Oligoclonal and polyclonal CD4 and CD8 lymphocytes in aplastic anemia and paroxysmal nocturnal hemoglobinuria measured by Vβ CDR3 spectratyping and flow cytometry

Abstract

SUMMARYWe have hypothesized that in aplastic anemia (AA) the presence of antigen-specific T-cells is reflected by their contribution to the expansion of a particular variable beta chain (V ) subfamily and also by clonal CDR3 skewing. To determine the role of disease-specific "signature" T-cell clones in AA, we studied preferential Vusage by flow cytometry and analyzed V -CDR3 regions for the presence of oligoclonality. We first established the contribution of each V family to the total CD4+ and CD8+ lymphocyte pool; in AA and PNH, a seemingly random over-representation of different V families was observed. On average, we found expansion in 3 (out of 22 examined) V families per patient. When the contribution of individual V families to the effector pool was examined, more striking V -skewing was found. V -CDR3 size distribution was analyzed for the expanded V families in isolated CD4+ and CD8+ populations; under-represented V families displayed more pronounced CDR3 skewing. Expanded CD4+V subfamilies showed mostly a polyclonal CDR3 size distribution with only 38% of skewing in expanded V families. In contrast, within over-represented CD8+V types, marked CDR3 skewing (82%) was seen, consistent with non-random expansion of specific CD8+ T-cell clones. No preferential expansion of particular V families was observed, in relation with HLA-type. In patients examined before and after immunosuppressive therapy, an abnormal V -distribution pattern was retained, but the degree of expansion of individual V was lower. As V -skewing may correlate with relative V size, oligoclonality in combination with numerical V expansion can be applied to recognition of disease-specific TCR.

Large granular lymphocyte (LGL)-like clonal expansions in paroxysmal nocturnal hemoglobinuria (PNH) patients

“…7 After a 20 min incubation at room temperature, erythrocytes were lysed, the remaining cells were fixed with a Q-prep apparatus (Beckman-Coulter). Then they were further analyzed by a Coulter XL flow cytometer equipped with an Epics Elite software.…”

“…A four color protocol was used: the individual percentage contribution of each of the 22 Vb-subfamilies mAb was determined as previously described. 7 All mAbs were obtained from Beckman-Coulter (Fullerton, CA, USA), except anti-Vb 6.7 (Endogen, Woburn, MA, USA); mAbs anti-CD55 and anti-CD59 were used for analysis of GPI-anchored proteins (Pharmigen, San Diego, CA, USA).…”

“…Our group has systematically studied cellular immune responses in different hematologic diseases characterized by bone marrow failure. We initially reported T-cell oligoclonality by CDR3 spectratyping in AA and PNH; 7 more recently, we identified patient-specific cytotoxic T-cells (CTL) clones in these syndromes. 13,14 Such clones were immunodominant in vivo and correlated with disease activity; thus they likely have a pathogenic role.…”

“…In support, association with particular HLA-antigens (HLA-DR1501) 5 and abnormalities of the immune system such as oligoclonality of the TCR repertoire have been described in PNH. 6,7 The TCR repertoire is physiologically extremely diverse, as a result of somatic V-(D)-J rearrangement and the contribution of nongermline N nucleotides; 8 dominance of a few TCRs over the heterogeneous repertoire is typical of oligoclonal T-cell immune response. 9 TCR clonality may be assessed by flow cytometric analysis of Vb usage 10 and by molecular studies, 11,12 including size-distribution analysis and sequencing of the highly variable complementarity determining region 3 (CDR3; the site of peptide binding), giving a unique signature of a T-cell clone or clonotype.…”

A patient with paroxysmal nocturnal hemoglobinuria, T cell large granular lymphocyte clonal expansion, and monoclonal gammopathy of undetermined significance

“…The association of PNH with clonal T cell populations has been reported recently, using flow cytometric and molecular analyses of the T cell receptor bchain variable region [6][7][8][9][10]. In our case, we detected T cell clones using a PCR-based method to analyze TCR-Vg.…”