Efficient protocol for the differentiation of kidney podocytes from induced pluripotent stem cells, involving the inhibition of mTOR.

The mechanistic/mammalian target of rapamycin (mTOR) is involved in a wide range of cellular processes. However, the role of mTOR in podocytes remains unclear. In this study, we aimed to clarify the role of mTOR in podocyte differentiation from human induced pluripotent stem cells (hiPSCs) and to establish an efficient differentiation protocol for human podocytes. We generated podocytes from hiPSCs by modifying protocol. The expression of the podocyte-specific slit membrane components nephrin and podocin was measured using PCR, western blotting, flow cytometry, and immunostaining; and the role of mTOR was evaluated using inhibitors of the mTOR pathway. Nephrin and podocin were found to be expressed in cells differentiated from hiPSCs, and their expression was increased by mTOR inhibitor treatment. S6, a downstream component of the mTOR pathway, was also found to be involved in podocyte differentiation. we evaluated its permeability to albumin, urea, and electrolytes. The induced podocytes were permeable to the small molecules, but only poorly permeable to albumin. We have shown that the mTOR pathway is involved in podocyte differentiation. Our monolayer podocyte differential protocol, using an mTOR inhibitor, provides a novel in vitro model for studies of kidney physiology and pathology.

The Induction of Parathyroid Cell Differentiation from Human Induced Pluripotent Stem Cells Promoted Via TGF-α/EGFR Signaling.

The parathyroid gland plays an essential role in mineral and bone metabolism. Cultivation of physiological human parathyroid cells has yet to be established and the method by which parathyroid cells differentiate from pluripotent stem cells remains uncertain. Therefore, it has been hard to clarify the mechanisms underlying the onset of parathyroid disorders, such as hyperparathyroidism. In this study, we developed a new method of parathyroid cell differentiation from human induced pluripotent stem (iPS) cells. Parathyroid cell differentiation occurred in accordance with embryologic development. Differentiated cells, which expressed the parathyroid hormone, adopted unique cell aggregation similar to the parathyroid gland. In addition, these differentiated cells were identified as calcium-sensing receptor (CaSR)/epithelial cell adhesion molecule (EpCAM) double-positive cells. Interestingly, stimulation with transforming growth factor-α (TGF-α), which is considered a causative molecule of parathyroid hyperplasia, increased the CaSR/EpCAM double-positive cells, but this effect was suppressed by erlotinib, which is an epidermal growth factor receptor (EGFR) inhibitor. These results suggest that TGF-α/EGFR signaling promotes parathyroid cell differentiation from iPS cells in a similar manner to parathyroid hyperplasia.

Low incidence of HHV-6 reactivation in haploidentical hematopoietic stem cell transplantation with corticosteroid as graft-vs-host disease prophylaxis compared with cord blood transplantation.

BACKGROUND: Human leukocyte antigen (HLA) mismatch and the administration of immunosuppressive agents are considered risks for human herpesvirus 6 (HHV-6) reactivation after stem cell transplantation (SCT). However, the incidence of HHV-6 reactivation in HLA-mismatched related SCT remains unknown. METHODS: We monitored plasma HHV-6 DNA loads weekly using real-time quantitative polymerase chain reaction for 5 weeks after SCT and compared serum IL-6 levels in HLA-mismatched SCT groups. RESULTS: Compared with detection in all 11 umbilical cord blood transplantation (CBT) patients (100%), plasma HHV-6 DNA was detected in only 3 of 42 haplo-SCT patients (7.1%) despite the use of methylprednisolone and antithymocyte globulin as graft-vs-host disease prophylaxis and a reduced-intensity conditioning regimen, respectively. Correspondingly, serum IL-6 levels in haplo-SCT patients were significantly lower than those in CBT patients. No HHV-6-associated encephalitis developed in either groups. CONCLUSIONS: Neither HLA disparity nor the use of methylprednisolone and antithymocyte globulin were risk factors for HHV-6 reactivation in our haplo-SCT patients. Rather than increasing risk, the administration of immunosuppressive agents potentially prevented HHV-6 reactivation after haplo-SCT by suppressing IL-6 production.

