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F A Lemonnier

Publications and source records attributed to F A Lemonnier.

At least 73 records · Page 4Linked to original sources
PubMed1985

HLA class I genes: from structure to expression, serology and function.

HLA class I genes have been isolated from phage and cosmid libraries and assayed by transfection into murine L cells. The transfection step proved to be very important because of the large number of genes (and pseudogenes) in this family. All functional genes characterized so far in this way are "classical" class I genes, i.e. members of the HLA-A, -B or -C families. Three of these have been sequenced (HLA-A3, -Aw24; HLA-Cw3) in addition to the pHLA 12.4 pseudogene. Sequence comparisons indicate, in particular, extreme conservation of the 3' non-coding region between allelic HLA-A locus genes; the general organization of all these genes (8 exons) is very similar. Restriction mapping around the functional genes has been performed to investigate the degree of conservation (e.g. between HLA-A3 regions from 2 different individuals) and examine allelism at the DNA level (e.g. between HLA-A3 and HLA-Aw24 regions). Exon shuffling experiments followed by serological analysis of the expressed product indicate that, as expected, specificities are determined by the first two domains of the molecule. However, further constructs show that as soon as a single exon is exchanged most specific reactivities disappear. CTL analysis of murine cells expressing HLA molecules has run into many difficulties but still holds promise for the study of structure-function relationships in this system.

Alleles↗
PubMed1985

The association between murine beta 2-microglobulin and HLA class I heavy chains results in serologically detectable conformational changes of both chains.

HLA-A3-, HLA-B7-, and HLA-CW3-transfected L cells, maintained in medium supplemented with murine serum so as to ensure that the human heavy chains were associated with murine beta 2-microglobulin, were subjected to a systematic serologic analysis for an evaluation of the structural consequences of such an heterologous association. The hybrid molecules exhibited alterations of their serologic reactivities that suggest the occurrence of structural modifications of both light and heavy chains. Thus, reactivity of HLA-A3-, HLA-B7-, and HLA-Cw3-transfected L cells with a monoclonal antibody (B1.1G6) directed at a human beta 2-microglobulin specific antigenic determinant was observed; this implies structural modifications of murine beta 2-microglobulin after its association with HLA class I heavy chains. Conversely, a profound reduction of the reactivity of the same transfectants with a monoclonal antibody (W6/32) directed at a monomorphic heavy chain related epitope was observed. The W6/32 reactivity was restored after replacement of the murine by the human light chain, indicating that the conformation adopted by the HLA class I heavy chain depends on the origin of the beta 2-microglobulin associated. Therefore it appears that the complex interactions that develop between the extracellular domains (including the one formed by the light chain) markedly influence the overall structure and the antigenic properties of HLA class I molecules.

Animals↗
PubMed1984

Absence of cell surface fixation of a monoclonal antibody detectable by conventional immunoassays does not exclude expression of and interaction with the corresponding antigenic determinant.

No specific binding of anti-HLA class I B.10.6 monoclonal antibody (mAb) could be demonstrated by cell surface radioimmunoassay and cytofluorographic studies at the surface of murine transformed L cells expressing HLA-A3 or Cw3 molecules. However, specific interaction of this antibody with these molecules at the surface of these transformed cells was indirectly established, since it inhibited specifically the binding to the same HLA class I molecules of other anti-HLA class I mAb. Therefore, the absence of detectable binding of mAb, in conventional immunoassays, does not exclude expression by these cells of the corresponding antigenic determinant.

Animals↗
PubMed1984

Transformation of LMTK- cells with purified HLA class I genes--IV. A determinant on beta 2-microglobulin is controlled by HLA heavy chain in a mouse-human hybrid complex: a biochemical analysis.

