolutely required for the signaling pathway leading to growth regulation, but has limited effects on apoptosis.
Antigen receptor‐triggered apoptosis in immature B cell lines is associated with the binding of a 44‐kDa phosphoprotein to the PTP1C tyrosine phosphatase
Authors: Wu Yongjian , Pani Giovanni , Siminovitch Katherine A. , Hozumi Nobumichi
Published: March 2007

While cross‐linking of the membrane IgM (mIgM) molecules expressed on WEHI 231 lymphoma cells induces these cells to undergo apoptosis, we have previously observed that ligation of the mIgD expressed on IgD‐transfected WEHI 231 (Wδ) cells is not associated with induction of cell death. Thus mIgM+IgD+ Wδ cells provide a valuable reagent for delineating the molecular events which modulate the physiologic outcome of B cell antigen receptor (BCR) engagement. In view of recent data implicating the cytosolic phosphotyrosine phosphatase PTP1C in the regulation of BCR signaling capacity, we used Wδ cells to investigate the potential role for PTP1C in modulating the cell response to BCR activation. The results of this analysis revealed PTP1C to undergo rapid tyrosine phosphorylation following mIgM or mIgD cross‐linking and to associate with a number of other phosphoproteins in stimulated Wδ cells. Among these latter phosphoproteins, one prominent species of about 44 kDa (pp44) which co‐precipitated with PTP1C in mIgM‐ligated cells was not detected in PTP1C immunoprecipitates from mIgD‐ligated cells. The association of PTP1C with this 44‐kDa phosphoprotein following mIgM cross‐linking was also observed in two additional B cell lines representing an immature state of differentiation, but was not detected after BCR engagement in two representative mature B cell lines or in splenic B cells. Initial data concerning the identity of pp44 indicate that this molecule does not represent the Shc, MAPK or Ig‐β proteins and may, therefore, constitute a previously unidentified signaling effector. While the structural and biochemical properties of pp44 require further definition, the findings suggest that BCR‐triggered interactions of PTP1C with pp44 occur only in the context of an immature state of cellular differentiation and the induction of apoptosis. These data therefore suggest that PTP1C interactions with pp44 may be relevant to the transduction of BCR signals which evoke cell death.
Characteristics of antigen‐independent and antigen‐dependent interaction of dendritic cells with CD4+ T cells
Authors: Hauss Pascale , Selz Françoise , Cavazzana‐Calvo Marina , Fischer Alain
Published: March 2007

Dendritic cells (DC) are the main antigen‐presenting cells for the initiation of primary T cell‐mediated immune responses. In the first stage of activation, T cells bind to DC in an antigen‐independent manner. We studied the adhesion characteristics of human CD4+ T cells to DC generated from CD34+ hematopoietic progenitors following 12 to 13 days of culture in the presence of granulo‐cyte/macrophage colony‐stimulating factor and tumor necrosis factor‐α. A majority of these cells had the morphology, phenotype and functions of DC. CD4+ T/DC adhesion was measured by means of fluorescence microscopy and flow cytometry. Four independent receptor/ligand pathways, LFA‐1/ICAM, ICAM/LFA‐1, CD2/LFA‐3 and CD28/CD80, were involved in the transient adhesion of DC to CD4+ T cells in antigen‐independent and specific alloantigen‐dependent situations, as shown by blocking experiments using monoclonal antibodies. The antibodies also blocked a primary mixed lymphocyte reaction (MLR) in which DC were used as stimulatory cells. Adhesion of alloreactive CD4+ T cells to antigen‐presenting DC was stronger than that of resting CD4+ T cells, while peak adhesion occurred after 5 and 20 min, respectively. The LFA‐1 ligands involved in adhesion of resting CD4 T cells to DC and alloreactive CD4+ T cells to specific DC differed in part, since ICAM‐3 on resting T cells and ICAM‐1 on alloreactive T lymphocytes preferentially bound LFA‐1. Studies of interactions between DC and phorbol ester‐activated T cells expressing the CD40 ligand revealed a fifth independent adhesion pathway, CD40/CD40 ligand. CD4‐mediated regulation of CD4+ T/DC adhesion was suggested by the observation that preincubation of CD4+ T cells and DC individually with anti‐CD4 antibodies inhibited adhesion. In addition, antibodies specific for HLA class II molecules inhibited adhesion when used to pretreat DC but not alloactivated CD4+ T cells.
Control of thymus‐independent intestinal intraepithelial lymphocytes by β2‐microglobulin
Authors: Neuhaus Oliver , Emoto Masashi , Blum Carmen , Yamamoto Shigeki , Kaufmann Stefan H. E.
Published: March 2007

