SEARCH

SEARCH BY CITATION

Keywords:

  • paroxysmal nocturnal haemoglobinuria;
  • hypoxanthine-guanine phosphoribosyltransferase gene;
  • GPI-anchored protein;
  • mutant frequency

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patient samples
  5. Peripheral blood cell preparation
  6. Phenotypic characterization of T-cells
  7. Cloning assay for HPRT mutant T-cells in vitro
  8. Methylcellulose colony-forming assay for HPRT mutation haematopoietic progenitor cells in vitro
  9. T cell receptor Vβ usage and complementarity determining region 3 (CDR3) size distribution analysis in HPRT mutation T cell clones
  10. Statistical analysis
  11. Results
  12. T lymphocytes phenotype in PNH
  13. 6-TG-resistant T cells in PNH
  14. Phenotype of HPRT mutant T cells
  15. TCR Vβ usage and CDR3 size distribution in HPRT mutant T cell clones
  16. Discussion
  17. References

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 Vβ2 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.

In paroxysmal nocturnal haemoglobinuria (PNH), acquired mutations in the PIG-A gene of an haematopoietic stem cell lead to defective biosynthesis of glycosylphosphatidylinositol (GPI) anchors and deficient expression of all GPI-anchored proteins (GPI-AP) on the surface of the cell's progeny; with sufficient expansion of the mutant clone, clinical intravascular haemolysis, venous thromboses, and defective haematopoiesis may result (Dunn et al, 1999; Rosse, 2000). Although well understood at the molecular and biochemical levels, the explanation for the crucial event of clonal expansion remains unclear. One provocative hypothesis has suggested that genomic instability underlies PNH. PNH can be associated with myelodysplasia, a preleukaemic bone marrow (BM) condition, and with the presence of cytogenetic abnormalities (Sloand et al, 2003; Tomonaga et al, 2003). Disease-causing mutations are present randomly throughout the PIG-A nucleic acid sequence, without hot spots; furthermore, multiple genetically-defined PNH clones may co-exist in a single patient (Nafa et al, 1998; Kawaguchi et al, 1999; Mortazavi et al, 2003). Direct evidence for genomic instability has come from measurements of somatic mutation rates in blood cell progenitors, especially for the glycophorin A loci and the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene (Hattori et al, 1997; Purow et al, 1999; Horikawa et al, 2002). An increased prevalence of specific gene mutations within PNH cells has been reported to contribute to clonal proliferation and selection (Inoue et al, 2003).

However, other observations of patients and laboratory experiments do not support the hypothesis that PNH is associated with an increase in mutability. First, most PNH patients do not show leukaemic progression and cytogenetic abnormalities usually occur outside the PNH clone (Rosse & Nishimura, 2003; Sloand et al, 2003). In ‘knock-out’ murine models, PNH cells do not appear to have an intrinsic proliferative advantage and they tend to disappear rather than to increase in number over time (Okuda et al, 1990; Dunn et al, 1996). PNH haematopoietic stem cells have been inferred to be present in most healthy individuals with small numbers of GPI-AP-deficient circulation granulocytes detectable (Araten et al, 1999). PNH is strongly associated with aplastic anaemia (AA), a disease that, in most cases, appears to be immune-mediated (Maciejewski et al, 2001a; Young et al, 2002), and the presence of a PNH clone is correlated with both a specific histocompatability locus and with the likelihood of response to immunosuppressive therapy (Maciejewski et al, 2001b; Wang et al, 2002). In AA, myelodysplasia, and PNH, our laboratory and others have demonstrated expanded populations of cytotoxic T cells, and oligoclonality within these populations suggests an antigen-driven process (Nakao et al, 1994; Kook et al, 2001; Maciejewski et al, 2001b; Risitano et al, 2002). Both functional (Chen et al, 2000, 2002a) and microarray experiments (Chen et al, 2002b) have suggested that PNH clones may expand due to escape from immune attack.

Measurement of the mutation rate in eukaryotic cells is problematic. Analysis of HPRT mutations, which confer a selective advantage for cells in culture, has been utilized for this purpose (Monteiro et al, 2000; Albertini, 2001). An increase in mutant frequency (Mf) detected by HPRT has been linked to environmental exposures to mutagens such as smoking, malignancy and in cancer postchemotherapy (Abramson et al, 1999; Monteiro et al, 2000; Albertini, 2001; Rice et al, 2003). However, the assay is dependent on the proliferation of lymphocytes, mainly T cells, in the culture system. As a result, the clonal expansion of T cells has also yielded apparent increases in Mf, presumably stochastically related to the increased numbers of cell proliferations rather than to genomic instability (Allegretta et al, 1990; Abramson et al, 1999; Falta et al, 1999). Therefore, we first attempted to confirm that HPRT mutations were more frequent in lymphocytes of PNH patients and, second, to determine whether the distribution of these mutations within the T cell compartment was due to an increase in mutagenesis as a whole or reflected the expansion of specific T cell clones.

Patient samples

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patient samples
  5. Peripheral blood cell preparation
  6. Phenotypic characterization of T-cells
  7. Cloning assay for HPRT mutant T-cells in vitro
  8. Methylcellulose colony-forming assay for HPRT mutation haematopoietic progenitor cells in vitro
  9. T cell receptor Vβ usage and complementarity determining region 3 (CDR3) size distribution analysis in HPRT mutation T cell clones
  10. Statistical analysis
  11. Results
  12. T lymphocytes phenotype in PNH
  13. 6-TG-resistant T cells in PNH
  14. Phenotype of HPRT mutant T cells
  15. TCR Vβ usage and CDR3 size distribution in HPRT mutant T cell clones
  16. Discussion
  17. References

We studied 11 patients with PNH and eight healthy, non-smoking volunteers who were approximately age-matched with the cases (Table I). Informed consent was obtained according to protocols approved by the Institutional Review Board of the National Heart, Lung and Blood Institute. Clinical features enabled the patients to be characterized as having predominantly haemolytic PNH or AA/PNH syndrome. Haemolytic PNH was defined by a hypercellular BM with normal karyotype and without dysplasia, and platelet count >60 × 109/l, absolute neutrophil count (ANC) >1·2 × 109/l, and reticulocyte count >100 × 109/l. In contrast, AA/PNH showed a normal or hypocellular BM and at least one blood count below the parameters described above. PNH itself was diagnosed by the absence of two GPI-AP, CD55 and CD59, on the surface of both granulocytes and red blood cells, as described previously (Dunn et al, 1999).