Transformation of LMTK- murine fibroblast cells with purified HLA class I heavy chain genes resulted in the expression of serologically detectable HLA-A3 molecules. Surprisingly, such cells also react with a murine monoclonal antibody specific for a serological determinant notably not expressed by murine but by human beta 2-microglobulin. The human HLA molecules expressed by the transformed cells were characterized on two-dimensional gels. The heavy chain was shown to be associated with a murine beta 2-microglobulin molecule, which could be distinguished from human beta 2-microglobulin by its higher isoelectric point. This heterodimer molecule was immunoprecipitated with the mouse anti-human beta 2-microglobulin monoclonal antibody showing that indeed the complex of mouse beta 2-microglobulin and human heavy chain expresses a human beta 2-microglobulin determinant.

Animals↗
PubMed1984

Transformation of LMTK- cells with purified HLA class I gene. VI. Serological characterization of HLA-B7 and AW24 molecules.

Serological characterization of HLA-B7 and HLA-AW24 class I molecules following transfection of murine LMTK- cells with purified HLA class I genes was performed using human alloantisera. Induction by murine alpha interferon of the expression of class I molecules was required to obtain unambiguous identification of these molecules which appear serologically identical to the HLA-B7 and HLA-AW24 molecules expressed at the surface of human peripheral blood lymphocytes of 20 unrelated individuals. Analysis of the transformed cells with 8 different anti-HLA class I monoclonal antibodies results in the definition of 3 separate clusters of antigenic determinants shared by all HLA class I molecules. These studies further suggest the existence of locus-specific serological reactivities associated either with the HLA-A or with the HLA-B and C gene products.

Animals↗
PubMed1984

Transformation of LMTK- cells with purified class I genes. V. Antibody-induced structural modification of HLA class I molecules results in potentiation of the fixation of a second monoclonal antibody.

A potentiation phenomenon was observed with HLA-A3 and CW3 transformed murine L cells between anti-HLA class I B10.6 (potentiated) and B10.8 (potentiating) monoclonal antibodies (m.Ab.). Further studies of this phenomenon with these transformed L cells indicated that: 1) no significant specific binding of B10.6 m.Ab. to HLA-A3 and CW3 transformed L cells could be demonstrated by conventional radioimmunoassay or cytofluorometric study in the absence of B10.8 m.Ab.; 2) potentiation of the fixation of B10.6 m.Ab. was induced by other anti-HLA class I m.Ab., which all reacted with the same cluster of antigenic determinants; 3) potentiation reflects an increased specific fixation of B10.6 m.Ab. to HLA class I molecules implicating its combining site; 4) potentiation was mediated by B10.8 Fab fragments. These results indicate that potentiation of the fixation of B10.6 m.Ab. to the HLA-A3 and CW3 molecules expressed by the transformed L cells reflects conformational changes of these molecules after interaction with B10.8 m.Ab.

Animals↗
PubMed1983

Quantification by flow cytofluorimetry of HLA class I molecules at the surface of murine cells transformed by cloned HLA genes.

A method allowing quantitative analysis of the expression of foreign antigens at the surface of transformed cells is described. Aminoethyl-Sephadex G-25 beads labelled with different amounts of fluorescein isothiocyanate (FITC) were used to calibrate an Epics V cell sorter. The quantity of FITC molecules bound per bead was found to be a linear function of relative fluorescence intensity expressed by channel number for intermediate and high levels of fluorescence and a non-linear function for low levels. The lowest limit of detectable fluorescence was 3400 FITC molecules bound per bead. Using FITC-conjugated monoclonal antibodies (m.Ab.) the number of HLA class I molecules expressed at the surface of murine LMTK- (H-2Kk) cells transfected by cloned HLA class I genes was estimated and compared with the amount expressed on human B (Raji) and T (MOLT 4) lymphoblastoid reference cells. Murine transformed cells expressed approximately 3 times less HLA class I determinants per surface unit (micrometer 2) than Raji cells and 1.4 times less than MOLT 4 cells. Similar results were obtained by Scatchard analysis of the same populations using radiolabelled m.Ab. A detailed analysis of fluorescent cells showed that 5-20% of transformed cells expressed less than 33,000 HLA class I molecules/cell whereas most MOLT 4 cells exhibited at least 280,000 molecules/cell and the majority of Raji cells more than 700,000 molecules/cell. The expression of foreign antigen did not reduce the amount of H-2Kk molecules at the surface of transformed cells (mean of 350,000 molecules/cell).

