OBJECTIVETo evaluate the effect and safety of Chinese herbal medicine Lingdankang Composite (LDK) combined dendritic cell-cytokine induced killer cells (DC-CIK) in treating leukemia.

METHODSSubjects were selected from leukemia patients who achieved hematological complete remission (HCR) but not achieved molecular biological remission (MBR), or with minimal residual leukemia (MRL) positive. Twenty patients, 19 of acute leukemia and 1 of chronic myelocytic leukemia, were enrolled. DC and CIK from patient's peripheral blood monocyte were separated respectively by blood cell separator, then DC-CIK was obtained through respective culture followed with mixed cultivation of them, and was infused back to the patient self via intravenous injection. The back infusion of DC-CIK was performed once every 15-20 days for 4-6 times in total. Meantime, LDK was administered orally every day.

RESULTSIn the 20 patients treated, 4 case of HCR achieved MBR, the negatively reversed marker gene was AML1/ETO in 1 case, CBFbeta/MYH11 in 1, bcr/abl in 1, and the other 1 was IgH gene rearrangement; 3 patients with positive MRL were reversed to negative. The 3-year CR rate was 75% with a medium CR period of 25 months (10-37 months). Except transient fever and chill in 5 cases, no other remarkable adverse reaction happened during or after DC-CIK infusion.

CONCLUSIONThe combined treatment of LDK and autologous DC-CIK in treating patients with HCR shows an obvious effect of clearing MRL, it is the appropriate choice for curing leukemia of HCR, and is safety for intravenous infusion, so it has potential clinical prospect.

Adolescent ; Adult ; Cell Differentiation ; Cells, Cultured ; Combined Modality Therapy ; Cytokine-Induced Killer Cells ; cytology ; transplantation ; Dendritic Cells ; cytology ; transplantation ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Leukemia ; immunology ; therapy ; Leukocytes, Mononuclear ; cytology ; Male ; Middle Aged ; Phytotherapy ; Young Adult

The study was aimed to explore the roles of exosomes derived from regulatory dendritic cells of mice in the induction of immune tolerance. Immature DC (iDC) from mouse bone marrow cells and regulatory DCs (rDC) were induced by treating iDC with TGF-beta1 and IL-10. The phenotype of regulatory DCs and normal DCs were assayed by flow cytometry. Exosomes from immature DCs (iDex) and regulatory DCs (rDex) were isolated by ultracentrifugation and ultrafiltration. A skin transplantation model was established with the recipients BALB/c mice and the donor C57BL/6 mice. Recipients were divided into PBS control group, iDex group (injection 10 microg iDex of donor C57BL/6 mice via tail vein at days 7 and 3 before skin transplantation), rDex group (injection 10 microg rDex of donor C57BL/6 mice via tail vein at days 7 and 3 before skin transplantation). The capacity of the donor mice and the unrelated allogeneic donor mice to stimulate allogeneic T lymphocyte proliferation was examined by mixed lymphocyte culture (MLR). The results showed that TGF-beta1 and IL-10 could down-regulate the expressions of costimulatory molecules, including CD80, CD86 and CD40. The graft mean survival time (MST) in control group, iDex group and rDex group was 7.8, 10.7 and 18.8 days, respectively. There was significant difference in MST between iDex group and control group (p<0.05), and between rDex group and iDex group (p<0.01). The results of MLR assays indicated donor-specific hyporeactivity especially in rDex group, while the tolerant B/C mice were still immunocompetent to unrelated allogeneic DBA mouse. It is concluded that injection iDex or rDex of donor mice via tail vein before skin transplantation induces immunotolerance, and the effect of rDex is more significant.
Animals ; Dendritic Cells ; cytology ; immunology ; transplantation ; Exosomes ; immunology ; transplantation ; Female ; Graft Enhancement, Immunologic ; methods ; Graft Survival ; Immune Tolerance ; immunology ; Lymphocyte Culture Test, Mixed ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Inbred DBA ; Skin Transplantation ; Transplantation Immunology ; Transplantation, Homologous