Table I.  Patients’ characteristics.
 Current diagnosisAge (years)GenderANC × 109/l Retics × 109/lPlatelets × 109/lGPI-AP-deficient granulocytes (%)Transfusion- dependent at samplingCurrent therapyPrevious-therapies
  1. ATG, antithymocyte globulin; CsA, ciclosporin A; Retics, reticulocytes; Steroids, corticosteroids.

 1PNH27F1·233035997·7NNoneATG, CsA
 2PNH/AA54F0·3132392·2YNoneSteroids
 3PNH31M2·276·521493·8YNoneATG, CsA, steroids
 4PNH27M3·537213799·3YSteroidsSteroids
 5PNH33F1·69632666·8YSteroidsSteroids
 6PNH43F1·42306992·8YNoneATG
 7PNH/AA31M0·91072431·7NNoneATG
 8PNH/AA65F0·528·42244YNoneATG, CsA
 9PNHAA54F0·3132398·7NDacluzimabNone
10PNH33M4·292·75050·6NSteroidsATG
11PNH/AA51M0·886·910051·7YNoneATG, CsA

Peripheral blood cell preparation

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patient samples
  5. Peripheral blood cell preparation
  6. Phenotypic characterization of T-cells
  7. Cloning assay for HPRT mutant T-cells in vitro
  8. Methylcellulose colony-forming assay for HPRT mutation haematopoietic progenitor cells in vitro
  9. T cell receptor Vβ usage and complementarity determining region 3 (CDR3) size distribution analysis in HPRT mutation T cell clones
  10. Statistical analysis
  11. Results
  12. T lymphocytes phenotype in PNH
  13. 6-TG-resistant T cells in PNH
  14. Phenotype of HPRT mutant T cells
  15. TCR Vβ usage and CDR3 size distribution in HPRT mutant T cell clones
  16. Discussion
  17. References

Blood was obtained through venipunctures into heparinized tubes (Becton Dickinson, San Diego, CA, USA). Peripheral blood (PB) mononuclear cells (PBMNC) were isolated by density gradient centrifugation using lymphocyte separation medium (Organon, Durham, NC, USA). After washing in Hank's balanced salt solution (Life Technologies, Gaithersburg, MD, USA), the cells were resuspended in Iscove's modified Dulbecco's medium (Life Technologies) supplemented with 20% fetal calf serum (Life Technologies).

Phenotypic characterization of T-cells

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patient samples
  5. Peripheral blood cell preparation
  6. Phenotypic characterization of T-cells
  7. Cloning assay for HPRT mutant T-cells in vitro
  8. Methylcellulose colony-forming assay for HPRT mutation haematopoietic progenitor cells in vitro
  9. T cell receptor Vβ usage and complementarity determining region 3 (CDR3) size distribution analysis in HPRT mutation T cell clones
  10. Statistical analysis
  11. Results
  12. T lymphocytes phenotype in PNH
  13. 6-TG-resistant T cells in PNH
  14. Phenotype of HPRT mutant T cells
  15. TCR Vβ usage and CDR3 size distribution in HPRT mutant T cell clones
  16. Discussion
  17. References

Peripheral blood T cells were stained with anti-CD4 fluoroscein isothyiocyanate (FITC)-conjugated, anti-CD8 FITC-conjugated, and anti-CD55 or CD59 phycoerythrin (PE)-conjugated monoclonal antibodies (mAb; all from PharMingen, San Diego, CA, USA) at 12°C for 20 min. Cells were analysed for expression of CD4, CD8, CD55, and CD59 by comparison with appropriate isotypic controls based on side and forward scatter properties using an EPICS Altra flow cytometer (Beckman Coulter, Fullerton, CA, USA).

Cloning assay for HPRT mutant T-cells in vitro

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patient samples
  5. Peripheral blood cell preparation
  6. Phenotypic characterization of T-cells
  7. Cloning assay for HPRT mutant T-cells in vitro
  8. Methylcellulose colony-forming assay for HPRT mutation haematopoietic progenitor cells in vitro
  9. T cell receptor Vβ usage and complementarity determining region 3 (CDR3) size distribution analysis in HPRT mutation T cell clones
  10. Statistical analysis
  11. Results
  12. T lymphocytes phenotype in PNH
  13. 6-TG-resistant T cells in PNH
  14. Phenotype of HPRT mutant T cells
  15. TCR Vβ usage and CDR3 size distribution in HPRT mutant T cell clones
  16. Discussion
  17. References

The prevalence of HPRT mutant lymphocytes was determined by standard protocols (Purow et al, 1999; Albertini, 2001). In brief, PBMNC were cultured under limiting dilution in 96-well round-bottomed microtitre plates at 1, 2, 5, and 10 cells per well, respectively, without 6-thioguanine (6-TG; Sigma, St Louis, MO, USA), and at 2 × 104 cells per well in the presence of 6-TG at a final concentration of 10 μmol/l. The total well volume of 0·2 ml contained Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies), 10% human AB serum (Valley Biomedical, Winchester, VA, USA), 1%l-glutamine (Life Technologies), 1% minimal essential amino acids, 1% sodium pyruvate, 1% penicillin-streptomycin (Life Technologies), 50 μg/l interleukin (IL) 2 (StemCell Technologies, Vancouver, Canada), 125 ng/ml phytohaemagglutinin (PHA; Sigma), and 1 × 104 irradiated (9K Grey) human lymphoblastoid feeder cells (36X4; the kind gift of Professor R.J. Albertini, University of Vermont, Montpelier, VT, USA). Clones were scored using an inverted microscope after 12–16 d of culture. The cloning efficiency (CE) was calculated using the Poisson relationship:

P0 = e−x of CE, CE = −(ln P0)/x, in which P0 is the fraction of negative wells, and x the average number of cells inoculated per well by limiting dilution.

Mf was equivalent to CE in the presence of 6-TG compared to CE in the absence of this drug.

The phenotypic characteristics of HPRT mutant T cells were determined by flow cytometry with antibodies as described above.

