RESUMEN

Individuals with fragile X syndrome (FXS) are cognitively impaired and have marked speech delays and deficits. Our goal was to characterize expression of fragile X mental retardation protein (FMRP), encoded by Fmr1 fragile X mental retardation 1 gene or transcript (FMR1), in an animal model that learns to vocalize, namely the zebra finch Taeniopygia guttata (Tgu). We cloned and sequenced the zebra finch ortholog of FMR1 (TguFmr1) and developed an antibody that recognizes TguFmrp specifically. TguFmrp has structural features similar to its human ortholog FMRP. Because FXS patients exhibit sensorimotor deficits, we examined TguFmrp expression prior to, during, and after sensorimotor song learning in zebra finches. We found that TguFmrp is expressed throughout the brain and in four major song nuclei of the male zebra finch brain, primarily in neurons. Additionally, prior to sensorimotor learning, we observed elevated TguFmrp expression in the robust nucleus of the arcopallium (RA) of post-hatch day 30 males, compared with the surrounding telencephalon, suggesting a preparation for this stage of song learning. Finally, we observed variable TguFmrp expression in the RA of adolescent and adult males: in some males it was elevated and in others it was comparable to the surrounding telencephalon. In summary, we have characterized the zebra finch ortholog of FMRP and found elevated levels in the premotor nucleus RA at a key developmental stage for vocal learning.

Asunto(s)

Envejecimiento/genética , Envejecimiento/metabolismo , Química Encefálica/genética , Pinzones/fisiología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/biosíntesis , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Vocalización Animal/fisiología , Secuencia de Aminoácidos , Animales , Northern Blotting , Western Blotting , Colorantes Fluorescentes , Inmunohistoquímica , Inmunoprecipitación , Aprendizaje/fisiología , Masculino , Vías Nerviosas/fisiología , Sistemas de Lectura Abierta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia

RESUMEN

Fragile X syndrome results from the absence of the RNA binding FMR protein. Here, mRNA was coimmunoprecipitated with the FMRP ribonucleoprotein complex and used to interrogate microarrays. We identified 432 associated mRNAs from mouse brain. Quantitative RT-PCR confirmed some to be >60-fold enriched in the immunoprecipitant. In parallel studies, mRNAs from polyribosomes of fragile X cells were used to probe microarrays. Despite equivalent cytoplasmic abundance, 251 mRNAs had an abnormal polyribosome profile in the absence of FMRP. Although this represents <2% of the total messages, 50% of the coimmunoprecipitated mRNAs with expressed human orthologs were found in this group. Nearly 70% of those transcripts found in both studies contain a G quartet structure, demonstrated as an in vitro FMRP target. We conclude that translational dysregulation of mRNAs normally associated with FMRP may be the proximal cause of fragile X syndrome, and we identify candidate genes relevant to this phenotype.

Asunto(s)

Química Encefálica , Síndrome del Cromosoma X Frágil/genética , Proteínas del Tejido Nervioso/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/fisiología , Secuencia de Aminoácidos , Animales , Centrifugación por Gradiente de Densidad , Modelos Animales de Enfermedad , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Humanos , Ligandos , Sustancias Macromoleculares , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Genéticos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Unión Proteica , ARN Mensajero/química , ARN Mensajero/aislamiento & purificación , Proteínas de Unión al ARN/genética , Secuencias Reguladoras de Ácidos Nucleicos , Ribosomas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

Fragile X mental retardation is caused by the absence of FMRP, an RNA-binding protein found in a large mRNP complex. Although there is evidence that FMRP exists as a homo-multimer, additional proteins have been identified that associate with FMRP in the mRNP. The autosomal paralogs of FMRP, FXR1P, and FXR2P, associate with FMRP, as do nucleolin and NUFIP1, all RNA binding proteins. Using cell lines that were stably transfected with Flag-Fmr1, we identified an additional protein that coimmunoprecipitates with FMRP. The approximately 50 kDa protein was identified by mass spectrometry as mouse Y box-binding protein 1 (YB1), which is 97% identical to the core mRNP protein p50, an RNA-binding protein. An anti-p50 antiserum recognized the 50 kDa protein, confirming the identification. The association of the FMRP-mRNP with a Y box protein, the latter commonly found in mRNPs, further suggests the involvement of FMRP in translation modulation.

