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1.
J Immunol ; 135(2): 1281-7, 1985 Aug.
Article in English | MEDLINE | ID: mdl-2409144

ABSTRACT

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.


Subject(s)
HLA Antigens/analysis , HLA-C Antigens , Immunoglobulin Heavy Chains/analysis , beta 2-Microglobulin/analysis , Animals , Antibodies, Monoclonal/immunology , Antigen-Antibody Reactions , Epitopes/analysis , Epitopes/genetics , Epitopes/immunology , HLA Antigens/genetics , HLA Antigens/immunology , HLA-A3 Antigen , HLA-B7 Antigen , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/genetics , L Cells/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred CBA , Protein Conformation , Transfection , beta 2-Microglobulin/genetics , beta 2-Microglobulin/immunology
2.
J Immunol ; 134(3): 1759-65, 1985 Mar.
Article in English | MEDLINE | ID: mdl-2578520

ABSTRACT

Three subsets of class I human T antigens (HTA) were serologically identified on the surface of the Molt-4 T lymphoma cell line. The HTA 1 subset is defined by NAI/34, D47, or 10H3.9 cross-reactive m.Ab. and by BL6 m.Ab. The HTA 2 and HTA 3 subsets are defined by M241 and 4A7.6 m.Ab., respectively. We obtained no evidence of any additional HTA subset. The different HTA antigens share only few epitopes with human leukocyte antigens (HLA-A, -B, and -C). Interestingly, these epitopes all belong to the same cluster defined on HLA class I molecules, but differ from one HTA subset to another. These results would therefore suggest that HTA and HLA class I antigens display a limited structural homology, but have a conserved epitopic area whose detailed structure differs for each HTA subset. Furthermore, the cell surface expression of each HTA class I molecule type is differently enhanced by natural interferon (IFN)-alpha or -gamma. This result additionally supports the serologic delineation of HTA subsets, and suggests that the corresponding genes in Molt-4 cells, are subjected to distinct regulations.


Subject(s)
Antigens, Surface/classification , HLA Antigens/classification , T-Lymphocytes/immunology , Animals , Antibodies, Monoclonal , Antigens, Surface/analysis , Antigens, Surface/immunology , Cell Line , Epitopes/analysis , Epitopes/immunology , HLA Antigens/analysis , HLA Antigens/immunology , HLA-A Antigens , HLA-B Antigens , HLA-C Antigens , Humans , Immunologic Capping , Interferon Type I/pharmacology , Interferon-gamma/pharmacology , Leukemia/immunology , Lymphocyte Function-Associated Antigen-1 , Mice , Radioimmunoassay
3.
Immunogenetics ; 21(4): 321-31, 1985.
Article in English | MEDLINE | ID: mdl-2581890

ABSTRACT

The serological reactivities of HLA-A3, -B7, and -CW3 heavy chains associated with either mouse, bovine, or human beta-2-microglobulin (beta 2m) and expressed on the surface of transfected mouse fibroblasts were analyzed. All reactivities associated with one cluster (defined by monoclonal antibody W6/32) of antigenic determinants expressed by these HLA class I molecules were lost, or profoundly reduced, after each heavy chain associated with mouse beta 2-m. Expression by the transfected fibroblasts of the HLA-A3, -B7, and -CW3 heavy chains in association with human beta 2m restores these reactivities. Since most of the amino acid differences between mouse and human beta 2m probably correspond to externally oriented hydrophilic residues, these results suggest that critical interactions in the three-dimensional structure of HLA class I molecules occur between the light chain and the first two external domains of the class I heavy chains, to which some of the altered reactivities have been mapped.


Subject(s)
HLA Antigens/immunology , beta 2-Microglobulin/immunology , Animals , Antibodies, Monoclonal/immunology , Binding Sites , Cells, Cultured , Cloning, Molecular , Epitopes/immunology , HLA Antigens/genetics , Humans , Mice , Molecular Conformation , Transfection
4.
Immunogenetics ; 21(5): 469-78, 1985.
Article in English | MEDLINE | ID: mdl-2581891

ABSTRACT

Radioimmunoassay of HLA-transformed mouse L cells expressing A3, A24, B7, or Cw3 HLA class I molecules with a set of monomorphic monoclonal antibodies distinguishes between A3-A24 and B7-Cw3 patterns of reactivity. Analyses with Bw6-specific monoclonal antibodies and a human alloantiserum demonstrate the expression by the HLA-Cw3 molecules of a Bw6 public specificity related to but not identical with that expressed by the HLA-B7 molecules. Exon-shuffling experiments and inhibition studies of monoclonal antibody cell-surface fixation indicate that similar parts of B7 and Cw3 molecules account for their serological cross-reactivity.


