Bone Marrow Transplantation ; immunology ; Case-Control Studies ; Haploidy ; Humans ; Immunity, Cellular ; immunology ; Recovery of Function ; Transplantation Conditioning ; Transplantation, Homologous

OBJECTIVETo detect the expression profile of NK ligands in acute leukemia cell lines and investigate the differential expression pattern between acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

METHODSUsing quantitative real-time PCR, 23 NK ligands (MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, HLA-E, HLA-G, CD48, NBTA, HLA-F, LLT-1, PVR, Nectin2, CD72, CD80, ICAM-1, LFA-3, CRACC, Fas, DR4, DR5, TNFR1) were detected in 6 acute leukemia cell lines, including 3 ALL cell lines (CEM, Jurkat T, Reh) and 3 AML cell lines (HL-60, KG-1a, NB4), respectively. Independent-samples t test analysis was performed to determine statistical significance.

RESULTSUsing β-actin as reference gene, the relative expression results showed that the expression of 4 NK ligands between ALL and AML is significantly different. Specifically, the level of ULBP-2 is higher in ALL (CEM: 1, Jurkat T: 0.617, Reh: 0.246) than that in AML (HL-60: 0.000, KG-1a: 0.003, NB4: 0.000)(P = 0.047). However, the expressions of CD48, PVR(PVR-1, PVR-2) and DR4 is higher in AML (HL-60: 13.987, 4.403, 10.334, 8.711; KG-1a: 5.387, 2.900, 7.315, 4.512; NB4: 7.763, 3.248, 7.049, 6.127) than that in ALL (CEM: 1, 1, 1, 1; Jurkat T: 2.035, 1.553, 3.888, 0.449; Reh: 1.559, 0.000, 0.000, 1.304) (P = 0.044, 0.014, 0.014, 0.011). And there're no significant differences between the rest 19 NK ligands.

CONCLUSIONSULBP-2, CD48, PVR and DR4 might play an important role in the distinct mechanisms in leukemogenesis between ALL and AML and could be potential targets for diagnosis and treatment.

Acute Disease ; Antigens, CD ; genetics ; metabolism ; CD48 Antigen ; Cell Line, Tumor ; GPI-Linked Proteins ; genetics ; metabolism ; HL-60 Cells ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Leukemia ; genetics ; metabolism ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Ligands ; Membrane Proteins ; genetics ; metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; Receptors, Virus ; genetics ; metabolism

Using transplantable erythroblastic leukemia cells of EL9611(H-2d), the cells were inoculated to CB6F(1)(H-2d/b) generation of BALB/c x C57BL/6 mouse, the biological characterization of erythroblastic leukemia in haploidentical mouse was studied, that provides an experimental model for the study of graft-versus leukemia (GVL) with bone marrow or stem cell transplantation. When 10(3) - 10(8) of the spleen cells of EL9611(H-2d) had been intravenously inoculated to CB6F(1) mouse, the erythroblastic leukemia cells were transplanted successively and the F(1) generation of erythroblastic leukemia model in mice was established with 100% incidence of erythroblastic leukemia. There was a linear relationship between the survival time and the number of leukemic cell. The survival time of the mice was (9.6 +/- 0.8) days when 10(6) cells were inoculated. If the CB6F(1) mouse was transplanted successively for four generations, the incidence was 100%. The main targets for the leukemic EL9611(H-2d) cells were liver, spleen and marrow. The reaction of the erythroblastic leukemia cells for hemoglobin staining was positive, while the peroxidase reaction was negative. These cells were sensitive to some chemotherapeutic drugs, such as cytosine arabinoside and cyclophosphamide. This study presents the convenience for the studies on the GVL with haplo-allogeneic transplantation, in the F(1) generation of erythroblastic leukemia model of the commonly-used CD57BL/6 x BALB/c mouse.
Animals ; Cell Division ; Disease Models, Animal ; H-2 Antigens ; analysis ; Leukemia, Erythroblastic, Acute ; immunology ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Inbred Strains ; Neoplasm Transplantation ; Survival Analysis ; Tumor Cells, Cultured

