Effects Of Hypoxia And CD137 Pathway On The Function Of DCs

PARTⅠEffects of Hypoxia and CD137 Pathway on the Function of DCsObjectiveHypoxia,a status of very low oxygen tension in local tissues or organs,is always found in various physiological and pathophysiological tissues,such as microenvironments that are located far from the ends of capillaries,cancerous or inflamed tissues,as well as organ transplantation,hypovolemic shock,liver surgery and cardiovascular diseases.It was reported that hypoxia could promote tumor cells progression and resistance to chemotherapy and radiotherapy,leading to poor prognosis. Though many researches reported the influence of hypoxia on tumor growth and metastasis,it's unclear that the change of immune cells and immune responses in such hypoxic microenvironments.Though limited data studied the effects of hypoxia on innate immunity such as macrophage,little reports referred to the effects on adaptive immunity,sporadic experiments showed that hypoxia could inhibit the fimction of T cells,the cell activation,proliferation,and the cytokine secretion decreased markedly, but detailed mechanism is largely unknown so far.Dendritic cells(DCs) are the critical antigen presenting cells linking innate and adaptive immunity,the status of DCs maturation can directly influence the ability of antigen presenting and stimulating T cell proliferation.Immature DCs mainly reside in non-lymphoid tissues,after uptake of antigen,these immature DCs migrate to the regional lymph nodes and differentiate into mature DCs,which express highly MHC classⅡmolecules,co-stimulatory molecules CD80,CD86,and have a potent capacity for antigen presentation,thereby initiate the specific immune response.The homing and migration of DCs need to go through the pathological tissues such as tumor and inflamed tissues,while the effects of hypoxic microenvironment in these tissues on DCs is unclear.Two reports suggested that human monocyte-derived DCs differentiated under hypoxia expressed decreased matrix metalloproteinase(MMP)-9 and have a reduced migratory capacity compared with those differentiated under normoxia,but further evidence for the effects of hypoxia on the function of DCs, especially on that of mouse DCs is lacking to date.In addition,the return of blood into ischemia tissues can result in the elevation of oxygen tension in hypoxic tissues,it is unclear whether the dramatic change of oxygen tension affects DCs as well as the T cell immune response mediated by these DCs.Therefore,in the current study,we focused on the effects of hypoxia on the phenotype and function of mouse DCs,further studied the effects of reoxygenation(from hypoxia to normoxia) on hypoxic DCs.It is much helpful for the control of ischemia reperfusion injury,graft rejection in organ transplantation,and the immunotherapy for tumor.CD137-CD137L is another pair of important co-stimulatory molecules besides CD28-B7 for T cells.As a member of tumor necrosis factor receptor superfamily, CD137 is an inducible T cell surface receptor and mainly expresses on activated T cells. CD137-CD137L pathway provides co-stimulatory signal for the activation, proliferation,differentiation of CD4~+ and CD8~+ T cells,and plays an important role in adaptive immunity,especially in the late phase of primary immune response and the secondary immune response.Recent studies showed that CD137 can be expressed on mouse splenic DCs and bone marrow-derived DCs(BM-DCs),agonistic mAb to CD137 increased the secretion of IL-6,IL-12 from DCs,importantly enhanced the ability of DCs to stimulate T cell proliferation,suggesting the important role of CD137 pathway on the modulation and enhancement of DCs function.Based on the above studies,we further explored the effects of CD137 pathway on hypoxic DCs,which aim to increase the function of hypoxic DCs,further enhance the adaptive immune response.It may provide a novel strategy for the immunotherapy for tumor and graft rejection.Methods1.Phenotye and function of DCs under normoxic or hypoxic conditions1) To isolate bone marrow cells from C57BL/6 mice,induce DCs using GM-CSF and IL-4,and stimulate maturation by LPS under normoxic or hypoxic conditions.2) To detect the expression of co-stimulatory molecules CD80,CD86,and MHC classⅡmolecules on DCs differentiated under normoxic or hypoxic conditions through Flow Cytometry.3) To