Asunto(s)

Proteínas Potenciadoras de Unión a CCAAT/aislamiento & purificación , Proteínas de Unión al ADN , Proteínas del Tejido Nervioso/análisis , Proteínas de Unión al ARN , Ribonucleoproteínas/análisis , Factores de Transcripción , Animales , Línea Celular , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil/metabolismo , Células HeLa , Humanos , Ratones , Peso Molecular , Factores de Transcripción NFI , Proteínas Nucleares , Proteína 1 de Unión a la Caja Y

RESUMEN

The loss of FMR1 expression due to trinucleotide repeat expansion leads to fragile X syndrome, a cause of mental retardation. The encoded protein, FMRP, is a member of a gene family that also contains the fragile X-related proteins, FXR1P and FXR2P. FMRP has been shown to be a nucleocytoplasmic shuttling protein that selectively binds a subset of mRNAs, forms messenger ribonucleoprotein (mRNP) complexes, and associates with translating ribosomes. Here we describe a cell culture system from which we can isolate epitope-tagged FMRP along with mRNA, including its own message, and at least six other proteins. We identify two of these proteins as FXR1P and FXR2P by using specific antisera and identify a third protein as nucleolin by using mass spectrometry. The presence of nucleolin is confirmed by both reactivity with a specific antiserum as well as reverse coimmunoprecipitation where antinucleolin antiserum immunoprecipitates endogenous FMRP from both cultured cells and mouse brain. The identification of nucleolin, a known component of other mRNPs, adds a new dimension to the analysis of FMRP function, and the approach described should also allow the identification of the remaining unknown proteins of this FMRP-associated mRNP as well as the other bound mRNAs.

Asunto(s)

Síndrome del Cromosoma X Frágil , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Animales , Línea Celular , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Expresión Génica , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/aislamiento & purificación , Oligopéptidos , Péptidos/genética , Fosfoproteínas/aislamiento & purificación , Pruebas de Precipitina , ARN Mensajero , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleasas/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/aislamiento & purificación , Nucleolina

RESUMEN

Determination of the crystal structure of class II: peptide complexes has shown that in addition to pocket interactions involving the side chains of the peptide, peptide binding to MHC class II molecules is characterized by a series of hydrogen bonds which are contributed by genetically conserved amino acid residues in the class II molecule to the main chain of the peptide. Our experiments have revealed an unexpectedly large contribution of hydrogen bonds at the periphery of the MHC peptide binding pocket to MHC class II function. Kinetic studies have shown that peptide dissociation rates are profoundly accelerated by loss of a single hydrogen bonding residue. The magnitude of the effects seen with the loss in potential for a single hydrogen bond support a co-operative model in which individual bonds between class II and peptide are dependent on the integrity of neighboring interactions. Collectively our studies have revealed that MHC class II structure, peptide binding and intracellular trafficking events are critically dependent on the integrity of the hydrogen bonding network between class II molecules and its bound peptide.

Asunto(s)

Antígenos de Histocompatibilidad Clase II/metabolismo , Péptidos/inmunología , Péptidos/metabolismo , Animales , Presentación de Antígeno , Sitios de Unión , Transporte Biológico Activo , Antígenos HLA-D/química , Antígenos HLA-D/metabolismo , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Enlace de Hidrógeno , Modelos Biológicos , Mutación , Péptidos/química

RESUMEN

To characterize the importance of a highly conserved region of the class II beta chain, we introduced an amino acid substitution that is predicted to eliminate a hydrogen bond formed between the class II molecule and peptide. We expressed the mutated beta chain with a wild-type alpha chain in a murine L cell by gene transfection. The mutant class II molecule (81betaH-) assembles normally in the endoplasmic reticulum and transits the Golgi complex. When invariant chain (Ii) is coexpressed with 81betaH-, the class II-Ii complex is degraded in the endosomes. Expression of 81betaH- in the absence of Ii results in a cell surface expressed molecule that is susceptible to proteolysis, a condition reversed by incubation with a peptide known to associate with 81betaH-. We propose that 81betaH- is protease sensitive because it is unable to productively associate with most peptides, including classII-associated invariant chain peptides. This model is supported by our data demonstrating protease sensitivity of peptide-free wild-type I-Ad molecules. Collectively, our results suggest both that the hydrogen bonds formed between the class II molecule and peptide are important for the integrity and stability of the complex, and that empty class II molecules are protease sensitive and degraded in endosomes. One function of DM may be to insure continuous groove occupancy of the class II molecule.

