ALBERT

Description: Typical acute promyelocytic leukemia (APL) is associated with expression of the PML/RARα fusion protein resulting from chromosomal translocation t(15;17) and responsiveness to treatment with all-trans retinoic acid (ATRA). A few population of APL is associated with variant chromosomal translocations, t(11;17), t(5;17) and t(17;17). PLZF/RARα is the chimeric fusion protein resulting from the chromosomal translocation, t(11;17)(q23;q21). APL cells with PLZF/RARα have been reported to be unresponsive to ATRA-induced terminal differentiation clinically and experimentally. The molecular basis of unresponsiveness in PLZF/RARα-derived APL cells against ATRA explained by a rigid interaction between BTB/POZ domain of PLZF and a transcriptional co-repressor, N-CoR. PLZF/RARα contains BTB/POZ domain in its N-terminus. BTB/POZ domain is developmentally conserved among various species, and recently several BTB/POZ-containing molecules have been reported to function as substrate-specific adaptors for Cul3-based E3 ubiquitin ligase. Here we examined the possibility that PLZF/RARα functions as an E3 ligase. We performed the series of transient transfection analysis. By immunoprecipitation assay, PLZF/RARα associated with Cul3, and PLZF/RARα also associated with RXRα. PLZF/RARα accelerated an ubiquitin-dependent degradation of RXRα, and resulted in the decreased expression of RXRα. This degradation of RXRα was dependent on the expression level of PLZF/RARα. On the contrary, co-expression of dominant negative form of Cul3 with PLZF/RARα resulted in the restored expression of RXRα. When we expressed PML/RARα, PLZF or PLZF/RARαΔBTB, the accelerated degradation of RXRα was not observed. In RARα-responsive luciferase assay, PLZF/RARα repressed ATRA response. Consistent with the result that PLZF/RARαΔBTB did not down-regulate the expression of RXRα, PLZF/RARαΔBTB did not repress ATRA response. In addition, the transduction of recombinant RXRα molecule into PLZF/RARα expressing cells partially restored ATRA-responsiveness. Collectively, we suggest that ATRA resistance in PLZF/RARα-positive cells is explained by the novel function of PLZF/RARα molecule.

Description: Appearance of anti-hepatitis B surface antigen antibody (anti-HBs) and clearance of hepatitis B virus (HBV) from serum usually indicates resolution of hepatitis in patients infected with HBV. However, in most patients in whom HBV has been eliminated from serum, HBV DNA is still detectable in the liver using polymerase chain reaction. Reactivation of this dormant HBV in the liver is known as reverse seroconversion (RS). Previously, we reported that HBV-RS after allogeneic hematopoietic stem cell transplantation (allo-HSCT) was a frequent late-onset complication that can be predicted by careful monitoring of progressive disappearance of anti-HBs. RS hepatitis after allo-HSCT is thought to be a phenomenon caused by naive donor immunity after loss of recipient-oriented immunity against HBV. We speculated that vaccination could prevent reactivation of HBV in allo-HSCT recipients. Safety and efficacy of recombinant HBV vaccine in allo-HSCT recipients have already been confirmed. We studied HBV serological markers in 23 patients with anti-HBs and/or anti-HBc before allo-HSCT who were followed for more than 1 year. Patients’ characters are following; Age at HSCT 22 to 65 (median, 38) years; M:F ratio 14:9; Hematological disorders CML 6, AML 1, ALL 4, MDS 4, SAA 2, NHL 4, MM 1 and CAEBV 1; Serological markers anti-HBc(+) and anti-HBs(+) 17, anti-HBc(+) and anti-HBs(−) 3, anti-HBc(−) and anti-HBs(+) 3. No patients had