ANTIBODY SELECTION AGAINST CD52 PRODUCES A PAROXYSMAL-NOCTURNAL HEMOGLOBINURIA PHENOTYPE IN HUMAN-LYMPHOCYTES BY A NOVEL MECHANISM

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. T reatment of rheumatoid arthritis patients with CAMPATH-1H, a humanized monoclonal antibody against CD52, resulted, in a small number of case s, in the appearance and persistence of CD52-negative T cells. Similar ly, CD52-negative B cells emerged following in vitro treatment of a CD 52-positive human B cell line with CAMPATH-1H. Both the B and T CD52-n egative cells were also found to be defective in surface expression of other GPI-anchored proteins. Biochemical analysis revealed a severe d efect in the synthesis of a mature GPI precursor in both the B and T c ell lines. Therefore the phenotype of these CD52-negative B and T cell s closely resembles that of lymphocytes from patients with paroxysmal nocturnal haemoglobinuria (PNH), in which the first step of the GPI-bi osynthetic 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 fr om the wild-type sequence were detected; therefore a classical PNH mut ation cannot be responsible for the GPI-biosynthesis defect in these c ell lines. Significantly, the GPI-negative phenotype of the B cells wa s reversible upon separation of the positive and negative cells, resul ting in a redistribution to a mixed population with either CD52-positi ve or -negative cells, whereas populations of 100% CD52-negative T cel ls were stably maintained during culture. Therefore, whereas the GPI-b iosynthesis 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 d emonstrate that the PNH phenotype can be generated without a mutation in the PIG-A structural gene, and thereby identify a novel mechanism f or the development of GPI deficiency.