The Role Of Marrow Derived Mesenchymal Stem Cells And ICOS-B7h Signal Blocking In The Prevention And Treatment Of Acute Graft-versus-host In Mice

The role of marrow derived mesenchymal stem cells and ICOS-B7h signal blocking in the prevention and treatment of acute graft-versus-host in miceBackground and objectiveMSCs are adult stem cells of mesodermal origin with self-renewal and multiple lineage differentiations potentials.Existing technology permitting in vitro expansion of MSCs without apparent loss of phenotype or function.A number of cytokines are secreted by MSCs,such as hematopoietic growth factors,nonhematopoietic factors and chemokines, which modulate the bone marrow microenviroment,and facilitate the proliferation and differentiation of hematopoietic cell.It has been documented that MSCs are not substantially immunogenic and may actually inhibit both primary and secondary mixed lymphocyte reaction,which has a great capacity of immunomodulatory.Data from animal models demonstrating the potential for MSCs to enhance engraftment of allogeneic HSCs have provided a rationale for clinical trials in the allogeneic setting.Acute graft-versus-host disease(aGVHD) is one of the major complications of allogenetic hematopoietic stem cell transplantation(allo-HSCT),accounting for more than 50%morbidity and mortality after transplant.Since MSCs process immunosuppressive properties in vivo,we supposed that there is the potential that MSCs could ameliorate or perhaps even prevent aGVHD reactions.On the other hand,the capacity of MSCs to repair or replace stromal matrix damaged by genetic mutation or intensive chemotherapy,may improve either the overall rate of engraftment or pace of hematopoietic recovery following HSCs transplantation.But till now,treat aGVHD with MSC is rarely reported.Most work foucusd on the prevention of MSC to GVHD.So in our present study,we investigated the properties about the prevention and treatment of aGVHD by mice marrow mesenchymal stem cells (mMSCs) in vivo.Inducible costimulator(ICOS) is a relatively new member of B7 family involved in the costimulation of T cells.Unlike CD28,ICOS expression is restricted to activated T cells present in lymphoid organs.The ICOS ligand(ICOSL) is constitutively expressed on unstimulated B cells,splenic and peritoneal macrophages,and blood-derived dendritic cells.The role of the ICOS-B7h interaction in the immune response is enhancing of T cell activation and altering the polarization of T helper cells.Since it has been assumed that Th1 cells contrubited to aGVHD,we also took the advantage to block ICOS-B7h signaling pathway with ICOS-Ig fusion protein and investigated its biological activity on allogene T lymphocyte and aGVHD.Part One The immunomodulatory potentials of mMSCs in aGVHD modelMethods:P5 mMSC were cultured in DMEM-LG medium supplemented with 10%fetal bovine serum(FBS).After serial plating,the phenotype,multilineage differentiation potential of P10 mMSCs were confirmed.Allogeneic aGVHD model was established as follows:lethally irradiated BALB/c recipients received allogeneic BM cell(BMC) and spleen cells(SP) of C57BL/6 origin with or without different dose of mMSCs with a time course.Groups:â‘ PBS group (irradiation control);â‘¡BM group(i.v.BMC only);â‘¢GVHD group(i.v.SP+ BMC);â‘£MSC-A group(i.v.SP+BMC+5 x 10⁶/kg mMSC at d₀);⑤MSC-B group(i.v.SP + BMC +2.5 x 10⁷/kg mMSC at d₀);â‘¥MSC-C group(i.v.SP + BMC +5 x 10⁶/kg mMSC at d₇).In addition,mMSCs transfected with adenoviral vector(Ad-GFP) were transplanted into aGVHD model to observe the distribution of mMSCs.Results:Flow-cytometric analysis showed that CD29,CD44 and Sca-1 were expressed on mMSCs with high