RESUMEN
Crossreactivity of anti-HLA antibodies with SLA alleles may limit the use of pig xenografts in some highly sensitized patients. An understanding of the molecular basis for this crossreactivity may allow better selection of xenograft donors. We have tested 68 human monoclonal HLA class I antibodies (mAbs) for reactivity with pig lymphocytes from SLA defined pigs and found nine to be crossreactive. Eight of nine were broadly HLA reactive IgM-mAbs. The putative HLA epitopes for seven mAbs. were conserved in the aminoacid sequence of the SLA alleles studied. The lack of reactivity of a large number of mAbs largely correlated with the absence of the putative epitopes in the SLA alleles studied. We conclude that most patients with anti-HLA class I antibodies should be able to find pig donors lacking SLA antigens that cross react with their antibodies and that many of the crossreacting epitopes can be defined by analysis of shared epitopes in the aminoacid sequence of human and pig MHC antigens.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Reacciones Cruzadas/inmunología , Epítopos/inmunología , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Sus scrofa/inmunología , Trasplante Heterólogo/inmunología , Alelos , Animales , Pruebas Inmunológicas de Citotoxicidad , Citometría de Flujo , Haplotipos/genética , Humanos , Ratones , Especificidad de la Especie , Sus scrofa/genéticaRESUMEN
The ability of tetrameric major histocompatibility complex (MHC) class I-peptide complexes (tetramers) to detect antigen-specific T lymphocyte responses has yielded significant information about the generation of in vivo immunity in numerous antigenic systems. Here we present a novel method for rapid validation of tetrameric HLA molecules based on the presence of allodeterminants. Human monoclonal antibodies (mAbs) recognizing polymorphic determinants on HLA class I were immobilized on polystyrene microparticles and used to probe the structural integrity of tetrameric HLA class I molecules by flow cytometry. A total of 22 tetramers, based on HLA-A1, A2, A3, A24, B7 and B8 were reactive with their counterpart mAbs, thus confirming their antigenic integrity. A positive outcome of this mAb test ensures that tetrameric HLA class I can be used with greater confidence in subsequent functional assays.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Antígenos HLA-A/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Especificidad de Anticuerpos , Células Cultivadas , Calor , Humanos , Microesferas , Polimorfismo Genético , Poliestirenos/químicaRESUMEN
MHC class I molecules expressed on cell surfaces are composed of H chain, beta2-microglobulin and any of a vast array of peptides. The role of peptide in the recognition of HLA class I by serum HLA Abs is unknown. In this study, the solid-phase assay of a series (n = 11) of HLA-A2-reactive, pregnancy-induced, human mAbs on a panel (n = 12) of recombinant monomeric HLA-A2 molecules, each containing a single peptide, revealed peptide selectivity of the mAbs. The flow cytometry membrane staining intensities on the HLA-A2-transduced cell line K562, caused by these mAbs, correlated with the number of monomer species detected by the mAbs. Flow cytometry staining on HLA-A2-bearing cell lines of a variety of lineages was indicative of tissue selectivity of these HLA-A2 mAbs. This tissue selectivity suggests that the deleterious effect on allografts is confined to alloantibodies recognizing only HLA class I loaded with peptides that are derived from tissue-specific and household proteins. Since Abs that are only reactive with HLA loaded with irrelevant peptides are expected to be harmless toward allografts, the practice of HLA Ab determination on lymphocyte-derived HLA deserves reconsideration.
Asunto(s)
Antígeno HLA-A2/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Isoanticuerpos/inmunología , Péptidos/inmunología , Anticuerpos Monoclonales/inmunología , Linaje de la Célula , HumanosRESUMEN
BACKGROUND: Human leukocyte antigen (HLA)-C is expressed on nucleated cells and platelets in lower levels than HLA-A,B, and its antigens are in linkage disequilibrium with HLA-B antigens. Therefore, HLA-C antibody detection is difficult. The authors questioned whether HLA-C could serve as a target in clinical kidney transplantation using a newly developed assay. METHODS: Flow cytometry was performed with sera from patients (n=34) awaiting a kidney retransplant using nine cell lines expressing a single HLA-C antigen (single-antigen lines [SAL]). RESULTS: The SAL were validated with HLA-C-specific alloantisera and human monoclonal antibodies against HLA-A, -B, and -C. The results were in agreement with the specificities previously reported. Exceptions, because of new HLA-C specificities used here, could be explained by epitope sharing between the antigens. With respect to patient sera, 15 of the 34 patients tested (44%) showed serum reactivity toward one or more HLA-C SAL. CONCLUSIONS: In contrast to peripheral blood lymphocytes, SAL are excellent targets for detecting HLA-C-reactive alloantibodies by flow cytometry. This preliminary analysis revealed that HLA-C-reactive antibodies are frequently present in sera of retransplant patients, serving as possible targets in clinical transplantation.