IntroductionAcquired aplastic anemia (AA), a bone marrow failure syndrome characterized by pancytopenia and bone marrow hypoplasia, has been the subject of study by hematologists for many years, as more than 70% of AA patients improve under immunosuppressive therapies such as antithymocyte globulin (ATG) and cyclosporine (CsA). [1][2][3] The dramatic effects of such T-cell suppressants on in vivo hematopoiesis suggest that immune system attack against hematopoietic stem cells plays an essential role in the development of AA. [4][5][6] However, despite extensive efforts to clarify the immune mechanisms of AA, the key antigens provoking immune response against hematopoietic stem cells remain unknown. This is largely due to a lack of animal models and the heterogeneity of pathogenesis in AA. Lack of good progenitor cell assays in humans has also hindered the elucidation of immune mechanisms in AA.In organ-specific autoimmune diseases, such as insulindependent diabetes mellitus (IDDM) and multiple sclerosis where autoreactive T cells play a primary role in pathogenesis, autoantibodies against target proteins of the pathogenic T cells are often detected. [7][8][9][10] Although such antibodies do not usually contribute to the pathogenesis of T-cell-mediated diseases, detection of the antibodies may prove useful in both identifying autoantigens and diagnosing immune mechanisms underlying the diseases. 11 We recently demonstrated that HLA-DRB1*1501 and increased paroxysmal nocturnal hemoglobinuria (PNH)-type cells represent prognostic markers for the immune mechanisms of AA. 12,13 Extensive investigation of antibodies in the sera of patients possessing HLA-DRB1*1501 and a minor population of PNH-type cells may be useful in identifying novel autoantigens in AA. Using immunofluorescent analysis, we previously found that antibodies to UT-7, a megakaryoblastic cell line, are frequently detectable in sera of AA patients who display increased PNH-type cells (PNH ϩ patients; unpublished observation, T.C. and S.N., May 2001). These antibodies may recognize antigens that elicit T-cell responses against hematopoietic stem cells, allowing expansion of PNH-type stem cells. 14,15 To examine these hypotheses, we screened proteins derived from UT-7 cDNA library using serum from a PNH ϩ patient with HLA-DRB1*1501. Serologic identification of antigens by recombinant expression cloning (SEREX) analysis identified diazepambinding inhibitor-related protein 1 (DRS-1) as an autoantigen that raises both antibody production and T-cell responses to antigenpresenting cells transfected with DRS-1 gene. AA and MDS were diagnosed in patients at Kanazawa University Hospital and other hospitals taking part in the bone marrow failure study group led by the Ministry of Health, Labor, and Welfare of Japan. MDS was diagnosed on the basis of cytopenia in peripheral blood, hypercellularity or normocellularity in the sternal or iliac bone marrow, and presence of dysplasia in at least 2 lineages of bone marrow cells. Cytogenetic abnormalities such as trisomy...

Valproic acid (VPA), a histone deacetylase inhibitor, upregulates NKG2D ligands (NKG2DLs) on some monocytic and lymphoid leukemic cells. However, its effect on myeloid leukemia cells and synergistic agents that can augment the effect of VPA remains unknown. Of the various myeloid cell lines examined, OUN-1, a chronic myelogenous leukemia cell line, showed the most prominent upregulation of MICA ⁄ B and ULBP2 in response to VPA. The NKG2DL upregulation was observed only in leukemic cells without apoptosis and the effect was abrogated by pretreatment of cells with caffeine, an inhibitor of ATM ⁄ ATR. Several activators of ATM ⁄ ATR were screened for their effect on NKG2DL expression, but only hydroxyurea (HU) efficiently upregulated both MICA ⁄ B and ULPB2 expression on the cell line. VPA and HU synergistically upregulated the NKG2DLs on OUN-1 cells as well as primary leukemic cells from some patients with acute myeloid leukemia. (1-3) Several lines of evidence indicate that the expression level of NKG2D ligand (NKG2DL) on leukemia cells affects the sensitivity of the leukemic cells to killing by NK cells.(4-11) Various agents have been evaluated for their inducibility of NKG2DLs on leukemic cells, to augment the NK cell-mediated antileukemia effect. (4,8,9,(12)(13)(14) Valproic acid (VPA), a histone deacetylase inhibitor, is a potent inducer of NKG2DLs such as MICA ⁄ B and ULBPs on malignant cells. (9,12,14) VPA augments the expression of MICA and ULBP2 on several monocytic and lymphoid leukemia cell lines and primary acute myeloid leukemia (AML) cells in vitro and in vivo.(9,12) However, the mechanisms for the upregulation of NKG2DL on AML cells by VPA has not been studied extensively due to the lack of myeloid leukemia cell lines that display an upregulation of NKG2DLs in response to VPA. Clarifying the mechanisms associated with upregulation could identify other reagents that synergize with VPA to augment the expression of NKG2DL by myeloid leukemia cells and thereby enhance the susceptibility of leukemic cells to NK cells.

To determine how immunosuppressant agents used for graft-versus-host disease (GVHD) prophylaxis affect natural killer (NK) cells, we examined the effects of cyclosporine (CSP), tacrolimus (TAC), mycophenolic acid (MPA, an active form of mycophenolate mofetil), and methotrexate (MTX) on the proliferation and cytotoxicity of NK cells. The proliferation of NK cells from healthy individuals in the presence of interleukin (IL)-2 and IL-15 was suppressed to 51% ± 16% of that of the controls with CSP, to 31% ± 19% with TAC, to 14% ± 6% with MPA, and to 87% ± 18% with MTX. Both CSP and TAC increased the proportion of CD16(-)CD56(bright) cells, a NK cell subset capable of secreting high amount of cytokines, and also enhanced NKp30 expression, whereas MPA markedly decreased the proportion of CD16(-)CD56(bright) cells and reduced the expression of all activating NK cell receptors, including NKG2D, NKp30, NKp44, and NKp46. MPA also reduced the cytotoxicity against K562 cells from 61% ± 15% to 17% ± 7% and that against Daudi cells from 44% ± 4% to 4% ± 4%, whereas the other 3 drugs did not diminish these cytotoxicities. The inhibition of NK cell proliferation and cytotoxicity against leukemic cell lines by MPA was partially abolished by the inclusion of guanosine in the culture. Similar to the effect of MPA on T cells, MPA inhibited the down-regulation of p27 on NK cells induced by the incubation of NK cells in the presence of IL-2. These results suggest that MPA is a potent inhibitor of NK cells, and that its inclusion in the GVHD prophylaxis regimen might diminish the graft-versus-leukemia effect of NK cells.