Veto activity was defined as the capacity of specifically downregulating cytotoxic T-precursor cell (CTL-p) directed against antigens of the veto cells themselves but not against third-party antigens. Many studies have shown that the most potent veto cells are CD8(+) cytotoxic T lymphocytes (CD8(+)CTLs). Effectively, CD8(+)CTLs of donor origin can facilitate engraftment of donor's stem cells by eliminating host-alloreactive T lymphocytes. In this article, effect mechanisms, depletion of GVH ex vivo, application in vivo, synergistic enhancement with rapamycin and regulatory T cells, and anti-tumor effect in the hematopoietic stem cell transplantation are summarized.
Hematopoietic Stem Cell Transplantation ; Humans ; Immune Tolerance ; T-Lymphocytes, Cytotoxic

This study was aimed to detect the changes of bcr/able gene level in ph+ CML patients at different stages after allo-HSCT by real-time quantitative PCR and to evaluate the significance of this detection. The serial detection of bcr/abl fusion gene levels in 21 cases of CML treated with allo-HSCT was performed by RQ-PCR. The results showed that the bcr/able fusion gene could not be detected in 7 out 21 CML cases with positive fusion gene after allo-HSCT, while the bcr/abl fusion gene of different levels could be detected in 14 cases within 1-6 months. Dynamic detection indicated that the bcr/abl fusion gene levels in 9 cases were lower with relative value 0.0074%-0.088% and then could not be detected within 3-7 months after allo-HSCT. The bcr/abl fusion gene levels in 5 cases diagnosed as molecular relapse were between 0.077%-75%. The bcr/abl fusion gene levels in 1 out of 5 cases were 0.95%, 1.5%, and 0.16% in month 1, 2 and 3, respectively, and turned to negative in the month 4 without any treatment after allo-HSCT. 2 cases received the donor peripheral blood stem cell infusion, and then their bcr/abl mRNA levels could not be detected in bone marrow. Another 2 cases developed to the hematologic relapse, 1 out of 2 cases reached CR again after infusion of donor peripheral blood stem cells and chemotherapy, the other one died. It is concluded that serial quantifications of bcr/abl mRNA levels by RQ-PCR are reliable and can be used to detect the MRD, to monitor the outcome and to predict the relapse.
Adolescent ; Adult ; Female ; Fusion Proteins, bcr-abl ; genetics ; Genes, abl ; Hematopoietic Stem Cell Transplantation ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; surgery ; Male ; Middle Aged ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult

This study was aimed to explore the difference of NK cell receptor NKG2D and NKG2A expression on NK cells and CD3(+) T cells and their ligand MHC-I A/B (major histocompatibility complex class I-related chains A/B) and HLA-E expression in leukemia cells, as well as its immunological significance. Flow cytometry was used to detect the killing rate of NK92 cells to 8 leukemia cell lines, and the expression of NKG2D and NKG2A on NK cells and CD3(+) T cells as well as their ligand MHC-I A/B and HLA-E expression on leukemia cells. The results indicated that the NK92 showed different killing activity to different leukemia cell lines. The positive expression rate of NKG2D and NKG2A on NK cells and CD3(+) T cells in ALL patients was no significantly different from that in AML patients (p > 0.05), but positive expression rate of MHC-I A/B and HLA-E in ALL patients was obviously higher than that in AML patients (p < 0.05). It is concluded that there is difference of immune cell function between ALL and AML patients, this difference may be associated with the expression difference of NKG2D and NKG2A ligands on leukemia cells while does not associated with the killing and inhibiting receptors expressed on NK cells and CD3(+) T cells.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Child, Preschool ; Female ; Histocompatibility Antigens Class I ; genetics ; metabolism ; Humans ; Infant ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Male ; Middle Aged ; NK Cell Lectin-Like Receptor Subfamily C ; genetics ; metabolism ; NK Cell Lectin-Like Receptor Subfamily K ; genetics ; metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; metabolism ; Young Adult