intensity,CD13 and CD90.2 with medium intensity, CD117,CD45,FLK-1 and MHC-â…¡were not expressed.With the induction reagents in vitro,mMSCs can be differentiated into osteoblast cells,adipocytes, and chonrdrocyte.With the intervention of mMSCs of donor origin controlled the lethal aGVHD in control group.The mean survival time(day) in PBS group was 13.5±2.63,GVHD group 11.1±3.96,MSC-A group:26.38±7.70,MSC-B group:22.69±9.15,MSC-C group:22.92±8.15,respectively.The difference between mMSCs and GVHD groups were statistically significant(P＜0.01),but no difference was observed among mMSCs groups(P=0.28);Pathologic evaluation demonstrated that there were less lymphocyte infiltration and tissue damage in the intestine,spleen and liver in MSCs group than those in control group,mMSCs significantly reduced the production of IFN-γand TNF-a in recipients.The concentration of IFN-γdetected in GVHD group(pg/mL)was 607.89±157.10,143.56±37.52 in MSC-A group,116.98±77.78 in MSC-B group and 131.45±63.35 in MSC-C group,respectively(P＜0.05);and the TNF-a (pg/mL) detected in GVHD group were 52.31±17.95,MSC-A group 6.02±3.99, MSC-B group 5.21±0.28 and MSC-C group 22.39±18.21,respectively(P＜0.05); (3) CFSE assay revealed that mMSCs had no effect on T cells of donor origin proliferation in GVHD model:GVHD group 98.4±1.32,MSC-A group 97.69±2.19,MSC-B group 97.44±2.31.The survival time of T cells of donor origin was shortened:Annexin-V positive lymphocyte at day10:GVHD group 10.33±6.61,MSC-A group 13.51±13.75,MSC-B group 19.68±5.98,MSC-C group16.56±7.27(P＜0.05) between MSC-B and GVHD group.The production of cytokines such as IFN-γ,IL-4 and IL-10 after in vitro stimulation with ConA were much less in spleen T cells from mMSCs groups than those from control group.IFN-γ(pg/mL) in GVHD group was 2030.65±876.36,MSC-A group 656.70±328.68,MSC-B group 547.38±278.79,and MSC-C group 306.07±143.59.IL-4(pg/mL) in GVHD group was 306.33±6.84,MSC-A group 38.82±0.33,MSC-B group 32.64±0.01,and MSC-C group 21.82±1.10. IL-10(pg/mL) in GVHD group was 456.03±69.61,MSC-A group 449.65±168.61, MSC-B group 319.07±33.37,and MSC-C group 239.27±46.43.In GVHD model, most GFP-MSCs lodged in lungs and intestines while rarely found in liver, spleen and kidney.Part two Blocking ICOS-B7h signal can inhibit allogeneic T lymphocytes activation and reduce the incidence and severity of aGVHDMethods:pSecTag2/Hygro A-ICOS-Ig was verified with restrictive enzyme digestion. The CHO cell lines were transfected with the plasmid through Lipoplast.Stable CHO lines expressing ICOS-Ig were selected with limiting dilution in RPMI1640 medium with 800ug/mL HygroB.Culture supernatants from drug-resistant clones were then assayed by IgG sandwich ELISA to screen for high-producing lines.Supernatants were harvested from large-scale cultures,and ICOS-Ig was purified by protein A affinity chromatography,analyzed with SDS-PAGE for endotoxin.Three lines were used to investigate the role of ICOS-Ig in proliferation and differentiation of allogene T lymphocytes.â‘ , splenic cells from C57BL/6 were cultured in 2×10⁵/mL and stimulated with 10ug/mL ConA;â‘¡CD4⁺ cells of C57BL/6 origin purified from the spleen were cultured in 1×10⁵/mL as responder,the irradiated splenic monocytes from BALB/C as stimulator in 2×10⁵/mL;â‘¢CD4⁺cells from the spleen of C57BL/6 mice were cultured in 1×10⁵/mL as respondor,irradiated dendritic cells from BALB/C as stimulate cells in 1×10⁴/mL.Different doses of ICOS-Ig or human-Ig(h-Ig) were used as controls.Proliferation rates were measured after 48-72 hours with CCK-8 kit.The proliferation history of allogeneic T cells was checked with FACS.Supernatants were collected on d₃ for measurement of