RESUMO
The aim of this study was to explore whether Thy-1, like other members of the Ig-like superfamily (e.g., CD2 and neural cell adhesion molecule), participates in cell-cell adhesion. This was investigated by measuring the binding of Thy-1+ probe cells (thymocytes or AKR1 T lymphoma cells) to Thy-1- cloned mouse thymic epithelial (MTE) cells using a quantitative cell adhesion assay. The results were as follows: (a) the thymo-epithelial cell interaction was found to be inhibitable (by 25-40%) by soluble Thy-1 molecules purified from phosphatidylinositol-specific phospholipase C-treated mouse thymocytes as well as by Fab' fragments of a Thy-1-specific mAb; (b) the binding of the Thy-1- AKR1 (Thy-1-d) mutant to MTE cells was found to be reduced (by 50%) as compared with that of the wild type T lymphoma; (c) the Thy-1-mediated adhesion pathway did not require Ca2+ and promoted the initial thymo-epithelial binding measured at 4 degrees C. These data provide the first direct evidence of an adhesive function of Thy-1 and suggest that this molecule, in addition to its T cell triggering properties, might play a role during the early T cell maturation by promoting thymocyte adhesion to thymic stroma.
Assuntos
Antígenos de Superfície/fisiologia , Cálcio/metabolismo , Linfócitos T/citologia , Timo/citologia , Animais , Anticorpos Monoclonais/imunologia , Ligação Competitiva/imunologia , Adesão Celular/fisiologia , Contagem de Células , Células Epiteliais , Camundongos , Ratos , Antígenos Thy-1RESUMO
Schistosomiasis, due to Schistosoma mansoni, is a major health problem in many subtropical countries, and major efforts are being made to define a vaccine. In this regard, we have reported that sera from subjects with low susceptibility to infection by S. mansoni react with a major larval surface antigen (P-37), having an apparent molecular mass of 37 kD, against which sera of susceptible individuals show little reactivity. We have now cloned the cDNA for this antigen by screening a schistosome cDNA expression library with antibodies against the purified protein. The selected cDNAs encode a protein that is specifically identified by immune human sera containing antibodies against P-37, while sera exhibiting low or no reactivity toward P-37 fail to recognize the recombinant protein. The cloned cDNAs hybridize with a 1.2-kb RNA that is the transcript of a single copy gene. This RNA directs the synthesis of a 36.5-kD polypeptide that is precipitated by sera from the most resistant subjects. The amino acid sequence of the encoded polypeptide shows homology with the glycolytic enzyme Glyceraldehyde-3P-dehydrogenase (72.5% of positional identity with human Glyceraldehyde-3P-dehydrogenase). Antibodies against the recombinant protein identified P-37 on the larva. These findings, together with other reports, indicate that a number of conserved proteins may be major targets of host-protective immunity against S. mansoni. The hypothesis is discussed that genetic restriction of the immune response to these antigens may occur in heterogeneous human populations because of the limited number of T cell epitopes carried by these host-like proteins. Such genetic effects might allow parasite transmission through nonresponder (susceptible) individuals. This hypothesis and the protective properties of P-37 can now be tested using the recombinant protein and synthetic peptides derived from selected regions of the polypeptide chain.
Assuntos
Antígenos de Helmintos/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Esquistossomose/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/análise , Antígenos de Helmintos/fisiologia , Antígenos de Superfície/genética , Sequência de Bases , Clonagem Molecular , DNA/análise , Gliceraldeído-3-Fosfato Desidrogenases/análise , Gliceraldeído-3-Fosfato Desidrogenases/imunologia , Humanos , Imunidade Inata , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , CoelhosRESUMO
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.
Assuntos
Citoplasma/imunologia , Isoantígenos , Polietilenoglicóis/farmacologia , Animais , Membrana Celular/imunologia , Membrana Celular/ultraestrutura , Sobrevivência Celular , Eritrócitos/ultraestrutura , Humanos , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBA , Neoplasias Experimentais/ultraestrutura , Proteína Estafilocócica A/farmacologiaRESUMO
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.
Assuntos
Epitopos/análise , Antígenos HLA/análise , Polietilenoglicóis/farmacologia , Radioimunoensaio/métodos , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular , Epitopos/imunologia , Antígenos HLA/imunologia , Antígenos HLA-B , Antígenos HLA-C , Antígenos HLA-DR , Antígenos de Histocompatibilidade Classe II/análise , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , SolubilidadeRESUMO
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.
