Asunto(s)
Proteínas del Citoesqueleto/análisis , Giardia/química , Proteínas Protozoarias/análisis , Secuencia de Aminoácidos , Animales , Proteínas del Citoesqueleto/química , Electroforesis en Gel Bidimensional , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Proteínas Protozoarias/química , Homología de Secuencia de Aminoácido , Tinción con Nitrato de PlataRESUMEN
Combinations of peripheral blood lymphocytes, matched or mismatched for HLA-DP, were analyzed in primary one-way mixed lymphocyte culture experiments. Proliferative responses as correlated with tritiated thymidine uptake were assessed over a kinetic range of 5-15 days. A proliferative response was observed between DP-mismatched combinations, whereas combinations matched for DP and all other HLA alloantigens did not elicit significant proliferation. Optimal responses were observed 9 days after the combination of 1 x 10(5) responder and stimulator cells. Responses were blocked by anti-DP monoclonal antibodies. These studies demonstrate the complexity of the primary mixed lymphocyte culture system and suggest that DP alloantigens should be considered when anomalous responses are obtained.
Asunto(s)
Antígenos HLA-DP/inmunología , Prueba de Cultivo Mixto de Linfocitos/métodos , Linfocitos T/inmunología , Anticuerpos Monoclonales , Antígenos HLA-D/inmunología , Prueba de Histocompatibilidad , Humanos , Factores de TiempoRESUMEN
Alloreactive T-cell clones were derived by limiting dilution following priming to allogeneic cells bearing HLA-DR1 alloantigens. Clonal specificities were determined by extensive testing on a panel of allogeneic lymphoblastoid cell lines and by blocking studies with monoclonal antibodies specific for HLA-DR, -DQ, and -DP class II molecules. Out of nine DR1-positive cell lines, three failed to stimulate a subset of the T-cell clones in conventional proliferation assays. Proliferation by all of the clones was blocked by anti-DR antibodies, not by anti-DQ or anti-DP, which was consistent with the conclusion that the HLA-DR molecule was recognized. This DR1-associated polymorphism has been identified as Dw20 by the Tenth International Histocompatibility Workshop. The molecular basis for this altered recognition of the DR1 molecule was determined by allele-specific oligonucleotide hybridization and by DNA sequencing studies. The first, second, and third hypervariable regions of all nine DR1-positive cell lines were identical. Valine and glycine were found at positions 85 and 86 of the DR1 beta 1 chain in DR1 molecules from six of the nine lymphoblastoid cell lines, whereas alanine and valine were found in the three variant (Dw20) DR1-positive cells. By analogy with class I structure, residues 85 and 86 would be located at the extreme C-terminal end of the beta-chain alpha helix. Together or separately, these amino acid differences may define a T-cell recognition element on the DR1 molecule serving to contact allospecific T-cell receptors. Alternatively, if allorecognition involves recognition of a self peptide complexed with an allogeneic MHC molecule, then it is possible that the differences T cells recognize on DR1 class II proteins arise from peptide-specific interactions with residues 85 and 86.
Asunto(s)
Aminoácidos/fisiología , Antígeno HLA-DR1/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Linfocitos B/inmunología , Secuencia de Bases , Sitios de Unión , Línea Celular , Epítopos , Antígeno HLA-DR1/genética , Humanos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Polimorfismo Genético/genética , Polimorfismo Genético/inmunología , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Human allospecific T-cell clones were generated against DR1 and DQw1 by limiting dilution. In proliferation experiments using a large panel of Epstein-Barr virus-transformed B-cell lines (LCL), eight T-cell clones (TLC) were found that responded only to the DR1+ LCLs* (9 of 9) and not the 94 other LCLs expressing DR specificities 2 through w9. TLCs* were analyzed further using monoclonal antibodies in blocking studies. As expected, most TLCs were blocked by anti-DR monoclonal antibodies (MoAbs)* and not by anti-DQ MoAbs. However, one clone, TLC 63.138, was not blocked by anti-DR MoAbs but was completely inhibited by anti-DQ MoAbs. This suggests that TLC 63.138 recognizes a private determinant on DQ molecules uniquely associated with DR1.
