Immunoregulation Efficacy Of Natural Killer T Cell In Acute Graft-versus-host Disease Of Allogeneic Hematopoietic Stem Cell Transplantation

Objective: To establish the culture system of murine type-1 natural killer (NKT) T cells in vitro and to explore the expansion efficacy ofα-GalCer-loaded dendritic cells (α-GalCer-loaded DCs) for murine NKT cells in vivo.Methods: Splenocytes derived from C57BL/6 mouse were cultured in complete RPMI-1640 medium containingα-GalCer (100 ng/ml),α-GalCer (100 ng/ml) + IL-2 (100 IU/ml) orα-GalCer (100 ng/ml) + IL-2 (100 IU/ml) + IL-7 (20 ng/ml) respectively. C57BL/6 mice were injected withα-GalCer (2μg /mouse) orα-GalCer-loaded DCs (1×106/mouse) respectively via tail vein for NKT cells expansion in vivo. The expanded cells were stained with PE-α-GalCer-loaded CD1d tetramer and FITC-TCRβmonoclonal antibody for percentage assay of NKT cells by flow cytometry. The expanded NKT cells in vivo were enriched by using microbeads conjugatedα-GalCer-loaded CD1d tetramer. The levels of IL-4 and IFNγin serum and supernatant were detected by ELISA on day 7.Results: The NKT cells percentage of splenocytes inα-GalCer group,α-GalCer + IL-2 group andα-GalCer + IL-2 + IL-7 group was (8.70±1.97)%, (27.28±5.26)% and (51.25±7.80)% respectively after 8 days culture. The percentage of NKT cells inα-GalCer + IL-2 group andα-GalCer + IL-2 + IL-7 group was remarkably higher than that inα-GalCer group (P<0.01). Comparison toα-GalCer + IL-2 group, the NKT cells percentage inα-GalCer + IL-2 + IL-7 group increased significantly (P=0.0327). The NKT cells percentage of splenocytes and thymocytes inα-GalCer group andα-GalCer-loaded DCs group was (15.46±2.63)%, (28.89±3.92)% and (38.41±3.91)%, (52.46±5.23)% respectively after 5 days stimulation byα-GalCer orα-GalCer-loaded DCs. The NKT cells percentage of splenocytes inα-GalCer-loaded DCs group increased obviously compared withα-GalCer group (P=0.0315). Although the NKT cells percentage of thymocytes inα-GalCer-loaded DCs group seemed to be higher than that inα-GalCer group, there was no statistical significance (P>0.05). The levels of IL-4 and IFNγin serum increased significantly inα-GalCer-loaded DCs group compared withα-GalCer group (P=0.0406, P=0.0129). In comparison ofα-GalCer group, the secretion of IL-4 and IFNγin supernatant increased remarkably inα-GalCer + IL-2 group andα-GalCer + IL-2 + IL-7 group. There was no statistical significance betweenα-GalCer + IL-2 group andα-GalCer + IL-2 + IL-7 group (P>0.05).Conclusion: Combinationα-GalCer with IL-2 could effectively expand type-1 NKT cells in vitro. Addition IL-7 in culture system containingα-GalCer and IL-2 could further improve the expansion efficiency for NKT cells. Administrationα-GalCer-loaded DCs in vivo could efficiently stimulate type-1 NKT cells expansion. The expanded and activated NKT cells could secrete a large amount of IL-4 and IFNγ.Objective: To establish a murine aGVHD model post myeloablative allogeneic bone marrow transplantation (allo-BMT), in which adult C57BL/6 mice were as donor and BALB/c mice were recipient.Methods: Twenty-five SPF grade BALB/c mice were randomly assigned to five groups, which received 7, 7.5, 8, 8.5 or 9 Gy X ray total body irradiation (TBI). Twenty SPF grade BALB/c mice were randomly assigned to four groups post TBI conditioning and were reconstituted with 1×106, 2.5×106, 5×106 or 10×106 bone marrow cells (BMCs) via lateral tail vein injection. The GVHD model was established by coinfusion of 5×106, 5×106, or 10×106 splenocytes via tail vein to induce the different grade of aGVHD. Clinical manifestations of aGVHD and survival were observed after allogeneic bone marrow transplantation. Histopathology and the state of chimera were detected in recipient