detect the secretion of cytokines(IL-1β,IL-6,TNF-α,TGF-β) and expression of matrix metal proteinases MMP9 by DCs differentiated under normoxic or hypoxic conditions through RT-PCR and ELISA.4) To assay the CD4~+T cell proliferation stimulated by DCs differentiated under normoxic or hypoxic conditions through CFSE labeling.2.Effects of reoxygenation on the phenotype and function of DCs differentiated under hypoxic conditions1) To reoxygenate DCs differentiated under hypoxic conditions for various periods of time.2) To detect the change of phenotype(CD80,CD86,and MHC classⅡmolecules) on reoxygenated DCs by Flow Cytometry.3) To assay the CD4~+T cell proliferation stimulated by reoxygenated DCs via CFSE labeling.4) To assay the CD4~+T cell differentiation stimulated by reoxygenated DCs via Flow Cytometry.5) To detect the cytokine expression in CD4~+T cell proliferation system stimulated by reoxygenated DCs through ELISA.3.Effects of CD137 pathway on the phenotype and function of DCs differentiated under hypoxic conditions1) To treat DCs differentiated under hypoxic conditions using agonistic anti-CD137 Ab.2) To detect the change of phenotype(CD80,CD86,and MHC classⅡmolecules) on hypoxic DCs after anti-CD137 Ab stimulation by Flow Cytometry.3) To detect the change of cytokine secretion and MMP9 expression by hypoxic DCs after anti-CD137 Ab stimulation through RT-PCR.4) To assay the CD4~+T cell proliferation stimulated by anti-CD137 Ab treated-hypoxic DCs via CFSE labelling.Results1.Phenotype and function of DCs differentiated under normoxic or hypoxic conditions 1) Induction and purity of DCs derived from bone marrow cells of C57BL/6 miceBone marrow cellls were isolated from C57BL/6 mice and were induced into immature DCs under normoxic or hypoxic conditions in the presence of GM-CSF and IL-4.To induce maturation,immature DCs were treated with LPS for 18-24h.After 5-7 days of induction,cells with typical dendritic morphology were observed under microscope;results from Flow Cytometry showed that the purity of DCs differentiated under normoxic or hypoxic conditions as examined by CD11c expression were both above 80%,suggesting hypoxia has no influence on the induction and differentiation of DCs.2) Hypoxia inhibits the phenotypic maturation of DCsTo examine the effect of hypoxia on the phenotypic maturation of DCs, murine DCs derived from bone marrow(BM-DC) differentiated under normoxic or hypoxic conditions were stimulated with LPS,related surface markers were detected by Flow Cytometry.The results showed that DCs differentiated under hypoxic conditions displayed immature or semi-mature phenotype though stimulated with LPS,the expression of CD80,CD86 and MHC classⅡmolecules on these DCs was markedly lower than that of normoxic mature DCs, especially the mean fluorescence intensity(MFI) of MHC classⅡmolecules (P＜0.01),thus LPS fails to induce the up-regulation of co-stimulatory and MHC classⅡmolecules on hypoxia-differentiated DCs,suggesting hypoxia inhibits the phenotypic maturation of DCs.3) Hypoxia inhibits the production of proinflammatory cytokines IL-1,IL-6, TNF-α,and MMP9,but up-regulate the secretion of immune suppressive cytokine TGF-βby DCsTo determine the effects of hypoxia on the cytokine production and migration of DCs,BM-DCs differentiated under normoxic or hypoxic conditions were stimulated with LPS,expression of cytokines and MMP9 on these DCs were assayed by RT-PCR and ELISA.The results showed that DCs differentiated under hypoxic conditions secreted much lower levels of proinflammatory cytokines IL-1β,IL-6 and TNF-α,but higher levels of immune suppressive cytokine TGF-βas compared to DCs differentiated under normoxic conditions,suggesting hypoxia inhibits the production of proinflammatory cytokines IL-1,IL-6 and TNF-α,but up-regulate the secretion of immune suppressive cytokine TGF-βby DCs.In addition,hypoxia can inhibit the expression of MMP9,indicating hypoxia may decrease the migration of DCs through down-regulating the expression of MMP9.4) Hypoxia inhibits the ability of DCs to stimulate CD4~+T cell proliferationTo study the effects of hypoxia on the ability of DCs to prime CD4~+T cells, normoxic or hypoxic DCs from C57BL/6 mice were used to stimulate CFSE-labeled CD4~+T cells from BALB/c mice,and CD4~+T cell proliferation was examined by Flow Cytometry.The results showed that