Immune-regulation Of Natural Killer Cells And Their Subtypes On Experimental Autoimmune Myasthenia Gravis

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Immune-regulation of natural killer cells on experimental autoimmune myasthenia gravisBackgroundMyasthenia gravis(MG)is an antibody mediated autoimmune disease.The pathogenesis of MG is mainly due to antibody targeting acetylcholine receptor(AchR),which binds to AchR and interferes with neuromuscular transmission,leading to muscle weakness.Immunization with peptide corresponding to rat AchR to establish experimental autoimmune myasthenia gravis(EAMG)is well accepted for studying the pathogenesis and treatment of MG.Follicular helper T(Tfh)cells are a kind of newly found CD4+T cell subtype,which are characterized by high expression levels of C-X-C chemokine receptor type 5(CXCR5),inducible costimulatory(ICOS),and programmed cell death 1(PD-1).Tfh cells paly critical roles in B cell proliferation and differentiation,as well as antibody class switch and affinity maturation.It has been said that Tfh cells participate in the pathogenesis and progression of MG.Natural killer(NK)cells are a kind of innate lymphocytes with the capacity to directly killer transformed cells.Other than responding to tumor and viral infection,NK cells play essential roles in autoimmune disease.However,the roles of NK cells in EAMG remain to be elucidated and effects of NK cells in regulating Tfh cells are also ambiguous.ObjectivesThe aim of this study is to investigate the roles of NK cells in EAMG and explore the effects and mechanism of NK cells in regulating Tfh cells.Methods1.EAMG rat model was induced by subcutaneous immunized with peptide(R97-116)corresponding to rat AchR a subunit at the base tail.After immunization,rats were injected with PBS(control group)or spleen isolated NK cells(NK cell group)via tail vein.The body weights and clinical scores were assessed every other day in a blind manner.At day 46 post immunization,EAMG rats were sacrificed,and blood serum and spleen were harvested.2.Serum antibody,antibody subtypes and antibody affinity were determined using ELISA.3.Flow cytometry was applied to analyze the percentages of splenic germinal center B cells,Tfh cells and their subtypes,regulatory T cells(Treg cells),natural killer T(NKT)cells and dendritic cells.4.Isolated spleen mononuclear cells were treated with interleukin(IL)-15,which could activate and expand NK cells.Then,cells were detected by flow cytometry for percentage,absolute number and apoptosis of CD4+T cells and Tfh cells.5.Isolated spleen monocytes or CD3+ T cells were co-cultured with NK cells.The percentage,absolute number and apoptosis of CD4+T cells and Tfh cells were analyzed using Flow cytometry.Results1.Compared with control group,treatment with splenic NK cells significantly ameliorated severity of EAMG.The clinical scores of NK-cell treated rats were significantly lower than control rats at day 38,day 40,day 42,day 44 and day 46 post immunization(p<0.05).2.Results from ELISA demonstrated NK cells reduced serum level of IgG2a antibody against R97-116(p<0.01),but not IgG,IgG1 or IgG2b antibody.In addition,a decreasing trend of antibody affinity was observed after NK cell treatment(p=0.09).3.Flow cytometry illustrated NK cell treatment led to a reduced percentage of germinal center B in spleen,accompanied by decreasing of Tfh cell percentage.There were no difference in the percentage of memory B cells,Tfh cell subtypes,Treg cells,NKT cells as well as dendritic cells.4.Co-cultured with NK cells increased the apoptosis of Tfh cells and resulted in diminished Tfh cell percentage.IL-15,which could expand NK cells,also inhibited Tfh cell differention and hence suppressed B cell proliferation.Conclusion1.NK cells inhibited serum antibody production by suppressing Tfh cell and germinal center B cells resulted in ameliorated EAMG symptoms.2.The inhibiting effects of NK cells on Tfh cell differentiation were mediated by cytotoxicity of NK cell against Tfh cells.Paper ?Phenotype,distribution and immunoregulatory function of CXCR5-and CXCR5+natural killer cellsBackgroundNatural killer(NK)cells are a kind of innate lymphocytes with the capacity to directly