Assuntos
Genes MHC da Classe II , Genes , Antígenos HLA/genética , Antígenos HLA-A , Complexo Principal de Histocompatibilidade , Timidina Quinase/genética , Transfecção , Animais , Complexo Antígeno-Anticorpo , Enzimas de Restrição do DNA , Epitopos/análise , Antígenos H-2/genética , Antígeno HLA-A24 , Antígeno HLA-B7 , Humanos , Células L/enzimologia , CamundongosRESUMO
We investigated the capacity of anti-Ia monoclonal antibody Fab and Fab'2 fragments to inhibit keyhole limpet haemocyanin (KLH) or concanavalin A (Con A)-induced T-cell proliferations. Both types of fragments of anti-I-Ak and anti-I-E/Ck monoclonal antibodies inhibited these responses. On a protein concentration basis, the inhibitory effects of fragmented antibodies were less pronounced than those of undigested molecules. However, once the differences in antigen-binding capacity were compensated, antibody fragments were as efficient inhibitors as undigested molecules. These results suggest (i) that masking of Ia antigenic determinants is the essential mechanism of anti-Ia antibody-mediated inhibitory effect; (ii) that some KLH-specific proliferating T lymphocytes are I-E/Ck restricted; (iii) that the Ia antigen role is not limited to restriction of cell interactions since the Con A-induced proliferation, a non-H-2 restricted response, is inhibited by anti-Ia Fab fragments.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Ativação Linfocitária , Animais , Concanavalina A/farmacologia , Hemocianinas/farmacologia , Imunoglobulinas/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Linfócitos T/imunologiaRESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos H-2/genética , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T/imunologia , Animais , Antígenos/imunologia , Concanavalina A/farmacologia , Isoantígenos/imunologia , Camundongos , Camundongos Endogâmicos/genéticaRESUMO
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.
Assuntos
Células Clonais/imunologia , Complexo Principal de Histocompatibilidade , Linfócitos T/imunologia , Alelos , Animais , Anticorpos/genética , Linhagem Celular , Células Cultivadas , Antígenos H-2/genética , Hemocianinas , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos , PlasmocitomaRESUMO
We have studied the role of the activin immediate-early response gene Mix.1 in mesoderm and endoderm formation. In early gastrulae, Mix.1 is expressed throughout the vegetal hemisphere, including marginal-zone cells expressing the trunk mesodermal marker Xbra. During gastrulation, the expression domains of Xbra and Mix.1 become progressively exclusive as a result of the establishment of a negative regulatory loop between these two genes. This mutual repression is important for the specification of the embryonic body plan as ectopic expression of Mix.1 in the Xbra domain suppresses mesoderm differentiation. The same effect was obtained by overexpressing VP16Mix.1, a fusion protein comprising the strong activator domain of viral VP16 and the homeodomain of Mix.1, suggesting that Mix.1 acts as a transcriptional activator. Mix.1 also has a role in endoderm formation. It cooperates with the dorsal vegetal homeobox gene Siamois to activate the endodermal markers edd, Xlhbox8 and cerberus in animal caps. Conversely, vegetal overexpression of enRMix.1, an antimorphic Mix.1 mutant, leads to a loss of endoderm differentiation. Finally, by targeting enRMix.1 expression to the anterior endoderm, we could test the role of this tissue during embryogenesis and show that it is required for head formation.
Assuntos
Embrião não Mamífero/fisiologia , Endoderma/fisiologia , Proteínas Fetais , Gástrula/fisiologia , Genes Homeobox , Proteínas de Homeodomínio/genética , Proteínas com Domínio T , Proteínas de Xenopus , Animais , Padronização Corporal , Diferenciação Celular , Proteínas de Ligação a DNA/biossíntese , Indução Embrionária , Endoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/biossíntese , Mesoderma/fisiologia , Reação em Cadeia da Polimerase , Capuzes de RNA/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Transativadores/biossíntese , Fatores de Transcrição/biossíntese , XenopusRESUMO
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.
Assuntos
Epitopos , Antígenos de Histocompatibilidade Classe II/genética , Biossíntese de Proteínas , Animais , Anticorpos , Precipitação Química , Células Clonais/imunologia , Reações Cruzadas , Citotoxicidade Imunológica , Feminino , Antígenos de Histocompatibilidade Classe II/imunologia , Imunidade Celular , Masculino , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos CBA , CoelhosRESUMO
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.