Asunto(s)

Antígenos de Histocompatibilidad Clase II , Sustitución de Aminoácidos , Animales , Línea Celular , Genes MHC Clase II , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Hidrógeno , Ratones , Mutación , Conformación Proteica , Relación Estructura-Actividad

RESUMEN

The molecular mechanisms that regulate sorting of major histocompatibility complex (MHC) class II molecules into the endocytic pathway are poorly understood. For many proteins, access to endosomal compartments is regulated by cytosolically expressed sequences. We present evidence that a sequence in the lumenal domain of the MHC class II molecule regulates a very late event in class II biogenesis. Class II molecules containing single amino acid changes in the highly conserved 80-82 region of the beta chain were introduced into invariant chain (Ii)-negative fibroblasts with wild-type alpha chain, and the derived transfectants were analyzed biochemically. Using an endosomal isolation technique, we have quantified the level of class II molecules expressed in endocytic compartments and found that in the absence of Ii, approximately 15% of total cellular class II molecules can be isolated from endosomal compartments. Mutation at position 80 enhances this localization, while changes at positions 81 and 82 ablate class II expression in endosomal compartments. In addition, we have evaluated whether the induced changes in intracellular distribution of class II molecules were due to alterations in early biosynthetic events, indicative of misfolding of the molecules, or to modulation of later trafficking events more likely to be a consequence of the modulation of a specific transport event. Despite the dramatic effects on endosomal localization induced by the mutations, early biosynthetic events and maturation of class II were unaffected by the mutations. Collectively, our data argue that late trafficking events that control the ability of the class II molecule to access antigens is regulated by the 80-82 segment of the MHC class II beta chains.

Asunto(s)

Endosomas/inmunología , Endosomas/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos de Histocompatibilidad Clase II/fisiología , Animales , Compartimento Celular/genética , Compartimento Celular/inmunología , Endocitosis/genética , Endocitosis/inmunología , Endosomas/genética , Antígenos de Histocompatibilidad Clase II/genética , Líquido Intracelular/inmunología , Líquido Intracelular/metabolismo , Cinética , Células L , Ratones , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Fracciones Subcelulares/química , Fracciones Subcelulares/inmunología , Fracciones Subcelulares/metabolismo , Transfección

RESUMEN

The role of invariant chain (Ii) in antigen presentation has been studied using two independent approaches. The first was to reconstitute cell lines with class II molecules and various forms of the Ii; the second has been to generate mice that lack functional expression of the Ii gene (Ii.). Both types of studies show that Ii facilitates assembly of class II molecules in the endoplasmic reticulum (ER), chaperones or retains properly folded class II complexes through endosomal compartments, and finally, serves as a T-cell ligand. How Ii facilitates assembly of antigenic class II-peptide complexes is less clear and seems to depend on a number of factors reviewed here: the antigen and epitope studied; the route and dose by which antigen is administered; the form of Ii present; and the responding T cell used in the experiment. During assembly of the class II-peptide complex, Ii may facilitate this process in a number of ways: specifically, by increasing the amount of class II available in endosomal vesicles; changing access or retention of class II molecules in the endosomal pathway; facilitating peptide exchange during CLIP dissociation, or finally, by modulating the antigen processing environment in the cell. In summary, we propose that instead of playing just one role in assembling the class II-peptide complex, Ii may perform all of the above described activities, functioning in different capacities along the pathway of antigen presentation.