The number of CD34+CD133+ hematopoietic stem cells residing in umbilical cord blood (UCB) units is not correlated with the numbers of total nucleated cells and CD34+ cells: a possible new indicator for quality evaluation of UCB units.

Umbilical cord blood transplantation (UCBT) is often associated with delayed neutrophil and platelet recovery. Engraftment failure is another major obstacle. Several factors influence these serious complications, including the numbers of total nucleated cells (TNCs) and CD34+ cells which have been used as reliable factors for selecting UCB units for transplantation. However, whether both factors are reliable indices of the hematopoietic stem cell (HSC) activity of UCB units remains unknown. To evaluate the quality of UCB units, we quantified the actual number of transplantable CD34+CD133+ HSCs (tHSCs) residing in UCB units. The number of tHSCs was not correlated with the numbers of TNCs or CD34+ cells. These results strongly suggest that neither factor reflects the numbers of tHSCs residing in UCB units. To validate the significance of the number of tHSCs, further analysis is required to determine whether the number of tHSCs residing in UCB units is useful as a new indicator for the quality assessment of UCB units.

A revised road map for the commitment of human cord blood CD34-negative hematopoietic stem cells.

We previously identified CD34-negative (CD34-) severe combined immunodeficiency (SCID)-repopulating cells as primitive hematopoietic stem cells (HSCs) in human cord blood. In this study, we develop a prospective ultra-high-resolution purification method by applying two positive markers, CD133 and GPI-80. Using this method, we succeed in purifying single long-term repopulating CD34- HSCs with self-renewing capability residing at the apex of the human HSC hierarchy from cord blood, as evidenced by a single-cell-initiated serial transplantation analysis. The gene expression profiles of individual CD34+ and CD34- HSCs and a global gene expression analysis demonstrate the unique molecular signature of CD34- HSCs. We find that the purified CD34- HSCs show a potent megakaryocyte/erythrocyte differentiation potential in vitro and in vivo. Megakaryocyte/erythrocyte progenitors may thus be generated directly via a bypass route from the CD34- HSCs. Based on these data, we propose a revised road map for the commitment of human CD34- HSCs in cord blood.

TGF-ß Signaling Accelerates Senescence of Human Bone-Derived CD271 and SSEA-4 Double-Positive Mesenchymal Stromal Cells.

It is generally thought that the proliferative capacity and differentiation potential of somatic stem cells, including mesenchymal stromal/stem cells (MSCs) and hematopoietic stem cells, decline with age. We investigated the effects of aging on human bone-derived MSCs expressing CD271 and SSEA-4 (double-positive MSCs [DPMSCs]). The percentages of DPMSCs in bone tissue decreased significantly with age. The DPMSCs from elderly patients (old DPMSCs) showed cellular senescence, which was evidenced by low growth potential, high senescence-associated ß-galactosidase activity, and elevated p16 and p21 CDK inhibitor levels. Moreover, old DPMSCs showed weak osteogenic differentiation potential and less hematopoiesis-supporting activity in comparison with young DPMSCs. Interestingly, the addition of transforming growth factor ß2 (TGF-ß2) induced cellular senescence in young DPMSCs. With the exception of the adipogenic differentiation potential, all of the aging phenomena observed in old DPMSCs were reversed by the addition of anti-TGF-ß antibodies. These results suggest that, in part, old DPMSCs accelerate cellular senescence through TGF-ß signaling.

CD34 Antigen and the MPL Receptor Expression Defines a Novel Class of Human Cord Blood-Derived Primitive Hematopoietic Stem Cells.

