A role of donor-specific HLA antibodies (DSA) in graft failure after SCT has been suggested, but the relevance of DSA in unmanipulated haploidentical SCT (haplo-SCT) remains unknown. We prospectively examined HLA antibodies using the Luminex-based single Ag assay for 79 adult patients undergoing unmanipulated haplo-SCT. Among them, 16 (20.2%) were HLA Ab-positive, including five patients with antibodies not corresponding to donor HLA Ags and 11 DSA-positive patients. Of the 11 DSA-positive patients, five received treatments to decrease DSA levels, including two, who received plasma exchange and rituximab, two who received platelet transfusions from healthy-related donors having DSAcorresponding HLA Ags and one who received bortezomib. Platelet transfusion was the most simple and effective treatment option for class I DSA. The cumulative incidence of neutrophil recovery was significantly lower in pretransplant (post-treatment) DSA-positive patients than in DSA-negative patients (61.9 vs 94.4%, P ¼ 0.026). Notably, three of five patients with high levels of DSA had graft failure. Donors should be selected on the basis of an evaluation of HLA antibodies. If haplo-SCT from donors with HLA Ags that correspond to high levels of DSA must be performed, then recipients should be treated for DSA to improve the chances of successful donor engraftment.

The product of the Wilms' tumor gene WT1 is a transcription factor overexpressed not only in leukemic blast cells of almost all patients with acute myeloid leukemia, acute lymphoid leukemia, and chronic myeloid leukemia, but also in various types of solid tumor cells. Thus, it is suggested that the WT1 gene plays an important role in both leukemogenesis and tumorigenesis. Here we tested the potential of WT1 to serve as a target for immunotherapy against leukemia and solid tumors. Four 9-mer WT1 peptides that contain HLA-A2.1-binding anchor motifs were synthesized. Two of them, Db126 and WH187, were determined to bind to HLA-A2.1 molecules in a binding assay using transporter associated with antigen processing-deficient T2 cells. Peripheral blood mononuclear cells from an HLA-A2.1-positive healthy donor were repeatedly sensitized in vitro with T2 cells pulsed with each of these two WT1 peptides, and CD8(+) cytotoxic T lymphocytes (CTLs) that specifically lyse WT1 peptide-pulsed T2 cells in an HLA-A2.1-restricted fashion were induced. The CTLs also exerted specific lysis against WT1-expressing, HLA-A2.1-positive leukemia cells, but not against WT1-expressing, HLA-A2.1-negative leukemia cells, or WT1-nonexpressing, HLA-A2. 1-positive B-lymphoblastoid cells. These data provide the first evidence of human CTL responses specific for the WT1 peptides, and provide a rationale for developing WT1 peptide-based adoptive T-cell therapy and vaccination against leukemia and solid tumors.

Expression of theThe Wilms' tumor gene WT1 was originally isolated as a tumorsuppressor gene responsible for Wilms' tumor, a kidney neoplasm of childhood. 1 However, we proposed that WT1 played an oncogenic role in leukemogenesis based on the following findings: 2 (i) the wild-type WT1 gene was expressed at high levels in leukemic blast cells, 3,4 (ii) there was a clear and inverse correlation between WT1 expression levels and prognosis in acute leukemia, 3 (iii) WT1 expression increased at relapse of acute leukemia, 5 (iv) growth of leukemic blast cells was inhibited by the treatment of WT1 antisense oligomers 6 and (v) constitutive expression of WT1 blocked differentiation and instead induced proliferation in response to granulocyte colony-stimulating factor in 32D cl3 myeloid progenitor cells 7 and normal myeloid progenitor cells. 8 Furthermore, we demonstrated that the wild-type WT1 as expressed in various types of cell line derived from lung cancer, gastric cancer, colon cancer and breast cancer and that growth of these WT1-expressing tumor cells was inhibited by the treatment of WT1 antisense oligomers. 9 These data suggested an oncogenic role of the WT1 gene in tumorigenesis. However, the involvement of the WT1 gene in de novo solid tumors remained unclear. In the present study, we examined WT1 expression in de novo lung cancer using quantitative real-time RT-PCR and immunohistochemistry and demonstrated that the wild-type WT1 was overexpressed in 54/56 (96%) de novo non-small cell lung cancers (NSCLCs) and 5/6 (83%) de novo small cell lung cancers (SCLCs) examined.

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In acute-type leukemia, no method for the prediction of relapse following allogeneic stem cell transplantation based on minimal residual disease (MRD) levels is established yet. In the present study, MRD in 72 cases of allogeneic transplantation for acute myeloid leukemia, acute lymphoid leukemia, and chronic myeloid leukemia (accelerated phase or blast crisis) was monitored frequently by quantitating the transcript of WT1 gene, a "panleukemic MRD marker," using reverse transcriptase-polymerase chain reaction. Based on the negativity of expression of chimeric genes, the background level of WT1 transcripts in bone marrow following allogeneic transplantation was significantly decreased compared with the level in healthy volunteers. The probability of relapse occurring within 40 days significantly increased step-by-step according to the increase in WT1 expression level (100% for 1.0 ؋ 10 ؊2 -5.0 ؋ 10 ؊2 , 44.4% for 4.0 ؋ 10 ؊3 -1.0 ؋ 10 ؊2 , 10.2% for 4.0 ؋ 10 ؊4 -4.0 ؋ 10 ؊3 , and 0.8% for < 4.0 ؋ 10 ؊4 ) when WT1 level in K562 was defined as 1.0). WT1 levels in patients having relapse increased exponentially with a constant doubling time. The doubling time of the WT1 level in patients for whom the discontinuation of immunosuppressive agents or donor leukocyte infusion was effective was significantly longer than that for patients in whom it was not (P < .05). No patients with a short doubling time of WT1 transcripts (< 13 days) responded to these immunomodulation therapies. These findings strongly suggest that the WT1 assay is very useful for the prediction and management of relapse following allogeneic stem cell transplantation regardless of the presence of chimeric gene

