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.

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.

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.

A 55,000 Mr surface antigen on activated human T lymphocytes defined by a monoclonal antibody.

A T cell growth factor-dependent alloreactive human T cell line has been used to generate a monoclonal antibody B1.49.9 that reacts with an antigen present on most if not all mitogen or alloantigen activated T cells but not on resting T cells. The T lymphoblastoid cell line HUT-102 is also strongly reactive with B1.49.9 but all other T and non-T leukemia-lymphoma cell lines tested were negative. The B1.49.9 antigen is a glycoprotein of 55,000 Mr on mitogen or alloantigen activated T cells and 50,000 Mr on the cell line HUT-102. Pulse labeling experiments showed that a 40,000 Mr precursor (at approximately 0.7 h) which does not bind to ricin lectin precedes the appearance of the ricin-binding 55,000 Mr form. Comparisons of the monoclonal antibody anti-Tac, which recognizes the IL-2 receptor, to B1.49.9 suggest that B1.49.9 also recognizes a structure similar or identical to the IL-2 receptor.

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).

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.

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.

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.

Induction of antibody-producing cell lines by Epstein-Barr virus.

This work makes a critical evaluation of EBV transformation as a tool for establishing human antibody producing lines. Since Steinitz et al. described the technique in 1977, at least 9 lymphoid cell lines with predetermined specificities have been reported with activity against NNP, TNP, A-CHO, phosphorylcholine, human Ig, Rh-D antigen and tetanus toxoid. Most successful attempts were based on the choice of immune donors and on the adequate selection of peripheral antigen-specific B cells with antigen-coated erythrocytes. When lines were established, a further selection of antigen-specific lymphoblastoid cells using several steps of rosetting or cloning proved to be necessary, in order to get mono- or oligoclonal lines, and thus to maintain an antibody production for a prolonged period (up to 18 months). Secretion ranged from 0.1 to 16 micrograms antibody per ml, depending on the lines. Two of the antibodies produced are used as biological reagents. When compared to hybridomas, EBV transformed lines have the disadvantage of a lower colony forming efficiency, and usually a lower level of antibody secretion. On the other hand, if fusion of EBV induced lymphoblastoid cell lines proves to be possible with human myeloma lines and results in the creation of hybridomas, EBV transformation might reveal a useful technique to raise minute amounts of antigen specific cells to the amount of cells required for the fusion techniques.

An antigenic determinant of human beta 2-microglobulin masked by the association with HLA heavy chains at the cell surface: analysis using monoclonal antibodies.

Four anti-human beta 2-microglobulin monoclonal antibodies were produced against whole lymphoid cells or against urinary beta 2-microglobulin. Their reactivity was fully inhibited by purified soluble beta 2-microglobulin. The B1.1G6, C23.24.2, and B2.62.2 antibodies bound either to free or to HLA-complexed beta 2m. They recognized the same or very close determinants, since they achieved mutual blocking. In contrast, the C21.48A1 antibody did not bind to the cell surface and did not recognize the same determinant on the purified beta 2-microglobulin molecule as the others. It was able to bind and to form a specific complex with membrane beta 2-microglobulin only, once separated from the HLA heavy chain. These data strongly suggest that the C21.48A1 antigenic determinant might be hidden when the beta 2-microglobulin is complexed with the HLA heavy chains at the cell surface.

Differences in the cell binding affinity of a cross-reactive monoclonal anti-Ia alloantibody in mice of different H-2 haplotypes.

The analysis of an Ia specificity was performed in several H-2 haplotypes by evaluating the affinity of the binding to mouse spleen cells of a monoclonal anti-Ia alloantibody. We used the monoclonal antibody 10-2.16 (Oi et al., Curr. Top. Microbiol. Immunol. 1978. 81: 115.) which recognizes on I-A gene products a public specificity common to several H-2 haplotypes (f, ja, k, r, s and u). One parameter of the affinity (dissociation rate constant) was determined by the kinetics of the dissociation of cellbound 125 I-labeled Fab fragments in the presence of homologous antibodies. The dissociation rate constants thus obtained were different when the antibody reacted with cells from mice bearing different haplotypes and were similar for reactions with different mouse strains bearing the same haplotype. These data strongly suggest that the Ia public determinants involved are not strictly identical in different haplotypes, and support the concept that a serological cross-reaction does not necessarily mean structural identity between public antigenic determinants.

