Biomedical subjects

C Marchiol-Fournigault

Publications and source records attributed to C Marchiol-Fournigault.

7 recordsLinked to original sources

PubMed1994

Further characterization of CD82/IA4 antigen (type III surface protein): an activation/differentiation marker of mononuclear cells.

The mononuclear cell surface protein IA4 was originally identified in our lab using a mAb selected because of its strong reactivity with three lymphoblastoid variant cell lines which are HLA class I deficient, are LAK susceptible, and form a high number of conjugates with LAK effectors. We previously cloned the cDNA of the IA4 protein, coding for a 267-amino-acid type III integral membrane protein, with four transmembrane domains and three possible N-glycosylation sites. The IA4 protein belongs to the tetra span transmembrane (TST) new family of surface molecules, which also includes CD9, CD37, CD53, CD63, and TAPA-1. IA4 antigen was recently recognized as belonging to a new cluster of differentiation CD82 (International CD Workshop, Boston 1993). The IA4 antigen expression pattern at the surface of immune cells from normal donors was studied. On T lymphocytes, IA4 was barely detectable on resting cells and increased 3.5- to 7-fold following PHA or PHA+PMA stimulation. This IA4 increased expression is correlated with the morphologic change in blast cells and with the expression of activation markers such as CD2 and MHC class II antigens, therefore suggesting that IA4 is an activation marker on T lymphocytes. The expression of IA4 was low on circulating resting monocytes collected by elutriation. However, these monocytes, cultured in medium alone or with GM-CSF, acquired the morphology of macrophage and simultaneously overexpressed MHC Class II, CD14, and IA4 antigens, suggesting that IA4 is a differentiation marker for macrophages, whatever the culture conditions, either adherent (plastic culture dishes) or nonadherent (Teflon culture bags). IA4 stable transfectants of the murine mastocytoma cell line P815 were obtained and used to generate a new mAb. Competitive epitope binding studies have shown that IA4 antigen presents a dominant epitope recognized by most of the mAb prepared either in our lab or elsewhere. This dominant epitope is not shared by any of the other antigens of the TST family. Using this new mAb we were able to biochemically characterize the IA4 antigen as a 28-kDa protein, highly N-glycosylated with different patterns on various cells.

Animals↗

PubMed1992

A member of the tetra spans transmembrane protein superfamily is recognized by a monoclonal antibody raised against an HLA class I-deficient, lymphokine-activated killer-susceptible, B lymphocyte line. Cloning and preliminary functional studies.

The IA4 mAb was identified among a series of antibodies raised in BALB/c mice after immunization against a HLA class I-deficient, lymphokine-activated killer (LAK)-susceptible EBV-B lymphocyte line. The IA4 antibody was selected because of its high expression, in the range of 10(5) to 25 x 10(5) sites/cell, on several B lymphocyte lines (EBV-transformed or Burkitt) and monocytic lines such as HL60 and U937, and because its expression was correlated with both target susceptibility to LAK lysis and reduced expression of HLA class I surface Ag on two pairs of EBV-B-transformed cell lines (721/721.134 and MM/10F2). Despite the strategy followed to raise the mAb and the correlation mentioned above, no direct role of the IA4 molecules in LAK susceptibility has been established, since the IA4 molecule is poorly expressed on the sensitive targets Daudi and K562; moreover, the IA4 antibody did not affect reproducibly the in vitro killing of positive target cells by LAK effectors. The IA4 antibody was poorly immunoprecipitating and the surface molecule recognized was identified by gene cloning following an expression strategy using a U937 cDNA library transfected in COS cells, and a screening strategy based on membrane expression of IA4 molecule. The IA4 cDNA is virtually identical to "R2," a mRNA species previously identified in activated human T cells by subtractive hybridization. The IA4 cDNA contains an open reading frame coding for a protein 267 amino acids long with four potential transmembrane domains and one large external hydrophilic domain of about 110 amino acids, possibly glycosylated. The encoded protein belongs to a family of surface molecules, the tetra spans transmembrane protein superfamily, all displaying the four transmembrane domains, expressed on various cell types including lymphocytes (CD9, CD37, CD53, TAPA-1), melanoma cells (ME491), and intestinal cells (CO-029). These molecules have been reported to be involved in cell activation and cell death. Surprisingly, the Schistosoma mansoni Ag Sm23 displays significant homologies with this family. The IA4 molecule is a widely distributed surface marker expressed on circulating lymphocytes and monocytes, newborn thymocytes, and the cell lines mentioned above. The IA4 molecule expression is up-regulated upon cell activation. Weakly expressed on resting peripheral T and B lymphocytes and large granular lymphocytes (NK), its expression roughly doubles after activation by PHA, staphylococcus aureus Cowan I, and IL-2, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1991