a prior history of vaccination or HBV-specific immunoglobulin usage. All patients were negative for hepatitis B surface antigen (HBsAg) and were considered to have previous HBV infection. The follow-up period varied from 12 to 116 (median, 36) months. Eighteen patients were followed without intervention. Five patients were vaccinated with recombinant HBV vaccine by the standard three-dose protocol after cessation of immunosuppressant administration. RS was defined as disappearance of anti-HBs and appearance of HBsAg and HBV-DNA with or without clinical hepatitis. Progressive decreases in anti-HBs titer were observed in all pre-HSCT anti-HBs-positive recipients. In 18 of the 20 patients with pre-HSCT anti-HBs, anti-HBs titer decreased to less than the protective value (

Description: Secondary clonal hematological disease in donor cells has rarely been reported as a complication of allogeneic stem cell transplantation in hematological disease. We report a case of myelodysplastic syndrome that showed cytogenetic abnormalities of t(2;3) and monosomy 7, which developed two years after peripheral blood stem cell transplantation for aplastic anemia and one year after liver transplantation for drug-induced hepatic failure. A 23-year-old female was diagnosed as having a severe-type aplastic anemia in April, 2000. Treatment with immunosuppressive drugs was not successful. Nineteen months later, she received allogeneic PBSCT from an HLA-matched elder on November 12, 2001. Her clinical course after PBSCT was marked by serious drug-induced hepatitis. Ten months after allogeneic PBSCT, she suddenly developed hepatic failure. On September 29, 2002, she was emergently transplanted with living donor-derived liver from the sibling who was the donor for allogeneic PBSCT. One month after liver transplantation (LT), pancytopenia had gradually progressed. At that time, chromosomal analysis showed 46, XX (20/20), and chimerism analysis of PB showed 100% donor-type. Late graft rejection of bone marrow was not suspected due to there being no relative lymphocytosis and the persistence of 100% complete donor-type chimerism. Thereafter, pancytopenia worsened, even with the stable complete donor-type chimerism, and G-CSF and EPO were administered. Bone marrow aspirate at seven months after the start of cytokine therapy showed trilineage dysplasia, suggesting secondary myelodysplasia. Cytogenetic analysis at that time showed t(2;3) in 17/20 cells. Eight months later, at 31 months post allogeneic PBSCT and 21 months post-LT, her bone marrow showed chromosomal abnormality with t(2;3) and additional monosomy 7 in 14/20 cells. There was no evidence of graft rejection because of sustained 100% donor-type chimerism. There was no possibility of second SCT because of hepatic dysfunction and hyperbilirubinemia even after LT as well as repeated intra-abdominal bacterial infection through a gastrostomy tube for nutrition. Bone marrow aspirations performed two and six months later showed an increased proportion of t(2;3) and monosomy 7 to 100%. She suddenly died of intra-abdominal bleeding due to profound thrombocytopenia 42 months after allogeneic PBSCT and 32 months after LT. The donor is currently alive and well with completely normal hematological data. This secondary malignancy of donor origin is most frequently seen in patients with leukemia. We suspect that the chromosomal abnormalities are related to hepatitis-associated aplastic anemia, administration of granulocyte colony-stimulating factor and erythropoietin for post-transplant pancytopenia, and repeated infections after liver transplantation.

