Production of soluble human alpha3-fucosyltransferase (FucT VII) by membrane targeting and in vivo proteolysis.

The rational design of fucosyltransferase (FucT VII) inhibitors as potential medication in the treatment of rheumatoid arthritis requires the three-dimensional structure of this member of the glycosyltransferase family. Structure determination by X-ray diffraction analysis needs purified, soluble enzyme protein. For this purpose we developed a novel method for the high-yield production of soluble FucT VII by in vivo proteolysis. To obtain a soluble form of FucT VII a mammalian expression construct was made encoding an N-terminal portion of FucT VI (amino acids 1-63) fused with the stem region and catalytic domain of FucT VII (amino acids 39-342). Chinese hamster ovary cells stably transfected with this construct produced FucT activity in the supernatant, which has the same catalytic properties as wild-type FucT VII. This soluble form of FucT VII can be obtained in high amounts (1 mg/L) and can be efficiently purified by GDP-hexanolamine affinity chromatography. In conclusion, it was demonstrated that the intrinsic properties of FucT VII could be transferred to secreted FucT VII constructs, which may open possibilities for production of soluble forms of other members of the glycosyltransferase family as well.

sLex is not responsible for the interaction of sLex-positive memory T lymphocytes with E-selectin.

E-selectin in an adhesion molecule that is transiently and exclusively expressed on endothelial cells in response to inflammatory cytokines. In addition, E-selectin participates in the initial interaction of leucocytes with activated endothelial cells. This role of E-selectin in cell adhesion has made it a potential target for modulation of inflammatory processes that, for example, are occurring in autoimmune diseases such as rheumatoid arthritis. Although on granulocytes the ligand for E-selectin has been identified as the tetrasaccharide sialyl Lewis x (sLex), the molecular nature of this ligand on T lymphocytes has not yet been identified. In the present study, it was shown by fluorescence-activated cell sorter (FACS) analysis with the anti-sLex antibody CSLEX1 that T lymphocytes stimulated with phytohaemagglutanin (PHA), interleukin-2 (IL-2) and transforming growth factor-beta 1 (TGF-beta 1) expressed sLex. Furthermore, in a cell adhesion assay these activated T cells of the memory phenotype bound specifically to E-selectin-transfected Chinese hamster ovary (E-CHO) cells. This adhesion could be blocked with an anti-E-selectin antibody but not with CSLEX1. In the same assay, the interaction of sLex-positive U937 cells with the E-CHO cells could be inhibited both with anti-E-selectin and CSLEX1 antibodies. From these results it can be inferred that sLex on activated T lymphocytes is not responsible for the interaction with E-selectin. Rather, these results suggest that stimulated T lymphocytes express an additional E-selectin ligand(s) with much higher avidity for E-selectin than sLex.

Glucosidase trimming inhibitors preferentially perturb T cell activation induced by CD2 mAb.

Glycosidase trimming inhibitors may be used to study contribution of N-linked glycan moieties in T cell function. We have studied the effects of castanospermine (Cas), swainsonine (Swain), 1-deoxynojirimycin (dNM), and 1-deoxymannojirimycin (dMM) on T cell activation and differentiation. Our analysis included a new dNM derivative, N-pentyl-1-deoxynojirimycin (pentyldNM). Previous reports showed inhibitory action of trimming inhibitors, such as Swain and Cas, on pokeweed mitogen-driven immunoglobulin (Ig) production. We extend these findings for pentyldNM and observed that glucosidase inhibitors, Cas and pentyldNM were effective in inhibiting CD2 and CD3 monoclonal antibody (mAb) driven Ig production. The pattern of inhibition by mannosidase and glucosidase inhibitors correlated with inhibitory action on T cell activation: only glucosidase trimming inhibitors (Cas and pentyldNM with comparable potency) perturbed mAb-induced T cell activation, in particular if induced by CD2 mAb. Expression of the early activation marker CD69 was not decreased in the presence of these inhibitors, while addition of exogenous recombinant interleukin-2 partially overcame inhibitory effects during proliferation. These findings suggest that glucosidase, but not mannosidase, trimming inhibitors interfere with a late phase of T cell activation. In addition, the enhanced sensitivity of CD2 mAb-induced proliferation for glucosidase trimming inhibitors suggests dependence on N-linked glycans during CD2-mediated adhesion and triggering functions.