Methylcellulose colony-forming assay for HPRT mutation haematopoietic progenitor cells in vitro

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patient samples
  5. Peripheral blood cell preparation
  6. Phenotypic characterization of T-cells
  7. Cloning assay for HPRT mutant T-cells in vitro
  8. Methylcellulose colony-forming assay for HPRT mutation haematopoietic progenitor cells in vitro
  9. T cell receptor Vβ usage and complementarity determining region 3 (CDR3) size distribution analysis in HPRT mutation T cell clones
  10. Statistical analysis
  11. Results
  12. T lymphocytes phenotype in PNH
  13. 6-TG-resistant T cells in PNH
  14. Phenotype of HPRT mutant T cells
  15. TCR Vβ usage and CDR3 size distribution in HPRT mutant T cell clones
  16. Discussion
  17. References

To assess the Mf of haematopoietic progenitor cells, fresh BM from four normal healthy volunteers and three PNH patients was studied. BM mononuclear cells (BMMNC) were isolated by density gradient centrifugation using lymphocyte separation medium (Organon), and stained with anti-CD55 and anti-CD59 (both PE-conjugated mAb from PharMingen) and FITC-labelled anti-CD34 mAb (PharMingen) at 12°C for 20 min. After washing, CD55+ CD59+ and CD55 CD59 CD34 cells were sorted using a flow cytometer (Moflo, Dako-Cytomation, Ft Collins, CO, USA). Gates were set based on appropriate isotypic controls. The purity of sorted cells ranged from 95–98%. Either population of CD34 cells were used for methylcellulose colony assay at 2 × 105 and 1 × 103 cells/ml in culture dishes (35 mm dish, Becton Dickinson, Franklin Lakes, NJ, USA) with and without 6-TG, respectively. Supraoptimal steady-state growth conditions were achieved with a cocktail of haematopoietic growth factors containing 50 ng/ml IL-3 (Genzyme, Boston, MA, USA), 2 U/ml erythropoietin (EPO, Amgen, Thousand Oaks, CA, USA), and 50 ng/ml granulocyte-macrophage colony stimulating factor (GM-CSF), 50 ng/ml stem cell factor (SCF), 20 ng/ml thrombopoietin (TPO), 50 ng/ml granulocyte colony-stimulating factor (G-CSF), 20 ng/ml Flt-3 (all from Stem Cell Technologies). Methycellulose was supplemented with 10% heat-inactivated (56°C for 30 min) AB serum (both reagents from StemCell Technologies). Cultures were performed in duplicate and were scored after 14 d of growth for colony formation including eyrthroid colony-forming units (CFU-E), erythroid burst-forming units (BFU-E), granulocyte-macrophage colony-forming units (GM-CFU), and granulocyte/erythroid/megakaryocyte CFU (CFU-GEMM).

T cell receptor Vβ usage and complementarity determining region 3 (CDR3) size distribution analysis in HPRT mutation T cell clones

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patient samples
  5. Peripheral blood cell preparation
  6. Phenotypic characterization of T-cells
  7. Cloning assay for HPRT mutant T-cells in vitro
  8. Methylcellulose colony-forming assay for HPRT mutation haematopoietic progenitor cells in vitro
  9. T cell receptor Vβ usage and complementarity determining region 3 (CDR3) size distribution analysis in HPRT mutation T cell clones
  10. Statistical analysis
  11. Results
  12. T lymphocytes phenotype in PNH
  13. 6-TG-resistant T cells in PNH
  14. Phenotype of HPRT mutant T cells
  15. TCR Vβ usage and CDR3 size distribution in HPRT mutant T cell clones
  16. Discussion
  17. References

Total RNA was extracted from HPRT mutant T cell clones using TRIzol reagent (Life Technologies) following the manufacturer's protocol (Zeng et al, 2001; Risitano et al, 2002). Briefly, 500 ng RNA from each sample was reverse-transcribed into cDNA primed with oligo (dT)12–18 using the SuperScript II RT kit (Life Technologies). cDNA was amplified using the polymerase chain reaction (PCR) with 22 different TCR Vβ families and a constant Vβ common primer. PCR products were revealed by 1·5% agarose gel electrophoresis.

For CDR3 size analysis, 1 μl of each amplified product from Vβ2 subfamilies was mixed with 12 μl deionized formamide (Sigma) and 0·5 μl size standard at 95°C for 2 min, chilled on ice, applied to an ABI 310 sequencer, and analysed using Genescan software (both from Perkin-Elmer Biosystems, Foster City, CA, USA).

T lymphocytes phenotype in PNH

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patient samples
  5. Peripheral blood cell preparation
  6. Phenotypic characterization of T-cells
  7. Cloning assay for HPRT mutant T-cells in vitro
  8. Methylcellulose colony-forming assay for HPRT mutation haematopoietic progenitor cells in vitro
  9. T cell receptor Vβ usage and complementarity determining region 3 (CDR3) size distribution analysis in HPRT mutation T cell clones
  10. Statistical analysis
  11. Results
  12. T lymphocytes phenotype in PNH
  13. 6-TG-resistant T cells in PNH
  14. Phenotype of HPRT mutant T cells
  15. TCR Vβ usage and CDR3 size distribution in HPRT mutant T cell clones
  16. Discussion
  17. References

As assessed by flow cytometry using directly conjugated fluorescent monoclonal antibodies, the majority of T cells in the circulation of patients with PNH were of CD8 phenotype (41 ± 4·9%), in comparison with a lower proportion of CD4 cells (36 ± 8·4%); thus, the ratio of CD4/CD8 was decreased relative to normal individuals (0·9 and 1·9, respectively, Fig 1A). On average, a large proportion of CD8 cells showed the absence of GPI-AP in the PNH patients (44 ± 12%), when CD55 and CD59 were combined as markers, in comparison with a much smaller proportion of CD4 cells (5 ± 4·3%), which appeared to derive from the PNH clone (Fig 1B).

image

Figure 1. Phenotype of PB T lymphocytes. CD4, CD8, and GPI-AP-deficient lymphocytes in individuals were determined by flow cytometry using appropriate mAbs. (A) Distribution of CD4 and CD8 cells in normal and PNH patients. (B) Proportion of GPI-AP-deficient CD4 and CD8 cells in individual PNH patients.