Cross-reactions between mouse Ia and human HLA-D/DR antigens analyzed with monoclonal alloantibodies.

Two mouse monoclonal anti-I-E/Ck alloantibodies (H7-8.26 and H10-81.10) directed against 2 distinct determinants of the specificity Ia-7 and 1 anti-I-Ak alloantibody (H8-15.9) directed against a public determinant common to the I-A subregion products of the H-2k, H-2b, H-2d, H-2q, and H-2ja haplotypes identified cross-reactive determinants on lymphoid cells from various mammalian species, including rat, dog, pig, cow, hamster, and guinea pig. In man, these antibodies detected nonpolymorphic determinants of DR antigens on B cell-enriched peripheral blood lymphocytes from 50 unrelated individuals. These cross-reactive DR determinants were also detected on lymphoblastoid B cell lines, on PHA-activated peripheral T lymphocytes, and on allospecific cytolytic T cell clones, but not on various DR-negative human T leukemia cell lines. Two chains of 29,000 and 35,000 daltons m.w., corresponding to DR antigens, were precipitated by H7-8.26 and H8-15.9 antibodies from radiolabeled membrane extracts of Raji cells. Competitive binding experiments indicated that the 3 mouse anti-Iak antibodies identified 3 distinct cross-reactive determinants on human cells. The results indicate that: a) The cross-reactivity described between mouse I-E/C gene products (Ia-7) and human DR antigen(s) involves, in fact, several distinct and topologically distant determinants. b) At least 1 determinant cross-reacting with DR can be identified on I-Ak gene products. c) The intriguing genetic problem of mouse MHC allotypic determinant(s) being nonpolymorphic in man cannot be simply explained by the deletion of an I-E alpha chain in some strains of mice.

Distinct epitopes of Ik gene products identified by monoclonal antibodies.

Analysis of reactivity of monoclonal anti-Iak alloantibodies, obtained by fusion between NS 1 myeloma and spleen cells from alloimmune A. TH mice, permitted the identification of 9 distinct determinants of the Ik gene products. Competitive binding experiments indicated that 2 private epitopes (defined by H8-109.13 and H8-138.4 antibodies) of the I-Ak product could be separated, thereby apparently splitting the Ia.2 specificity. A public determinant of the I-Ak molecule (identified by H8-15.9 antibody) was found expressed not only on the I-A products of the H-2b, H-2d, H-2ja, H-2p and H-2q murine haplotypes, but also on human HLA-DR antigens. Four determinants of the I-E/Ck antigen (defined by H7-8.26, H10-81.10, H10-93.2 and H8-86.2 antibodies) had a strain distribution analogues to the Ia.7 specificity. However, competitive binding experiments, and the cross-reactivity pattern with Ia-like antigens from other species (e.g. human HLA-DR antigens) indicated that these antibodies detected distinct determinants on the I-E/Ck molecule, thereby subdividing the broad Ia. 7 specificity. Two other determinants (defined by H9-14.8 and H9-15.4 antibodies) had a strain distribution that did not permit a precise assignment to a given Ia antigen, even though preliminary data suggested that they could detect separate determinants on the I-E/Ck product. All these monoclonal antibodies identified membrane antigens with the expected Ia tissue distribution pattern, and most of them could precipitate a molecule containing two chains of 28kD and 35kD, from mouse spleen cell lysates.

[Comparative study of anti EBV antibodies in non-Hodgkin's lymphomams and angio-immunoblastic lymphadenopathies (author's transl)]. / Anticorps dirigés contre le virus d'Epstein Barr. Etude comparative au cours des lymphosarcomes et des adénopathies angio-immunoblastiques.