Assuntos
Antígenos HLA/análise , Antígenos HLA-C , Cadeias Pesadas de Imunoglobulinas/análise , Microglobulina beta-2/análise , Animais , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Epitopos/análise , Epitopos/genética , Epitopos/imunologia , Antígenos HLA/genética , Antígenos HLA/imunologia , Antígeno HLA-A3 , Antígeno HLA-B7 , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Células L/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos CBA , Conformação Proteica , Transfecção , Microglobulina beta-2/genética , Microglobulina beta-2/imunologiaRESUMO
To address the role of the TEA germline transcription, which initiates upstream of the TCR-J alpha S, in the regulation of TCR-J alpha locus accessibility, we created a mouse in which this region has been removed by homologous recombination. Normal development of T alpha beta cells and the expression of other TCR alpha germline transcripts in TEA-/- mice ruled out an exclusive role for TEA in the overall accessibility of the J alpha cluster. However, the rearrangement of the most 5' J alpha (J alpha 61 to J alpha 53) was severely impaired, indicating that TEA may control the DNA accessibility of a particular J alpha window. Moreover, the relative usage of every J alpha segment was affected. These results are consistent with TEA acting as a "rearrangement-focusing" element, targeting the primary waves of V alpha-J alpha recombination to the most 5' J alpha S in an ongoing TCR-J alpha rearrangement model.
Assuntos
Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/fisiologia , Animais , DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Sequências Reguladoras de Ácido Nucleico , Deleção de Sequência , Transcrição GênicaRESUMO
The immunoglobulin heavy chain intronic transcriptional enhancer (E mu) is part of a complex cis-regulatory DNA region which has notably been shown to modulate V(D)J rearrangements of associated variable gene segments. We have used recombination substrates comprised of the E mu enhancer together with various lengths of additional downstream mu sequences to assess the individual contribution of those sequences to the V(D)J recombinational regulatory activity. Surprisingly, in the absence of large amounts of mu sequences, substrate rearrangements were not detected in Southern blot analyses of the lymphoid tissues from independent transgenic mice, but were readily detectable following transfection into cultured pre-B cells. A short mu segment which includes matrix association regions (MARs) was not sufficient to restore high levels of rearrangements within the reporter transgenes. However, additional experiments demonstrated that the mu sequences are dispensable for V(D)J recombination in transgenic thymuses, implying a suppressive effect exerted by the vector sequences left in the transgenic insert, when they are attached near the E mu regulatory region. This suppression of V(D)J recombination, which correlates with an hypermethylation of the transgenes, is discussed in view of previously reported transgenic and gene targeting experiments.
Assuntos
Elementos Facilitadores Genéticos , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Cadeias Pesadas de Imunoglobulinas/genética , Íntrons , Animais , Sequência de Bases , Linhagem Celular , DNA , DNA Nucleotidiltransferases/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Metilação , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Recombinação Genética , Especificidade por Substrato , VDJ RecombinasesRESUMO
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.
Assuntos
Antígenos de Superfície/classificação , Antígenos HLA/classificação , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais , Antígenos de Superfície/análise , Antígenos de Superfície/imunologia , Linhagem Celular , Epitopos/análise , Epitopos/imunologia , Antígenos HLA/análise , Antígenos HLA/imunologia , Antígenos HLA-A , Antígenos HLA-B , Antígenos HLA-C , Humanos , Capeamento Imunológico , Interferon Tipo I/farmacologia , Interferon gama/farmacologia , Leucemia/imunologia , Antígeno-1 Associado à Função Linfocitária , Camundongos , RadioimunoensaioRESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Antígenos de Histocompatibilidade Classe II/imunologia , Linfócitos T/imunologia , Animais , Epitopos , Hemocianinas/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologiaRESUMO
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.
Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Membrana Celular/imunologia , Epitopos , Tolerância Imunológica , Substâncias Macromoleculares , CamundongosRESUMO
By using a calibrated cell sorter and monoclonal antibodies conjugated to fluorochromes, a quantitative analysis of the levels of expression of HLA class I molecules at the surface of cloned murine L cells transfected with purified A3, B7, or CW3 genes was performed and compared with radioimmunoassay data. We selected clones of heterogeneous levels of HLA class I expression, which were shown to remain constant over a period of 4 mo in absence of HAT selection and not to be correlated to the DNA copy number of the corresponding integrated gene.
Assuntos
Citometria de Fluxo/métodos , Antígenos HLA/genética , Animais , Anticorpos Monoclonais , Separação Celular/métodos , DNA Recombinante , Regulação da Expressão Gênica , Genes , Antígenos HLA/análise , Humanos , Camundongos , Hibridização de Ácido Nucleico , TransfecçãoRESUMO
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.
Assuntos
Antígenos HLA/imunologia , Microglobulina beta-2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Células Cultivadas , Clonagem Molecular , Epitopos/imunologia , Antígenos HLA/genética , Humanos , Camundongos , Conformação Molecular , TransfecçãoRESUMO
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.