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

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