Both interleukin-1 alpha and interleukin-1 beta are involved as accessory signals in primary antigen (tetanus toxoid) induced human T-cell activation.

The function of interleukin-1 alpha and interleukin-1 beta (IL-1 alpha, IL-1 beta) in tetanus toxoid (TT) induced T-cell proliferation in cultures of peripheral blood mononuclear cells (PBL) obtained from healthy donors was assessed by using neutralizing antisera to IL-1 alpha and IL-1 beta. The neutralizing capacity and the specificity of the IL-1 antisera were tested by the use of the thymoma EL-4 NOB-1 cell line. Antisera to IL-1 beta effectively neutralized the proliferative capacity of human recombinant IL-1 beta but not of human recombinant IL-1 alpha and vice versa. Addition of either anti-IL-1 beta or anti-IL-1 alpha antiserum to the culture medium hardly affected TT induced T-cell proliferation. However, the proliferative T-cell response was consistently attenuated when a combination of IL-1 alpha and IL-1 beta antiserum was used. The antisera were never capable of completely abolishing the T-cell response to TT. We conclude that (a) IL-1 alpha and IL-1 beta are both necessary accessory signals for T-cell proliferation to antigen in vitro; (b) in T-cell proliferation IL-1 alpha and IL-1 beta are interchangeable; and (c) T-cell proliferation to antigen is only partially dependent on IL-1 as signal.

Relative increase of inflammatory CD4+ T cells in the cerebrospinal fluid of multiple sclerosis patients and control individuals.

Of three patients with multiple sclerosis (MS) and two non-MS individuals a large number of CD4+ T cell clones was obtained from the cerebrospinal fluid and peripheral blood by direct limiting dilution. The CD4+ T cell clones from cerebrospinal fluid and peripheral blood lymphocytes were compared for their cytotoxic activity and lymphokine production. Cytotoxic capacity of cloned T cells was analysed with the use of anti-CD3 antibodies and target cells bearing Fc receptors for murine IgG. Recently, we demonstrated the existence of two different subsets of human CD4+ T cell clones by phenotypic and functional criteria. One type of CD4+ T cell with anti-CD3 mediated cytotoxic activity, in analogy with murine studies, is the inflammatory or TH1 subtype, the main producer of interleukin (IL-2), interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF)-alpha, -beta, whereas the other type of CD4+ T cell clone lacked anti-CD3 mediated cytotoxicity and produced minimal amounts of IL-2 concomitant with reduced levels of IFN-gamma and TNF-alpha, -beta. The present study demonstrates that among three MS patients, relatively more inflammatory CD4+ T cell clones with cytotoxic activity and IFN-gamma and TNF-alpha, -beta production were derived from the cerebrospinal fluid as compared with peripheral blood lymphocytes. Also among control individuals more inflammatory CD4+ T cell clones could be obtained from the cerebrospinal fluid as from the peripheral blood. The enrichment of inflammatory CD4+ T cells, therefore, appears to be physiological rather than associated with the disease.

Influenza virus changes cell-surface glycoproteins including major histocompatibility complex determinants on lymphocytes.

The effect of influenza virus infection on the expression of major histocompatibility complex (MHC) antigens was investigated. Infection with influenza virus resulted in an increase of the binding of anti-MHC class I and class II antibodies to resting T cells. The binding of anti-MHC class II antibodies to activated T cells was increased approximately threefold. The binding of anti-MHC class I and class II antibodies to Epstein-Barr virus-transformed B cells appeared unaffected after influenza virus infection. Recombinant human interferon-alpha and/or -gamma added to T cells did not enhance the binding of anti-MHC antibodies. Biochemical analysis revealed no increase in the amount of class I and class II antigens as a consequence of viral infection, but a marked decrease in sialic acid content was found, most probably caused by the viral neuraminidase. Pulse-chase experiments suggest that the viral neuraminidase can catalyze the removal of sialic acids both en route to and at the cell surface. The absence of sialic acid residues can explain the increased binding of anti-MHC antibodies, because neuraminidase (clostridium perfringens) treatment of T and Epstein-Barr virus-transformed B cells resulted in a shift in both isoelectric point and antibody binding similar to that observed after influenza virus infection.