Target lysis by human LAK cells is critically dependent upon target binding properties, but LFA-1, LFA-3 and ICAM-1 are not the major adhesion ligands on targets.

The cytotoxicity mediated by the CD2+ CD3- lymphocyte subset, either NK or LAK, is puzzling since no specific antigen recognition structures, equivalent to the CD3-associated heterodimer T-cell receptor, have been recognized on these cells so far. The possibility exists that the CD3- cytotoxic effectors recognize their targets through non-specific adhesion mechanisms. The goal of this study was: (a) to examine the correlation between binding properties and susceptibility to lysis of 6 informative target cell lines; (b) to evaluate the role, as ligands on these targets, of adhesion molecules such as LFA-1, LFA-3 and ICAM-1. The effectors used in this study were IL-2-activated LGL, predominantly CD3-, or highly purified CD3- lymphocytes from normal human donors. The 6 target lines studied included 2 pairs of EBV-transformed B-cell lines (721 LCL vs. 721.134, and MM vs. MM-10F2) in which the parental lines were resistant to lysis while HLA variants were susceptible. A third pair was the Daudi Burkitt cell line, susceptible to LAK lysis, and an HLA-positive transfected Daudi line which was more resistant to lysis. The binding properties of these targets to LAK effectors (conjugate formation) were evaluated using a sensitive double fluorescence flow cytometry method. In each pair examined, the susceptible targets formed more conjugates and were surrounded by more cytotoxic LAK effectors than their resistant counterparts, indicating that the conjugation properties of targets are closely correlated with their susceptibility to LAK lysis. The expression of adhesion molecules on the informative targets was examined by indirect immunofluorescence and their role was evaluated by inhibition of lysis after pre-coating the targets with the relevant antibodies. The differences in the expression of the classical cell-cell adhesion molecules LFA-1, LFA-3 and ICAM-1 on the target surfaces were only marginal, insufficient to explain the striking differences in susceptibility to lysis and in binding properties. Coating the target cells with antibodies directed against these adhesion determinants had no effects on the lysis of susceptible target cells. The same antibodies reacting with the LAK effectors did inhibit lysis. Taken together, these results suggest that, on the targets, presently undefined membrane adhesion structures may have a major role in conjugate formation between target and CD3- effectors and determine the susceptibility of the targets to lysis.

Antibodies, Monoclonal↗

PubMed1988

Allogeneic T cell activation triggering by MHC class I antigens.

The role of MHC-encoded class I molecules in allogeneic activation and proliferation of human T lymphocytes was investigated. The study was performed by using primary mixed culture of lymphocytes from MHC recombinant siblings identical for MHC class II Ag (DR, DP, DQ) and displaying MHC class I disparity. The results indicate that such allogeneic combination is sufficient to trigger early activation steps within responder T cells without promoting a significant proliferation. After MHC class I allosensitization, a significant proportion of cells entered the cell cycle (G0----G1). The stimulatory potential of MHC class I Ag was further stressed by the specific induction on responder cells of IL-2R (22% T cell activation Ag positive). Under the same experimental conditions, transferrin receptor expression and IL-2 activity were not detectable. This is consistent with the low T cell proliferation. Exogenous rIL-1 did not improve IL-2 production and the subsequent T cell proliferation indicating that these two events were not associated with a defective accessory cell function involving IL-1 release. MHC class I disparity can also prime precursor CTL to differentiate into IL-2-dependent functional MHC restricted cytotoxic T cells. Conversely IFN-gamma had no effect. Addition to the culture of W6/32, a mAb specifically directed against a monomorphic determinant on human class I HLA-A, -B, and -C Ag was able to block all these activation events. These data clearly indicate a role of HLA class I Ag involvement in the early events triggering allogeneic T cell activation.