mice after two to three weeks and three to four weeks post allo-BMT.Results: The survival rate and the median survival time of mice conditioned with 7Gy TBI was 20% and 22 days. All mice received 7.5, 8, 8.5 or 9 Gy TBI died within 30 days due to hematogenesis failure, and the median survival time of them was 18, 12, 9 and 7 days respectively. The survival time was compared by Log-rank test, withχ2=18.85, P<0.0001. The survival rate at 60 days was 60% and 40% respectively when mice were injected with 1×106, 2.5×106 BMCs. It indicated these dosages of BMCs could not fully reconstitute hematogenesis. All mice survived at 60 days post allo-BMT when reconstituted with 5×106, 10×106 BMCs. No obvious aGVHD was observed when mice were reconstituted with BMCs alone. The death related aGVHD was only 20% in mice injected with 1×106 splenocytes and 100% in mice received 5×106 or 10×106 splenocytes (P<0.0001). The obvious aGVHD lesion could be detected in skin, liver and ileum in mice of severe GVHD. The complete chimera derived from donor was detected in recipient mice on day 28 after allo-BMT.Conclusion: It was lethal conditioning when BALB/c mice received more than 7.5 Gy TBI. Hematopoietic recovery could achieve when BALB/c mice were reconstituted with greater than 5×106 BMCs. An obvious aGVHD could be induced when mice were injected with more than 5×106 splenocytes. We determined to establish an aGVHD model with myeloablative conditioning of 8.5 Gy TBI, 1×107 BMCs and 5×106 splenocytes.Objective: To explore the effects ofα-GalCer-loaded DCs infusion to expand and active host residual type-1 NKT cells on mouse aGVHD of allo-BMT. To observe the immunoregulation efficacy of host type-1 NKT cells infusion on mouse aGVHD of allo-BMT.Methods: Groups of allogeneic bone marrow transplantation aGVHD model as follow: (1) groups ofα-GalCer andα-GalCer-loaded DCs infusion.①BMT control: BMCs 1×107;②aGVHD control: BMCs 1×107 + SCs 5×106;③α-GalCer group: BMCs 1×107 + SCs 5×106 +α-GalCer (2μg/mouse);④α-GalCer-loaded DCs group 1: BMCs 1×107 + SCs 5×106 +α-GalCer-loaded DCs (1×105);⑤α-GalCer-loaded DCs group 2: BMCs 1×107 + SCs 5×106 +α-GalCer-loaded DCs (5×105);⑥α-GalCer-loaded DCs group 3: BMCs 1×107 + SCs 5×106 +α-GalCer-loaded DCs (1×106). (2) groups of host NKT cells infusion.①BMT control: BMCs 1×107;②aGVHD control: BMCs 1×107 + SCs 5×106;③host NKT cells infusion group 1: BMCs 1×107 + SCs 5×106 + host NKT cells (5×105);④host NKT cells infusion group 2: BMCs 1×107 + SCs 5×106 + host NKT cells (1×106). The manifestations of aGVHD were evaluated by using clinical aGVHD score and histopathologic examination. The survival rate was compared by Log-rank test. The levels of IL-4, IL-10, IFNγ, TNFαand CCL8 in serum were assaied by ELISA on day 7. We used H2b splenocytes from C57BL/6 mice as responder cells, and H2d SCs stimulated by DCs, H2d SCs without stimulation from BALB/c mice, H2d host NKT cells and H2d host NKT- T cells as stimulator cells which treated by mitomycin C to set up an in vitro mixed lymphocyte reaction (MLR) system. Tritiated thymidine was pulsed to detect the proliferation of responder cells.Results: No aGVHD manifestations were observed in BMT control group. All mice in aGVHD control goup showed the middle or severe aGVHD, andα-GalCer-loaded DCs (1×106) group 3 presented the severe aGVHD. The clinical GVHD scores ofα-GalCer group,α-GalCer-loaded DCs (1×105) group 1,α-GalCer-loaded DCs (5×105) group 2, host NKT cells infusion (5×105) group 1 and host NKT cells infusion (1×106) group 2 were lower than that of GVHD control group (P<0.05), in which, the clinical GVHD scores decreased dramatically inα-GalCer-loaded DCs (5×105) group 2 and NKT cells infusion (1×106) group 2. The lesion of skin, liver and ileum