Description: It has recently been shown that inhibitory natural killer cell receptors (NKRs) on not only NK cells but also on T cells negatively regulate NK cell and T cell functions through their binding to MHC class I molecules. The C-type lectin superfamily inhibitory NKR CD94/NKG2A heterodimer recognizes an HLA-E that preferably bound to a peptide derived from the signal sequences of most HLA class I. Therefore, CD94-expressing cells can monitor the global status of HLA class I on the tumor and leukemic cells and induce cytolytic attack without inhibitory signal against HLA class I decreased target cells resulting induction of graft-versus-leukemia (GVL) effect but does not attack normal cells with HLA class I expression resulting no enhancement of graft-versus-host disease (GVHD). On the other hand, CD4+ CD25+ regulatory T cells (Treg) contribute to suppress allogeneic immune responses and prevent transplant rejection and GVHD. In this study, we tried to expand CD94-expressing T cells and Treg cells from the same cord blood cells and then investigated their cytolytic characteristics and immunoregulatory function in order to develop a potential strategy of cell therapy for hematological malignancy. After CD4 enrichment by negative selection using magnetic cell sorting (MACS) (Miltenyi Biotec)(CD4-enriched fraction) from cord blood, CD4+ CD25+ cells were isolated by positive selection with anti-CD25 magnetic microbeads. We could get more than 1,000 fold expansion of CD94-expressing CD8 T cells from CD4-depleted fraction after 8 days culture with immobilized anti-CD3 monoclonal antibody (mAb) (1 μg/mL) and IL-15 (5 ng/mL). Isolated CD4+ CD25+ cells were cultured with anti-CD3/CD28 mAb-coated dynabeads and IL-15 (5 ng/mL) and we could get about 50 fold expansion of Treg cells for 8 days. These expanded Treg cells could suppress allogeneic mixed lymphocyte culture more than 80% (effector cells: Treg cells= 2:1) and expressed FoxP3 mRNA about 100 fold compared with isolated CD25-negative cells. Cytolytic activities of purified CD94-expressing cells (CD94 〉 90%) detected by 4 hours 51Cr release assay against K562 were 68.8 ± 16.8 % (n=5). Coculture of CD94-expressing cells with expanded Treg cells (CD94-expressing cell: Treg cells= 1:1, preincubation 4 hours) did not have any effect on cytolytic activities of purified CD94-expressing cells against K562 cells (66.1 ± 19.8 %, n=5). CD94-expressing CD8 T cells with cytolytic activity could be expanded from CD4-deplted fractions and Treg cells with immunosuppressive activity and increased expression level of FoxP3 mRNA could be expanded from CD4-enriched fractions of the same cord blood. Expanded these cytolytic CD94-expressing CD8 cells might be able to induce GVL effect without enhancing GVHD and Treg cells might be able to suppress allogeneic response including GVHD and graft rejection. Therefore, this strategy may be useful to differentiate lymphocytes in cord blood to two different kinds of effector cells exhibiting cytolytic or immunoreguratoly characters.

Description: Thalidomide (Thal) alone or in combination with steroids achieves responses even in the setting of refractory multiple myeloma (MM), however, responses are still limited. The precise mechanism of Thal action is unknown, further, no distinct marker, which could prognosticate the efficacy of Thal, is known. Therefore, we evaluated the correlation between the efficacy of Thal and the potent prognostic factors in patients with refractory MM. Ten patients with refractory MM received Thal at doses of 50 or 100 mg per day and steroids, either dexamethasone (Dex) or prednisolone (PSL). Dex was administrated 20 mg per day, 4 days every 28 days, and PSL was administrated 10 mg per day. The median age was 71.5 years (range, 62–79 years) and 20 % were man, and all patients were diagnosed as clinical stage IIIA based on the Durie and Salmon classification. The therapeutic response was assessed according to the modified criteria of Southwest Oncology Group (SWOG). Among 10 patients, 7 patients were the responders; 2 had complete remission, 3 had partial remission, and 2 had minimal remission. There were no differences in the pretreatment characteristics of responders and nonresponders (age, sex, type and concentration of serum and/or urine monoclonal component, international prognostic index, presence of bone lesion, and chromosomal abnormalities). However, flow cytometric evaluation of the myeloma cells revealed that CD56, which is one of the adhesion molecules N-CAM, expressed more than 45 % in all responders, while those expressed less than 5 % in all nonresponders (84 ± 19 (±SD) % v/s 4 ± 2 %, P=0.017). Furthermore, CD56 expression of the myeloma cells was reduced from 84% to 70 ± 32 % after Thal therapy in all evaluated responders (P =0.048). These results suggest that CD56 expression of the myeloma cells could be the potent prognostic marker of the Thal efficacy. Moreover, it was reported that Thal reduced the expression of cell adhesion molecules, such as LFA-1 and ICAM-1, and abrogated the binding of MM cells to bone marrow stromal cells, that triggered the secretion of interleukin-6 and vascular endothelial growth factor. Taken together, it was suggested that Thal reduced the expression of CD56 and altered the MM cell adhesion to bone marrow stromal cells, and that could be one of the pathogenesis of anti-MM activity of Thal.