In the murine hematopoietic stem cell (HSC) compartment, thrombopoietin (THPO)/MPL (THPO receptor) signaling plays an important role in the maintenance of adult quiescent HSCs. However, the role of THPO/MPL signaling in the human primitive HSC compartment has not yet been elucidated. We have identified very primitive human cord blood (CB)-derived CD34- severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method. In this study, we investigated the roles of the MPL expression in the human primitive HSC compartment. The SRC activities of the highly purified CB-derived 18Lin-CD34+/-MPL+/- cells were analyzed using NOG mice. In the primary recipient mice, nearly all mice that received CD34+/-MPL+/- cells were repopulated with human CD45+ cells. Nearly all of these mice that received CD34+MPL+/- and CD34-MPL- cells showed a secondary repopulation. Interestingly, the secondary recipient mice that received CD34+/-MPL- cells showed a distinct tertiary repopulation. These results clearly indicate that the CD34+/- SRCs not expressing MPL sustain a long-term (LT) (>1 year) human cell repopulation in NOG mice. Moreover, CD34- SRCs generate CD34+CD38-CD90+ SRCs in vitro and in vivo. These findings provide a new concept that CD34-MPL- SRCs reside at the apex of the human HSC hierarchy.

Identification and Characterization of Lineage(-)CD45(-)Sca-1(+) VSEL Phenotypic Cells Residing in Adult Mouse Bone Tissue.

Murine bone marrow (BM)-derived very small embryonic-like stem cells (BM VSELs), defined by a lineage-negative (Lin(-)), CD45-negative (CD45(-)), Sca-1-positive (Sca-1(+)) immunophenotype, were previously reported as postnatal pluripotent stem cells (SCs). We developed a highly efficient method for isolating Lin(-)CD45(-)Sca-1(+) small cells using enzymatic treatment of murine bone. We designated these cells as bone-derived VSELs (BD VSELs). The incidences of BM VSELs in the BM-derived nucleated cells and that of BD VSELs in bone-derived nucleated cells were 0.002% and 0.15%, respectively. These BD VSELs expressed a variety of hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and endothelial cell markers. The gene expression profile of the BD VSELs was clearly distinct from those of HSCs, MSCs, and ES cells. In the steady state, the BD VSELs proliferated slowly, however, the number of BD VSELs significantly increased in the bone after acute liver injury. Moreover, green fluorescent protein-mouse derived BD VSELs transplanted via tail vein injection after acute liver injury were detected in the liver parenchyma of recipient mice. Immunohistological analyses suggested that these BD VSELs might transdifferentiate into hepatocytes. This study demonstrated that the majority of the Lin(-)CD45(-)Sca-1(+) VSEL phenotypic cells reside in the bone rather than the BM. However, the immunophenotype and the gene expression profile of BD VSELs were clearly different from those of other types of SCs, including BM VSELs, MSCs, HSCs, and ES cells. Further studies will therefore be required to elucidate their cellular and/or SC characteristics and the potential relationship between BD VSELs and BM VSELs.

Early detection of cytomegalovirus-specific cytotoxic T lymphocytes against cytomegalovirus antigenemia in human leukocyte antigen haploidentical hematopoietic stem cell transplantation.

Human leukocyte antigen (HLA)-haploidentical stem cell transplantation (haplo-SCT) is associated with a high incidence of cytomegalovirus (CMV) infection, probably originating from the delayed reconstitution of CMV-specific T cell immunity. There have been few reports on the presence of CMV-specific cytotoxic T lymphocytes (CMV-CTLs) after haplo-SCT. We have studied CMV-specific immune reconstitution by measuring the absolute number of CMV-CTLs using a flow cytometry method with HLA-A2-restricted NLVPMVATV peptide dextramers. We examined the association between reconstitution patterns of CMV-CTLs and the duration of CMV antigenemia in 15 patients who underwent first allogeneic SCT from HLA-haploidentical-related donors with HLA-A2. In seven and eight patients, CMV antigenemia consecutively resolved for more than 4 weeks (the CMV antigenemia 'resolved' group) and intermittently persisted (the CMV antigenemia 'persistent' group) during a 100-day observation period, respectively. The group of the seven patients, in whom levels of CMV antigenemia were reduced to zero, had a significantly lower maximum level of CMV antigenemia than the CMV antigenemia persistent group. In contrast, the CMV antigenemia persistent group had a significantly higher maximum level of CMV-CTLs, but the levels took longer to peak. Despite no difference in general lymphocyte recovery between the two groups, the CMV antigenemia resolved group had significantly higher median CMV-CTL counts than the CMV antigenemia persistent group at 6 weeks after onset of CMV infection. Flow cytometry analysis of CMV-CTLs is a convenient method of monitoring reconstitution of CMV-specific lymphocyte immunity following haplo-SCT.