Asunto(s)

Presentación de Antígeno , Antígenos de Diferenciación de Linfocitos B/fisiología , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/fisiología , Animales , Retículo Endoplásmico/fisiología , Endosomas/metabolismo , Epítopos , Antígenos de Histocompatibilidad Clase II/biosíntesis , Humanos , Ratones , Modelos Inmunológicos , Chaperonas Moleculares , Péptidos/inmunología , Unión Proteica , Pliegue de Proteína

RESUMEN

Previous studies have shown that homozygous mutations between the LMP2 and DNA loci in the human MHC cause class II molecules to be abnormally conformed and unstable in the presence of SDS at low temperature, and impede class II-associated Ag processing and presentation. These abnormalities result from impaired ability to form intracellular class II/peptide complexes that predominate in normal cells. We show in this work that this defect results from deficient expression of either the DMA or the DMB gene. Human B-LCL.174 (DR3) cells, which have a deletion of all known expressible genes in the class II region, express transgene-encoded HLA-DR3, but have the abnormalities. Transfer of cosmid HA14, which contains the DMA and DMB genes, into .174 (DR3) cells restored normal DR3 conformation, stability in 0.4% SDS at 0 degree, and ability to process and present tetanus toxoid, but only when both DMA and DMB mRNAs were present. The requirement for both genetic expressions in engendering normal phenotypes was confirmed by transferring the cloned genes into .174 (DR3) cells separately or together. Because normal phenotypes were fully restored in transferent cells expressing DMA plus DMB, other genes in the approximately 1-mb homozygous class II region deletion in .174 (DR3) cells either do not participate in or are dispensable for apparently normal production of intracellular class II/peptide complexes. The properties of DM-deficient EBV-transformed B lymphoblastoid cell lines (LCLs) suggest ways of identifying humans in whom DM deficiency contributes to congenital immunodeficiency and malignancy.

Asunto(s)

Presentación de Antígeno/genética , Antígenos HLA-D/genética , Antígeno HLA-DR3/genética , Antígenos de Histocompatibilidad Clase II , Secuencia de Bases , Células Cultivadas , Frío , ADN Complementario/genética , Biblioteca de Genes , Humanos , Activación de Linfocitos/inmunología , Datos de Secuencia Molecular , Dodecil Sulfato de Sodio , Linfocitos T/inmunología , Toxoide Tetánico/inmunología , Transfección/genética

RESUMEN

In certain mutant human B cell lines, MHC-encoded class II molecules displayed at the cell surface have an abnormal conformation and are unstable in the presence of SDS. The mutants cannot present exogenous protein Ags to T cells but elicit responses with exogenous antigenic peptides; thus, formation of intracellular complexes between antigenic peptides and class II molecules is impaired. Previous analysis of LCL deletion mutants, .82, .174, and 5.2.4, showed that genes needed for this function must be present in approximately 230 kb of DNA in the class II region of the MHC. We now describe a new deletion mutant, .61, which has normal class II-mediated Ag processing/presentation. The TAP1, TAP2, LMP2, and LMP7 genes are deleted from .61, demonstrating that those genes are not needed for normal formation of intracellular class II/peptide complexes. The genes in question must be located in DNA that is present in .61 and .82 (both normal) and absent from .174 and 5.2.4. (both defective). Mapping of the deletion breakpoints indicates that genes needed for normal class II-associated Ag processing/presentation are either: 1) in an approximately 40 kb L DNA segment located between the DMB and LMP2 loci or 2) in an R region between the DQA2 and DQB1 loci and are completely included on a 5.1-kb fragment formed by joining of DNA that flanks the deletion in .61. The evidence favors location of the genes in the L DNA segment.

Asunto(s)

Transportadoras de Casetes de Unión a ATP , Sistemas de Transporte de Aminoácidos , Mapeo Cromosómico , Cisteína Endopeptidasas , ADN/análisis , Exorribonucleasas , Genes MHC Clase II , Antígenos de Histocompatibilidad Clase II/análisis , Complejos Multienzimáticos , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Proteínas Portadoras/genética , Proteínas Fúngicas/genética , Eliminación de Gen , Antígenos HLA-DR/análisis , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Mutación , Complejo de la Endopetidasa Proteasomal , Proteínas/genética , Proteínas de Saccharomyces cerevisiae , Transactivadores/genética