Description: CD94 is one of the C-type lectin family members, forms a heterodimer with NKG2 gene family, and CD94 /NKG2A are inhibitory receptors. Not only NK cells but a subset of T cells express CD94/NKG2A, and previously we revealed the proportion of CD94/NKG2A expressing CD8 T cells were higher in patients with chronic graft versus host disease (GVHD), and CD94 expressing T cells have suppressive effects on mixed lymphocyte culture(MLC). We focus on CD94 positive T cell during T cell reconstitution after allogeneic hematopietic stem cell transplantation (allo-HSCT). T cell receptor excision circles (TREC) are suggested to be a useful marker of recent thymic output. In this study, we attempt to study TREC-containing CD8 T cell subset expressing CD94, and to examine the relation of TREC DNA level in CD94 expressing CD8 T cell and GVHD. We analyzed peripheral blood mononuclear cells (PBMCs) isolated from 24 patients (82 samples) undergone allo-HSCT including 15 patients with bone marrow transplantation and 9 patients with non-myeloablative stem cell transplantation. Informed consent was obtained from all patients. CD4 positive T cells were separated from PBMCs by magnetic cell sorting, and CD4 negative cell population was divided into CD94 positive CD8 T cells and CD94 negative CD8 T cells by fluorescence activated cell sorter. Genomic DNA was extracted from these separated T cell subsets. TREC DNA copy numbers per 105 isolated T cells (TREC level) were quantified by real time PCR. We investigated TREC levels in clinical status with pre-allo-HSCT, no episodes of GVHD or before manifestation of GVHD (No GVHD), chronic GVHD on disease (C-GVHD), and no symptoms and remission status of GVHD after immunosuppressive therapy (R-GVHD). Statical analyses were carried out by Mann-Whitney U test. There were no significant differences in TREC level of sorted CD4 positive T cells in C-GVHD compared with No GVHD (p=0.75) and R-GVHD (p=0.61), and also CD94 negative CD8 T cells in C-GVHD compared with No GVHD (p=0.79) and R-GVHD (p=0.20). On the other hand, TREC level of CD94 positive CD8 T cells in C-GVHD decreased in comparison with No GVHD (p=0.015) and R-GVHD (p=0.0019). The reduction of TREC level is thought to be induced not only by low thymic output but also by dilution of TREC concentration due to peripheral T cell expansion without duplications of TREC. These results may suggest that CD94 positive T cells play a role in modulation of GVHD, and proliferate during chronic GVHD with dilution of TREC in CD94 positive CD8 T cells. It is suggested that TREC level of CD94 expressing CD8 T cells may be useful markers of chronic GVHD.

Description: Innate immune cells such as natural killer (NK) cells play a crucial rule in antitumor immune responses. NK cells have functionally opposite receptors for activating and inhibiting signals to exert cytotoxicity. NKG2D is a C-type lectin-like activating receptor and is expressed in various immune cells, including NK cells, NKT cells, CD8+α/β+T cells, and γ/δ+T cells. NKG2D recognizes its ligands, MHC class I-related chain A and B (MICA, MICB). NKG2D ligands are not present on normal cells but are induced by various stresses such as viral infection. Moreover NKG2D ligands are expressed in many malignant cells including hematological malignancies. It has been suggested that involvement of NKG2D in NK or CD8+Tcell-mediated cytotoxicity correlates with the expression levels of ligands on target cells. Therefore induction of NKG2D ligands may lead to enhance the sensitivity to NKG2D-mediated cytotoxicity. In this study, we could enhance expression levels of MICA and MICB by treatment with the histone deacetylase inhibitor trichostatin A (TsA) in lymphoid leukemic cell line BALL1 and some primary patients’ lymphoid leukemic cells. Treatment of BALL1 and patients’ leukemic cells with TsA for 12 hr increased MICA and MICB mRNA expression at least by more than 2 fold by Real-time PCR. Treatment of BALL1 with TsA for 12 hr increased MICA and MICB protein expression on the cell surface by more than 2 fold by flow cytometry analysis. These results suggested that expression of MICA and MICB is partly regulated by histone acetylation. Chromatin immunoprecipitation assay revealed that treatment with TsA resulted in increased acetylation of histone H3 and decreased association with the counteracting enzymes of histone acetyltransferases HDAC1 at the MICA and MICB promoter in BALL1 cell and patients’ leukemic cells. To examine the impact of the cytolytic activity of NKG2D-expressing cells on leukemic cells in which expression of NKG2D ligands was induced by TsA treatment, we performed standard 4 hr 51Cr release assays using BALL1 cells and patients’ leukemic cells. Up-regulation of NKG2D ligands by TsA treatment led to enhance the susceptibility of BALL1 and patients’ leukemic cells by 2 or 3 times to the cytolytic activity of NKG2D-expressing cells. Blocking experiment using specific antibodies for MICA and MICB inhibited the NKG2D-expressing cell-mediated cytolytic activity against BALL1 cells. Our results suggest that regulation of NKG2D ligands expression by treatment with chromatin-remodeling drugs may be an effective strategy to enhance the susceptibility of leukemic cells to the cytolytic activity of NKG2D-expressing cells for immunotherapy.