BACKGROUNDSemi-mature dendritic cells (DCs) may induce tolerance rather than immunity. However, little is known about the regulatory mechanism by which these DCs induce transplant tolerance. Myeloid differentiation factor 88 (MyD88) is a key adaptor of Toll-like receptor signaling, which plays a critical role in DC maturation. Activation of MyD88-silenced immature DCs results in the generation of semi-mature DCs. We explored the possibility of using these DCs to induce intestinal transplant tolerance in rats.

METHODSMyD88 expression was silenced in bone marrow DCs (F344 rats) using small interfering RNAs for 24 hours, at which point, lipopolysaccharide (LPS) was added to the culture for another 48 hours. These cells were analyzed for their in vitro and in vivo tolerizing capacities.

RESULTSSemi-mature DCs expressing moderate levels of MHC class II and low levels of co-stimulatory molecules were found to produce interleukin (IL)-10, while IL-12 production was decreased. In vitro co-culture with completely allogeneic T cells from Wistar rats led to a significant decrease in alloreactive T-cell responses. In vivo, the transfer of semi-mature DCs (1 × 10(6) cells) followed by the transplantation of fully mismatched intestinal grafts (F344 rats) led to significantly prolonged survival compared to rats receiving immature and mature DCs. Serum from semi-mature DC-treated rats contained lower concentrations of the pro-inflammatory cytokines IL-2 and interferon-γ 5 days after transplantation.

CONCLUSIONSemi-mature DCs may promote inducible allograft tolerance and this study suggests a new strategy by which to facilitate the induction of transplant tolerance.

Animals ; Blotting, Western ; Bone Marrow Cells ; cytology ; Cell Proliferation ; Dendritic Cells ; cytology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Intestines ; transplantation ; Male ; Myeloid Differentiation Factor 88 ; genetics ; metabolism ; Polymerase Chain Reaction ; Rats ; Rats, Inbred F344 ; T-Lymphocytes ; metabolism ; Transplantation, Homologous ; methods

OBJECTIVETo observe the dynamic changes of dendritic cells (DCs) in the renal graft of rats within 72 h after renal transplantation.

METHODSUsing SD rats as the donors and Wistar rats as the recipients, renal transplantation was performed in 30 pairs of rats, with another 5 donor kidneys that were not transplanted serving as the sham operation group. The transplanted kidneys were harvested at 1, 6, 12, 24, 48 and 72 h after recovery of blood circulation, paraffin-embedded and sectioned ,followed by HE staining and immunohistochemical staining for S-100 protein for DC identification. The pathological changes and the DC density per glomerulus in the renal graft were observed with optical microscope.

RESULTSNo signs of acute rejection were found in these sections. Few DCs were observed in the sham operation group and in the renal graft 1 h after transplantation. The number of DCs in the renal graft increased with time and reached the maximum 24 h after transplantation followed by gradual decrease.

CONCLUSIONSWithin 72 h after renal transplantation, the number of DCs in the graft varies following a curve with a single peak. Increased DC density in the graft may result from recipient DC migration into the graft, and accordingly, decreased recipient DC migration results in decrease of DC density in the graft. The pattern of DC number variation in the graft can be helpful to further improve the therapy against graft rejection.

Animals ; Cell Count ; Cell Movement ; immunology ; Dendritic Cells ; cytology ; immunology ; Female ; Graft Rejection ; prevention & control ; Kidney Glomerulus ; immunology ; Kidney Transplantation ; immunology ; Male ; Rats ; Rats, Wistar ; Time Factors

OBJECTIVETo explore the biological characteristic of third-party-derived tolerogenic DC(tDC) and the influence of third-party-derived tDC on acute graft-versus-host-disease (aGVHD) following allogeneic bone marrow transplantation (allo-BMT) in mice.

