The CD52 antigen is a lymphocyte glycoprotein with an extremely short polypeptide backbone and a single N-linked glycan, and it is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Treatment of rheumatoid arthritis patients with CAMPATH-1H, a humanized monoclonal antibody against CD52, resulted, in a small number of cases, in the appearance and persistence of CD52-negative T cells. Similarly, CD52-negative B cells emerged following in vitro treatment of a CD52-positive human B cell line with CAMPATH-1H. Both the B and T CD52-negative cells were also found to be defective in surface expression of other GPI-anchored proteins. Biochemical analysis revealed a severe defect in the synthesis of a mature GPI precursor in both the B and T cell lines. Therefore the phenotype of these CD52-negative B and T cells closely resembles that of lymphocytes from patients with paroxysmal nocturnal haemoglobinuria (PNH), in which the first step of the GPI-biosynthetic pathway, i.e. synthesis of GlcNAc-phosphatidylinositol, is blocked. In all cases studied to date, this defect maps to a mutation of the phosphatidylinositolglycan class A (PIG-A) structural gene. We therefore amplified the PIG-A gene from both the GPI-negative B and T cells by PCR and determined the nucleotide sequence. No differences from the wild-type sequence were detected; therefore a classical PNH mutation cannot be responsible for the GPI-biosynthesis defect in these cell lines. Significantly, the GPI-negative phenotype of the B cells was reversible upon separation of the positive and negative cells, resulting in a redistribution to a mixed population with either CD52-positive or -negative cells, whereas populations of 100% CD52-negative T cells were stably maintained during culture. Therefore, whereas the GPI-biosynthesis deficiency in the T cell lines may be due to a mutation in another gene required by the GPI-biosynthetic pathway, the reversible nature of this block in the B cell lines suggests a less direct cause, possibly an alteration in a regulatory factor. Overall, these data demonstrate that the PNH phenotype can be generated without a mutation in the PIG-A structural gene, and thereby identify a novel mechanism for the development of GPI deficiency.