Murine intestinal intraepithelial lymphocytes (i‐IEL) comprise thymusdependent cells such as T cell receptor (TcR) α/β CD8α/β+ i‐IEL, as well as thymus‐independent ones such as TcRα/β CD8α/α+ and TcRγ/δ CD8α/α+ i‐IEL. Whilst the development of the CD8α/β expressing i‐IEL is strictly contingent on major histocompatibility complex (MHC) class I surface expression, that of CD8α/α i‐IEL appears largely MHC class I independent. We have used β2‐microglobulin (β2m)−/− mutant mice lacking surface‐expressed MHC class I and TcRα/β CD8α/β+ i‐IEL to analyze the potential impact of MHC class I on regional activation of thymus‐independent i‐IEL. To analyze the role of TcRγ/δ i‐IEL in regional cell interactions, these mice were treated with the anti‐TcRγ/δ mAb, GL3. Whilst numbers of TcRα/β CD8α/α i‐IEL were markedly reduced in βm+/− mice, those of TcRγ/δ i‐IEL were elevated. Administration of GL3 in vivo caused TcR down‐modulation and functional inactivation of TcRγ/δ i‐IEL in β2m+/− mice. In contrast, TcR expression and functional activities of TcRγ/δ i‐IEL from β2m−/− mice were not impaired by GL3 treatment. The TcRα/β CD8β i‐IEL from β2m−/− mice were expanded and functionally activated as a consequence of TcRγ/δ engagement. The TcRγ/δ i‐IEL and TcRα/β CD8α/α+ i‐IEL from athymic nu/nu mice which express MHC class I, but lack TcRα/β CD8α/β+ i‐IEL, responded to TcRγ/δ engagement as those from the β2m+/− controls. In addition, the TcRγ/δ i‐IEL from TcRβ−/− and TcRβ+/− mutants were equally affected by GL3. We conclude that the absence of β2m renders TcRγ/δ i‐IEL resistant to TcR‐mediated inactivation and promotes activation of TcRα/β CD8β− i‐IEL. The activation of TcRγ/δ i‐IEL seems to be directly controlled by β2m/MHC class I expression and independent from TcRα/β CD8β+ i‐IEL. Regulation of self‐reactive thymus‐independent i‐IEL through β2m/MHC class I may contribute to control of autoreactive immune responses in the intestine.
CD34‐positive early human thymocytes: T cell receptor and cytokine receptor gene expression
Authors: Ktorza Sandra , Sarun Sukhéna , Rieux‐Laucat Frédéric , de Villartay Jean‐Pierre , Debré Patrice , Schmitt Christian
Published: March 2007

CD34, a stem cell marker, has been shown to be expressed on human CD3−CD4−CD8− (triple‐negative; TN) thymocytes. Phenotypic and functional analyses suggest the following differentiation sequence: CD34+ 1−3−4−8− → CD34+ 1+3−4→8− → CD34−1++3−4+8 → CD34−1++3−4+8+. In this report, we examined cytokine receptor gene expression on these subsets by reverse transcription‐polymerase chain reaction analysis (RT‐PCR). We were able to detect interleukin‐7 receptor (IL‐7R), c‐kit and IL‐2Rγ in all CD34+ thymocyte subsets, consistent with previous functional studies. We found IL‐1R, granulocyte/macrophage colony‐stimulating factor receptor‐α and IL‐4R transcripts in CD3− and CD34+ subsets. Secondly, we investigated T cell receptor (TCR)‐δ and ‐β gene rearrangement and transcription in CD34+ thymocytes. Our results show that a full‐length TCR‐δ transcript and the recombination activating genes RAG‐1 and RAG‐2 mRNA were already expressed in the CD34+1− subset. Mature Vβ‐containing TCR transcripts were also detected in the CD34+ 1+ subset, but not in the CD1− fraction. Furthermore, PCR analysis of D‐Jβ gene rearrangements showed that ≥ 70% of CD34+ 1− cells are in a TCRβ germ‐line configuration, although D‐Jβ recombination had already started in this population.
T cell receptor diversity in alloreactive responses against HLA‐B27 (B*2705) is limited by multiple‐level restrictions in both α and β chains
Authors: Barber Domingo F. , López Daniel , de Castro José A. López
Published: March 2007