Mouse dental pulp stem cells support human umbilical cord blood-derived hematopoietic stem/progenitor cells in vitro.

It is well documented that specialized mesenchymal stem/stromal cells (MSCs) constitute the hematopoietic stem cell (HSC) niche in the bone marrow (BM), and these MSCs support/maintain the HSCs in an undifferentiated state. A number of studies have demonstrated that BM-derived MSCs (BM-MSCs) can support HSCs in vitro. However, it remains unclear whether nonhematopoietic tissue-derived MSC-like cells, such as dental pulp stem cells (DPSCs), have the ability to support HSCs. In this study, we prospectively isolated DPSCs from mouse mandibular incisors by fluorescence-activated cell sorting (FACS) using BM-MSC markers, such as PDGFRα and Sca-1. The PDGFRα and Sca-1 double-positive DPSCs and BM-MSCs showed similar morphologies and expression patterns of MSC markers. The ability of the DPSCs to support hematopoietic stem/progenitor cells (HSPCs) was then analyzed by an in vitro coculture system. Moreover, their HSC-supporting activity was evaluated by in vivo xenotransplantation assays using NOD/Shi-scid/IL-2Rγc(null) (NOG) mice. Interestingly, the DPSCs supported human cord blood (CB)-derived CD34-positive (CD34(+)), as well as CD34-negative (CD34(-)), HSCs. The supporting activities of DPSCs for human CB-derived CD34(+) and CD34(-) HSCs were comparable to those of BM-MSCs. The results of the present study demonstrated, for the first time, that prospectively isolated murine PDGFRα and Sca-1 double-positive DPSCs could support primitive human CD34(+) and CD34(-) HSCs in vitro.

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, TGFß3, 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.

Detection of donor-derived CMV-specific T cells in cerebrospinal fluid in a case of CMV meningoencephalitis after cord blood stem cell transplantation.

Cytomegalovirus (CMV) meningoencephalitis is a rather rare complication after allogeneic stem cell transplantation. We describe here the case of a 59-year-old man with acute myeloid leukemia who developed CMV meningoencephalitis after cord blood transplantation. The patient presented with a sudden onset of neurological symptoms, such as convulsion, on day 37. The analysis of cerebrospinal fluid (CSF) sample revealed an increase in the number of cells, which were of donor (cord blood) origin, consisting mainly of T cells. No bacteria were detected in the CSF sample. Real-time PCR analysis revealed that the CSF sample was positive for CMV, but was negative for HHV-6, adenovirus, or BK virus. The patient was diagnosed with CMV meningoencephalitis and received cidofovir. His neurological symptoms were gradually improved and completely disappeared by day 60. CMV-specific dextramer-positive CD8(+) T cells were detected in the peripheral blood and CSF samples, with the frequency being much higher in the CSF. To our knowledge, this is the first report on the appearance of CMV-specific T cells in CSF samples from a patient with CMV meningoencephalitis. Cord blood-derived CMV-specific T cells may develop early after transplantation, enter the intrathecal compartment, and likely contribute to the regulation of CMV-meningoencephalitis.

Different mechanisms causing loss of mismatched human leukocyte antigens in relapsing t(6;11)(q27;q23) acute myeloid leukemia after haploidentical transplantation.

