Tatsuya Fujioka

Risk and prevention of graft failure in patients with preexisting donor-specific HLA antibodies undergoing unmanipulated haploidentical SCT.

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 DSA-corresponding 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=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.

Monitoring Minimal Residual Disease in Leukemia Using Real-time Quantitative Polymerase Chain Reaction for Wilms Tumor Gene (WT1)

We previously showed that Wilms tumor gene (WT1) expression level, measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), was useful as an indicator of minimal residual disease (MRD) in leukemia and myelodysplastic syndrome. However, in conventional quantitative RT-PCR (CQ-PCR), RT-PCR must be performed for various numbers of cycles depending onWT1 expression level. In the present study, we developed a new real-time quantitative RT-PCR (RQ-PCR) method for quantitatingWT1 transcripts. Results of intraassay and interassay variability tests demonstrated that the real-timeWT1 assay had high reproducibility.WT1 expression levels measured by the RQ- and the CQ-PCR methods were strongly correlated (r = 0.998). Furthermore, a strong correlation was observed amongWT1 transcript values normalized with 3 different control genes (β-actin,ABL, andglyceraldehyde-3-phosphate dehydrogenase) and between relativeWT1 transcript values withWT1 expression in K562 cells as the reference and absoluteWT1 transcript copy numbers per microgram RNA. WhenWT1 expression andminor bcr-abl expression were concurrently monitored in 2 patients withbcr-abl-positive acute lymphoblastic leukemia, both MRDs changed mostly in parallel, indicating the reliability and validity of our RQ-PCR method. In conclusion, this RQ-PCR method is convenient and reliable for monitoring MRD and enables routine clinical use of aWT1 assay.

Identification and characterization of a WT1 (Wilms Tumor Gene) protein-derived HLA-DRB1*0405-restricted 16-mer helper peptide that promotes the induction and activation of WT1-specific cytotoxic T lymphocytes.

Effective tumor vaccine may be required to induce both cytotoxic T lymphocyte (CTL) and CD4+ helper T-cell responses against tumor-associated antigens. CD4+ helper T cells that recognize HLA class II-restricted epitopes play a central role in the initiation and maintenance of antitumor immune responses. The Wilms tumor gene WT1 is overexpressed in both leukemias and solid tumors, and the WT1 protein was demonstrated to be an attractive target antigen for cancer immunotherapy. In this study, we identified a WT1 protein-derived 16-mer peptide, WT1332 (KRYFKLSHLQMHSRKH), which was restricted with HLA-DRB1*0405, one of the most common HLA class II types in Japanese, as a helper epitope that could elicit WT1-specific CD4+ T-cell responses. We established a WT1332-specific CD4+ helper T-cell clone (E04.1), which could respond to both HLA-DRB1*0405-positive, WT1-expressing transformed hematopoietic cells and autologous dendritic cells pulsed with apoptosis-induced WT1-expressing cells, indicating that the WT1332 was a naturally processed helper epitope. Stimulation of peripheral blood mononuclear cells with both the CTL epitope (WT1235) and the helper epitope (WT1332) in the presence of WT1332-specific TH1-type CD4+ T cell clone strikingly enhanced the induction and the functional activity of WT1235-specific CTLs compared with that of peripheral blood mononuclear cells with the WT1235 alone. These results indicated that a helper epitope, WT1332 should be useful for improvement of the efficacy of CTL epitope-based cancer vaccine targeting WT1 in the clinical setting.