The T cell receptors (TCR) in HLA‐B27 (B*2705) alloreactivity were analyzed in cytotoxic T lymphocytes (CTL) from two individuals. Non‐random usage was found in Vβ, N+Dβ, Vα, and Jα, but not in Jβ segments or Nα‐regions. Vβ segments from homology subgroup 4 were predominant and not associated to a particular donor or fine specificity, suggesting involvement in recognizing the HLA‐B27 molecule. In contrast, preferential Vα usage was associated with particular individuals and fine specificities, indicating distinct Vβ and Vα recruitment and contribution to allorecognition. Recurrent N+Dβ motifs and Jα segments, even from different donors, limited junctional diversity, suggesting that CDR3 usage was determined by the alloantigenic epitope independently of individuals. TCR were selected differently at various levels, as indicated by the following findings. Four clonotypes with similar fine specificity had identical β and unrelated α chains. Similar α were associated with unrelated β chains, and vice versa. CTL using Vβ subgroup 4 did not globally show concomitant predominance of other TCR elements. Vα7, one of the preferred Vα segments, was always associated with Vβ subgroups other than 4. Sometimes, a TCR showed homology in elements of one chain to a second TCR or group of TCR, and to another in the other chain. These results are best explained by differential selection of TCR elements by different epitopes, providing a key to the inner structure of allospecific TCR repertoires.
Superantigens initiate cognate CD4+ T cell/ B cell interactions leading to early activation and proliferation of B cells
Authors: Al‐Daccak Reem , Damaj Bassam , Naccache Paul H. , Mourad Walid
Published: March 2007

Dimerization or even multimerization of various receptors is commonly required for signal transduction. We report here that clustering of major histocompatibility complex class II molecules in human B cells by biotinylated staphylococcal enterotoxin A (SEA) cross‐linked with avidin induces an increase in the level of intracellular calcium. This response was abolished by prior treatment with protein tyrosine kinase (PTK) inhibitors, suggesting that SEA‐triggered calcium mobilization in B cells is probably dependent on the activation of PTK. The implication of PTK in SEA‐induced early B cell activation was then confirmed by demonstrating that cross‐linked SEA induces a significant increase in the level of tyrosine phosphorylation in B cells. The requirement of biotinavidin cross‐linking in SEA‐induced calcium mobilization in B cells can be fulfilled by the addition CD4+ T cells, suggesting a role for CD4 molecules. Using the murine CD4− T cell hybridoma 3DT, or its derivative 11B3 transfected with human CD4 that both express SEA‐specific TCR, we confirmed the CD4 requirement for B cell calcium mobilization and that both specific TCR and CD4 molecules are required in early events of B cell activation induced by SEA. The role of CD4 in SEA‐induced B cell proliferation was then investigated. SEA‐stimulated B cells proliferated in the presence of CD4+ T cells, whereas no response was observed in the presence of CD8+ T cells. The addition of clone 11B3 CD4+ T cells failed to fulfill the requirement of CD4+ T cells in SEA‐induced B cell proliferation, indicating the possible involvement of other CD4+ T cell surface molecules in this response. This issue is currently under investigation.
Sialyl LewisX carbohydrate is expressed differentially during avian lymphoid cell development
Authors: Palojoki Eeva , Jalkanen Sirpa , Toivanen Paavo
Published: March 2007