Clonal analysis of functionally distinct human CD4+ T cell subsets.

A large number of CD4+ T cell clones, obtained from peripheral blood T lymphocytes by direct limiting dilution, allowed us to address the question whether functional heterogeneity exists within the human CD4+ T cell subset. Cytotoxic capacity of cloned T cells was analyzed with the use of anti-CD3 antibodies and target cells bearing FcR for murine IgG. 6 of 12 CD4+ clones obtained were able to lyse Daudi or P815 cells in the presence of anti-CD3 antibodies. The remaining six CD4+ T cell clones tested did not display anti-CD3-mediated cytotoxic activity and did not acquire this cytotoxic capacity during a culture period of 20 wk. In the absence of anti-CD3 mAb, no lytic activity against Daudi, P815, and K562 target cells was observed under normal culture conditions. Phenotypic analysis of these two distinct types of CD4+ T cells did not reveal differences with regard to reactivity with CDw29 (4B4) and CD45R (2H4) mAbs that have been described to recognize antigens associated with helper suppressor/inducer (respectively) CD4+ cells. The CD4+ clones without anti-CD3-mediated cytotoxic activities (Th2) consistently showed a high expression level of CD28 antigens, whereas the cytotoxic clones (Th1) expressed low amounts of CD28. Th1 CD4+ clones did produce IL-2, IFN-gamma, and TNF-alpha/beta, whereas the Th2 T cell clones produced minimal amounts of IL-2 and only low levels of INF-gamma and TNF-alpha/beta in response to anti-CD3 mAbs and PMA. Although not all CD4+ clones did release IL-4, there was no correlation with cytotoxic activity. Moreover, as compared with the Th1 CD4+ clones, Th2 CD4+ T cell clones proliferated moderately in response to immobilized anti-CD3 mAbs. However, proliferation reached the level of the cytotoxic clones when anti-CD28 mABs were present during culture. Both CD4+ subsets provided help for B cell differentiation upon stimulation with anti-CD3 mAbs. Our data suggest that the human CD4+ subset, in analogy to the murine system, comprises two functionally distinct T cell subpopulations, both of which are able to exert helper activity for polyclonal B cell differentiation, but which differ in cytotoxic capacity, lymphokine production, and requirements for proliferation. A function for these two types of T cells in the immune response is discussed.

Recognition of influenza virus-infected B-cell lines by human influenza virus-specific CTL.

The cytotoxic activity on influenza virus-infected Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (LCL-Flu) and influenza virus-infected phytohemagglutinin lymphoblasts (PHA-Flu) was compared with the use of influenza-A virus-specific cytotoxic T lymphocytes (CTL), generated in short-term bulk cultures. Cold-target inhibition experiments showed that the lysis of PHA-Flu was completely blocked by both cold LCL-Flu and cold PHA-Flu whereas the lysis of LCL-Flu was completely inhibited by cold LCL-Flu, but only partially by cold PHA-Flu, indicating that structures can be recognized on LCL-Flu which are absent from PHA-Flu. Monoclonal antibody (McAb) directed against a monomorphic determinant of major histocompatibility complex (MHC) class I molecules inhibited the lysis of PHA-Flu more strongly than the lysis of LCL-Flu. Since LCL have a high expression of MHC class II molecules compared to PHA lymphoblasts, we examined whether class II-restricted CTL activity was responsible for the (anti)class I McAb-resistant lysis of LCL-Flu. Neither anti-CD4 McAb nor anti-class II McAb inhibited the lysis of LCL-Flu which argues against a contribution of MHC class II-restricted CTL. Depletion of CD16+ cells, containing the majority of the nonspecific cytotoxic cells, did not affect the lysis of LCL-Flu, indicating that the remaining lysis on LCL-Flu was also not due to a nonspecific component. We suggest that cell-type-dependent variations exist in the nature of the immunogenic determinants to which CTL respond.