Animals↗
PubMed1983

HLA class I genes integrated into murine cells are inducible by interferon.

In human cells treated with interferon, there is an increase in the amount of HLA-A, B, C mRNA and, to a lower extent, membrane-bound antigen. However, the mechanism of this mRNA enhancement is still unknown. Using mouse L cells transfected with a unique class I HLA gene, we were able to show that both the related HLA mRNA and protein are increased after murine but not human interferon treatment. Moreover, the discrepancy between interferon-directed HLA mRNA and protein enhancement is also observed. The mouse transfected cells allowed us to study more precisely the origin of this discrepancy.

Animals↗
PubMed1983

Endocytosis of HLA and H-2 molecules on transformed murine cells measured by fluorescence dequenching of liposome-encapsulated carboxyfluorescein.

We have studied internalization of cell surface proteins encoded by genes of the human major histocompatibility complex (HLA) transferred into murine L cells, in comparison with mouse (H-2) histocompatibility determinants. This internalization was measured by the use of monoclonal antibodies directed at these determinants, to which were bound methotrexate- and carboxyfluorescein-containing liposomes covalently coupled to protein A. In addition to the effect of methotrexate on the cells, the technique of fluorescence self-quenching release was used to measure the kinetics of internalization for single cells using a flow cytofluorometer. The native and foreign gene products were internalized in an apparently identical manner. These techniques can be applied to cells expressing genes with altered or deleted segments thus providing a basis for the analysis of the effect of sequence modifications on the internalization of the encoded molecule.

Animals↗
PubMed1983

Transformation of LMTK- cells with purified HLA class I genes. II. Serologic characterization of HLA-A3 and CW3 molecules.

The expression of two different HLA class I genes was observed after transformation of LMTK- cells. The corresponding class I molecules reacted differentially with monomorphic monoclonal antibodies (m.Ab). Absorption and elution studies of the human alloantibodies reacting with the transformed cells and cellular radioimmunoassay of these cells with polymorphic m.Ab resulted in the identification of HLA-A3 and CW3 molecules. These transformed cells were used to immunize C3H mice and induce the production of xenogeneic antisera, which, following absorption, showed polymorphic reactivity with human cells, suggesting that some of these sera could be used as typing reagents.

Animals↗
PubMed1983

Dual parameter, quantitative cytofluorometric analysis of endogenous H-2Kk and foreign HLA class I molecules expressed at the surface of murine transformed L cells.

By using a calibrated dual laser cell sorter and monoclonal antibodies directly conjugated to fluorescein and rhodamine and specific for H-2Kk and HLA class I antigens, quantitative cytofluorometric analysis was performed on individual HLA-A3 or -CW3 transformed mouse L cells (H-2k). More than 80% of these cells expressed both HLA class I and H-2Kk molecules. Their respective levels of expression were calculated: a mean of 4 X 10(5) HLA class I and 2.3 X 10(5) H-2Kk molecules per single cell. Quantitative comparison with control untransformed L cells and double fluorescence contour maps showed a positive correlation between the levels of expression of HLA class I and H-2Kk molecules suggesting that expression of foreign class I molecules did not occur at the expense of the endogenous H-2k product.

Animals↗
PubMed1983

Transformation of murine LMTK- cells with purified HLA class I genes. I. Modification of conformation of murine beta 2-microglobulin upon its association with HLA heavy chains.