Description: Buckgrand. Graft-versus host disease (GVHD) is a major complication after hematopoietic stem cell transplantation (HSCT). Macrophage migration inhibitory factor (MIF) plays a pivotal role in systemic as well as local inflammatory and immune responses. Recent reports that MIF expression is up-regulated in the allo-immune reaction during renal transplantation. Otherwise, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein belong to the TNF family. The level of TRAIL expression in T cells as well as NK cells can be markedly up-regulated after cell activations. In this study we report that kinetics of serum level of MIF and TRAIL in GVHD patients before and after HSCT. Patients and Methods. Date randomly obtained from 16 patients (10 males and 6 females) who underwent allo-SCT for treatment of hematological malignancies at Hokkaido University Hospital during the period May 2001 to January 2005. All patients were informed consent of peripheral blood smpling. Eight patients were received conventional transplantation and the others were reduced intensity stem cell transplantation (RIST). Seven patients have HLA identical sibling donor, but the others were received unrelated donor. Twelve of the 16 patients was achieved acute GVHD (aGVHD), gradeIto IIin 8 patients. Twelve patients survived day 100 after allo-SCT, 9 of those 12 patients developed chronic GVHD(cGVHD). Serum MIF and TRAIL concentration were measured at various time points using enzyme-linked immunosorbent assays (ELISAs). Results. Serum MIF concentration analysis by ELISA showed that only patients who developed aGVHD significantly increased (two folds) before and after allo-SCT (avelage, from 7.34 ng/ml before allo-SCT to 14.7 ng/ml after allo-SCT, p=0.018). However, we could not detect any correlation of MIF levels and aGVHD severity, donor sources. On the other hand, serum TRAIL concentration analysis by ELISA showed that patients who developed aGVHD were not associated (avelage, from 458.6 pg/ml before allo-SCT to 484.12 pg/ml after allo-SCT, p=0.632). We could not detect association aGVHD severity, donor sources. However, peak titer in aGVHD patients tends to decrease in unrelated transplantation (related 580.86pg/ml, unrelated 415.59pg/ml, p=0.22). Interestingly, we showed that average serum TRAIL concentration before allo-SCT associated with aGVHD and cGVHD. Serum TRAIL concentration with aGVHD patients (n=12, 458.85pg/ml) was tended to increase than without aGVHD (n=4, 330.45pg/ml, P=0.063) and with cGVHD patients (n=9, 535.21pg/ml) was significantly increase without cGVHD patients (n=3, 282.0 ng/ml, P=0.007). Discussion. The present study demonstrated the kinetics of MIF and TRAIL. Systemic up-regulation of MIF expression is associated with the occurrence of aGVHD. This data suggested that MIF after allo-SCT might play a pathogenetic role in aGVHD. On the other hand, we suggested that high level of TRAIL before allo-SCT associated acute and cGVHD. Maybe we might be to estimate acute and cGVHD in examining TRAIL level before allo-SCT. In conclusion our data are the first to establish an association TRAIL and GVHD in allogeneic stem cell transplantation.