Antibody titers to Epstein-Barr virus (EBV)--related antigens (viral capsid antigen : VCA; early antigen : EA; and EBV associated nuclear antigen : EBNA) were determined in the sera of 86 patients and 150 matched control subjects. The patients belonged to four histological groups : diffuse and nodular non-hodgkin's lymphomas angio-immunoblastic lymphadenopathies and apparented syndroms. The incidence of antibodies to other herpes-viruses (cytomégalovirus, herpes simplex virus, and varicella zoster virus) was compared. There was a significantly higher incidence of anti VCA and anti EA titers in some patients, not associated with an increase in titres of antibodies to other herpes viruses.

Preliminary characterization of a glycoprotein having Fc receptor properties extracted from a T cell lymphoma (L-5178-Y).

After incorporation of 14C-labeled amino acids and/or of [3H]fucose during in vitro culture of the Thy-1.1-bearing, Fc receptor-positive T lymphoma, L-5187-Y, attempts were made to purify the Fc binding structure(s). Following solubilization of a crude membrane pellet using sodium deoxycholate, the 110 000 x g supernatant was filtered on Sephadex G-200, and the fractions containing IgG-binding material were further purified by affinity chromatography (on IgG Sepharose 4B.) Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the final Fc-binding product showed an apparent molecular weight of 110 000 daltons. On subsequent reduction with 2-mercaptoethanol, five bands (mol. wts. 56 000, 36, 000, 25 000, 18 000 and 15 000) were observed, the latter two being probably degradation products. These results are in accord with some of the published data concerning B cell Fc receptors.

[IgG levels in the sera of melanoma patients (author's transl)]. / Etude des IgG dans les sérums des sujets porteurs de mélanomes.

397 sera from 185 melanoma patients have been tested. We classified our subjects into three groups, according to the stage of disease. An alteration of the level of IgG 4 sub-class was found and related to the extension of the disease. The percentage of abnormalities was more frequent in stage II and III (55 p. 100 and 53 p. 100) than in stage I (19 p. 100). High titers of IgG 4 subclass were essentially detected in advanced disease. The biological significance is discussed.

[IgG levels in the sera of melanoma patients (author's transl)]. / Etude des IgG dans les sérums de sujets porteurs de mélanomes.

397 sera from 185 melanoma patients have been tested. We classified our subjects into three groups, according to the stage of disease. An alteration of the level of IgG4 subclass was found and related to the extension of the disease. The percentage of abnormalities was more frequent in stage II and III (55 p. 100 and 53 p. 100) than in stage I (19 p. 100). High titers of IgG 4 subclass were essentially detected in advanced disease. The biological significance is discussed.

IgG4 subclass in malignant melanoma.

Three hundred and ninety-seven sera from 185 melanoma patients were studied. These sera were classified into three groups according to stage of disease. An alteration in the level of the IgG4 subclass was found. It was related to the dissemination of disease. The percentage of abnormalities (either increased or decreased levels of IgG4) was more frequent in patients with stage II and III diseases (55 and 53%, respectively) than in patients with stage I(19%). The higher frequencies of high titers of IgG4 were essentially detected in advanced disease. The biologic significance of the increase of IgG4 in melanoma remains obscure. The increase may be related to the development of facilitating antibodies of the IgG4 subclass.

[IgG subclasses in malignant melanoma]. / Etude des allotypes Gm et des sous-classes d'IgG chez des sujets porteurs de mélanomes malins

The 397 sera from 185 melanoma patients have been studied and classified in three groups according to the stage of disease. Our findings revealed an alteration of the level of IgG4 subclass which is related to the dissemination of disease. The percentage of abnormalities (either increased or decreased levels of IgG4) was more frequent in stage II and III (55% and 53% respectively) than in stage I (19%), The higher frequencies of high titers of IgG4 were essentially detected in advanced disease. The biological significance of the increase of IgG4 in melanoma remains obscure. It may be related to the development of facilitating antibodies of IgG4 subclass.