Studies Of In Vitro Activation Of Bone Marrow Nature Killer T Cells And Its Implication In Aplastic Anemia

Aplastic anemia(AA)is a syndrome of bone-marrow failure characterized by peripheral pancytopenia and marrow hypoplasia.Ontologically,threr are two kinds of aplastic anemia,namely,acquired AA and hereditary AA.Of these,acquired AA is the more common type.Acquired AA can be triggered by multiple factors including chemical agents such as exposure to toxic chemicals or chemotherapy drugs,physical agents such as ionizing radiation and biological agents mainly virus infections. However,in most of the cases,the cause of acquired AA is not known.Acquired AA is,therefore,referred to as idiopathic AA and abbrivated to AA throughourt the paper. Immunosuppressive therapy leads to remission in the majority of AA patients, suggesting that failure in hematopoiesis,at lease in some cases,is likely to be mediated by the aberrance in the immune system.Reduced CD4~+/CD8~+ ratio and elevated Th1/Th2 ratio are detected in both the blood and bone marrow of AA. Increased inhibitory cytokines,such as IFN-γ,TNF and IL-2 have also been detected. As a result,hematopietic stem/progenitor cells(HSPCs)in BM of AA patients are both quantitatively and qualitatively damaged by the abnormal cellular immunity and finally leads to BM failure.Natural killer T(NKT)cells are a unique T lymphocyte sub-population that has been implicated in the regulation of immune responses associated with a broad range of diseases,including autoimmunity,infectious diseases,and cancer.In human,NKT cells express a Vα24-Jα18-rearranged TCRαchain and a Vβ11-containingβchain,as well as NK cell-specific CD161.When activated,NKT cells result in a vigorous response evidenced by cytokine production within 1-2 hours.These cells release Th1-type cytokines including IFN-γ,and TNF,as well as Th2-type cytokines including IL-4 and IL-13.The mechanisms that determine the cytokine polarity of NKT cells,and the contribution of NKT cells to the systemic immune response,are not well understood,and indeed,this question represents one of the important challenges in the understanding of NKT cell roles in the regulation of cellular immunity.As an important immunoregulator in Th1/Th2 balance,NKT cells is, therefore,an attractable and interesting research area in the pathogenesis of AA.In the present study,we investigated the quantitative and qualitative changes of NKT cells in BMMNCs of AA patients in response to in vitro stimulation ofα-galactosylceramide(α-Galcer),and the effect of activated NKT cells on the expansion potential of HSPCs of AA patients.The objective of the study is to primarily look into the roles of NKT cells in the pathogenesis of AA,and to explore the possible application ofα-Galcer,as a new immuno-modulation agent,in the treatment of AA.PartⅠStudy of the Function of Expansion and Cytokine Releasing of TCRVα24~+Vβ11~+NKT Cells in Bone Marrow of Aplastic AnemiaObjective:To investigate the quantitative and qualitative changes of TCRVα24~+Vβ11~+NKT cells from BM of AA to the in vitro stimulation ofα-Galcer.Methods:TCRVα24~+Vβ11~+ NKT cells in the bone marrow mononuclear cells (BMMNCs)from either AA patients or healthy controls were enumerated with flow cytometry.BMMNCs were cultured in RPMI1640 medium supplemented with eitherα-Galcer and rhIL-2 orα-Galcer,rhIL-2 and rhG-CSE The proliferative capacity of NKT cells was expressed by fold expansion,as determined by NKT cell numbers after in vitro culture relative to those before the culture.Expression of intracellular IFN-γand IL-4 in activated NKT cells was analyzed with flow cytometry.Result:The percentage of NKT cells in BMMNCs from AA was significantly lower than that from healthy controls.Upon stimulation in vitro with eitherα-Galcer and rhIL-2 orα-Galcer,rhIL-2 and rhG-CSF,compared to those from healthy controls, NKT cells from AA showed reduced potential to expand,with a higher fraction of NKT cells expressing IFN-γ,(NKT1).When added to the culture medium,rhG-CSE in combination withα-Galcer and rhIL-2,decreased the expansion of NKT cells in both AA and control groups.It also induced polarization of NKT cells towards the NKT2 subpopulation,resulting in an elevated percentage of IL-4~+ NKT2 cells and a decreased percentage of IFN-γ~+ NKT1 cells in both AA and healthy control groups.Conclusion:Compared to those from healthy controls,BMMNCs from AA have a reduced fraction of NKT cells,which possesses a decreased potential to expand in vitro in response toα-Galcer stimulation,producing more IFN-γ~+ NKT1 cells. rhG-CSF,in combination withα-Galcer,confers polarization of NKT cells towards IL-4~+ NKT2 subpopulation.PartⅡStudy of the Effects of Activated NKT Cells on in vitro Hematopoietic Recovery in Aplastic AnemiaObjective:To investigate the effects of activated NKT cells on the expansion potential of HSPCs from AA.Methods:BMMNCs from AA patients and healthy controls were plated in IMDM medium supplemented with either rhSCF and rhG-CSF or rhSCF,rhG-CSF andα-Galcer.The level of cytokine IFN-γ/IL-4 in supernatant were determined by ELISA 3 days after culture.Colony-forming assay was conducted by culturing BMMNCs in methylcellulose medium supplemented with either rhSCF and rhG-CSF or rhSCF, rhG-CSF andα-Galcer.Yields of granulocyte/monocyte colony-forming unit (CFU-GM)were analyzed 7 days after plating.Result:Whenα-Galcer was added to the IMDM medium in combination with rhSCF and rhG-CSF,an elevated level of IL-4 and decreased level of IFN-γwere observed in supernatant in both AA and control groups.Meanwhile,with the addition ofα-Galcer to the methylcellulose medium,a significant increase in the number of CFU-GM was observed in both AA and control groups.Conclusion:An increased number of CFU-GM was generated in the BMMNCs from both AA and control groups whenα-Galcer was added in combination with rhG-CSF. These results indicate that NKT cells activated byα-Galcer and rhG-CSF are capable of modulating the abnormal T-cell mediated immunity and improving the in vitro expansion potential of HSPCs in AA.