This study was purposed to investigate the therapeutic effects of early transfusion of immunized donor lymphocytes after haploidentical transplantation by means of mouse model of nonmyeloablative haploidentical bone marrow transplantation. CB6F1 female mouse was served as recipient and C57BL/6 male mouse was served as donor. Each CB6F1 female mouse was subjected to intravenous transfusion with 1×10(6) erythroleukemia (EL9611) cells at day 4 before transplantation, followed with intraperitoneal injection of Ara-C (0.015 g) respectively at day 2 and day 1, then conditioned for BMT with TBI (450 cGy) at day 1 before transplantation. After conditioning (day 0), each of recipients was transplanted with 6×10(7) mixture of bone marrow and spleen cells from the C57BL/6 mice, and was infused with 6 × 10(7) immunized donor lymphocytes at day 15 after transplantation. All treated animals were evaluated for survival, development of leukemia and aGVHD. The donor CD3(+) cell chimerism and sex determining region Y gene (SRY)in recipients were monitored periodically after transplantation. The results showed tht all mice with only inoculation of 10(6) EL9611 cells survived for 15 ± 1 days (n = 4); all mice of other groups obtained the varying degrees of implantation. SRY could be detected at day 30 and 60 after transplantation. The chimerism of donor CD3(+) cells in mixed bone marrow transplantation (MT) group at day 14, 30 and 60 respectively reached 17.95% ± 12.03%, 37.34% ± 2.78% and 47.06% ± 6.1%. In donor lymphocyte infusion (DLI) group it reached 69.78% ± 12.62%, 75% ± 15.97%, 83.41% ± 16.07% at day 30, 45 and 60 after transplantation. The mice of MT and DLI group survived for 66.66 ± 1.47 days and 78.2 ± 7.82 days. It is concluded that the high tumor burden before transplantation can affect donor cell engraftment and prognosis.Early post-transplanted infusion of immunized lymphocytes from donor can help to improve the therapeutic efficacy and survival.
Animals ; Bone Marrow Transplantation ; methods ; Female ; Haplotypes ; Leukemia, Erythroblastic, Acute ; therapy ; Lymphocyte Transfusion ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Tissue Donors ; Transplantation Conditioning ; methods ; Transplantation, Homologous

This study was purposed to explore the efficacy of hematopoietic reconstitution and survival of patients with myelodysplastic syndrome (MDS) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Allo-HSCT without T lymphocyte depletion was used in 6 patients with MDS from November 1999 to June 2007. 4 cases out of them received allo-PBSCT from HLA matched sibling donors with conditioning regimen of cyclophosphamide (CTX) and Bu. Graft versus host disease (GVHD) was prevented by the administration of immunosuppressive drugs of cyclosporine A (CsA) and short-course MTX. 2 patients received haploidentical allogeneic bone marrow transplantation (hi-alloBMT) after preconditioning with cytosine arabinoside (Ara-C), CTX and total body irradiation (TBI) with a linear accelerator. GVHD was prevented by the administration of immunosuppressive drugs including CSA, short-course MTX, MMF, anti-CD25 monoclonal antibody and ATG. The results showed that all of the patients were engrafted successfully. The median time of granulocyte recovery exceeding 0.5 x 10(9)/L and platelets exceeding 20 x 10(9)/L were days 15 and 20.3 respectively, and 100% donor hematological cells were detected by cytogenetic analysis. All patients did not experience serious acute graft-versus-host disease (aGVHD). During 18 - 108 months of following-up, 2 cases died of pulmonary complication and of relapse; the other 4 cases survive in a disease-free situation. In conclusion, allo-HSCT was an effective approach for the treatment of MDS.
Adolescent ; Adult ; Female ; Graft vs Host Disease ; prevention & control ; Hematopoietic Stem Cell Transplantation ; Humans ; Male ; Myelodysplastic Syndromes ; surgery ; Transplantation, Homologous ; Young Adult

OBJECTIVETo investigate the effect and its mechanism of reducing graft-versus-host disease (GVHD) by in vitro blockade of CD(40)-CD(40)L pathway in vitro, the donor T lymphocytes cultured in vitro with anti-CD(40)L mAb were transfused in bone marrow transplantation (BMT) GVHD mouse model.

METHODSC57BL/6(H-2b) spleen T cells were isolated as responder cells, and BALB/c(H-2d) spleen cells as stimulator cells. They were cocultured with or without Anti-CD(40)L mAb as anti-CD(40)L mAb group and control group, respectively. At day 5, the mixed lymphocyte response (MLR)-culture cells mixed with bone marrow cells and transfused respectively into the TBI conditioned recipient mice. The mice were divided into two groups: group A, bone marrow cells (2 x 10(6)) and spleen T lymphocytes (2 x 10(6)) from MLR control group; group B, bone marrow cells (2 x 10(6)) and spleen T lymphocytes (2 x 10(6)) from MLR anti-CD(40)L mAb group. The GVHD incidence and hematopoietic reconstitution were observed. Peripheral blood sera and spleen cells of the recipients mice were harvested at scheduled time points for the measurement of cytokines and T cell immunophenotyping with flow cytometry.