Murine LMTK- cells were unexpectedly found to cross-react with a murine anti-human beta 2-microglobulin (beta 2-m)monoclonal antibody (m.Ab) after transformation with cosmid clones containing different purified HLA class I genes. The same cross-reactivity was observed with CTP 34 B4 (murine x human) somatic hybrid cells, which express class I molecules constituted of human HLA heavy chains and murine beta 2-m. Inhibition studies of the complement-dependent cytolysis mediated by the cross-reacting m.Ab indicated that isolated murine beta 2-m does not express the cross-reacting determinant, suggesting that its expression by the transformed cells reflects conformational modification of murine beta 2-m upon its association with HLA heavy chains. These results illustrate one of the possible post-translational mechanisms through which the antigenicity of a polypeptide chain can be modified. They might provide a serologic marker of the third domain of HLA class I heavy chains. Finally, because quantitative differences of reactivity with the anti-human beta 2-m m.Ab were observed, depending on the HLA class I genes used for transformation, these results individualize two families of HLA class I heavy chains responsible for different conformational modifications of murine beta 2-m.

Animals↗
PubMed1982

Epitopic analysis of detergent-solubilized HLA molecules by solid-phase radioimmunoassay.

A solid-phase radioimmunoanalysis of plasma membrane molecules solubilized in the presence of detergent was developed. From a crude cell lysate, membrane molecules were specifically immobilized by solid-phase adsorbed monoclonal antibodies. Analysis of these molecules was easily performed with radiolabeled monoclonal antibodies of different specificities. This technique was used to analyze the HLA-DR and HLA-A,B,C molecules expressed by RAJI cells. It was possible (a) to determine which epitopes are expressed on the same molecules; (b) to define subsets of HLA molecules according to the epitopes which they express, and (c) to perform quantitative analysis of HLA molecules on a crude cell lysate.

Animals↗
PubMed1982

Identification on I-Ak molecules of a functional site recognized by proliferating T-lymphocytes.

The inhibitory capacity of 17 monoclonal antibodies (m.Ab.) specific for the products of the I-Ak subregion was evaluated in proliferative responses of B10.BR T-lymphocytes to GAT, Keyhole limpet hemocyanin, and ovalbumin. Considered in isolation, each m.Ab. mediated inhibitory effects of comparable magnitude on these three different proliferative responses. On the other hand, clear differences were observed when the magnitude of the inhibitory effects was compared from one m.Ab. to another. The m.Ab. were consequently classified as strong or moderate-to-weak inhibitors of T-cell proliferative responses. Evidence was simultaneously gained indicating the following: (a) the determinants recognized by different m.Ab. were expressed on the same molecules; (b) the differences in affinity of the m.Ab. for I-Ak positive cells did not explain their differences in inhibitory capacities; (c) conversely, the inhibitory capacity of each m.Ab. followed its ability to inhibit the cell surface fixation of Ia.17-specific 10-2.16 m.Ab; (d) the strong inhibitory capacity of some m.Ab. was not related to a special ability to modulate cell surface Ia molecules. These results suggest that antigen recognition by T lymphocytes is preferentially restricted by a functional site of the I-Ak molecules related to the Ia.17 and Ia.1 specificities.

Animals↗
PubMed1982

Affinity and inhibitory capacity of T-cell proliferation of monoclonal anti-Ia antibodies.

The rates of dissociation from the surface of Ia-positive spleen cells of three different anti-Ia monoclonal antibodies were evaluated and compared with their inhibitory effects on T-lymphocyte proliferative responses involving the same spleen cell population, either as a source of accessory cells (responses to soluble antigens) or as allogeneic stimulating cells. In general, a direct relationship was observed between the affinity with which these monoclonal antibodies interact with Ia-positive cells and the magnitude of their inhibitory effects, whether soluble antigen- or alloantigen-induced responses were tested. These results indicate that the affinity of monoclonal antibodies is a factor that has to be considered to the same extent as their fine specificity when interpreting the results of inhibitory studies of T-cell responses by anti-Ia monoclonal antibodies.

Animals↗