Antibodies, Monoclonal↗

PubMed1988

Increased resistance to non-MHC-restricted cytotoxicity related to HLA A, B expression. Direct demonstration using beta 2-microglobulin-transfected Daudi cells.

Experiments in several laboratories have shown that target susceptibility to NK and lymphokine-activated killer (LAK) cytotoxicity is inversely correlated with the target expression of HLA Class I molecules. We present the first direct evidence, obtained by gene transfection, that target cell HLA, A, B expression increases the resistance to the "so-called" non-MHC-restricted cytotoxicity. We have co-transfected, by electroporation, the human beta 2-microglobulin gene and the gene carrying the resistance to geneticin into Daudi cell line. Geneticin selection in culture followed by FACS sorting on the basis of strong positivity with the mAb W6/32 (which is specific for the HLA class I H chain associated to beta 2-microglobulin) have led to the establishment of a HLA+ Daudi cell line permanently expressing HLA A10, A11, and B17 molecules. Studies were performed in vitro to evaluate the susceptibility of these cells to either NK and LAK cytotoxicity. The HLA class I+ Daudi cells exhibit an increased resistance to killing by non-MHC-restricted killer cells (both NK and LAK) as compared with their HLA-Daudi counterpart.

Cytotoxicity, Immunologic↗

PubMed1988

An improved electrotransfection method using square shaped electric impulsions.

Transfection of DNA into non adherent cells can be achieved by electropermeation. Previously published results, partially successful, were obtained using exponential decaying electric impulsions. However, one limitation of this technique has been the damaging effect of this type of impulsions resulting in poor cell recovery. We report hereby the electropermeation of human lymphoblastoid cell lines using a commercially available electropulsator delivering repeated, short, high voltage, square shaped, electric pulses. The parameters of transfection have been optimized using the "Lucifer Yellow Permeation Assay". With the optimum electric parameters, virtually all the cells were permeated and at least 70% survived the shocking conditions. Both transient expression and permanent integration and expression of DNA was observed.

Cell Line↗

PubMed1988

Generation of lymphokine-activated killer cells: synergy between tumor necrosis factor and interleukin 2.

Large granular lymphocytes (LGL) can be activated by interleukin 2 (IL-2) to lymphokine-activated killers (LAK). The effect of tumor necrosis factor (TNF) on LAK generation was investigated. TNF was found to act synergistically with low concentrations of IL-2 (0.10-0.25 ng/ml), which were ineffective by themselves in inducing LAK activity, to promote the differentiation of LGL into non-major histocompatibility complex-restricted killers. When IL-2 was used at concentrations optimal for LAK generation, TNF did not further enhance this phenomenon. Specific binding of 125I-labeled TNF to LGL was increased by IL-2 stimulation. Scatchard analysis of TNF binding revealed the existence of two classes of binding sites with markedly different affinities (Kd values of 57 and 600 pM). We also demonstrated that the IL-2/TNF synergistic induction of LAK activity did not involve either IL-1 or interferon-gamma. This IL-2/TNF synergistic effect was blocked by anti-Tac antibodies. Immunofluorescence analysis revealed that IL-2/TNF selectively up-regulated Tac antigen expression on LAK precursors. Our results suggest a functional interaction between IL-2 and TNF on LAK precursors, which results in a reduction of the IL-2 concentration required for differentiation of LGL into LAK killers.

Drug Synergism↗