Sialyl LewisX is a carbohydrate moiety involved in the regulation of white blood cell adhesion to endothelial cells. In this work, we have studied the expression, localization and function of sialyl LewisX carbohydrate on maturing B and T cells and the stroma of avian bursa, thymus and spleen as well as the role of sialyl LewisX in the generation of immune response and formation of germinal centers in the spleen. The expression of sialyl LewisX on bursal B cells decreases during the embryonic period, while on T cells it remains at a stable low level throughout their thymic development. Immediately after hatching, by which time the expression of sialyl LewisX on bursal B cells has already decreased, the stromal microenvironment of the bursa starts to express it. After hatching, sialyl LewisX is localized exclusively in the medullae of the follicles and the number of sialyl LewisX‐positive follicles declines gradually during the following weeks. Thymic stroma does not express sialyl LewisX at any stage of development. In spleen, sialyl LewisX is expressed on a number of B and T cells as well as on other cell types in a way that seems not to be restricted to any structurally defined area. The cells or stroma of the germinal centers, however, do not express sialyl LewisX. When given simultaneously with an immunizing agent, anti‐sialyl LewisX monoclonal antibody reduced the formation of germinal centers and suppressed antibody response in young birds. These results demonstrate that sialyl LewisX‐bearing cell surface molecules are required during the generation of immune responses. They also suggest a role for sialyl LewisX in B cell maturation.
Fetal VDJ gene repertoire in rabbit: evidence for preferential rearrangement of VH1
Authors: Tunyaplin Chainarong , Knight Katherine L.
Published: March 2007

B cells in rabbit preferentially utilize the 3'‐most VH gene, VH1, in VDJ genes. To determine whether the preferential utilization of VH1 results from preferential rearrangement, we examined VH gene usage in nonproductive VDJ gene rearrangements, genes that would not be influenced by antigen selection. Since nonproductive VDJ gene rearrangements are found infrequently in neonatal and adult rabbits, we examined the VDJ gene rearrangements during early fetal development. According to nested‐primer polymerase chain reaction analyses, VDJ genes in fetal liver appeared around day 14 of gestation and nucleotide sequence analyses of VDJ genes cloned from 14‐ to 28‐day‐old fetuses showed that many of the genes were nonproductive. We found that most of these non‐productive genes had utilized VH1 indicating to us that the preferential utilization of VH1 is due to preferential rearrangement. We compared the nucleotide sequence of the promoter region of VH1 with sequences of several other unutilized and infrequently utilized VH genes and found that the core transcriptional factor motifs were similar in all VH promoters examined. We suggest that transcriptional activity of the VH1 promoter region does not explain the preferential rearrangement of VH1.
Monocyte chemotactic protein‐3 (MCP3) interacts with multiple leukocyte receptors: binding and signaling of MCP3 through shared as well as unique receptors on monocytes and neutrophils
Authors: Xu Luo Ling , McVicar Daniel W. , Ben‐Baruch Adit , Kuhns Douglas B. , Johnston James , Oppenheim Joost J. , Wang Ji Ming
Published: March 2007