ofα-GalCer-loaded DCs (5×105) group 2 and host NKT cells infusion (1×106) group 2 was less severe than that of GVHD control group. All mice in aGVHD control group andα-GalCer-loaded DCs (1×106) group 3 died within 30 days. Comparison to aGVHD control group, the survival rate ofα-GalCer group (28.6%, 2/7),α-GalCer-loaded DCs (1×105) group 1 (16.7%, 1/6),α-GalCer-loaded DCs (5×105) group 2 (50.0%, 4/8), host NKT cells infusion (5×105) group 1 (28.6%, 2/7) and host NKT cells infusion (1×106) group 2 (37.5%, 3/8) improved remarkably (P<0.0001). The survival rate ofα-GalCer-loaded DCs (5×105) group 2 increased obviously compared withα-GalCer group andα-GalCer-loaded DCs (1×105) group 1 (P=0.0243,P=0.0097). There was no statistical significance between host NKT cells infusion (5×105) group 1 and host NKT cells infusion (1×106) group 2 (P>0.05). The levels of IL-4 and IL-10 in serum increased dramatically inα-GalCer group and α-GalCer-loaded DCs (5×105) group compared with aGVHD control group (P<0.0001). The secretion of IL-4 promoted obviously inα-GalCer-loaded DCs (5×105) group in comparison ofα-GalCer group (P=0.0176). The secretion of IFNγand TNFαinα-GalCer group,α-GalCer-loaded DCs (5×105) group and aGVHD control group appeared high level, no statistical significance was found between these groups (P>0.05). The levels of IFNγand TNFαinα-GalCer-loaded DCs (1×106) group were higher than that ofα-GalCer-loaded DCs (5×105) group (P<0.05). In host NKT cells infusion (1×106) group, the levels of IL-4 and IL-10 increased remarkably while the secretion of IFNγdecreased significantly compared with aGVHD control group (P<0.0001, P=0.0361). The secretion of CCL8 increased dramatically in each group, and there was no statistical significance (P>0.05). H2d SCs stimulated by DCs and H2d host NKT cells could obviously suppress the proliferation of H2b splenocytes from C57BL/6 mice in mixed lymphocyte responses compared with H2d SCs without stimulation and H2d host NKT- T cells respectively (P<0.05), and the suppression effects depended on the dose of stimulator cells.Conclusion: Administration of a suitable dose ofα-GalCer-loaded DCs infusion or host type-1 NKT cells infusion in recipient mice post allo-BMT could significantly suppress the pathology of aGVHD and remarkably improve the survival through activation of host residual type-1 NKT cells byα-GalCer-loaded DCs, secreting a large amount of Th2 cytokines by NKT cells and suppressing the proliferation of donor Tlymphocytes.Objective: To explore the immunoregulation efficacy of donor type-1 NKT cells infusion in mouse aGVHD of allogeneic bone marrow transplantation.Methods: Groups of allogeneic bone marrow transplantation aGVHD model as follow:①BMT control: BMCs 1×107;②GVHD control: BMCs 1×107 + SCs 5×106;③donor NKT cells infusion group 1: BMCs 1×107 + SCs 5×106 + donor NKT cells (5×105);④donor NKT cells infusion group 2: BMCs 1×107 + SCs 5×106 + donor NKT cells (1×106). The manifestations of aGVHD were evaluated by using clinical aGVHD score and histopathologic examination. The survival rate was compared by Log-rank test. The levels of IL-4, IL-10, IFNγ, TNFαand CCL8 in serum were assaied by ELISA on day 7. We used H2b splenocytes from C57BL/6 mice as responder cells, and H2d splenocytes from BALB/c mice as stimulator cells which treated by mitomycin C to set up an in vitro mixed lymphocyte reaction (MLR) system. The different dose of donor NKT cells or donor NKT- T cells which treated by mitomycin C added in the MLR system. Tritiated thymidine was pulsed to detect the proliferation of responder cells.Results: No mice in BMT control group occured the aGVHD manifestations. All mice in aGVHD control goup showed the middle or severe aGVHD and died within 30 days. The