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

Abstract

Abstract We 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 (Vβ) subfamily and also by clonal CDR3 skewing. To determine the role of disease-specific “signature” T-cell clones in AA, we studied preferential Vβ usage by flow cytometry and analyzed Vβ-CDR3 regions for the presence of oligoclonality. We first established the contribution of each Vβ family to the total CD4+ and CD8+ lymphocyte pool; in AA and paroxysmal nocturnal hemoglobinuria, a seemingly random overrepresentation of different Vβ families was observed. On average, we found expansion in 3 (of 22 examined) Vβ families per patient. When the contribution of individual Vβ families to the effector pool was examined, more striking Vβ skewing was found. Vβ-CDR3 size distribution was analyzed for the expanded Vβ families in isolated CD4+ and CD8+ populations; underrepresented Vβ families displayed more pronounced CDR3 skewing. Expanded CD4+Vβ subfamilies showed mostly a polyclonal CDR3 size distribution with only 38% of skewing in expanded Vβ families. In contrast, within overrepresented CD8+Vβ types, marked CDR3 skewing (82%) was seen, consistent with nonrandom expansion of specific CD8+ T-cell clones. No preferential expansion of particular Vβ families was observed, in relation to HLA-type. In patients examined after immunosuppressive therapy, an abnormal Vβ-distribution pattern was retained, but the degree of expansion of individual Vβ was lower. As Vβ skewing may correlate with relative Vβ size, oligoclonality in combination with numerical Vβ expansion can be applied to recognition of disease-specific T-cell receptors.