CD133 is a positive marker for a distinct class of primitive human cord blood-derived CD34-negative hematopoietic stem cells

The identification of human CD34-negative (CD34−) hematopoietic stem cells (HSCs) provides a new concept for the hierarchy in the human HSC compartment. Previous studies demonstrated that CD34− severe combined immunodeficiency (SCID)-repopulating cells (SRCs) are a distinct class of primitive HSCs in comparison to the well-characterized CD34+CD38− SRCs. However, the purification level of rare CD34− SRCs in 18 lineage marker-negative (Lin−) CD34− cells (1/1000) is still very low compared with that of CD34+CD38− SRCs (1/40). As in the mouse, it will be necessary to identify useful positive markers for a high degree of purification of rare human CD34− SRCs. Using 18Lin−CD34− cells, we analyzed the expression of candidate positive markers by flow cytometric analysis. We finally identified CD133 as a reliable positive marker of human CB-derived CD34− SRCs and succeeded in highly purifying primitive human CD34− HSCs. The limiting dilution analysis demonstrated that the incidence of CD34− SRCs in 18Lin−CD34−CD133+ cells was 1/142, which is the highest level of purification of these unique CD34− HSCs to date. Furthermore, CD133 expression clearly segregated the SRC activities of 18Lin−CD34− cells, as well as 18Lin−CD34+ cells, in their positive fractions, indicating its functional significance as a common cell surface maker to isolate effectively both CD34+ and CD34− SRCs.

Frequency of CD4(+)FOXP3(+) regulatory T-cells at early stages after HLA-mismatched allogeneic hematopoietic SCT predicts the incidence of acute GVHD.

Acute GVHD (aGVHD) is a major obstacle to allogeneic hematopoietic SCT (alloHSCT). Although it is thought that aGVHD is initiated in secondary lymphoid organs at a very early stage of alloHSCT, whether CD4+FOXP3+ regulatory T-cells (Tregs) have an impact on aGVHD development during this period remains unclear. Here, we measured Tregs in peripheral blood as early as possible after HLA-mismatched alloHSCT, and assessed the incidence of aGVHD. Flow cytometric analyses revealed that at the second week after HSCT, patients with aGVHD had significantly (P=0.018) lower Treg:CD4+T-cell ratios than those without aGVHD. As these differences were seen before the development of aGVHD, these ratios can predict the incidence of aGVHD. The cumulative incidence of aGVHD in patients with ratios of <9% was significantly higher than that in patients with ratios of ⩾9% (P=0.0082, log-rank test). Additionally, the specific ratio of Tregs:CD4+T-cells was the most significant value among all other possible lymphocyte-associated ratios and absolute cell counts. These findings suggest that the ratio of Tregs:CD4+T-cells at the second week post HLA-mismatched alloHSCT might be a potent predictor of aGVHD in these patients. The practical efficacy of this finding should be verified in further interventional studies.

Successful engraftment of HLA-haploidentical related transplants using nonmyeloablative conditioning with fludarabine, busulfan and anti-T-lymphocyte globulin

Successful engraftment of HLA-haploidentical related transplants using nonmyeloablative conditioning with fludarabine, busulfan and anti-T-lymphocyte globulin

Prospectively Isolated Human Bone Marrow Cell‐Derived MSCs Support Primitive Human CD34‐Negative Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) niche, which consists of osteoblasts, endothelial cells, and a variety of mesenchymal stem/stromal cells (MSCs). However, precisely what types of MSCs support human HSCs in the BM remain to be elucidated because of their heterogeneity. In this study, we succeeded in prospectively isolating/establishing three types of MSCs from human BM‐derived lineage‐ and CD45‐negative cells, according to their cell surface expression of CD271 and stage‐specific embryonic antigen (SSEA)−4. Among them, the MSCs established from the Lineage−CD45−CD271+SSEA‐4+ fraction (DP MSC) could differentiate into osteoblasts and chondrocytes, but they lacked adipogenic differentiation potential. The DP MSCs expressed significantly higher levels of well‐characterized HSC‐supportive genes, including IGF‐2, Wnt3a, Jagged1, TGFβ3, nestin, CXCL12, and Foxc1, compared with other MSCs. Interestingly, these osteo‐chondrogenic DP MSCs possessed the ability to support cord blood‐derived primitive human CD34‐negative severe combined immunodeficiency‐repopulating cells. The HSC‐supportive actions of DP MSCs were partially carried out by soluble factors, including IGF‐2, Wnt3a, and Jagged1. Moreover, contact between DP MSCs and CD34‐positive (CD34+) as well as CD34‐negative (CD34−) HSCs was important for the support/maintenance of the CD34+/− HSCs in vitro. These data suggest that DP MSCs might play an important role in the maintenance of human primitive HSCs in the BM niche. Therefore, the establishment of DP MSCs provides a new tool for the elucidation of the human HSC/niche interaction in vitro as well as in vivo. Stem Cells 2015;33:1554–1565