RESUMEN

The processing pathway for the MHC class II-restricted presentation of endogenous cytosolic Ag is distinct from the class I pathway since a cytosolic form of the influenza virus A hemagglutinin, expressed by a recombinant vaccinia virus, was presented by HLA-DR in a B cell mutant lacking the TAP1 subunit of the transporter for Ag presentation (TAP). In this report, two additional B cell mutants have been used to define the requirements of this TAP1-independent processing pathway. The first mutant, .61, lacks expression of both TAP1 and TAP2 genes, and of both LMP2 and LMP7 genes encoding proteasome subunits. As expected, class I-restricted presentation of the influenza virus matrix protein was totally deficient in mutant .61. In contrast, class II-restricted presentation of both the natural cytosolic matrix and the engineered cytosolic hemagglutinin proteins was functional in mutant .61. Thus, presentation of cytosolic Ag by class II molecules is independent of both TAP subunits and of the two MHC-encoded proteasome subunits. However, this endogenous processing pathway is dependent on at least one other function encoded in the class II region of the MHC as demonstrated with the second mutant, .174, in which a large deletion eliminates all expressed class II genes. Mutant .174 transfected with HLA-DR1 genes was previously shown to be defective in the presentation of exogenous Ag but normal in the presentation of short exogenous peptides. We show here that .174(DR1) is also defective in the presentation of cytosolic matrix and hemagglutinin proteins. This similar requirement for the class II-restricted presentation of either cytosolic Ag or internalized exogenous Ag suggests that both forms of Ag are ultimately targeted to the same cellular compartment for association with class II molecules.

Asunto(s)

Presentación de Antígeno , Citosol/inmunología , Antígenos HLA-DR/metabolismo , Presentación de Antígeno/genética , Linfocitos B/inmunología , Línea Celular , Citotoxicidad Inmunológica , Genes MHC Clase II , Antígenos HLA-DR/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza , Hemaglutininas Virales/genética , Hemaglutininas Virales/inmunología , Humanos , Mutación , Transfección

RESUMEN

Class II major histocompatibility complexes bind peptides in an endosome-like compartment. When the class II null cell line 721.174 was transfected with class II DR3 genes, DR molecules were produced in normal amounts. However, the DR molecules were abnormally conformed and unstable because deletion of an antigen-processing gene had impaired intracellular formation of most class II-peptide complexes. Yet, 70 percent of the DR molecules still bore peptides, 80 percent of which were 21- to 24-amino acid fragments of the class II-associated invariant chain. These peptides were rare on DR3 from control cells. Thus, a defect in the main antigen-processing pathway revealed a process in which DR molecules bind long peptides derived from proteins present in the same compartment.

Asunto(s)

Genes MHC Clase II , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Antígeno HLA-DR3/genética , Péptidos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Eliminación de Gen , Antígeno HLA-DR3/metabolismo , Humanos , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Transfección

RESUMEN

Successive transfers of HLA-DR alpha and beta genes restored expression of HLA-DR antigens to human B-lymphoblastoid cell line, LCL .174, from which all known expressible class II genes are deleted. While transferent cells displayed large amounts of DR on their surfaces, transgene-encoded DR3 molecules lacked a conformation-dependent epitope. DR1-restricted CTL lysis of DR1-expressing transferents pulsed with native influenza virus proteins was greatly reduced; the same cells were efficiently lysed in the presence of CTL-recognized influenza peptides. The properties of DR-expressing transferents of .174 suggest they are defective in producing peptides from exogenous proteins or in forming DR/peptide complexes. Comparison with other DR-expressing deletion mutants indicates that at least one gene in an approximately 230 kb DNA segment between the DQ1 and Ring 7 loci is needed for normal DR-mediated processing and presentation. Production of DR3 molecules having the conformation-dependent 16.23 epitope and efficient DR1-restricted presentation of influenza viral epitopes occurred in a B cell line that has a mutation specifically eliminating expression of the TAP1 transporter gene, which is in the approximately 230 kb interval and is needed for production of HLA class I/peptide complexes.