Download figure to PowerPoint

6-TG-resistant T cells in PNH

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patient samples
  5. Peripheral blood cell preparation
  6. Phenotypic characterization of T-cells
  7. Cloning assay for HPRT mutant T-cells in vitro
  8. Methylcellulose colony-forming assay for HPRT mutation haematopoietic progenitor cells in vitro
  9. T cell receptor Vβ usage and complementarity determining region 3 (CDR3) size distribution analysis in HPRT mutation T cell clones
  10. Statistical analysis
  11. Results
  12. T lymphocytes phenotype in PNH
  13. 6-TG-resistant T cells in PNH
  14. Phenotype of HPRT mutant T cells
  15. TCR Vβ usage and CDR3 size distribution in HPRT mutant T cell clones
  16. Discussion
  17. References

In the absence of 6-TG, under normal lymphocyte culture conditions, the CE of T cells for PNH patients was lower than that of healthy controls (21 ± 0·07 compared with 38 ± 0·03, P < 0·05, Fig 2A); nevertheless, PNH blood T cells yielded relatively more 6-TG-resistant colonies (Fig 2B). As a result, calculations showed an approximate 20-fold higher average Mf based on HPRT mutants for PNH patients compared with controls, a significant difference (P < 0·05). There was no correlation between Mf and patient age, gender, or the proportion of GPI-AP-deficient granulocytes or erythrocytes.

image

Figure 2. Distribution of Mfs in PNH patients. The Mf of HPRT T lymphocytes in PB of patients and normal controls was quantified using a negative selection strategy with 6-TG. (A) CE value of T lymphocytes without 6-TG in normal healthy controls was significantly higher than that of PNH patients. (B) Mf values for T lymphocytes with 6-TG were found to be significantly higher in PNH patients compared with normal controls (2·36 × 10−5 and 1·06 × 10−6, respectively, P < 0·05).

Download figure to PowerPoint

Phenotype of HPRT mutant T cells

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patient samples
  5. Peripheral blood cell preparation
  6. Phenotypic characterization of T-cells
  7. Cloning assay for HPRT mutant T-cells in vitro
  8. Methylcellulose colony-forming assay for HPRT mutation haematopoietic progenitor cells in vitro
  9. T cell receptor Vβ usage and complementarity determining region 3 (CDR3) size distribution analysis in HPRT mutation T cell clones
  10. Statistical analysis
  11. Results
  12. T lymphocytes phenotype in PNH
  13. 6-TG-resistant T cells in PNH
  14. Phenotype of HPRT mutant T cells
  15. TCR Vβ usage and CDR3 size distribution in HPRT mutant T cell clones
  16. Discussion
  17. References

In order to characterize HPRT mutant T cells, we determined the phenotype of 138 6-TG-resistant colonies from eight PNH patients and 21 colonies from three healthy donors. The control colonies were all CD4 phenotype, but PNH colonies showed three distinct phenotypes: CD4 homogeneous, CD8 homogeneous, and CD4/CD8 heterogeneous. Most HPRT mutant colonies expressed the CD4 phenotype (110/138), while 16% (22/138) and 4% (6/138) colonies were CD8 and CD4/CD8 mixed, respectively, in our culture system (Fig 3A). Additionally, the great majority of HPRT mutant colonies were of normal phenotype for GPI-AP expression (based on CD55 and CD59 expression), and only a few CD4 (8%) and CD8 (9%) colonies showed low GPI-AP representation, consistent with PNH clonal derivation (Fig 3B). There was a higher proportion of GPI-AP-deficient CD4 T cells among the HPRT mutant clones (11%) compared with PB (3%), consistent with a modest selective advantage for PNH-type CD4 cells in vitro under 6-TG selection. In contrast, only a few GPI-AP-deficient CD8 T cells of HPRT mutant phenotype were identified, despite a large proportion of such cells in the fresh PB of patients.

image

Figure 3. Characterization of HPRT T-cell mutation clones. The phenotypic characteristics of HPRT mutant T cells were identified using mAbs to CD4, CD8, and CD55/CD59 and flow cytometry. (A) Distribution of HPRT mutant T-cell clones; most mutant clones were CD4 phenotype. (B) Phenotype of HPRT mutant T-cell clones.

Download figure to PowerPoint

An attempt was made to detect 6-TG-resistant HPRT mutant haematopoietic cells in standard haematopoietic colony culture assays. We utilized very purified CD34 cells at high concentration. The average total colony formation for CD34 cells without 6-TG was 198 ± 21·6 in normal healthy volunteers; decreased numbers of colonies were observed for GPI-AP-normal and GPI-AP-deficient CD34 cells from PNH patients (66 ± 24·7 and 121 ± 31·6, respectively). However, in three patients for whom large numbers of dishes were assessed, we were unable to detect mutant haematopoietic colonies of any type from the BM of either PNH or healthy volunteers.

TCR Vβ usage and CDR3 size distribution in HPRT mutant T cell clones

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patient samples
  5. Peripheral blood cell preparation
  6. Phenotypic characterization of T-cells
  7. Cloning assay for HPRT mutant T-cells in vitro
  8. Methylcellulose colony-forming assay for HPRT mutation haematopoietic progenitor cells in vitro
  9. T cell receptor Vβ usage and complementarity determining region 3 (CDR3) size distribution analysis in HPRT mutation T cell clones
  10. Statistical analysis
  11. Results
  12. T lymphocytes phenotype in PNH
  13. 6-TG-resistant T cells in PNH
  14. Phenotype of HPRT mutant T cells
  15. TCR Vβ usage and CDR3 size distribution in HPRT mutant T cell clones
  16. Discussion
  17. References

By the method of reverse gene amplification and utilization of specific Vβ primers, we determined TCR Vβ in mutant T cell colonies. Multiple Vβ gene families were represented in a large proportion of the colonies (17/32), probably due to the inoculum of many thousand lymphocytes and the detection of DNA sequences from contaminating residual T cells. Nevertheless, there was a strong preference for Vβ2 usage among the mutant clones, which was found in almost all colonies (31/32; Fig 4). Vβ9, Vβ5, and Vβ13 were also frequently detected. Analysis of Vβ2 CDR3 revealed a skewed profile and same size distribution, highly suggestive that the mutant colonies share the same CDR3 of TCR Vβ2.