IL-10,IL-4,TNF-a and IFN-γ,.Allogeneic aGVHD model was established with lethally irradiated BALB/c recipients receiving allogeneic BM and spleen T cells from C57BL/6 with 100ug ICOS-Ig or h-Ig intraperitoneally 4 times at d₀,d_2,4,6 of transplant.Results:The plasmid was comfirmed in our hands.CHO cells were transfected with ICOS-Ig cDNA in pSecTag2/HygroA,then selected with HygroB,and isolated for high-producing clones with IgG sandwich ELISA.The concentrantion of protein in supernatants from stable CHO lines were 563.022ng/mL.Supernatants were harvested from large-scale cultures,and ICOSIg was purified by protein A affinity chromatography.The protein weight was 54KDa confirmed by SDS-PAGE.And the contents of endotoxin was less than 10EU/mL.ICOS-Ig inhibited splenic cells activation responsing to ConA when the concentration reached more than 20ug/mL(P＜0.05);ICOS-Ig inhibited allogenetic CD4⁺ cells proliferation and reduced the production of IFN-γin the supernatants inducing by alloantigen when the concentration reached more than 40ug/mL(P＜0.05); 10ug/mL ICOS-Ig significantly inhibited CD4⁺T cells proliferation(P＜0.01), 40ug/mL ICOS-Ig reduced the production of TNF-a(h-Ig: 1712.62±313.93;ICOS-Ig 1025.07±156.51 pg/mL),20ug/mL ICOS-Ig increased the production of IL-4(h-Ig:187.65±27.54;ICOS-Ig 527.20±26.54 pg/mL) in the supernatant of CD4⁺ T cells responsing to allogeneic mature DCs but had no effect on IFN-γproduction;the apoptotic splenic CD4⁺ T cells increased when ICOS-Ig pathway was blocked while no effect was observed on T cell activation. Block ICOS-Ig pathway effectively controlled the severe aGVHD occurred in control group.The median survival time was 10 days in h-Ig group over 20 days in ICOS-Ig group(P=0.0217,n=14).Pathologic evaluation revealed that the liver and intestine in ICOS-Ig group had less lymphocyte infiltration and tissue damage than those in control group.In vivo,ICOS-Ig had no obvious effect on allogeneic T cells proliferation(h-Ig:98.40±1.32,ICOS-Ig:97.69±2.19 in CD4⁺ T cells,h-Ig:97.93±1.04,ICOS-Ig:98.94±1.64 in CD8⁺ T cells by CFSE tracing assay at d₃) and no effect was observed in the CD4⁺/CD8⁺populaton:h-Ig: 26.35±0.07,ICOS-Ig:22.12±0.21(P=0.44,n=5),but increased apoptosis of allogeneic CD8⁺T cells in GVHD model at d₁₀: h-Ig:15.72±7.34,ICOS-Ig:22.78±6.94 in CD4⁺ T cells(P=0.078,n=5);h-Ig: 15.7±9.59,ICOS-Ig:25.92±5.66 in CD8⁺ T cells(P=0.032,n=5).ICOS-Ig did not influence the distrubition of allogeneic CD4⁺ and CD8⁺ T cells post transplantation:h-Ig:12.45±3.04,ICOS-Ig:10.71±2.88 in CD4⁺ T cells(P=0.56, n=5);h-Ig:47.31±0.45,ICOS-Ig:47.76±4.81 in CD8⁺ T cells(P=0.91,n=5).T cells from spleen were stimulated with ConA in vitro at d₁₀.T cells in ICOS-Ig group shown less proliferative potential than control(h-Ig:0.86±0.04,ICOS-Ig: 0.69±0.12,P＜0.05).CONCLUSIONS:(1) mMSCs not only can prevent but also can treat severe aGVHD;(2) mMSCs did not influence the allo-T cells' proliferative potential but shortened the survival time of T cells;(3) mMSCs significantly reduced the production of cytokine of Thl cells(IFN-γand TNF-a);(4) In aGVHD model,mMSCs can lodge to and repair the damaged tissue such as lung,intestine,liver,spleen and kidney;which accounted for the role of mMSCs in aGVHD;(5) The ICOS-Ig fusion protein had bioactivity property of inhibiting T cell proliferation and alternating the polarization state of T helper cells;shortening the survival time of allo-reactive T cells of donor origin,but not influencing the activation of allo-reactive CD4⁺T cells;(6) Block ICOS-Ig pathway can prevent aGVHD through attenuating the function of the allo-reactive T cells and shortening the survival time of allo-T cells.