Asunto(s)

Genes MHC Clase II , Antígenos HLA-DR/genética , Complejo Mayor de Histocompatibilidad , Anticuerpos Monoclonales/inmunología , Células Presentadoras de Antígenos/inmunología , Proteínas Portadoras/genética , Deleción Cromosómica , Antígenos HLA-DR/ultraestructura , Humanos , Técnicas In Vitro , Mutación , Conformación Proteica , Transfección

RESUMEN

B-lymphoblastoid cell line (LCL) 721.221 lacks HLA-A, -B, and -C class I antigens and transcripts as a result of gamma-ray-induced mutations. LCL 721, from which mutant .221 was derived, produces membrane and secreted forms of IgG1(kappa). In contrast, IgG expression in .221 had these characteristics: (i) gamma 1 heavy chains were diminished by 98% but were detectable with chain-specific antibodies in cell lysates; (ii) kappa light chains were present at normal levels in cell lysates and free kappa chains were secreted; (iii) cell-surface-associated IgG and secreted IgG were absent. Mutants that had partially reduced amounts of class I antigens continued to secrete IgG; however, both the absolute amount of IgG secreted and the relative amount of kappa vs. intact IgG secreted were abnormal in such partially class I-deficient cells. The failure to export IgG and the deficiency of HLA-A, -B, and -C were not merely coincidental in mutant .221, since production of IgG was restored by transferring a functional HLA-A, -B, or -C gene into .221. Cell surface antigen expression of cloned HLA-A, -B, and -C transgenes introduced into .221 was comparable to that of the same genes in their normal chromosomal locations. These observations reveal a relation between production of HLA class I gene products and production of IgG.

Asunto(s)

Linfocitos B/inmunología , Antígenos HLA-A/genética , Antígenos HLA-B/genética , Antígenos HLA-C/genética , Inmunoglobulina G/biosíntesis , Receptores de Antígenos de Linfocitos B/biosíntesis , Anticuerpos Monoclonales , Línea Celular , Citometría de Flujo , Genes MHC Clase I , Humanos , Inmunoglobulina G/genética , Mutación , Fenotipo , Receptores de Antígenos de Linfocitos B/genética

RESUMEN

A patient (HLA-A2,3; B35,60) with end-stage renal disease and a high level of pretransfusion (t0) anti-HLA cytotoxic antibodies (60% positive to a random panel), but lacking cytotoxic antibodies against her HLA haploidentical sister (HLA-A2,3; B35,44), received 3 donor-specific transfusions (DST) from the latter: 200 cc fresh whole blood at biweekly intervals, while being treated with azathioprine (AZA, 1 mg/kg/day). Her serum remained negative for antidonor antibodies both by standard cytotoxicity assay and by immunofluorescence flow cytometry after DST + AZA treatment, and she experienced no acute rejection episodes following donor kidney transplantation. Microcytotoxicity inhibition tests were performed using standard HLA-typing sera as a source of Ab-1, and pre- and posttransfusion sera were added to serial dilutions of Ab-1 to test for the presence of Ab-2 (antiidiotype) to donor HLA class I specificities. Although both pre- and posttransfusion sera inhibited cytotoxicity toward HLA-A2 antigens expressed on recipient target cells, only posttransfusion serum was found to inhibit cytotoxicity against the HLA-A2 antigens expressed on donor target cells. Absorption of soluble HLA class I antigens present in pre- or posttransfusion sera removed the inhibition of cytotoxicity toward recipient HLA-A2 but did not affect the inhibition of cytotoxicity toward donor HLA-A2 by posttransfusion sera. The F(ab')2 fragment of the IgG fraction of posttransfusion sera contained the inhibitory activity, suggesting induction of Ab-2 toward idiotypes specific for donor HLA-A2 antigens encoded on the unshared haplotype.

Asunto(s)

Anticuerpos Antiidiotipos/biosíntesis , Suero Antilinfocítico/biosíntesis , Antígenos HLA/inmunología , Idiotipos de Inmunoglobulinas/inmunología , Donantes de Tejidos , Reacción a la Transfusión , Anticuerpos Antiidiotipos/fisiología , Especificidad de Anticuerpos , Suero Antilinfocítico/fisiología , Azatioprina/uso terapéutico , Unión Competitiva , Pruebas Inmunológicas de Citotoxicidad , Femenino , Antígenos HLA/genética , Antígeno HLA-A2 , Antígenos HLA-B , Humanos , Sueros Inmunes/farmacología , Trasplante de Riñón , Fenotipo