image

Figure 4. Frequency of Vβ usage in T-cell mutant clones. To determine TCR Vβ usage, 32 random HPRT mutant T-cell clones were analysed by reverse transcription polymerase chain reaction with 22 specific Vβ primers and BC primer. Almost all mutant T-cell clones exhibited a strong preference for the Vβ2 gene (31/32). Following Vβ2, the most frequent Vβ gene families were Vβ9, Vβ5, and Vβ13. Vβ8 and Vβ24 genes were not present in any mutant clones studied.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patient samples
  5. Peripheral blood cell preparation
  6. Phenotypic characterization of T-cells
  7. Cloning assay for HPRT mutant T-cells in vitro
  8. Methylcellulose colony-forming assay for HPRT mutation haematopoietic progenitor cells in vitro
  9. T cell receptor Vβ usage and complementarity determining region 3 (CDR3) size distribution analysis in HPRT mutation T cell clones
  10. Statistical analysis
  11. Results
  12. T lymphocytes phenotype in PNH
  13. 6-TG-resistant T cells in PNH
  14. Phenotype of HPRT mutant T cells
  15. TCR Vβ usage and CDR3 size distribution in HPRT mutant T cell clones
  16. Discussion
  17. References

That T cells might be involved in the pathophysiology of PNH was initially implied by reduced expression of decay accelerating factor (DAF) on the surface not only of erythrocytes, granulocytes, monocytes, and platelets but also of lymphocytes (Kinoshita et al, 1985; Nicholson-Weller et al, 1985; Selvaraj et al, 1987). Variably sized but sometimes large discrete populations of DAF- or CD59-deficient CD4 and CD8 T cells were apparent from cytofluorometric histograms of patients with PNH (Nagakura et al, 1993). Cells from the PB of 22 PNH patients were studied by two colour immunophenotypic analysis: CD8 T cells showed a high percentage of GPI-AP-deficient compared to CD4 cells, and several patients’ cytotoxic lymphocytes, apparently derived from the PNH clone, predominated in the circulation (Tseng et al, 1995). Similarly, substantial proportions of GPI-AP-deficient CD3, CD4, and CD8 cells were detected in the great majority of 19 PNH patients from the UK (Richards et al, 1998). Smaller numbers of negative lymphocytes were present in AA patients as described in the first report of subtle PNH clones in this BM failure syndrome (Schrezenmeier et al, 1995). GPI-AP-deficient lymphocyte progenitors may also exist in normal individuals, as suggested by the early identification of such tiny populations by flow cytometry and then dramatically demonstrated by apparent in vivo selection of PNH-like lymphocyte clones during treatment of lymphoma patients with a mAb directed against the GPI-AP CD52 (Hertenstein et al, 1995; Rawstron et al, 1999). Our results therefore are entirely consistent with these earlier studies that so clearly implicate the PNH clone in the production of GPI-AP-deficient lymphocyte progeny, leading to a high proportion of PNH-type T cells in some patients. Similarly, an inverted CD4/CD8 ratio has been observed in PNH as in other BM failure syndromes, and was confirmed in this study.

In PNH, evidence for genetic instability was first sought by the analysis of microsatellites, HPRT mutations, and ‘illegitimate’ VDJ genetic recombination events in the TCR (Purow et al, 1999). While the majority of experiments in a group of 29 patients were negative, two patients showed a markedly elevated number of HPRT mutant colonies, and in one case the Mf was greater than 10-fold normal (Purow et al, 1999). More consistent and dramatic results were described by Horikawa et al (2002) who studied 12 PNH patients and 17 age-matched controls; 6-TG-resistant T cell colonies were present in two-thirds of the patients but in less than 20% of the healthy volunteers, and the incidence of resistant colonies was, on average, 20 times higher in PNH. T lymphocyte clones were grown after treatment with aerolysin, a bacterial toxin that induces cell lysis in cells bearing GPI anchors (Ware et al, 2001): residual GPI-AP-deficient cells were isolated at a much higher rate from patients with PNH than from normal samples, and the calculated Mf for patients with PNH was an astonishingly 10 000-fold greater than in normal samples! An Mf for cells of the BM, where the PNH clone originates, has not been calculated, but data described by Horikawa et al, (2002) would also be consistent with increased orders of magnitude; in two of their patients, the frequency of 6-TG-resistant BM colonies appeared equivalent or higher than the average CE for untreated PNH BMMNC, compatible only with derivation of the great majority or all of the marrow cells from an HPRT mutant clone.

Our data broadly confirm an increase in the number of T cells from PNH patients that are resistant to 6-TG in vitro, and therefore likely to harbour acquired HPRT mutations. In contrast to the data of Horikawa et al (2002), we were unable to detect evidence of HPRT mutations in BM, even when purified CD34 cells were plated at high concentrations. We were not surprised by this latter result, as the normally low frequency of HPRT mutants and the much lower CE for BM compared with PB T cells combined would make it extraordinarily difficult to detect even an enormous increase in Mf without culture of extremely large numbers of haematopoietic progenitors, which are particularly scarce in this BM failure syndrome. Additionally, our experiments show that the HPRT mutants generally did not occur in cells derived from the PNH clone, against an intrinsic increase in mutability related to the acquired PIG-A mutation. Furthermore, and more important, HPRT mutations were present in oligoclonally expanded T cell populations and did not occur randomly among many different T cell Vβ subtypes. These results argue against genomic instability in either the PNH clone or in PNH patients’ BM in general.

We interpret our results as consistent with previously published evidence of abnormal T cell clonal expansion in PNH and related BM failure syndromes. T cells have been strongly implicated in the pathophysiology of most AA, which itself has also been shown to be associated with PNH (Maciejewski et al, 2001b; Shichishima et al, 2002; Young et al, 2002). T cell clonal expansion, directly measured in the BM failure syndromes by combinations of immunophenotyping for specific Vβ subfamilies and spectratyping for CDR3 size distribution, appears to be frequent in AA (Risitano et al, 2002), myelodysplasia (Saunthararajah et al, 2001), and in PNH (Karadimitris et al, 2000; Risitano et al, 2003). Increase in HPRT mutant T cells has been observed in analyses of other autoimmune diseases in humans, including systemic lupus erythematosus, rheumatoid arthritis, diabetes mellitus and multiple sclerosis (Allegretta et al, 1994; Lodge et al, 1994; Van den Berg et al, 1995; Cannons et al, 1998; Abramson et al, 1999; Falta et al, 1999). The usual interpretation of such results has been that the marked increase in cell divisions required for antigen-driven T cell expansion stochastically increases the probability of observing HPRT mutants, that 6-TG-resistant colonies were thus all derived from a single HPRT mutant T cell progenitor, and that the Mf itself is not increased in autoimmune diseases.