METHODStDC from bone marrow cells of D1 mice was cultured with low doses of GM-CSF, IL-10 and TGF-β1D1. The phenotype, expression of cytokines and function associated molecules were identified with FACS and RT-PCR. Mixed lymphocyte reaction was applied to analyze the influence of third-party-derived tDC on allo-CD4(+)T cells proliferation in vitro. Different doses of D1-tDC were adoptive transferred in the aGVHD model in allogeneic BMT which B6 mice as donors and D2 mice as recipients. Survival time, clinical GVHD score and the levels of Th1/2 cytokines in serum were monitored after allo-BMT using the aGVHD model as control.

RESULTStDC expressed lower levels of MHC II and co-stimulatory molecules, such as CD80, CD86 and CD40, even when stimulated by LPS. The results by RT-PCR indicated that tDC expressed low levels of IL-12p40 and high levels of immunosuppressive molecules, such as IL-10, TGF-β, Fas Ligand, indoleamine 2, 3-dioxygenase (IDO) and arginase. In the allogeneic MLR, third-party tDC suppressed allo-CD4(+)T cells proliferation, which was relative to the dose of tDC. In the B6→D2 mouse model, all aGVHD mice died within 18 days. Remarkably, if 10(4) third-party tDC were transferred, 60% mice survived at least 60 days. When the doses of tDC were reduced to 10(3) cells, only 20% of mice survived day 60, and when increased tDC to 10(5), all of the mice died within day 37 after allo-BMT. The cytokine levels in serum indicated that 10(4) tDC-treated mice secreted in vivo high level of IL-10 21d after BMT (P < 0.05), the levels of IL-10 in 10(3), 10(4) and 10(5) tDC-treated mice were (114.23 ± 7.78), (646.18 ± 212.02), (121.97 ± 10.47) ng/L, respectively.

CONCLUSIONThird-party tDC could suppress allo-CD4(+)T cells proliferation in vitro and prevent aGVHD in allogeneic BMT mode, which may be mediated by modulating tolerogenic cytokines secretion, such as IL-10. And this effect was associated with the dose of tDC. Adoptive therapy by transfusing third-party tDC cultured with low doses of GM-CSF, IL-10 and TGF-β1 could significantly prolong the survival of recipients and prevent aGVHD in allogeneic BMT.

Animals ; Bone Marrow Transplantation ; adverse effects ; CD4-Positive T-Lymphocytes ; cytology ; Cell Proliferation ; Dendritic Cells ; cytology ; immunology ; metabolism ; Graft vs Host Disease ; prevention & control ; Interleukin-10 ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Transforming Growth Factor beta1 ; immunology ; Transplantation, Homologous

OBJECTIVETo observe the effects of NBD-peptide pretreatment of the donor dendritic cells in immune tolerance induction in mouse allograft recipients and investigate the mechanisms.

METHODSBALB/c mouse DCs pretreated with NBD-peptide (NBD-Peptide-DC) were injected into the recipient C57BL/6 mice 7 days before transplantation. Cervical heterotopic heart transplantation model was established using the cuff technique and the cardiac allograft survival time was observed. Pathological analysis were performed to examine the graft injection and the responsiveness of the recipient spleen T cell to the donor alloantigen was determined by mixed lymphocyte reaction (MLR). The serum levels of cytokines were determined using ELISA.

RESULTSThe cardiac allograft survival time in the NBD-Peptide-DC-treated group (21.83-/+3.54 days) was significantly longer than that in the Day9-DC group (13.33-/+2.58 days) and PBS-treated group (6.66-/+1.21 days) (P<0.01), with also significantly lower pathological grade for graft rejection (P<0.01). The donor-derived NBD-Peptide-DCs induced alloantigen-specific T-cell hyporesponsiveness. In the NBD-Peptide-DC-treated group, the serum levels of IL-12 and IFN-gamma decreased significantly (P<0.01), but the levels of IL-4 and IL-10 increased significantly (P<0.01).