kill transformed cells.Other than responding to tumor and viral infection,NK cells play essential roles in autoimmune diseases.NK cells are heterogenous population and can be divided into several subtypes based on the differences in phonotypes and function,which exert different impacts on immune response.C-X-C chemokine receptor type 5(CXCR5)belongs to C-X-C chemokine receptor family and its ligand is C-X-C chemokine ligand 13(CXCL13).CXCR5 is expressed on B lymphocytes,follicular helper T(Tfh)cells,some CD8+T cells and natural killer T(NKT)cells.Guided by CXCL13 expressed by stromal cells in B cell follicles,CXCR5-positive cells(such as Tfh cells,CD8+CXCR5+T cells and CXCR5+NKT cells)can migrate into B cell follicles and modulate germinal center reaction and antibody production.Studies in the part I of this paper revealed the immunoregulation of NK cells on germinal center B cells as well as antibody production.However,whether CXCR5 is expressed on NK cells and the function of CXCR5+NK cells remain to be illustrated.ObjectivesThe aim of this study is to investigate the phenotypes,distribution,migration and function of CXCR5-and CXCR5+ NK cells,and to illustrate the roles of CXCR5-and CXCR5+ NK in regulating Tfh cells.Methods1.Isolated mononuclear cells from peripheral blood,spleen,and lymph node of Lewis rats were analyzed for the percentages of CXCR5-and CXCR5+ NK cells using flow cytometry.2.Detect distribution of NK cells and their subtypes in B cell zone or germinal center by immunofluorescent staining.3.NK cells or CXCR5-and CXCR5+NK cells were isolated,labeled with CFSE and transferred into recipient rats.The migration of each NK cells were addressed by immunofluorescent staining.4.Compare the phenotype differences of CXCR5-and CXCR5+NK cells by detecting the expression levels of inducible co-stimulator(ICOS),CD25,CD27,interferon(IFN)-y and interleukin(IL)-17 by flow cytometry.5.Animal models of experimental autoimmune myasthenia gravis(EAMG)and experimental autoimmune neuritis(EAN)were induced.Linear regression was carried out to reveal the correlation of CXCR5+NK cells and Tfh cells or germinal center B cells.6.Isolated splenic mononuclear cells or CD3+ T cells were co-cultured with CXCR5-and CXCR5+NK cells or not.The number and percentages of CD4+T cells and Tfh cells were analyzed using Flow cytometry.Results1.Based on the expression of CXCR5,rat peripheral blood,spleen and lymph node NK cells were divided into CXCR5-and CXCR5+NK cells.The percentages of CXCR5+NK cells in NK cells are 0.63±0.03%,2.44±0.17%,and 9.75±0.96%in rat peripheral blood,spleen and lymph node,respectively.2.Results from immunofluorescence and in vivo tracing experiment revealed that there were NK cells in the B cell follicles.Compared with CXCR5-NK cells,there were more CXCR5+NK cells migrating into the B cell follicles.3.Compared with CXCR5-NK cells,CXCR5+NK cells exhibited higher levels of ICOS as well as IL-17,and lower levels of CD27.There were no differences of CD25 expression between these two subsets.In addition,the volume and granularity of CXCR5+NK cells are higher than their CXCR5-counterparts.4.Linear regression analysis illustrated that the percentages of CXCR5+NK cells are positively correlated with the percentages of Tfh cells or germinal center B cells.In contrast,the ratios of CXCR5-to CXCR5+ NK cells are inversely correlated with the percentages of Tfh cells or germinal center B cells.5.CXCR5-NK cells but not CXCR5+NK cells dampened the number of CD4+T cells and Tfh cells when co-cultured with splenic mononuclear cells.Similarly,when isolated CD3+ T cells were co-cultured with CXCR5-or CXCR5+ NK cells,only CXCR5-NK cells inhibited the percentages of Tfh cells.Furthermore,CXCR5+NK cells rather than CXCR5-NK cells enhanced ICOS expression on Tfh cells.Conclusion1.Based on the expression of CXCR5,rat peripheral blood,spleen and lymph node NK cells can be divided into CXCR5-and CXCR5+ NK cells.2.CXCR5-and CXCR5+ NK cells exhibit different phenotypes in regarding to surface molecules and cytokine production3.CXCR5-and CXCR5+ NK cells have different functions.CXCR5-NK inhibit Tfh cell differentiation,while CXCR5-NK cells enhanced ICOS expression on Tfh cells.