The diversity of monocyte chemotactic protein (MCP)3 target cell types, as well as the capacity of MCP3 to desensitize leukocyte responses to other CC chemokines, suggested that MCP3 may interact with multiple CC chemokine receptors. The purpose of this study is to establish how MCP3 binds and activates monocytes and neutrophils. We show that human monocytes exhibit high‐affinity binding for 125I‐MCP3 with an estimated Kd of 1–3 nM and about 10000 binding sites/cell. The binding of 125I‐MCP3 to monocytes was progressively less well competed by CC chemokines macrophage inflammatory protein (MIP)lα (Kd = 5–10 nM), RANTES (Kd = 5–10 nM), MCP1 (monocyte chemoattractant and activating factor, or MCAF) (Kd = 60 nM) and MIP1β (Kd > 100 nM). On the other hand, unlabeled MCP3 displaced the binding of radiolabeled MIP1α, RANTES, MCP1 and MIP1β as effectively as the isologous CC chemokines. In agreement with the binding data, pretreatment of monocytes with MCP3 completely desensitized the calcium flux in response to MIP1α and RANTES. However, MIP1α and RANTES failed to desensitize the response of monocytes to MCP3. MCP3 and MCP1 partially desensitized each other's effects on monocytes. These binding and cross‐desensitization results suggest that MCP3 binds and signals through other binding sites in addition to those shared with MIP1α, RANTES and MCP1. The unidirectional competition for MIP1β binding and signaling by MCP3 suggests the existence of an as‐yet unidentified site for MCP3 shared with MIP1β. The existence of another unique binding site(s) for MCP3 was further shown by the failure of any of the other CC chemokines to compete effectively for MCP3 binding on neutrophils. MCP3 in our study was also the only human CC chemokine that consistently chemoattracted neutrophils. These results suggest that MCP3 is a ligand that can bind and activate a broad range of target cells through receptors shared by other CC chemokines as well as its own receptor.
Physical association and functional relationship between protein kinase Cζ and the actin cytoskeleton
Authors: Gómez Javier , de Aragón Ana Martínez , Bonay Pedro , Pitton Christina , García Alphonse , Silva Augusto , Fresno Manuel , Alvarez Fernando , Rebollo Angelita
Published: March 2007

Protein kinase C (PKC) was initially identified as a serine/threonine protein kinase dependent on calcium and phospholipids and shown to be involved in intracellular signaling pathways. PKC isoforms have been classified into four groups: Ca2+‐dependent conventional PKC α, βI, βII, γ; Ca2+‐independent, novel PKC δ, ϵ, η, θ; atypical PKCζ, λ, ι which are not activated by Ca2+ or diacylglycerol, and the recently discovered PKCμ. We reported that activation of the ζ PKC isoform is an important step in interleukin‐2 (IL‐2)‐mediated proliferation (Gómez, J., Pitton, C., García, A., Martínez, A., Silva, A. and Rebollo, A., Exp. Cell Res. 1995. 218: 105.). ζPKC is also required for mitogenic activation of fibroblasts and for the maturation pathway activated by insulin and Ras. Contradictory results have been reported regarding the subcellular redistribution of ζPKC upon activation. We report here, using confocal microscopy, that IL‐2 induces expression, translocation and association of ζPKC to a structure coincident with the actin cytoskeleton. Furthermore, we show that ζPKC has a role in maintaining the integrity of the actin cytoskeletal structure in IL‐2‐stimulated cells. On the contrary, ζPKC is not involved in the actin cytoskeleton organization when cells are maintained in IL‐4, confirming our previous results showing that IL‐4‐induced signal transduction is PKC independent.
Evidence that productive rearrangements of TCR γ genes influence the commitment of progenitor cells to differentiate into αβ or γδ T cells
Authors: Kang Joonsoo , Baker Jeanne , Raulet David H.
Published: March 2007

Two models have been considered to account for the differentiation of γδ and αβ T cells from a common hematopoietic progenitor cell. In one model, progenitor cells commit to a lineage before T cell receptor (TCR) rearrangement occurs. In the other model, progenitor cells first undergo rearrangement of TCRγ, δ, or both genes, and cells that succeed in generating a functional receptor commit to the γδ lineage, while those that do not proceed to attempt complete β and subsequently α gene rearrangements. A prediction of the latter model is that TCRγ rearrangements present in αβ T cells will be nonproductive. We tested this hypothesis by examining Vγ2‐Jγ1Cγ1 rearrangements, which are commonly found in αβ T cells. The results indicate that Vγ2‐Jγ1Cγ1 rearrangements in purified αβ T cell populations are almost all nonproductive. The low frequency of productive rearrangements of Vγ2 in αβ T cells is apparently not due to a property of the rearrangement machinery, because a transgenic rearrangement substrate, in which the Vγ2 gene harbored a frame‐shift mutation that prevents expression at the protein level, was often rearranged in a productive configuration in αβ T cells. The results suggest that progenitor cells which undergo productive rearrangement of their endogenous Vγ2 gene are selectively excluded from the αβ T cell lineage.