CONCLUSIONInjection of donor-derived NBD-Peptide-DCs can leads to donor-specific tolerance in the transplant recipients, and the induction of recipient T-cell hyporesponsiveness and polarization of Th2 response may play important roles in immune tolerance to cardiac allografts.

Animals ; Dendritic Cells ; cytology ; immunology ; transplantation ; Graft Rejection ; immunology ; Graft Survival ; immunology ; Heart Transplantation ; immunology ; methods ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Peptides ; immunology ; Transplantation, Homologous

OBJECTIVE: To explore the effects of granulocyte macrophage-colony-stimulating factor (GM-CSF) on the proliferation and activation of dendritic cells (DC) in vivo. METHODS: Balb/c mice were randomly assigned into the control and GM-CSF treatment group. After the lethal dosage of irridiation, green fluorescence protein labelled hematopoietic stem cells (HSC) were injected into the tail veins of the mice. The dosage of 0.1 micorg GM-CSF was administer subcutaneously every 2 days after the HSC infusion. The numbers and activation status of splenic DC were observed by flow cytometry 1, 2, 3, and 4 weeks after the HSC transplantation. RESULTS: The numbers of splenic DC in the GM-CSF treatment group were significantly higher than those of the control group. The expressions of surface marker CD40, CD80, and CD86 in the GM-CSF treatment group were also higher than those of the control group. CONCLUSION GM-CSF can enhance the proliferation and activation of DC in vivo.
Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; Female ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cell Transplantation ; Male ; Mice ; Mice, Inbred BALB C ; Random Allocation ; Spleen ; cytology ; Whole-Body Irradiation

OBJECTIVETo explore the feasibility and efficiency of immunotherapy with dendritic cell (DC) in leukemic mice model after allogeneic bone marrow transplantation (allo-BMT).

METHODSMature DC were expanded from mice bone marrow mononuclear cells (MNC) by adding mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) and interleukin-4 (mIL-4). Three days later they were pulsed with frozen thawing L7212 leukemia-related antigen. Mice bearing leukemia received allo-BMT at d 0, and then were divided into control group (A), T cells group (B) and DC + T cells group (C) to receive respective immune therapy at d 14. The survival rate, survival time, occurrence of graft-versus-host disease (GVHD), cytotoxicity of spleen cells and serum cytokine level were observed. The survivors in each group were rechallenged with L7212 cells to observe the immune response to the leukemia.

RESULTSMature DC were successfully induced from bone marrow MNC. In groups B and C, the relapse rates were 30% and 0%, while the long term survival rates after BMT was 30% and 70% respectively. Both of the differences were statistically significant (P < 0.05). However, the incidence of GVHD in these two groups were similar. The mean survival times were (32.95 +/- 13.29) days and (41.15 +/- 13.88) days, respectively (P < 0.01). MTT assay indicated that spleen cells from group C had specific killing activity to L7212 cells. Enzyme-labeled immunosorbent assay (ELISA) showed that the serum IL-2 level in group C was (419.75 +/- 26.66) pg/ml, being significantly higher than that in the other two groups (P < 0.01). When the survivors were rechallenged with L7212 cells, there was difference between the survival rates of groups C and B (85.7% vs 33.3%, P < 0.05).

CONCLUSIONImmunotherapy with leukemia related antigen-pulsed DC in combination with donor lymphocyte infusions is an effective approach to reinforce GVL effect and decrease relapse after allo-BMT.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; immunology ; Bone Marrow Transplantation ; Cancer Vaccines ; immunology ; Cell Differentiation ; Dendritic Cells ; immunology ; Female ; Graft vs Leukemia Effect ; Immunotherapy ; Leukemia, Experimental ; immunology ; surgery ; therapy ; Male ; Mice ; Mice, Inbred BALB C ; Survival Rate ; Transplantation, Homologous

OBJECTIVETo investigate the synergic effects of rapamycin and donor bone marrow-derived immature dendritic cells (DCs) in inducing skin allograft tolerance in mice.

