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Paroxysmal nocturnal haemoglobinuria (PNH) results from acquired mutations in the PIG-A gene of an haematopoietic stem cell, leading to defective biosynthesis of glycosylphosphatidylinositol (GPI) anchors and deficient expression of GPI-anchored proteins on the surface of the cell's progeny. Some laboratory and clinical findings have suggested genomic instability to be intrinsic in PNH; this possibility has been supported by mutation analysis of hypoxanthine-guanine phosphoribosyltransferase ( HPRT) gene abnormalities. However, the HPRT assay examines lymphocytes in peripheral blood (PB), and T cells may be related to the pathophysiology of PNH. We analysed the molecular and functional features of HPRT mutants in PB mononuclear cells from eleven PNH patients. CD8 T cells predominated in these samples; approximately half of the CD8 cells lacked GPI-anchored protein expression, while only a small proportion of CD4 cells appeared to derive from the PNH clone. The HPRT mutant frequency (Mf) in T lymphocytes from PNH patients was significantly higher than in healthy controls. The majority of the mutant T lymphocyte clones were of CD4 phenotype, and they had phenotypically normal GPI-anchored protein expression. In PNH patients, the majority of HPRT mutant clones were contained within the V β2 T cell receptor (TCR) subfamily, which was oligoclonal by complementarity-determining region three (CDR3) size analysis. Our results are more consistent with detection of uniform populations of expanded T cell clones, which presumably acquired HPRT mutations during antigen-driven cell proliferation, and not due to an increased Mf in PNH. HPRT mutant analysis does not support underlying genomic instability in PNH. [ABSTRACT FROM AUTHOR]

Summary. T cell-mediated suppression of haematopoiesis is believed to play an important role in the pathophysiology of aplastic anaemia (AA) and in the pancytopenia of some myelodysplastic syndromes (MDS). Natural-killer T (NKT) cells belong to a unique lymphocyte subset that expresses an invariant T-cell receptor (TCR), consisting of Vα24JαQ, and common NK cell surface markers. NKT cells have been hypothesized to play a role in immune regulation, and many human autoimmune conditions are associated with NKT cell deficiency. Here we investigate the role of NKT cells in AA and MDS patients. Flow cytometry demonstrated that NKT cells, unlike other T-lymphocyte subpopulations, were disproportionally decreased in AA and MDS marrow. When we compared variability within the CDR3 region of Vα24 in CD4– CD8– T cells derived from AA and healthy individuals, the CDR3 size of Vα24 cells showed a polyclonal distribution in AA patients, while in control subjects a typical oligoclonal or monoclonal pattern was found. Southern blot and sequence analysis of Vα24 polymerase chain reaction products revealed that the NKT cell-specific JαQ region was predominant in control subjects, whereas it was not, or only very weakly, detected in AA and MDS patients. These results show that NKT cells are profoundly decreased in AA and MDS, and their deficiency may, as in other human autoimmune diseases, play a role in the local immune dysregulation in AA and MDS. [ABSTRACT FROM AUTHOR]

In an attempt to elucidate the pathogenic role of a CD4+ cytotoxic T-cell clone NT4.2 isolated from the bone marrow of a patient with cyclosporine-dependent aplastic anaemia, we characterized the T-cell clone as well as its cytotoxicity against an autologous Epstein-Barr (EB) virus-transformed B-lymphoblastoid cell line (LCL). NT4.2 expressed BV21+ BJ2.7+ with a complementarity-determining region (CDR) 3 motif of SQGQGEVEQY which was homologous to that of a T-cell clone isolated from a patient with connective tissue disease. NT4.2 started to lyse LCL cells within 2 h and exerted maximal cytotoxicity within 3 h of incubation. The cytotoxicity required the presence of divalent cations and was not associated with DNA fragmentation of the target cells. Anti-CD59 monoclonal antibodies (MoAb) blocked the cytotoxicity to the same degree as anti-CD3, HLA-DR or CD2 mAb. Flow cytometric analysis of the peripheral blood of this patient during remission after cyclosporine therapy revealed 1.7% of granulocytes to be deficient in CD59. These findings indicate that NT4.2 exerts its cytotoxicity through a perforin-mediated pathway, not a Fas/Fas ligand-dependent pathway, and that haemopoietic stem cells lacking CD59 may evade cytotoxic T lymphocytes, leading to the in vivo expansion of a paroxysmal nocturnal haemoglobinuria clone. [ABSTRACT FROM AUTHOR]

Background Aplastic anaemia is a bone-marrow-failure syndrome characterised by low blood-cell counts and fatty bone marrow. In most cases, no obvious aetiological factor can be identified. However, clinical responses to immunosuppression strongly suggest an immune pathophysiology. Methods To test the hypothesis that aplastic anaemia results from antigen-specific lymphocyte attack against haemopoietic tissue, we analysed effector immunity, seeking especially dominant specific T-cell responses. Blood samples from 54 patients with aplastic anaemia were subjected to flow cytometry to define T-cell-receptor V -chain usage and expansion of particular V subsets. We measured the size distribution of the complementarity determining region 3 (CDR3) for expanded V subsets, then cloned and sequenced skewed, oligoclonal, or monoclonal peaks. Findings Expanded V subsets were identified in almost all the patients. Over-represented V subsets from CD8-positive cells showed oligoclonal or monoclonal CDR3 size patterns. The CDR3 sequence repertoire in aplastic anaemia showed much redundancy compared with healthy donors. We identified patient-specific putative pathogenetic clonotypes that were not detectable in controls. In selected patients who were assessed longitudinally, these clonotypes were quantitatively related to disease activity. Selective killing of autologous haemopoietic progenitors by the V -specific lymphocyte population was shown in one patient. These apparently pathogenetic CDR3 sequences showed homology between individuals, suggesting a role for a "semi-public" immune response in the pathophysiology of aplastic anaemia. Interpretation In-vivo dominant clonal immune response can be identified in many patients with aplastic anaemia, which is evidence for an underlying antigen-driven immune process. [ABSTRACT FROM AUTHOR]