Analysis of T cell receptor-gene rearrangement in T cells from the cerebrospinal fluid of patients with multiple sclerosis.

Characterization of T cells present in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) may contribute to an understanding of the immunopathologic role of these cells. To analyze the T cells in the CSF of MS patients, 30 cloned T cell lines from each of two MS patients were surveyed for their patterns of T cell receptor (TcR) beta-chain gene rearrangement. DNA from the (CSF-derived) T cell clones was digested with a number of restriction endonucleases and the gene rearrangement patterns were analyzed with a T cell receptor beta-chain probe. Southern blot analysis of the DNA of these T cell clones indicated that all had rearrangements of the TcR beta-chain genes, but none of the rearrangements were identical. These results suggest that, if a few clones of specific T cells are involved, they must form a tiny minority in comparison to the total number of T cells in the CSF of MS patients.

Are MHC class II-restricted cytotoxic T lymphocytes important?

Although most cytotoxic T lymphocytes (CTL) are MHC class I restricted and most helper T cells class II restricted, some CTL are MHC class II restricted. The function of these cells is unclear. Here, Eric Braakman and co-workers suggest that they are an important regulatory influence, appearing late in the immune response and destroying cells presenting antigen in the context of class II molecules. The elimination of these cells inhibits the generation of the helper T response and consequently terminates the immune response to that antigen.

Topography of binding sites for neurohypophyseal hormones in rat brain.

Binding sites for arginine-vasopressin (AVP) and oxytocin (OT) were detected in neuroanatomically defined regions of rat brain by using in vitro incubation of 32 micron brain sections with the tritium-labelled ligands and autoradiography with LKB Ultrofilm. Sites were quantified by computerized densitometry of the film images. The highest amount of [3H]AVP-labelled binding sites were observed in the ventral subiculum of the hippocampus, central amygdala nucleus, lateral septum, olfactory nucleus and nucleus tractus solitarii, while the dentate gyrus contained a large amount of low affinity binding. The binding pattern of [3H]OT was more discrete. Relatively high concentrations of OT binding sites were observed in the central amygdala, ventral subiculum and olfactory nucleus. The lateral septum and the nucleus tractus solitarii were virtually devoid of displaceable OT binding while no significant association of OT with the dentate gyrus was found. The topography of binding sites for neurohypophyseal hormones corresponds to the localization of responsive sites in behavioural, biochemical and electrophysiological studies.

Immunogenic properties of octasaccharide-protein conjugates derived from Klebsiella serotype 11 capsular polysaccharide.

The tetrasaccharide repeating unit of the capsular polysaccharide of Klebsiella serotype 11, K11PS, comprises the following sequence: [----3)-beta-D-GlcpA-(1----3)-alpha-D-Galp-(1----3)-beta-D-Glcp-(1 ----] with a 4,6-O-(1-carboxyethylidene)-alpha-D-galactopyranosyl residue linked to O-4 of the glucuronic acid residue. Octasaccharide (OS) derived from K11PS by bacteriophage phi 11-associated glycanase, was coupled to bovine serum albumin and to keyhole limpet hemocyanin. The immunogenicity of various antigens after intraperitoneal immunization was studied by measuring the levels of circulating antibodies. Injection of BALB/c mice with K11PS resulted in induction of 2-mercaptoethanol-sensitive immunoglobulin M antibodies. The responses observed in BALB/c nu/nu mice and in male (CBA/N X C3H/HeN)F1 mice indicate that K11PS is a thymus-independent type 2 antigen. Immunization of BALB/c mice with either OS-bovine serum albumin or OS-keyhole limpet hemocyanin resulted in the induction of circulating 2-mercaptoethanol-resistant immunoglobulin G antibodies. Results in BALB/c nu/nu mice indicate that the OS-protein conjugates are thymus-dependent antigens. Since the OS-keyhole limpet hemocyanin conjugate induced antibodies in both (CBA/N X C3H/HeN)F1 females and males, we propose to refer to this kind of antigen as a thymus-dependent type 1 antigen, whereas OS-bovine serum albumin, which evoked immunoglobulins in (CBA/N X C3H/HeN)F1 females only, can be referred to as a thymus-dependent type 2 antigen.