METHODSThe recipient BALB/c mice receiving transplantation of skin allograft from C57BL/6 mice were divided into control group (without perioperative treatments), rapamycin group (receiving rapamycin at 1 mg.kg(-1).d(-1) by gavage for 7 consecutive 7 days after skin transplantation), immature DC group (receiving an injection of donor bone marrow-derived immature DCs of 2 x 10(6) via tail vein before skin transplantation), combined group (receiving an injection of the DCs of 2 x 10(6) before transplantation and rapamycin at 1 mg.kg(-1).d(-1) for 7 consecutive days after transplantation). The survival time of the skin allograft was observed in each group.

RESULTSThe survival time of the skin allograft in the control, rapamycin, immature DC and immature DC +rapamycin groups were 6.9-/+1.9, 12.3-/+3.0, 17.0-/+3.4 and 20.8-/+3.6 days, respectively, showing significant differences among the groups (P<0.05), and SNK test also indicated significant differences between every two groups.

CONCLUSIONSRapamycin and donor bone marrow-derived immature DCs have synergic effects in inducing skin allograft tolerance in mice.

Animals ; Bone Marrow Cells ; cytology ; immunology ; Dendritic Cells ; immunology ; Graft Survival ; drug effects ; immunology ; Immunosuppressive Agents ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Sirolimus ; pharmacology ; Skin Transplantation ; immunology ; methods ; Transplantation, Homologous

OBJECTIVETo investigate the effects of immature dendritic cells (imDC) expressing chemokine receptor-7 (CCR7) on acute graft-versus-host disease (aGVHD) in allogeneic bone marrow transposed (allo-BMT) mouse model.

METHODSWe constructed the lentiviral vectors carrying mouse CCR7 gene and infect imDC effectively in vitro. GVHD model was established with C57BL/6(H-2b) donor mice and BALB/c (H-2d) recipient mice. After irradiation, recipients were injected with donor bone marrow and spleen cells along with CCR7-modified dendritic cells. Mice were randomized into irradiation, transplant control, pXZ9-imDC (empty vector control) and CCR7-imDC groups. Survival, GVHD score, histopathological analysis and plasma levels of inflammatory cytokines were observed.

RESULTSThe mean survival in irradiation, transplantation, pXZ9-imDC and CCR7-imDC groups were (8.20±1.48)d, (12.20±2.78)d, (20.70±6.01)d and (27.5±7.55)d respectively. The survival in CCR7- imDC group was significantly improved compared with other groups (P<0.05). GVHD scores in transplantation, pXZ9-imDC and CCR7-imDC groups were (6.90±1.66), (5.60±0.97) and (4.10±1.79) respectively. CCR7-imDC group had significantly lower GVHD score and minor tissue damages shown by histopathological analysis than the other groups. Plasma IFN-γ level increased and reached the peak at +10 day in transplant group, while it gradually decreased in pXZ9-imDC and CCR7-imDC groups, and then reached the nadir at +20 day post-allo-BMT, with the lowest level in CCR7-imDC group (P<0.01). Plasma IL-4 decreased in transplant group, while it gradually increased in pXZ9-imDC and CCR7-imDC groups and reached the highest level at + 10 day in CCR7- imDC group (P<0.01). The 95%-100% of H-2b positive cells in recipient mice on + 30 day post-allo-BMT demonstrated the complete donor- type implantation.

CONCLUSIONGenetically modified immature DC by CCR7 gene could alleviate damages by GVHD and prolong survival of recipient mice after allo-BMT.

Animals ; Bone Marrow Transplantation ; adverse effects ; Dendritic Cells ; cytology ; Genetic Vectors ; Graft vs Host Disease ; prevention & control ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Receptors, CCR7 ; genetics ; Transplantation, Homologous