Subject(s)
HLA Antigens/immunology , HLA-B Antigens , HLA-C Antigens , Animals , Antibodies, Monoclonal/immunology , Cross Reactions , Epitopes/immunology , HLA Antigens/genetics , Humans , Immunochemistry , Isoantibodies/immunology , L Cells/immunology , Mice , Transformation, Genetic
5.
Hum Immunol ; 11(1): 31-45, 1984 Sep.
Article in English | MEDLINE | ID: mdl-6207154

ABSTRACT

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.


Subject(s)
Genes, MHC Class II , Genes , HLA Antigens/genetics , HLA-A Antigens , Major Histocompatibility Complex , Thymidine Kinase/genetics , Transfection , Animals , Antigen-Antibody Complex , DNA Restriction Enzymes , Epitopes/analysis , H-2 Antigens/genetics , HLA-A24 Antigen , HLA-B7 Antigen , Humans , L Cells/enzymology , Mice
6.
J Immunol ; 132(3): 1176-82, 1984 Mar.
Article in English | MEDLINE | ID: mdl-6198381

ABSTRACT

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.


Subject(s)
Binding Sites, Antibody , HLA Antigens/immunology , HLA-C Antigens , L Cells/immunology , Transformation, Genetic , Animals , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/physiology , Antigen-Antibody Reactions , Binding, Competitive , Cell Line , Drug Synergism , Epitopes/immunology , HLA Antigens/genetics , HLA-A3 Antigen , Humans , Immunoglobulin Fab Fragments/immunology , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred CBA
7.
J Immunol ; 130(3): 1432-8, 1983 Mar.
Article in English | MEDLINE | ID: mdl-6185587

ABSTRACT

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.


Subject(s)
Beta-Globulins/immunology , HLA Antigens/immunology , Hybrid Cells/immunology , Immunoglobulin Heavy Chains/immunology , Lymphocyte Activation , beta 2-Microglobulin/immunology , Animals , Cell Membrane/analysis , Cross Reactions , Epitopes/analysis , Fluorescent Antibody Technique , Genes, MHC Class II , HLA Antigens/genetics , Humans , Immunoglobulin Heavy Chains/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Protein Conformation , Radioimmunoassay , Species Specificity , beta 2-Microglobulin/genetics
9.
Immunogenetics ; 18(1): 65-77, 1983.
Article in English | MEDLINE | ID: mdl-6190745

ABSTRACT

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.


Subject(s)
Genes , HLA Antigens/genetics , HLA-C Antigens , Major Histocompatibility Complex , Thymidine Kinase/deficiency , Animals , Antibodies, Monoclonal , DNA Restriction Enzymes , DNA Transposable Elements , Epitopes/analysis , HLA-A3 Antigen , L Cells/enzymology , L Cells/immunology , Mice , Mice, Inbred BALB C , Radioimmunoassay , Species Specificity , Thymidine Kinase/genetics
10.
J Immunol Methods ; 54(1): 9-22, 1982 Oct 15.
Article in English | MEDLINE | ID: mdl-6183366

ABSTRACT

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.


Subject(s)
Epitopes/analysis , HLA Antigens/analysis , Polyethylene Glycols/pharmacology , Radioimmunoassay/methods , Animals , Antibodies, Monoclonal/immunology , Cell Line , Epitopes/immunology , HLA Antigens/immunology , HLA-B Antigens , HLA-C Antigens , HLA-DR Antigens , Histocompatibility Antigens Class II/analysis , Histocompatibility Antigens Class II/immunology , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Solubility
11.
Scand J Immunol ; 16(3): 233-41, 1982 Sep.
Article in English | MEDLINE | ID: mdl-6183735

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/immunology , Antibody Affinity , Histocompatibility Antigens Class II/immunology , T-Lymphocytes/immunology , Animals , Epitopes , Hemocyanins/immunology , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Ovalbumin/immunology
12.
J Immunogenet ; 9(1): 11-24, 1982 Feb.
Article in English | MEDLINE | ID: mdl-6978913

ABSTRACT

We investigated the capacity of anti-I-Ak and anti-I-E/Ck monoclonal antibodies (m.Ab.) to inhibit T lymphocyte proliferative responses to soluble antigen (Keyhole limpet hemocyanin), alloantigens (H-2 or non-H-2 related) and a mitogen (Concanavalin A). Surprisingly, specific inhibition was observed in all circumstances, and with both anti-I-Ak and anti-I-E/Ck m.Ab., whether the responses tested were I restricted in cell mixing experiments or not. The significance of the inhibition by anti-Ia m.Ab. of non-Ia-restricted responses is still not completely understood. These results, however, strongly suggest that in vitro inhibition by anti-Ia antibodies of T cell proliferative responses does not necessarily indicate I restriction of the presentation to T lymphocytes of the corresponding antigen.


