PNH is an acquired clonal disorder of the hematopoietic stem cell (HSC) characterized by intravascular hemolysis, venous thrombosis, and variable degrees of bone marrow failure. In PNH a somatic mutation of the X-linked PIG-A gene in HSC results in complete or partial deficiency of all proteins anchored by the glycosylphosphatidylinositol (GPI) on the membrane of the mutated HSC and in its mature progeny. The close association between PNH and Idiopathic Aplastic Anemia (IAA), and other lines of evidence support the hypothesis that auto-reactive T cells might be responsible for the expansion of hematopoietic PNH clone(s), which is required to cause clinical PNH.
Stemming from our observation of a unique patient with PNH and with a large granular lymphocyte (LGL) leukemia with NKT phenotype (
Karadimitris et al, Br J Haematol 115:1010, 2001
), we have measured systematically the percentage of NKT [CD3+ CD8+(bright) CD57+] cells in the peripheral blood of PNH patients. The proportion of NKT cells was quite variable and very similar in 18 patients (6.9±5.9; range: 0.8 – 22.3%) and in 18 healthy individuals (6.5±5.2; range: 0.9 – 21.2; P>0.5). However, when we analyzed the size distribution of the complementarity-determining region 3 (CDR3) of the TCR-beta chain genes in sorted NKT cells, there was a sharp difference. In healthy individuals we observed a normally distributed ladder of bands of different sizes. By contrast, in 14 out of 15 PNH patients we found a markedly non-random (“oligoclonal”) pattern; and in each patient some clones were predominant. In 6 out of 6 patients followed-up longitudinally over 6–12 months the “oligoclonal” pattern was consistent and persistent. In each of 10 patients in whom we carried out systematic sequencing of the TCR-beta CDR3 of sorted NKT cells we have observed an average of 25 different TCR-beta CDR3 sequences (out of an average of 80 total sequences obtained): but only one or two sequences were predominant. Interestingly, an identical or quasi-identical (single amino acid difference) sequence was found in 4 patients; and in two of these the sequence belonged to one of the predominant clones. In addition, in 5 cases a sequence found in one patient was subsequently found also in another patient. These data are reminiscent of recent findings reported in patients with IAA (Risitano et al, Lancet 364:355, 2004
): in these patients, however, no identity of sequence was detected. We surmise that in both groups of patients specific T cells clones may be responsible for damage to normal HSCs. However, it is possible that in IAA a number of different antigens are recognized on HSCs in individual patients; whereas in PNH the range of potential target antigens is much more restricted, because they must be present on normal HSC but not on PNH HSCs, thus enabling them to survive the auto immune attack and to expand.