LPS-treated hypoxia-differentiated DCs had poor ability to promote CD4~+T cell proliferation compared with LPS-treated normoxia-differentiated DCs(about 10%vs.about 40-50%),suggesting hypoxia inhibits functional maturation of DCs,especially the ability to mediate adaptive immune response.2.Effects of reoxygenation on the phenotype and function of hypoxia-differentiated DCs1) Reoxygenation promotes the phenotypic maturation of DCsTo determine whether oxygen delivery can influence the phenotypic maturation of hypoxic DCs,reoxygenation was performed on hypoxic DCs for various periods of time,related surface markers were examined by Flow Cytometry.The results showed that reoxygenation of hypoxic DCs obviously up-regulated the expression of CD80,CD86 and MHC classⅡmolecules on hypoxic DCs with increased time.Compared with normoxic mature DCs, these DCs expressed higher levels of CD80,CD86 and MHC classⅡmolecules after 6h of reoxygenation,and peaked after 24-48 h of reoxygenation,displaying a full mature phenotype.These results indicate that hypoxia-differentiated DCs possess fully or even more potential to become mature DCs when sufficient oxygen is available.2) Reoxygenation enhances the ability of hypoxic DCs to stimulate CD4~+T cell proliferationSince reoxygenation promoted the phenotypic maturation of hypoxic DCs, we further analyzed the effects of reoxygenated DCs on the proliferation of allogeneic or syngeneic CD4~+T cells.In the proliferation assay, mitomycin-treated DCs from C57BL/6 mice were co-cultured with CD4~+T cells from BALB/c(allogeneic) or C57BL/6 mice plus stimulation of CD3-specific antibody(syngeneic),respectively.DCs reoxygenated for 6 h stimulated vigorous CD4~+T cell proliferation,which was even stronger than normoxic mature DCs(for allogeneic CD4~+T cells,P＜0.01;for syngeneic CD4~+T cells,P＜0.05),suggesting reoxygenation markedly enhances the ability of hypoxic DCs to stimulate CD4~+T cell.3) Reoxygenated DCs induced Th1 cell differentiationWe then investigated CD4~+T cell differentiation driven by reoxygenated DCs in the MLR culture systems.Reoxygenated DCs induced significantly higher number of IFN-γ~+CD4~+T(Th1) cells than DCs differentiated under hypoxic or normoxic conditions did.The levels of IFN-γin the supernatants of MLR systems was also higher in reoxygenated DCs-mediated stimulation system than those in immature DCs(P＜0.05) and normoxia-differentiated mature DCs-mediated stimulation system.IL-4~+CD4~+T(Th2) cells induced by reoxygenated DCs were also elevated.These data indicate that reoxygenated DCs induce Th1 cell differentiation.Taken together,these results suggest that reoxygenated DCs have strong capacity to drive immune response toward a proinflammatory direction.3.Effects of CD137 pathway on phenotype and function of hypoxic DCs1) Anti-CD137 mAb partly promoted the phenotypic maturation of hypoxic DCsTo study the role of CD137 pathway on hypoxic DCs,CD137 expression on DCs differentiated under normoxic or hypoxic conditions were detected via Flow Cytometry.The results showed that CD137 expression was detected on both normoxic and hypoxic DCs,though hypoxia had a trend to down-regulate CD137 expression on DCs,no statistical difference was observed,indicating hypoxia has no obvious influence on the CD137 expression on DCs.To further explore the effects of CD137 on the phenotypic maturation of hypoxic DCs,anti-CD137 mAb was used to stimulate hypoxic DCs,result from Flow Cytometry showed that anti-CD137 mAb treatment markedly increased the expression of co-stimulatory molecules CD80,CD86,and MHC classⅡmolecules on hypoxic DCs,but the levels were still lower than that on normoxic mature DCs,suggesting anti-CD137 mAb may promote the switch of hypoxic DCs toward a more mature direction,but has a limited ability to promote the full phenotypic maturation of hypoxic DCs.2) Anti-CD137 mAb promoted the production of proinflammatory cytokines and MMP9 by hypoxic DCs To clarify the effects of CD137 pathway on the cytokine secretion and migration of hypoxic DCs,mRNA levels for IL-1β,IL-6,IL-12p40,TNF-αand MMP9 in hypoxic DCs stimulated with anti-CD137 mAb were detected by RT-PCR.The results showed that anti-CD137 mAb treatment increased mRNA levels for IL-1β,IL-6,IL-12p40,TNF-αand MMP9,suggesting anti-CD137 mAb may not only promotes the production of proinflammatory cytokines,but