High Frequencies of Increased Interferon-γ-Producing and/or Skewed CD8⁺ T Lymphocyte Subfamilies in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH).

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematological disorder affecting all hematopoietic lineages, which lack glycosylphosphatidylinositol (GPI)-anchored membrane proteins due to somatic mutations in the phosphatidylinositol glycan-class A gene, and is one disorder of bone marrow failure (BMF) syndromes. Autoreactive T lymphocytes are implicated in some of the immune mechanisms involved in PNH. In fact, we reported recently that the HLA-DRB1^*1501 allele and HLA-A^*0206 allele is frequent and is related to grading of hemolysis, respectively, in PNH patients (Shichishima T et al, Blood, 2002 and Haematologica, 2006, respectively). However, some characteristics of CD4⁺ and CD8⁺ T lymphocytes, including GPI-negative CD4⁺ and CD8⁺ T lymphocytes, in PNH patients remain unknown. To know some characteristics of CD4⁺ and CD8⁺ T lymphocytes with and without expressions of GPI proteins in PNH, we examined preferential variable beta chain (Vβ) repertoires of the T-cell receptor (TCR) and expressions of interferon-γ (IFN-γ) by flow cytometry and the TCR Vβ complementarity-determining region 3 (CDR3) spectratypes by genetic methods at the same time in CD4⁺CD59⁺, CD4⁺CD59⁻, CD8⁺CD59⁺, and/or CD8⁺CD59⁻ T lymphocytes from 10 Japanese patients, including 6 and 4 with the HLA-DRB1^*1501 allele and HLA-A^*0206 allele, respectively, and from 5 age-matched healthy individuals. In the analyses of TCR Vβ repertoires, over-expressed TCR Vβ subfamilies were found in any T lymphocytes subsets from all the patients. We found significantly higher numbers (mean ± standard deviation; 1.9 ± 1.2) of over-expressed TCR Vβ subfamilies in CD8⁺CD59⁺ T lymphocytes from PNH patients compared with those (0 ± 0, p <0.01) from healthy individuals. In the TCR Vβ CDR3 spectratyping, skewed TCR Vβ CDR3 spectatypes were found in more than one TCR Vβ subfamilies of CD3⁺CD4⁺CD59⁻, CD3⁺CD8⁺CD59⁺, and CD3⁺CD8⁺CD59⁻ T lymphocytes from all the PNH patients. The numbers of skewed TCR Vβ CDR3 spectatypes in CD3⁺CD8⁺CD59⁺ (4.0 ± 3.3) and CD3⁺CD8⁺CD59⁻ (7.5 ± 3.9) T lymphocytes from one PNH patient were significantly greater than those in CD3⁺CD4⁺CD59⁺ T lymphocytes (0.6 ± 1.0, p <0.005) and CD3⁺CD4⁺CD59⁻ (2.5 ± 1.5, p <0.002), respectively. Skewed TCR Vβ CDR3 spectatypes were found commonly in Vβ 25 subfamily of CD3⁺CD8⁺CD59⁺ and CD3⁺CD8⁺CD59⁻ T lymphocytes from all of 4 PNH patients with the HLA-A^*0206 allele. In the analyses of IFN-γ expressions, more than one TCR Vβ subfamiliy with over-expression of IFN-γ was found in CD8⁺CD59⁺ and/or CD8⁺CD59⁻ T lymphocytes from 9 patients and in CD4⁺CD59⁺ and/or CD4⁺CD59⁻ T lymphocytes from 8 patients. The numbers of TCR Vβ subfamiliy with over-expression of IFN-γ in CD8⁺CD59⁺ T lymphocytes (5.2 ± 4.3), but not in the other T lymphocyte subsets, from one PNH patient were significantly greater than those from healthy individuals (0 ± 0, p <0.05). However, there were no specific Vβ subfamilies determined by any analyses, described above, in PNH. In conclusion, we found high frequencies of increased IFN-γ-producing and/or skewed CD8⁺ T lymphocyte subfamilies with and/or without CD59 expression in PNH patients, suggesting that these cells may contribute to the occurrence of BMF rather than negative selection of PNH clones through action of IFN-γ in PNH.