The Important Role of T Regulatory Cells in Bone Marrow Failure Syndromes.

Autoreactive T lymphocytes are implicated in the immune mechanisms involved in the bone marrow failure syndromes (BMFS), including aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH), and myelodysplastic syndrome (MDS). CD4⁺CD25^highFoxP3⁺ T regulatory cells (Treg) play crucial roles in regulating immune responses to autoreactive T lymphocytes via interferon (IFN)-gamma- and interleukin (IL)-10-dependent pathways. Recently, it has also been reported that a stimulation of CD4⁺ T cells by anti-CD3/CD46 antibodies induces Treg that kill autoreactive T lymphocytes activated (Grossman WJ et al, Blood, 2004). Then, we hypothesized that some distributional and/or functional abnormalities of Treg may lead to an inappropriate proliferation or reduction of autoreactive T lymphocytes in BMFS. In this study, to clarify some roles of Treg in an immune pathophysiology of BMFS, we examined the proportions of Treg in CD4⁺ T cells from BMFS patients using flow cytometry. Subsequently, we evaluated autologous cytotoxicity of cultured CD4⁺ T cells (target cells), which were stimulated by the addition of anti-CD3/CD28 antibodies, by cultured CD4⁺ T cells (effector cells), which were activated by the addition of anti-CD3/CD46 antibodies, at an effector versus target ratio of 1:1 by flow cytometry using carboxyfluorescein diacetate succinimidyl ester and 7-amino-actinomycin D and then calculated the percentages of specific lysis of the targeted CD4⁺ T cells from BMFS patients (Lecoeur H et al., J Immunol Methods, 2001). We also measured the concentrations of IFN-gamma and IL-10 in aliquots of supernatant of cultured CD4⁺ T cells from BMFS patients, stimulated by the addition of anti-CD3/CD46 antibodies, using enzyme-linked immunosorbent assay. We found significantly higher proportions of Treg in CD4⁺ T cells from MDS patients (n=10; mean ± standard deviation; 5.66 ± 2.70%; p<0.05), but not from AA (n=5; 3.88 ± 1.37%) and PNH patients (n=5; 2.81 ± 1.20%), than those from healthy individuals (n=10; 3.36 ± 1.39%). We also found that cytotoxicity of anti-CD3/CD46 antibodies-stimulated Treg from MDS patients (n=3; 10.82 ± 3.52%) and AA patients (n=3; 1.06 ± 0.89%) were significantly higher and lower, respectively, than those from healthy individuals (n=5; 4.35 ± 1.92%; p<0.01 and p<0.05, respectively). In contrast, the cytotoxicity of them from PNH patients (n=3; 1.61 ± 1.16%) was not different from that from healthy individuals. The concentrations of IFN-gamma in aliquots of supernatant of anti-CD3/CD46 antibodies-stimulated Treg from 3 MDS (1290 ± 380 IU/ml) and 3 PNH (1058 ± 173 IU/ml) patients, but not from 3 AA patients (326 ± 366 IU/ml), were higher than those from 5 healthy individuals (410 ± 175 IU/ml). In addition, the concentrations of IL-10 in aliquots of supernatant of anti-CD3/CD46 antibodies-stimulated Treg from 3 MDS (2250 ± 420 IU/ml) and 3 PNH (1440 ± 1340 IU/ml) patients, but not from 3 AA patients (428 ± 110 IU/ml), were higher than those from 5 healthy individuals (659 ± 282 IU/ml). Our results suggest that insufficiency or excess in function and/or distribution of Treg may partly contribute to marrow injury of AA or immunologic selection of PNH and MDS clones, respectively. In conclusion, some abnormalities in Treg may play a crucial role in an immunologic pathophysiology of BMFS.