clinical GVHD scores of donor NKT cells infusion (5×105) group 1 and donor NKT cells infusion (1×106) group 2 were lower than that of GVHD control group (P<0.05), The lesion of skin, liver and ileum of donor NKT cells infusion (1×106) group was less severe than that of GVHD control group. Comparison to aGVHD control goup, the survival rate of donor NKT cells infusion (5×105) group 1 (25.0%,2/8) and donor NKT cells infusion (1×106) group 2 (42.9%,3/7) improved remarkably (P<0.0001). The survival rate of donor NKT cells infusion (1×106) group 2 increased significantly compared with donor NKT cells infusion (5×105) group 1 (P=0.0375). The levels of IL-4 and IL-10 in serum increased dramatically in donor NKT cells infusion (1×106) group, and the secretion of IFNγ, TNFαand CCL8 showed no statistical significance in comparison of aGVHD control group (P>0.05). Donor NKT cells at a dose of 2×105 could obviously suppress the proliferation of H2b splenocytes from C57BL/6 mice in mixed lymphocyte responses compared with donor NKT- T cells (P=0.0382).Conclusion: Donor type-1 NKT cells infusion in recipient mice post allo-BMT could significantly suppress the pathology of aGVHD and remarkably improve the survival through secreting a large amount of Th2 cytokines and suppressing the proliferation of donor T lymphocytes. Objective: To explore the reconstruction and the influence to aGVHD of Vα24+Vβ11+ NKT cells in patients of allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods: There were 38 patients including 20 males and 18 females received allo-HSCT in Department of Hematology, First Affiliated Hospital of Soochow University from May 2010 to October 2010. The median age was 33 years old (range from 13 to 55). Matched sibling donor bone marrow transplantation (MSD-BMT) performed in 20 patients, and matched sibling donor peripheral blood stem cell transplantation (MSD-PBSCT) carried out in four patients. Eleven patients received matched unrelated donor peripheral blood stem cell transplantation (MUD-PBSCT) and three patients received haploidentical donor bone marrow transplantation (HID-BMT). Mononuclear cells were separated from bone marrow grafts, peripheral blood grafts, peripheral blood at day 30, day 60 and day 90 post allo-HSCT, peripheral blood of the patients when occurred aGVHD. Mononuclear cells were labeled with PE-TCRVα24, FITC-TCRVβ11 and APC-CD3 monoclonal antibody for percentage assay of Vα24+Vβ11+ NKT cells by flow cytometry. The absolute number of NKT cells was counted to analyze the influence to aGVHD in patients with aGVHD or without aGVHD.Results: All patients recovered hematogenesis post allo-HSCT. The incidence of aGVHD was 63%, comprised gradeⅠ~Ⅰ( 50%) and gradeⅢ~Ⅲ( 13%). The percentage of Vα24+Vβ11+ NKT cells at day 30, day 60 and day 90 post allo-HSCT was (0.37±0.10)%, (0.34±0.09)%, (0.48±0.16)% in BMT group, and (0.33±0.07)%, (0.63±0.19)%, (0.55±0.15)% in PBSCT group respectively. The percentage of NKT cells in PBSCT group at day 60 was significantly higher than that in BMT group (P=0.0462). In patients with aGVHD or without aGVHD who received MSD-BMT, the absolute number of NKT cells in graft and peripheral blood was (0.33±0.06)×106/kg, (0.40±0.10)×106/kg (P>0.05), and (0.9±0.18)×106/L, (1.1±0.25)×106/L (P>0.05) respectively. In patients with aGVHD or without aGVHD who received MUD-PBSCT, the absolute number of NKT cells in graft and peripheral blood was (0.29±0.07)×106/kg, (0.29±0.07)×106/kg (P>0.05), and (1.2±0.24)×106/L, (1.0±0.21)×106/L (P>0.05) respectively.Conclusion: The recovery of Vα24+Vβ11+ NKT cells in BMT patients was slower than that in PBSCT patients. The absolute number of NKT cells in graft and peripheral blood did not influence the incidence and the grade of aGVHD.