also increase the migration of hypoxic DCs through up-regulation of MMP9 expression.3) Effects ofanti-CD137 mAb on the ability of hypoxic DCs to stimulate CD4~+T cell proliferationSince anti-CD137 mAb partly promoted the phenotypic maturation of hypoxic DCs,we further studied the effects of anti-CD137 mAb on the ability of hypoxic DCs to stimulate CD4~+T cell proliferation.Hypoxic DCs from C57BL/6 were treated with anti-CD137 mAb,and then were used to stimulate CD4~+T cells from BALB/c mice,CD4~+T cell proliferation were assayed by Flow Cytometry,no obviously increased CD4~+T cell proliferation was observed when hypoxic DCs were stimulated with anti-CD137 mAb, suggesting anti-CD137 mAb has little influence on the ability of hypoxic DCs to stimulate CD4~+T cell proliferation.Conclusion1.Hypoxia inhibits the maturation and function of DCs induced by LPS1) Hypoxia inhibits the phenotypic maturation of DCs.2) Hypoxia inhibits the secretion of proinflammatory cytokines IL-1,IL-6 and TNF-α,but increases the production of immune suppressive cytokine TGF-βby DCs.3) Hypoxia impairs the ability of DCs to stimulate CD4~+T cell proliferation.2.Reoxygenation promotes the maturation and function of hypoxic DCs1) Reoxygenation promotes the phenotypic maturation of hypoxic DCs.2) Reoxygenation enhances the ability of DCs to stimulate CD4~+T cell proliferation.3) Reoxygenated DCs induced Th1 cell differentiation.3.CD137 pathway partly promotes the maturation and function of hypoxie DCs1) Anti-CD137 mAb partly promotes the phenotypic maturation of DCs.2) Anti-CD137 mAb promotes the secretion of proinflammatory cytokines IL-1, IL-6,IL-12 and TNF-αby DCs3) Anti-CD137 mAb has little influence on the ability of hypoxic DCs to stimulate CD4~+T cell proliferationOriginality1.Hypoxia can inhibit the maturation and function of DCsIt's the first time to provide evidence that hypoxia can inhibit the phenotypic maturation,proinflammatory cytokines secretion,and the ability to stimulate CD4~+T cell proliferation of DCs.It provides novel experimental basis for studying the effects of hypoxic microenvironments on immune function.2.Reoxygenation can promote the maturation and function of hyoxic DCsIt's the first time to provide evidence that reoxygenation can promote the phenotypic maturation and the ability to stimulate CD4~+T cells proliferation and Th1 cell differentiation of DCs,suggesting reoxygenation can reverse the inhibitory effects of hypoxic DCs.It is much helpful for the control of ischemia reperfusion injury,graft rejection in organ transplantation,and the immunotherapy for tumor.3.CD137 pathway partly promote the maturation and function of hypoxic DCsWe demonstrate that anti-CD137 mAb can partly promote the phenotypic maturation and proinflammatory cytokine of secretion of DCs.It's helpful to interpret the mechanisms of in vivo anti-tumor immunity mediated by anti-CD137 mAb.Limitation1.Reoxygenation promotes the maturation and function of hypoxic DCs,but detailed mechanism need to be further studied.2.Mechanism for the effect of anti-CD137 mAb on the maturation and function of hypoxic DCs need to be further explored. PARTⅡAnalysis of CD137 and CD137L Expression in Human Primary Tumor TissuesObjectiveCD137,a member of the tumor necrosis factor receptor family,is expressed primarily on activated T lymphocytes and natural killer cells.In contrast,its ligand CD 137L is mainly expressed on antigen-presenting cells,such as mature dendritic cells, activated B cells,and macrophages.The co-stimulation through CD137-CD137L enhances T cell activation,promotes the rejection of cardiac allografts and skin transplants,and eradicates experimentally induced tumors in mice.However,expression of human CD137 and CD137L is not restricted to immune cells,and the functions of human CD137-CD137L pathway are more complex than that of mice.Expression of CD137 protein has been verified on blood vessel walls in primary malignant tumors.Expression of CD137L has also been found on several human carcinoma cell lines and its function has been analyzed in vitro.Up to now, however,no studies have reported the expression of CD137L in human primary tumor tissues.Therefore,the aim of this study was to assess the expression of CD137 and CD137L in human primary tumor tissues and their potential role in tumor immunity.Methods1.Surgical specimens:A total