Asunto(s)
Antígenos HLA-DQ/análisis , Antígenos HLA-DR/análisis , Linfocitos T/clasificación , Anticuerpos Monoclonales , Células Cultivadas , Humanos , Leucocitos Mononucleares/inmunologíaRESUMEN
A human fibroblast expressing HLA-DR1 antigen on its surface was generated by transfection with DR alpha and DR beta cDNAs. The ability of this transfected fibroblast line to process and to present antigens was analyzed by using human T-lymphocyte clones (TLCs) specific for HLA-DR1 alloantigen or restricted by DR1 in their recognition of influenza virus. TLC responses were measured in proliferative assays and were tested for blocking by monoclonal antibodies specific for MHC antigens. Two TLCs specific for a discrete segment (aa 306-320) of the influenza hemagglutinin molecule responded to the antigen added in peptide form but not as intact virion. The transfected fibroblast line thus appears unable to process antigen properly. A DR1-alloreactive TLC was able to respond to the transfected fibroblast. However, 24 other DR1-alloreactive TLCs and oligoclonal T-cell lines were unable to respond. These results suggest either that the conformation of the DR1 molecule on a transfected fibroblast allows peptide presentation but not allorecognition, or that self antigen processing and subsequent presentation by MHC antigens is necessary for allorecognition.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Antígenos HLA-D/genética , Antígenos HLA-DR/genética , Hemaglutininas Virales/inmunología , Transfección , Anticuerpos Monoclonales , Epítopos , Fibroblastos/inmunología , Antígenos HLA-DR/inmunología , Antígeno HLA-DR1 , Glicoproteínas Hemaglutininas del Virus de la Influenza , Humanos , Técnicas In Vitro , Activación de Linfocitos , Linfocitos T/inmunologíaRESUMEN
Human T-lymphocyte clones ( TLCs ) were generated against the hemagglutinin (HA) of A/Texas/1/77 influenza virus by limiting dilution. TLCs were then screened for antigen specificity on chemically synthesized peptides representing the HA1 molecule. It has been hypothesized that different T cells that recognize the identical antigenic determinant are controlled by (restricted by) the same class II epitope. Two TLCs , HA1.4 and HA1.7, both recognized the same HA peptide and in proliferation studies exhibited identical restriction patterns. Two other clones, HA 1.9 and HA 2.43, recognized different HA determinants and also had distinct restriction patterns. Proliferation inhibition studies with monoclonal antibodies against human class II molecules demonstrated three unique patterns of blocking with the clones, suggesting that clones may be restricted to a unique class II epitope depending on the HA determinant recognized. These data can be interpreted as supporting the argument that human immune responses to influenza hemagglutinin are under Ir gene control exerted at the level of the viral antigenic determinant recognized in association with particular D-region restricting elements. The determinant selection and clonal deletion theories are compared for their capacity to best explain these findings.
Asunto(s)
Epítopos/análisis , Genes MHC Clase II , Hemaglutininas Virales/inmunología , Virus de la Influenza A/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Anticuerpos Monoclonales , Células Cultivadas , Células Clonales , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , FenotipoRESUMEN
We studied the effects of xenoantiserum to human nonpolymorphic Ia-like antigens upon in vitro antigen-specific T cell proliferative responses in unfractionated PBL populations and at the monoclonal level. Our findings suggest that the xenoantiserum, although it inhibits the antigen-specific response of unfractionated PBL and allospecific T cell clones, does not inhibit the proliferative response to cloned influenza virus immune human T lymphocytes, and therefore may be mediating inhibition by dual mechanisms: direct inhibition of alloantigen recognition and induction of nonspecific suppression. Kinetic differences may explain these phenomena. In cocultivation experiments with a virus-specific clone, the RaIa antiserum appears to induce an OKT3+,8+,4-, radiosensitive regulatory subset of lymphocytes. When adoptively transferred, these induced cells inhibit the TLC response in an antigen-nonspecific and genetically nonrestricted manner. We discuss the various modes and levels of inhibition of antigen-specific proliferation by anti-Ia antisera and their multiple activities.