The relationship between abnormalities in T cells derived from the PNH clone and the explanation for T cell clonal expansion in the setting of an autoimmune disease of the BM remains elusive. Incidentally, seen in our experiments and observed also by others (Tseng et al, 1995), GPI-AP-deficient T cells may have a growth advantage in vitro when stimulated with a lectin such as phytohaemmagglutinin. PIG-A mutant T cells are functionally different from their normal counterparts, although this difference does not result in increased clinical infections. Deficiency in red cell rosetting was correlated with defective presentation of CD2 (Selvaraj et al, 1987). There is diminished TCR signalling in T cells of the PNH phenotype; these cells display decreased tyrosine kinase activation, delayed capping and TCR internalization, and differences in substrate-specific phosphorylation (Romagnoli & Bron, 1999), and PNH lymphocyte population, derived from a patient during treatment with anti-CD52 mAb, showed abnormalities in cytokine expression (Fracchiolla et al, 2001). These results are even more provocative considering that such a large proportion of CD8 cells, believed to be the immediate effector cells in many BM failure syndromes, are derived from the PNH phenotype and that PNH clone-derived T cells may persist for decades (Nakakuma et al, 1994; Richards et al, 1999). Multiple GPI-AP on the T cell surface are important for ligation of antigen presenting cells and target cells, and their absence on a large proportion of T cells may be important in the immune destruction of haematopoietic cells and clonal evolution.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Patient samples
  5. Peripheral blood cell preparation
  6. Phenotypic characterization of T-cells
  7. Cloning assay for HPRT mutant T-cells in vitro
  8. Methylcellulose colony-forming assay for HPRT mutation haematopoietic progenitor cells in vitro
  9. T cell receptor Vβ usage and complementarity determining region 3 (CDR3) size distribution analysis in HPRT mutation T cell clones
  10. Statistical analysis
  11. Results
  12. T lymphocytes phenotype in PNH
  13. 6-TG-resistant T cells in PNH
  14. Phenotype of HPRT mutant T cells
  15. TCR Vβ usage and CDR3 size distribution in HPRT mutant T cell clones
  16. Discussion
  17. References
  • Abramson, L.S., Albertini, R.J., Pachman, L.M. & Finette, B.A. (1999) Association among somatic HPRT mutant frequency, peripheral blood T-lymphocyte clonality, and serologic parameters of disease activity in children with juvenile onset dermatomyositis. Clinical Immunology, 91, 6167.
  • Albertini, R.J. (2001) HPRT mutations in humans: biomarkers for mechanistic studies. Mutation Research, 489, 116.
  • Allegretta, M., Nicklas, J.A., Sriram, S. & Albertini, R.J. (1990) T cells responsive to myelin basic protein in patients with multiple sclerosis. Science, 247, 718721.
  • Allegretta, M., Albertini, R.J., Howell, M.D., Smith, L.R., Martin, R., McFarland, H.F., Sriram, S., Brostoff, S. & Steinman, L. (1994) Homologies between T cell receptor junctional sequences unique to multiple sclerosis and T cells mediating experimental allergic encephalomyelitis. Journal of Clinical Investigation, 94, 105109.
  • Araten, D.J., Nafa, K., Pakdeesuwan, K. & Luzzatto, L. (1999) Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria genotype and phenotype are present in normal individuals. Proceedings of the National Acadamy of Sciences of the United States of America, 96, 52095214.
  • Cannons, J.L., Karsh, J., Birnboim, H.C. & Goldstein, R. (1998) HPRT- mutant T cells in the peripheral blood and synovial tissue of patients with rheumatoid arthritis. Arthritis and Rheumatism, 41, 17721782.
  • Chen, R., Nagarajan, S., Prince, G.M., Maheshwari, U., Terstappen, L.W., Kaplan, D.R., Gerson, S.L., Albert, J.M., Dunn, D.E., Lazarus, H.M. & Medof, M.E. (2000) Impaired growth and elevated fas receptor expression in PIGA(+) stem cells in primary paroxysmal nocturnal hemoglobinuria. Journal of Clinical Investigation, 106, 689696.
  • Chen, G., Kirby, M., Zeng, W., Young, N.S. & Maciejewski, J.P. (2002a) Superior growth of glycophosphatidy linositol-anchored protein-deficient progenitor cells in vitro is due to the higher apoptotic rate of progenitors with normal phenotype in vivo. Experimental Hematology, 30, 774782.
  • Chen, G., Zeng, W., Keyvanfar, K., Billings, E. & Young, S.N. (2002b) Differential gene expression profile in hematopoietic progenitor cells from paroxysmal nocturnal hemoglobinuria (PNH) patients. Blood, 100, 228a.
  • Dunn, D.E., Yu, J., Nagarajan, S., Devetten, M., Weichold, F.F., Medof, M.E., Young, N.S. & Liu, J.M. (1996) A knock-out model of paroxysmal nocturnal hemoglobinuria: Pig-a(-) hematopoiesis is reconstituted following intercellular transfer of GPI-anchored proteins. Proceedings of the National Acadamy of Sciences of the United States of America, 93, 79387943.
  • Dunn, D.E., Tanawattanacharoen, P., Boccuni, P., Nagakura, S., Green, S.W., Kirby, M.R., Kumar, M.S., Rosenfeld, S. & Young, N.S. (1999) Paroxysmal nocturnal hemoglobinuria cells in patients with bone marrow failure syndromes. Annals of Internal Medicine, 131, 401408.
  • Falta, M.T., Magin, G.K., Allegretta, M., Steinman, L., Atkinson, M.A., Brostoff, S.W. & Albertini, R.J. (1999) Selection of hprt mutant T cells as surrogates for dividing cells reveals a restricted T cell receptor BV repertoire in insulin-dependent diabetes mellitus. Clinical Immunology, 90, 340351.