RESULTSThe incidence of GVHD in group A was 100% (10/10), and in group B was 20% (2/10). The percentage of H-2D(b) positive cells in group B (n = 8) was (93.54 +/- 2.32)% at day 40 after transplantation. The levels of cytokines in serum from group B were significantly lower than those from group A (P < 0.05). The expressions of CD(4)(+), CD(8)(+), CD(4)(+)CD(25)(+), CD(8)(+)CD(25)(+), CD(4)(+)CD(69)(+), CD(8)(+)CD(69)(+) and CD(4)(+)CD(40)L(+) were lower in group B than in group A (P < 0.05). The expressions of CD(8)(+)CD(40)L(+) and CD(4)(+)CD(45)RA(+) were similar in the two groups (P > 0.05).

CONCLUSIONBlockade of CD(40)-CD(40)L interaction in vitro could induce immune tolerance in vivo, reduce aGVHD in aGVHD mice model and form chimerism, which was mediated by inhibiting the Th1 and Th2 cytokines production, inducing tolerance of CD(4)(+) and CD(8)(+) cells to alloantigens. The obstruction of T cells activation after tolerance happened mainly at the early and mature phase of T cells activation. These provided the experimental basis for the use of anti-CD(40)L mAb in the clinical transplantation to prevent aGVHD.

Animals ; Antibodies, Monoclonal ; therapeutic use ; Bone Marrow Transplantation ; adverse effects ; CD40 Antigens ; physiology ; CD40 Ligand ; immunology ; physiology ; Graft vs Host Disease ; prevention & control ; Interleukin-10 ; blood ; Interleukin-4 ; blood ; Male ; Mice ; Mice, Inbred C57BL

The purpose of this study was to investigate the feasibility and clinical outcome of granulocyte colony stimulating factor (G-CSF)-mobilized haploidentical bone marrow transplantation combined with peripheral blood stem cells (hiBM+PBSCT) for therapy of leukemia. 125 leukemia patients underwent G-CSF primed haploidentical stem cell transplantation without ex-vivo T cell depletion. All haploidentical donors were injected with G-CSF at dose of 5 microg/(kg.d) for 7 days. The patients were divided into groups A and B. 29 patients in group A underwent hiBM+PBSCT at 7th and 8th days of mobilization in donors with G-CSF respectively; 96 patients in group B underwent hiBMT. All patients received the same GVHD prophylaxis regimen, the clinical outcomes were investigated. The results showed that all patients except one CML-myelofibrosis patient achieved trilineage engraftment. Engraftment median times were 15 and 19 days for neutrophil and platelet in group A respectively, while engraftment median times were 18 and 23 days for neutrophil and platelet in group B respectively. The incidences of grade II-IV aGVHD were 31.03% in group A and 12.5% in group B respectively (p<0.05). The incidences of grade III-IV aGVHD was 13.79% and 10.41% in group A and group B (p>0.05). The aGVHD-related death incidence was 3.45% and 5.21% in group A and group B (p>0.05). The incidence of grade II-IV cGVHD was 48.2% and 35.4% in group A and group B respectively (p>0.05). The incidence of extensive cGVHD was 23.3% and 15.6% in group A and group B respectively (p>0.05). The disease relapse rate was 6.8% (2/29) and 18.75% (18/96) in group A and group B respectively (p<0.05). It is concluded that the G-CSF-mobilized allogeneic haploidentical BM plus peripheral blood HSCT without T cell depletion provides a rapid and sustained engraftment without increase of severe GVHD, furthermore, the relapse rate of disease is reduced remarkably, thus this method can be used in clinic.
Adolescent ; Adult ; Bone Marrow Transplantation ; methods ; Child ; Child, Preschool ; Female ; Graft Survival ; Graft vs Host Disease ; prevention & control ; Haploidy ; Hematopoietic Stem Cell Mobilization ; methods ; Humans ; Leukemia ; surgery ; Middle Aged ; Peripheral Blood Stem Cell Transplantation ; methods ; Treatment Outcome ; Young Adult