Mismatched human leukocyte antigens (HLAs) on leukemic cells can be targeted by donor T cells in HLA-mismatched/haploidentical stem cell transplantation. In two cases of acute myeloid leukemia with t(6;11)(q27;q23) abnormality presented here, flow cytometry analysis showed a lack of HLA-A unshared between recipients and donors in relapsing leukemic cells after HLA-haploidentical transplantation. However, high-resolution HLA genotyping showed that one case lacked a corresponding HLA haplotype, whereas the other preserved it. These cases suggest that leukemic cells, which lacked mismatched HLA expression, might have an advantage in selective expansion under donor T-cell immune surveillance after HLA-haploidentical transplantation. Most importantly, down-regulation of unshared HLA expression potentially occurs by genetic alterations other than loss of HLA alleles.

Feasibility of unmanipulated haploidentical stem cell transplantation using standard GVHD prophylaxis for HLA-homozygous patients.

HLA-haploidentical hematopoietic stem cell transplantation (haplo-SCT) in HLA-homozygous patients is accompanied by HLA mismatches only in the host-versus-graft vector, and thus theoretically could be performed with standard graft-versus-host disease (GVHD) prophylaxis. However, the risk of GVHD remains uncertain, and graft failure could be a problem. In this study, we assessed nine HLA-homozygous patients who underwent haplo-SCT. Preparative treatment was cyclophosphamide/total body irradiation-based regimen in five patients, fludarabine/busulfan-based regimen in two, and other regimens in two. GVHD prophylaxis consisted of cyclosporine and methotrexate in seven patients, cyclosporine and mycophenolate mofetil in one, and cyclosporine alone in one. Seven patients achieved neutrophil engraftment and platelet recovery. The median times to neutrophil engraftment and platelet recovery were 15 and 44 days, respectively. Two patients developed graft failure, including one who achieved engraftment with a second SCT from the same donor. Grade II GVHD was observed in half of the evaluable patients; grades III and IV were not observed. Two patients died from treatment-related causes. Five patients were alive after a median follow-up period of 563 days. The probability of overall survival at 5 years was 65 %. These findings may serve as a rationale for considering haplo-SCT as a treatment option for HLA-homozygous patients.

Incidence and treatment strategy for disseminated adenovirus disease after haploidentical stem cell transplantation.

Adenovirus (AdV) infection is an emerging complication in patients undergoing allogeneic stem cell transplantation (SCT) and is closely associated with delayed immune reconstitution. In particular, disseminated AdV disease accompanies a high mortality. We retrospectively examined the incidence of AdV infection in patients undergoing unmanipulated haploidentical SCT. Following 121 transplantations in 110 patients, three had asymptomatic AdV viremia, three had localized AdV disease (hemorrhagic cystitis, HC), and seven had disseminated AdV disease (HC + viremia). The median time from transplantation to the onset of AdV-associated HC was 15 days (range 4-39), and the median time to the onset of disseminated AdV disease was 23 days (range 7-38). The cumulative incidence of AdV-associated HC was 8.3 %, and that of disseminated AdV disease was 5.8 %. AdV group B (type 11, type 34, or type 35) was detected in plasma samples from all the patients with disseminated AdV disease. Among them, three patients who received either cidofovir or donor lymphocyte infusion (DLI) alone progressed to pneumonia and died. The remaining four patients were treated with the combination of cidofovir and low-dose unmanipulated DLI, and all survived. We showed that disseminated AdV disease is a significant complication after haplo-SCT and that the combination of cidofovir and DLI is a promising treatment option.