To clarify whether the expression of the WT1 gene in leukemic cells is aberrant or merely reflects that in normal counterparts, the expression levels of the WT1 gene were quantitated for normal hematopoietic progenitor cells. Bone marrow (BM) and umbilical cord blood (CB) cells were fluorescence-activated cell sorting (FACS)-sorted into CD34+ and CD34− cell populations, and the CD34+ cells into nine subsets (CD34+CD33−, CD34+CD33+, CD34+CD38−, CD34+CD38+, CD34+HLA-DR−, CD34+HLA-DR+, CD34+c-kithigh, CD34+c-kitlow, and CD34+c-kit−) according to the expression levels of CD34, CD33, CD38, HLA-DR, and c-kit. Moreover, acute myeloid leukemic cells were also FACS-sorted into four populations (CD34+CD33−, CD34+CD33+, CD34− CD33+, and CD34− CD33−). FACS-sorted normal hematopoietic progenitor and leukemic cells and FACS-unsorted leukemic cells were examined for the WT1 expression by quantitative reverse transcriptase-polymerase chain reaction. The WT1 expression in the CD34+ and CD34− cell populations and in the nine CD34+ subsets of BM and CB was at either very low (1.0 to 2.4 × 10−2) or undetectable (<10−2) levels (the WT1 expression level of K562 cells was defined as 1.0), whereas the average levels of WT1 expression in FACS-sorted and -unsorted leukemic cells were 2.4 to 9.3 × 10−1. Thus, the WT1 expression levels in normal hematopoietic progenitor cells were at least 10 times less than those in leukemic cells. Therefore, we could not find any normal counterparts of BM or CB that expressed the WT1 at levels comparable with those in leukemic cells. These results indicate an aberrant overexpression of the WT1 gene in leukemic cells and imply the involvement of this gene in human leukemogenesis.

The Wilms' tumor gene, WT1, is a tumor marker for leukemic blast cells. The WT1 expression levels were examined for 57 patients with myelodysplastic syndromes (MDS) (refractory anemia (RA), 35; RA with excess of blasts (RAEB) 14; RAEB in transformation (RAEB-t), six; and MDS with fibrosis, two) and 12 patients with acute myeloid leukemia (AML) evolved from MDS. These levels significantly increased in proportion to the disease progression of MDS from RA to overt AML via RAEB and RAEB-t in both bone marrow (BM) and peripheral blood (PB). WT1 expression levels in PB significantly correlated with the evolution of RAEB or RAEB-t to overt AML within 6 months. Therefore, WT1 expression levels in PB were superior to those in BM for early prediction of the evolution to AML by means of quantitation of the WT1 expression levels. Furthermore, WT1 expression in PB of patients with overt AML evolved from MDS was significantly decreased by effective chemotherapy or allogeneic stem cell transplantation and became undetectable in long-term survivors. These results clearly showed that WT1 expression levels are a tumor marker for preleukemic or leukemic blast cells of MDS and thus reflect the disease progression of MDS. Therefore, monitoring of WT1 expression levels has made continuous assessment of the disease progression of MDS possible, as well as the prediction of the evolution of RAEB or RAEB-t to overt AML within 6 months. The results also showed that quantitation of WT1 expression levels is useful for diagnosis of minimal residual disease of MDS with high sensitivity, thus making it possible to evaluate the efficacy of treatment for MDS.

• There is a role for the posttranslational modification, neddylation, in regulation of immune responses mediated by dendritic cells.• A role for neddylation in NF-kB signaling in dendritic cells was identified.Posttranslational protein modifications (PTMs) are necessary for cells to function properly. The role of PTMs in regulating immune responses, specifically those mediated by dendritic cells (DCs), which are critical for both innate and adaptive immunity, is not well understood. Utilizing multiple but complementary approaches, we determined the role of an important but less understood type of PTM, namely, neddylation, in regulating DC functions. Inhibition of neddylation suppressed the release of proinflammatory cytokines by DCs in response to Toll-like receptor, nucleotide oligomerization domain-like receptor, and noninfectious CD40L stimulation. These effects were more profound than those mediated by the proteasome inhibitor bortezomib or a commonly used antiinflammatory agent, dexamethasone. Targeting neddylation also suppressed the ability of DCs to stimulate murine allogeneic T cells in vitro and in vivo and human allogeneic T-cell responses in vitro. Mechanistic studies demonstrated that inhibition of neddylation reduced both canonical and noncanonical nuclear factor-kB (NF-kB) activity. Neddylation inhibition prevented the degradation of inhibitor-kB and thus reduced the translocation and activation of NF-kB, but without perturbation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Thus, blocking neddylation could be a novel strategy for mitigating immune-mediated disease processes. (Blood. 2013;122(12):2062-2073