Surface CD4 density remains constant on lymphocytes of HIV-infected patients in the progression of disease.

In an attempt to question the influence of circulating virus, soluble gp120 or CD4 self-reacting antibodies upon results of CD4+ T-cell immunophenotyping in AIDS patients, five anti-CD4 mAb defining several epitopes of the V1 and V2 domains of the CD4 molecule were used to analyse the epitopic density of CD4 on lymphocytes of seropositive patients taken at stages II, III and IV of HIV infection, according to the Centers for Disease Control (CDC, Atlanta) classification. Our results demonstrate that each CD4 epitopic density measured on circulating lymphocytes remains constant at a mean level of 46,000 epitopes per cell whatever the stage of the disease and whatever the serum p25 concentration. These data provide evidence that antibody accessibility to several CD4 epitopes is not altered by putative interactions between CD4 molecules and circulating virus, soluble gp120 or anti-CD4 autoantibodies. If such binding events, as expected, do occur in vivo, they are of too low a magnitude to influence the immunophenotyping. Furthermore, we show that mAb specific for different epitopes in the V1 and V2 domains of the CD4 molecule can be used interchangeably for the biological followup of the CD4+ cell population in blood samples of HIV-infected patients.

Characterization of monoclonal antibodies directed against distinct conserved epitopes of human immunodeficiency virus type 1 core proteins.

Two mouse hybridoma cell lines secreting antibodies to the Human Immunodeficiency Virus (HIV) p25 major core protein and its precursors p55 and p41, were developed after immunization with the highly cytopathic Zaïrian HIV-1 isolate, NDK. These monoclonal antibodies also react with the gag gene products from HIV-1-BRU prototype and present cross reaction with HIV-2-ROD, and SIV-AGM. They map into topographically distinct areas of p25 and define epitopic regions topographically separated from those recognized by four other anti-p25 mAb suggesting the existence of at least 6 spatially distinct epitopic regions on HIV-1-p25 core protein.

Anti-beta 2-microglobulin monoclonal antibodies mediate a delay in HIV1 cytopathic effect on MT4 cells.

We attempted to select monoclonal antibodies (mAb) which reacted with T-cell surface molecules and were able to interfere with the human immunodeficiency virus type 1 (HIV1) replicative cycle in the MT4 T-leukaemic cell line. In comparison with OKT4A, an anti-CD4 mAb, only one mAb, HC11.151.1, was found to significantly delay HIV-induced cytopathic effect on MT4 cells among the 15 mAb tested which reacted with MT4 cell surface antigens. Biochemical and immunological characterization of HC11.151.1 demonstrated its specificity for beta 2-microglobulin (beta 2m), the light chain of human leukocyte antigen (HLA) class I molecules. Other beta 2m-specific mAb were tested in order to assess whether this effect represented an intrinsic capacity of HC11.151.1 or whether it was a common feature shared by all anti-beta 2m mAb. Three (B1.1G6, B2.62.2 and BBM1) of the four anti-beta 2m mAb demonstrated the same protective effect, whereas C21.48A, which was devoid of a functional effect, was directed towards a beta 2m epitope involved in binding to the HLA class I heavy chain molecule. The physiological relevance of this observation is discussed.

An early postinfection signal mediated by monoclonal anti-beta 2 microglobulin antibody is responsible for delayed production of human immunodeficiency virus type 1 in peripheral blood mononuclear cells.

We recently found (C. Devaux, J. Boucraut, G. Poirier, P. Corbeau, F. Rey, M. Benkirane, B. Perarneau, F. Kourilsky, and J.C. Chermann, submitted for publication) a latency in the human immunodeficiency virus (HIV) type 1 cytopathic effect in the human T-cell lymphotropic virus type I immortalized T-cell line MT4 that was mediated by anti-beta 2 microglobulin (beta 2m) monoclonal antibodies (MAb). Here we describe a delay in viral particle production in peripheral blood mononuclear cells (PBMC) that was mediated by three (B1-1G6, B2-62-2, and HC11-151-1) of four anti-beta 2m MAb tested, the nonefficient MAb (C21-48A) being specific for an epitope on beta 2m that was masked by association with the human leukocyte antigen class I heavy chain. Experiments were designed to determine the mechanism of interference. PBMC incubated with anti-beta 2m MAb before viral exposure were not protected from HIV infection. In addition, anti-beta 2m MAb were not efficient in preventing syncytium formation between HIV-infected PBMC and CD4-positive MT4 cells. In contrast, anti-beta 2m MAb treatment of freshly infected PBMC significantly delayed HIV production in these cells. The window of cell sensitivity to anti-beta 2m MAb treatment took place during a very early post-HIV-binding stage. The possible mechanism of anti-beta 2m MAb action is discussed.

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.

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.

Specificity of monoclonal antibodies against human thyroglobulin; comparison with autoimmune antibodies.

Ten monoclonal antibodies (mAb) directed against human thyroglobulin (hTgb) were produced, purified and characterized. The mAb avidity for hTgb ranged from 10(-10) to 10(-6) M. The species specificity of the mAb was as follows: eight mAb reacted with monkey Tgb, three with dog Tgb and one with pig Tgb; none with bovine and ovine Tgb. The binding of mAb to hTgb was not significantly inhibited in the presence of Tgb carbohydrate moieties, tyrosine, iodotyrosines and iodothyronines. The topology of the antigenic determinants recognized by the 10 mAb on hTgb was explored by inhibition of Tgb binding of radiolabeled mAb by the other antibodies. Six distinct clusters of reactivity were described. Localization of the antigenic determinants recognized by mAb on hTgb was attempted using tryptic fragments of hTgb to inhibit the binding of mAb to hTgb. The inhibitory effect of hydrolysis products was different for each mAb but exhibited partial analogies between mAb of the same cluster of reactivity. Anti-hTgb autoimmune antibodies (aAb) purified from sera of Graves patients cross-reacted essentially with mAb of one out of the six clusters. These results demonstrate that the large number of antigenic determinants presented by the hTgb are not disseminated on the molecule but are clustered in antigenic regions. Furthermore, from the six antigenic regions evidenced in this paper, only one is involved in autoimmune antibody production in Grave's disease.

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.