  • Fracchiolla, N.S., Barcellini, W., Bianchi, P., Motta, M., Fermo, E. & Cortelezzi, A. (2001) Biological and molecular characterization of PNH-like lymphocytes emerging after Campath-1H therapy. British Journal of Haematology, 112, 969971.
  • Hattori, H., Machii, T., Ueda, E., Shibano, M., Kageyama, T. & Kitani, T. (1997) Increased frequency of somatic mutations at glycophorin A loci in patients with aplastic anaemia, myelodysplastic syndrome and paroxysmal nocturnal haemoglobinuria. British Journal of Haematology, 98, 384391.
  • Hertenstein, B., Wagner, B., Bunjes, D., Duncker, C., Raghavachar, A., Arnold, R., Heimpel, H. & Schrezenmeier, H. (1995) Emergence of CD52-, phosphatidylinositolglycan-anchor-deficient T lymphocytes after in vivo application of Campath-1H for refractory B-cell non-Hodgkin lymphoma. Blood, 86, 14871492.
  • Horikawa, K., Kawaguchi, T., Ishihara, S., Nagakura, S., Hidaka, M., Kagimoto, T., Mitsuya, H. & Nakakuma, H. (2002) Frequent detection of T cells with mutations of the hypoxanthine-guanine phosphoribosyl transferase gene in patients with paroxysmal nocturnal hemoglobinuria. Blood, 99, 2429.
  • Inoue, N., Murakami, Y. & Kinoshita, T. (2003) Molecular genetics of paroxysmal nocturnal hemoglobinuria. International Journal of Hematology, 77, 107112.
  • Karadimitris, A., Notaro, R., Koehne, G., Roberts, I.A. & Luzzatto, L. (2000) PNH cells are as sensitive to T-cell-mediated lysis as their normal counterparts: implications for the pathogenesis of paroxysmal nocturnal haemoglobinuria. British Journal of Haematology, 111, 11581163.
  • Kawaguchi, K., Wada, H., Mori, A., Takemoto, Y., Kakishita, E. & Kanamaru, A. (1999) Detection of GPI-anchored protein-deficient cells in patients with aplastic anaemia and evidence for clonal expansion during the clinical course. British Journal of Haematology, 105, 80.
  • Kinoshita, T., Medof, M.E., Silber, R. & Nussenzweig, V. (1985) Distribution of decay-accelerating factor in the peripheral blood of normal individuals and patients with paroxysmal nocturnal hemoglobinuria. Journal of Experimental Medicine, 162, 7592.
  • Kook, H., Zeng, W., Guibin, C., Kirby, M., Young, N.S. & Maciejewski, J.P. (2001) Increased cytotoxic T cells with effector phenotype in aplastic anemia and myelodysplasia. Experimental Hematology, 29, 12701277.
  • Lodge, P.A., Allegretta, M., Steinman, L. & Sriram, S. (1994) Myelin basic protein peptide specificity and T-cell receptor gene usage of HPRT mutant T-cell clones in patients with multiple sclerosis. Annals of Neurology, 36, 734740.
  • Maciejewski, J.P., Rivera, C., Kook, H., Dunn, D. & Young, N.S. (2001a) Relationship between bone marrow failure syndromes and the presence of glycophosphatidyl inositol-anchored protein-deficient clones. British Journal of Haematology, 115, 10151022.
  • Maciejewski, J.P., Follmann, D., Nakamura, R., Saunthararajah, Y., Rivera, C.E., Simonis, T., Brown, K.E., Barrett, J.A. & Young, N.S. (2001b) Increased frequency of HLA-DR2 in patients with paroxysmal nocturnal hemoglobinuria and the PNH/aplastic anemia syndrome. Blood, 98, 35133519.
  • Monteiro, C., Marcelino, L.A., Conde, A.R., Saraiva, C., Giphart-Gassler, M., De Nooij-van Dalen A.G., Buuren-van Seggelen, V., Van der, K.M., May, C.A., Cole, J., Lehmann, A.R., Steinsgrimsdottir, H., Beare, D., Capulas, E. & Armour, J.A. (2000) Molecular methods for the detection of mutations. Teratogenesis, Carcinogenesis and Mutagenesis, 20, 357386.
  • Mortazavi, Y., Merk, B., McIntosh, J., Marsh, J.C., Schrezenmeier, H. & Rutherford, T.R. (2003) The spectrum of PIG-A gene mutations in aplastic anemia/paroxysmal nocturnal hemoglobinuria (AA/PNH): a high incidence of multiple mutations and evidence of a mutational hot spot. Blood, 101, 28332841.
  • Nafa, K., Bessler, M., Castro-Malaspina, H., Jhanwar, S. & Luzzatto, L. (1998) The spectrum of somatic mutations in the PIG-A gene in paroxysmal nocturnal hemoglobinuria includes large deletions and small duplications. Blood Cells, Molecules and Diseases, 24, 370384.
  • Nagakura, S., Nakakuma, H., Horikawa, K., Hidaka, M., Kagimoto, T., Kawakita, M., Tomita, M. & Takatsuki, K. (1993) Expression of decay-accelerating factor and CD59 in lymphocyte subsets of healthy individuals and paroxysmal nocturnal hemoglobinuria patients. American Journal of Hematology, 43, 1418.
  • Nakakuma, H., Nagakura, S., Kawaguchi, T., Iwamoto, N., Hidaka, M., Horikawa, K., Kagimoto, T., Tsuruzaki, R. & Takatsuki, K. (1994) Persistence of affected T lymphocytes in long-term clinical remission in paroxysmal nocturnal hemoglobinuria. Blood, 84, 39253928.
  • Nakao, S., Takamatsu, H., Chuhjo, T., Ueda, M., Shiobara, S., Matsuda, T., Kaneshige, T. & Mizoguchi, H. (1994) Identification of a specific HLA class II haplotype strongly associated with susceptibility to cyclosporine-dependent aplastic anemia. Blood, 84, 42574261.
  • Nicholson-Weller, A., March, J.P., Rosen, C.E., Spicer, D.B. & Austen, K.F. (1985) Surface membrane expression by human blood leukocytes and platelets of decay-accelerating factor, a regulatory protein of the complement system. Blood, 65, 12371244.
  • Okuda, K., Kanamaru, A., Ueda, E., Kitani, T., Okada, N., Okada, H., Kakishita, E. & Nagai, K. (1990) Expression of decay-accelerating factor on hematopoietic progenitors and their progeny cells grown in cultures with fractionated bone marrow cells from normal individuals and patients with paroxysmal nocturnal hemoglobinuria. Experimental Hematology, 18, 11321136.
