OBJECTIVE: Similar immune mechanisms have been suggested to operate in aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH), and the presence of PNH clones in AA may indicate that an immune reaction directed against hematopoietic stem cells may be responsible for the immune selection pressure leading to PNH evolution. We previously described expansions of selective cytotoxic T-lymphocyte (CTL) clones in AA patients. MATERIALS AND METHODS: We applied a molecular analysis of the T-cell receptor repertoire to study the characteristics of CTL response in patients with various forms of PNH. Immunodominant T-cell clones were detected using combined flow cytometric and molecular analysis of the variable beta (VB) chain and CDR3 representation, followed by determination of the frequency of individual CDR3 clonotypes. Clonotypic polymerase chain reaction (PCR) was performed to establish clonotypic utilization pattern. RESULTS: In patients with a past history of AA, and when subgrouped by current blood counts as "hypoproliferative" PNH patients (in contrast to purely hemolytic form of PNH), more pronounced skewing of VB family utilization was found, consistent with T-cell responses involving several immunodominant CTL clones. Sequences of the PNH-derived clonotypes were used to design PCR-based assays for the utilization analysis of individual clones in PNH patients. The clonotypic distribution pattern established by this PCR method indicated that immunodominant T-cell specificities were shared between some patients but also may be found at low frequencies in controls. CONCLUSION: Analysis of the CDR3 sequence pattern as a marker for expanded immunodominant clonotypes may have an application in the study of T-cell responses in PNH.
OBJECTIVE: Similar immune mechanisms have been suggested to operate in aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH), and the presence of PNH clones in AA may indicate that an immune reaction directed against hematopoietic stem cells may be responsible for the immune selection pressure leading to PNH evolution. We previously described expansions of selective cytotoxic T-lymphocyte (CTL) clones in AA patients. MATERIALS AND METHODS: We applied a molecular analysis of the T-cell receptor repertoire to study the characteristics of CTL response in patients with various forms of PNH. Immunodominant T-cell clones were detected using combined flow cytometric and molecular analysis of the variable beta (VB) chain and CDR3 representation, followed by determination of the frequency of individual CDR3 clonotypes. Clonotypic polymerase chain reaction (PCR) was performed to establish clonotypic utilization pattern. RESULTS: In patients with a past history of AA, and when subgrouped by current blood counts as "hypoproliferative" PNH patients (in contrast to purely hemolytic form of PNH), more pronounced skewing of VB family utilization was found, consistent with T-cell responses involving several immunodominant CTL clones. Sequences of the PNH-derived clonotypes were used to design PCR-based assays for the utilization analysis of individual clones in PNH patients. The clonotypic distribution pattern established by this PCR method indicated that immunodominant T-cell specificities were shared between some patients but also may be found at low frequencies in controls. CONCLUSION: Analysis of the CDR3 sequence pattern as a marker for expanded immunodominant clonotypes may have an application in the study of T-cell responses in PNH.
Authors: Marcin W Wlodarski; Lukasz P Gondek; Zachary P Nearman; Magdalena Plasilova; Matt Kalaycio; Eric D Hsi; Jaroslaw P Maciejewski Journal: Blood Date: 2006-04-13 Impact factor: 22.113