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. STUDY DESIGN AND METHODS: The data on 123 granulocyte collections were retrospectively investigated for the effect of the mobilization regimen and the harvesting technique. After a single subcutaneous dose (600 µg) of granulocyte-colony-stimulating factor (G-CSF) with (n = 68) or without (n = 40) 8 mg of orally administered dexamethasone, 108 granulocyte donors underwent granulocyte collections. Moreover, 15 peripheral blood stem cell (PBSC) donors who had received 400 µg/m2 or 10 µg/kg G-CSF for 5 days underwent granulocyte collections on the day after the last PBSC collections (PBSC-GTX donors). Granulocyte harvesting was performed by leukapheresis with (n = 108) or without (n = 15) using high-molecular-weight hydroxyethyl starch (HES). RESULTS: Granulocyte donors who received mobilization with G-CSF plus dexamethasone produced significantly higher granulocyte yields than those who received G-CSF alone (7.2 × 10(10) ± 2.0 × 10(10) vs. 5.7 × 10(10) ± 1.7 × 10(10) , p = 0.006). PBSC-GTX donors produced a remarkably high granulocyte yield (9.7 × 10(10) ± 2.3 × 10(10) ). The use of HES was associated with better granulocyte collection efficiency (42 ± 7.8% vs. 10 ± 9.1%, p < 0.0001). CONCLUSION: G-CSF plus dexamethasone produces higher granulocyte yields than G-CSF alone. Granulocyte collection from PBSC donors appears to be a rational strategy, since it produces high granulocyte yields when the related patients are at a high risk for infection and reduces difficulties in finding granulocyte donors. HES should be used in apheresis procedures.

Allogeneic stem cell transplantation as treatment for heavily treated, refractory acute graft-versus-host disease after HLA-mismatched stem cell transplantation.

OBJECTIVE: No effective treatment has been established for patients with steroid-refractory acute graft-versus-host disease (GVHD). Recently, we demonstrated in a murine tandem bone marrow transplantation model that life-threatening GVHD established by the first bone marrow transplantation was successfully treated by engraftment of a second donor graft after reduced-intensity conditioning. We named the effect by which allografts counteract GVHD "graft-versus-GVHD." MATERIALS AND METHODS: To investigate the efficacy of graft-versus-GVHD treatment clinically, 16 patients who developed, after human leukocyte antigen-mismatched stem cell transplantation, severe GVHD, refractory to three to five lines of GVHD-specific treatments, underwent 17 allogeneic stem cell transplantations using reduced-intensity conditioning regimens with grafts from a second donor. RESULTS: Among the 15 transplantations that could be evaluated, rescue donor grafts were engrafted in 11 cases and rejected in 4 cases. For patients who achieved rescue donor engraftment, the response rate was 90.9% (eight complete response, two partial response, and one stable disease). Six of the eight patients with complete response survived without GVHD symptoms, with a median follow-up of 2128 days. No new development of GVHD by the second graft was observed. No patients had recurrence of the original malignant disease. In contrast, no long-term survivors were observed in patients who rejected rescue donor grafts. CONCLUSIONS: We propose here a novel graft-versus-GVHD treatment to treat refractory GVHD, and these results strongly suggest that GVHD can be successfully treated by eliminating the harmful lymphocytes responsible for GVHD by a second allogeneic stem cell transplantation.

Separation of antileukemic effects from graft-versus-host disease in MHC-haploidentical murine bone marrow transplantation: participation of host immune cells.

Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects. In clinical studies of HLA-mismatched HSCT, strong GVL effects have been reported. In the present study, we addressed the mechanism of the GVL and GVH response using MHC-haploidentical murine bone marrow transplantation (BMT) models. Recipient BDF1 (H-2(b/d)) mice received T cell-depleted bone marrow and spleen cells from B6C3F1 (H-2(b/k)) or C57BL/6 (H-2(b)) mice with or without P815 mastocytoma cells (H-2(d)) after receiving lethal total body irradiation. B6C3F1 --> BDF1 (hetero-to-hetero type) recipients showed more powerful antileukemic effects with less severe GVHD than C57BL/6 --> BDF1 (parent-to-F1 type) recipients. Compared with C57BL/6 --> BDF1 recipients, significantly higher in vitro cytotoxic activity against P815 cells was observed in B6C3F1 --> BDF1 recipients. Significantly lower CXCR3 expression on donor T cells and higher interferon (IFN)-gamma expression were considered to be associated with strong antileukemic effects with less severe GVHD in B6C3F1 --> BDF1 recipients. Furthermore, host immune cells, especially natural killer cells and CD8(+) T cells, were found to contribute remarkably to high IFN-gamma production in B6C3F1 --> BDF1 recipients. Thus, in MHC-haploidentical HSCT, host immune cells may change the balance between GVH and GVL response through IFN-gamma production.