  • Purow, D.B., Howard, T.A., Marcus, S.J., Rosse, W.F. & Ware, R.E. (1999) Genetic instability and the etiology of somatic PIG-A mutations in paroxysmal nocturnal hemoglobinuria. Blood Cells, Molecules, and Disease, 25, 8191.
  • Rawstron, A.C., Rollinson, S.J., Richards, S., Short, M.A., English, A., Morgan, G.J., Hale, G. & Hillmen, P. (1999) The PNH phenotype cells that emerge in most patients after CAMPATH-1H therapy are present prior to treatment. British Journal of Haematology, 107, 148153.
  • Rice, S.C., Vacek, P.M., Homans, A.H., Kendall, H., Rivers, J., Messier, T. & Finette, B.A. (2003) Comparative analysis of HPRT mutant frequency in children with cancer. Environmental and Molecular Mutagenesis, 42, 4449.
  • Richards, S.J., Norfolk, D.R., Swirsky, D.M. & Hillmen, P. (1998) Lymphocyte subset analysis and glycosylphosphatidylinositol phenotype in patients with paroxysmal nocturnal hemoglobinuria. Blood, 92, 17991806.
  • Richards, S.J., Morgan, G.J. & Hillmen, P. (1999) Analysis of T cells in paroxysmal nocturnal hemoglobinuria provides direct evidence that thymic T-cell production declines with age. Blood, 94, 27902799.
  • Risitano, A.M., Kook, H., Zeng, W., Chen, G., Young, N.S. & Maciejewski, J.P. (2002) Oligoclonal and polyclonal CD4 and CD8 lymphocytes in aplastic anemia and paroxysmal nocturnal hemoglobinuria measured by V beta CDR3 spectratyping and flow cytometry. Blood, 100, 178183.
  • Risitano, A.M., Plasilova, M., O'Keefe, C., Muranski, P., Sloand, E.M., Maciejewski, J.P. & Young, N.S. (2003) Clonal T cell expansions in paroxysmal nocturnal hemoglobinuria (PNH) detected by T cell receptor analysis suggest a strong pathophysiologic relationship among PNH, aplastic anemia, and large granular lymphocytic syndrome. Blood, 102, 206a.
  • Romagnoli, P. & Bron, C. (1999) Defective TCR signaling events in glycosylphosphatidylinositol-deficient T cells derived from paroxysmal nocturnal hemoglobinuria patients. International Immunology, 11, 14111422.
  • Rosse, W. (2000) A brief history of PNH. In: PNH and the GPI-linked Proteins (ed. by N.S.Young & J.Moss), pp. 1. Academic Press, San Diego, CA.
  • Rosse, W.F. & Nishimura, J. (2003) Clinical manifestations of paroxysmal nocturnal hemoglobinuria: present state and future problems. International Journal of Hematology, 77, 113120.
  • Saunthararajah, Y., Molldrem, J.L., Rivera, M., Williams, A., Stetler-Stevenson, M., Sorbara, L., Young, N.S. & Barrett, J.A. (2001) Coincident myelodysplastic syndrome and T-cell large granular lymphocytic disease: clinical and pathophysiological features. British Journal of Haematology, 112, 195200.
  • Schrezenmeier, H., Hertenstein, B., Wagner, B., Raghavachar, A. & Heimpel, H. (1995) A pathogenetic link between aplastic anemia and paroxysmal nocturnal hemoglobinuria is suggested by a high frequency of aplastic anemia patients with a deficiency of phosphatidylinositol glycan anchored proteins. Experimental Hematology, 23, 8187.
  • Selvaraj, P., Dustin, M.L., Silber, R., Low, M.G. & Springer, T.A. (1987) Deficiency of lymphocyte function-associated antigen 3 (LFA-3) in paroxysmal nocturnal hemoglobinuria. Functional correlates and evidence for a phosphatidylinositol membrane anchor. Journal of Experimental Medicine, 166, 10111025.
  • Shichishima, T., Okamoto, M., Ikeda, K., Kaneshige, T., Sugiyama, H., Terasawa, T., Osumi, K. & Maruyama, Y. (2002) HLA class II haplotype and quantitation of WT1 RNA in Japanese patients with paroxysmal nocturnal hemoglobinuria. Blood, 100, 2228.
  • Sloand, E.M., Fuhrer, M., Keyvanfar, K., Mainwaring, L., Maciejewski, J., Wang, Y., Johnson, S., Barrett, A.J. & Young, N.S. (2003) Cytogenetic abnormalities in paroxysmal nocturnal haemoglobinuria usually occur in haematopoietic cells that are glycosylphosphatidylinositol-anchored protein (GPI-AP) positive. British Journal of Haematology, 123, 173176.
  • Tomonaga, M., Iwanaga, M., Fuchigami, K., Inoue, Y., Joh, T. & Jinnai, I. (2003) Incidence and Clinical Significance of PNH Clone in Myelodysplastic Syndromes. In: Paroxysmal nocturnal hemoglobinuria and related disorders/molecular aspects of pathogenesis (ed. by K.T.Omine), pp. 139148. Springer, Tokyo.
  • Tseng, J.E., Hall, S.E., Howard, T.A. & Ware, R.E. (1995) Phenotypic and functional analysis of lymphocytes in paroxysmal nocturnal hemoglobinuria. American Journal of Hematology, 50, 244253.
  • Van den Berg, L.H., Mollee, I., Wokke, J.H. & Logtenberg, T. (1995) Increased frequencies of HPRT mutant T lymphocytes in patients with Guillain-Barre syndrome and chronic inflammatory demyelinating polyneuropathy: further evidence for a role of T cells in the etiopathogenesis of peripheral demyelinating diseases. Journal of Neuroimmunology, 58, 3742.
  • Wang, H., Chuhjo, T., Yasue, S., Omine, M. & Nakao, S. (2002) Clinical significance of a minor population of paroxysmal nocturnal hemoglobinuria-type cells in bone marrow failure syndrome. Blood, 100, 38973902.
  • Ware, R.E., Pickens, C.V., DeCastro, C.M. & Howard, T.A. (2001) Circulating PIG-A mutant T lymphocytes in healthy adults and patients with bone marrow failure syndromes. Experimental Hematology, 29, 14031409.
  • Young, N.S., Maciejewski, J.P., Sloand, E., Chen, G., Zeng, W., Risitano, A. & Miyazato, A. (2002) The relationship of aplastic anemia and PNH. International Journal of Hematology, 76 (Suppl. 2), 168172.
  • Zeng, W., Maciejewski, J.P., Chen, G. & Young, N.S. (2001) Limited heterogeneity of T cell receptor BV usage in aplastic anemia. Journal of Clinical Investigation, 108, 765773.