Subject(s)
Antibodies, Monoclonal/immunology , H-2 Antigens/genetics , Lymphocyte Activation/drug effects , T-Lymphocytes/immunology , Animals , Antigens/immunology , Concanavalin A/pharmacology , Isoantigens/immunology , Mice , Mice, Inbred Strains/genetics
13.
Immunogenetics ; 16(5): 407-24, 1982.
Article in English | MEDLINE | ID: mdl-6185416

ABSTRACT

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.


Subject(s)
Histocompatibility Antigens Class II/immunology , Lymphocyte Activation , T-Lymphocytes/immunology , Animals , Antibodies, Monoclonal , Antibody Specificity , Cell Membrane/immunology , Epitopes , Immune Tolerance , Macromolecular Substances , Mice
14.
Immunogenetics ; 14(6): 469-79, 1981 Dec.
Article in English | MEDLINE | ID: mdl-6976316

ABSTRACT

The recognition of keyhole limpet hemocyanin by a substantial proportion of proliferating clones of murine T lymphocytes was found to be restricted by the I-E/Ck molecule, which is a combinatorial product of genes located in the I-A (Ae) and I-E/C (E alpha) subregions of the murine major histocompatibility complex. The respective roles of the Ae (polymorphic) and E alpha (oligomorphic) gene products in the expression of the structures which are used as restriction elements by these T-cell clones was analyzed by mating parental strains unable to present the antigen and bearing selected Ae and E alpha alleles. Efficient complementation for antigen presentation was found to require the expression by accessory cells of the Aek-gene product, whereas all E alpha allelic molecules were functionally equivalent. These results (a) indicate that the immunoregulatory role of I-region gene products, initially described for molecules selected for their limited number of antigenic epitopes, also applies to complex multiepitopic antigens; (b) illustrate the advantage which results from the diversity of the Ia molecules expressed by accessory cells for the development of potent immune responses; and (c) suggest that a correlation might exist between the degree of polymorphism of a given family of H-2 allelic molecules and their ability to be used as restriction elements for antigen recognition by T lymphocytes.


Subject(s)
Clone Cells/immunology , Major Histocompatibility Complex , T-Lymphocytes/immunology , Alleles , Animals , Antibodies/genetics , Cell Line , Cells, Cultured , H-2 Antigens/genetics , Hemocyanins , Lymphocyte Activation , Mice , Mice, Inbred Strains , Plasmacytoma
15.
J Immunol Methods ; 44(3): 285-99, 1981.
Article in English | MEDLINE | ID: mdl-7276581

ABSTRACT

The fixation of plasma vesicles at the surface of intact mouse spleen or tumor cells was studied in order to introduce the foreign alloantigens of the vesicles into the plasma membrane of these cells. A 3-6-fold increase of fixation of radioiodinated vesicles was obtained when cells and vesicles were incubated in the presence of polyethylene glycol 1500 (PEG 1500). The fixation of vesicles on the surface of cells was demonstrated by scanning electron microscopy. Cells treated with vesicles in the presence of PEG acquired the corresponding membrane alloantigens, as demonstrated by cellular binding radioimmunoassay. However, sensitivity to antibody-dependent lysis was obtained only when vesicle fixation was achieved in the presence of both wheat germ agglutinin and polyethylene glycol. The introduction of foreign alloantigens in the plasma membrane of the treated cells might help to define the functional properties of these molecules.


Subject(s)
Cytoplasm/immunology , Isoantigens , Polyethylene Glycols/pharmacology , Animals , Cell Membrane/immunology , Cell Membrane/ultrastructure , Cell Survival , Erythrocytes/ultrastructure , Humans , Macrophages/ultrastructure , Mice , Mice, Inbred AKR , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Inbred DBA , Neoplasms, Experimental/ultrastructure , Staphylococcal Protein A/pharmacology
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