Salvage haploidentical transplantation for graft failure using reduced-intensity conditioning

Graft failure is a major concern after cord blood transplantation (CBT) or HLA-haploidentical transplantation (haplo-SCT). As patients who undergo CBT or haplo-SCT almost always lack both matched-related and -unrelated donors, salvage transplantation would also be limited to either CBT or haplo-SCT. In this study, we assessed eight patients who received haplo-SCT as salvage therapy for graft failure. Five and three patients had received haplo-SCT and CBT, respectively, which resulted in graft failure. The median interval from the failed transplantation to salvage transplantation in six patients with primary graft failure was 33.5 days. The reduced-intensity conditioning regimen consisted of fludarabine, thiotepa, rabbit antithymocyte globulin and low-dose TBI. All eight patients achieved neutrophil engraftment, and seven patients achieved platelet recovery. The median times to neutrophil recovery and platelet recovery were 10 and 20 days, respectively. Three patients died from treatment-related causes: two from GVHD and one from rupture of carotid artery aneurysm. Five patients are alive, at a median follow-up of 946 days. The probability of overall survival at 5 years was 75%. These findings may serve as a rationale for giving precedence to haplo-SCT over CBT in salvage SCT after graft failure.

Combination of tacrolimus, methotrexate, and methylprednisolone prevents acute but not chronic Graft-Versus-Host disease in unrelated bone marrow transplantation

Background. Graft-versus-host disease (GVHD) is still a major problem in allogeneic bone marrow transplantation (BMT). Prophylactic regimens used against GVHD in unrelated BMT, including cyclosporine (CsA)-plus-methotrexate (MTX), CsA-plus-MTX-plus-prednisone, and tacrolimus (FK506)-plus-MTX, are still unsatisfactory (34–70% occurrence of grades II–IV GVHD). To address this problem, we examined the efficacy of FK506-plus-MTX-plus-methylprednisolone (mPSL) in 20 patients who underwent BMT from unrelated donors. Methods. All patients received FK506 beginning the day before transplantation at a dose of 0.03 mg/kg per day by continuous intravenous (IV) infusion. MTX was administered at a dose of 10 mg/m2 IV on day 1, and 7 mg/m2 on days 3, 6, and 11. Intravenous administration of mPSL was started at a dose of 2 mg/kg per day on day 1. In the absence of acute GVHD, mPSL was gradually tapered from day 29. Results. Development of acute GVHD was almost completely suppressed (one patient with grade I, none with grades II–IV). However, the incidence and severity of chronic GVHD did not decrease. Eight of 12 patients with extensive chronic GVHD died of thrombotic microangiopathy or infection. A vigorous fluctuation (>100 U/mL per 10 days) of the soluble interleukin 2 receptor level in the serum after engraftment was highly related to the occurrence of chronic GVHD. Conclusions. An FK506-plus(+)-MTX-plus(+)-mPSL prophylactic regimen could almost completely suppress acute GVHD but not chronic GVHD in unrelated BMT. In this GVHD prophylactic system, the extent of the change of soluble interleukin 2 receptor level may be a good predictor of development of chronic GVHD.

Impact of the mobilization regimen and the harvesting technique on the granulocyte yield in healthy donors for granulocyte transfusion therapy

BACKGROUND: Granulocyte mobilization and harvesting, the two major phases of granulocyte collection, have not been standardized.