of 63 tissue specimens were obtained from patients who underwent operations at Qilu Hospital,Shandong University,including 12 human normal tissues,15 benign tumors of epithelial or mesenchymal origin (adenoma and leiomyoma),and 36 malignant tumors of epithelial origin (squamous cell carcinoma and adenocarcinoma).2.Expression of CD137 and CD137L was assessed by immunohistochemistry in the above frozen sections.3.The expression of CD137L on 9 human tumor cell lines(3 hepatocarcinoma,2 lung carcinoma,2 colon carcinoma,1 lymphoma,and 1 leukemia) was detected by RT-PCR.4.To analyze the role of CD137L expressed on tumor cells,tumor cells expressing CD137L were co-cultured with activated T lymphocytes expressing CD137 or with Chinese hamster ovary cells expressing CD137 and then detected by ELISA the levels of cytokines(IL-8,IFN-γ) secreted by tumor cells or activated T cells.Results1.Human primary tumor tissues express CD137 and CD137L at different locationsThe expression of CD137 and CD137L was observed only in human benign (2/15,3/15) or malignant tumors(15/36,21/36),but not in normal tissues(0/12, 0/12).CD137 was expressed on the endothelial cells and smooth muscle cells of the vessel walls within tumor tissues,whereas CD137L was expressed on tumor cells.Though the samples were derived from many different tissue types,the expression of CD137 and CD137L was more common in malignant tumor tissues, especially in moderate or low-differentiated tumor tissues.2.Human tumor cell lines express high levels of CD137LOur results showed that tumor cells in human primary tumor tissues expressed CD137L.We also detected CD137L expression in human tumor cell lines derived from different primary tissues by RT-PCR.Normal un-stimulated peripheral blood mononuclear cells did not express CD137L mRNA but all investigated tumor cell lines(HepG2.2.15,BEL7402,HLE,HT29,L78,A2, U937,HL60,and H6) showed high levels of CD137L expression.Unexpectedly, non-tumor cell line,embryonic venous endotheliocytes ECV304,also expressed CD137L.These results indicated that CD137L was not only expressed in primary tumors but also in tumor cell lines and some non-tumor cell lines,suggesting CD137L expression may be related to rapid cell growth.3.CD137L expressed on tumor cells is functionalTo investigate the role of CD137L on tumor cells,we co-cultured tumor cells expressing CD137L with activated human T cells expressing CD137 in the presence of anti-CD3 mAb,and detected IFN-γsecretion by ELISA.The results showed that CD137-expressing T cells co-cultured with L78 cells produced higher levels of IFN-γthan CD137-1acking T cells(P＜0.001).After blocking CD137-CD137L pathway using anti-CD137L mAb,the levels of IFN-γsignificantly decreased(P＜0.001).This suggests that CD137L on L78 cells could conduct a co-stimulatory signal into T cells and this action could be specifically blocked by anti-CD 137L mAb.To confirm whether CD137L expressed on tumor cells could also signal back into tumor cells,different tumor cells were co-cultured with fixed CD137-CHO cells expressing CD137,and IL-8 was detected by ELISA.The results showed ligation of CD137L on tumor cells by CD137 expressed on CD137-CHO cells elevated the levels of IL-8 produced by HepG2.2.15 cells(P=0.038).Conclusion1.CD137 and CD137L are expressed in different human primary tumor tissues, but with different locations.CD137 was expressed on the endothelial cells and smooth muscle cells of the vessel walls within tumor tissues,whereas CD 137L was expressed on tumor cells.2.CD 137L expression found on tumor cell lines was functional because the ligation of CD137L on lung squamous carcinoma cells L78 with CD137 on T cells induced IFN-γproduction by T cells,and ligation of CD137L on hepatocarcinoma cells HepG2.2.15 with CD137 triggered tumor cells to produce IL-8,Suggesting the expression of CD137 and CD137L in tumor tissues may influence the progression of tumors.OriginalityIt is the first time to prove that CD137 and CD137L can be expressed by different human primary tumor tissues,and the expression of CD137L on tumor cells is functional,suggesting the expression of CD137 and CD137L in tumor tissues may influence the progression of tumors.LimitationMore cases need to be expanded for further study,and specimens derived from the same tissues should be collected for the CD 137 and CD 137L expression assay.