Regulation Of Chemokines Expression And Function In Tumor Microenvironment

IntroductionChemokines are structurally related,small(8-14 kDa)polypeptide signaling molecules that bind to the chemokine receptors,a family of seven transmembrane G protein-coupled receptors.The chemokines and their respective receptors are divided into the CXC,CC,C,and CX₃C families,based upon the positions of their conserved two N-terminal cys residues.The chemokines genes are clustered on genomic loci, including chromosome 4q12-q13(CXC acting mainly on neutrophils),4q21,and 17qll.2(CC chemokines acting mainly on monocytes).The chemokine receptors gene clusters exist on chromosomes 2 and 3.Many chemokines share common receptors and bind to multiple receptors.Chemokines were originally characterized by their ability to induce chemotaxis of leukocytes.They have since been shown to act on multiple cell types,including T lymphocytes.Chemokines lead to the directed migration of leukocytes especially T lymphocytes to the inflammation and tumor sites.Chapter one Oncogenic signaling pathways promoting tumor growth and invasion also affect antitumor immune responses including immune cell recruitment. Constitutive activation of Notch signaling was found in leukaemia,breast cancer, lung cancer,mucoepidermoid tumors,Kaposi's sarcoma,neuroblastoma,pancreas cancer,cervical cancers and melanoma,providing a basis to explore Notch signaling as a therapeutic target in cancers.However,the chemokine expression profile affected by Notch signaling activation has not been investigated.Since the Notch signaling pathway conveys the ligands-binding signal from the cell surface to the transcriptional machinery through multiple proteolytic cleavage and activation signaling,pharmacological inhibitors of gamma-secretase and small siRNA specifically taregtting Notch transcriptional signaling activities including the co-activitor mastermind-like proteins(MAMLs).We investigated broad gene expressions affected by specific small siMAML1 transfection.We found a significant upregulation of Tweak receptor(TweakR,Fn14) and several chemokines including CCL2 after siMAML1 transfection in melanoma cells.Chapter two As a transmembrane chemokine,CXCL16 has been detected in various tissues and organs under normal and pathological conditions,also plays an important role in macrophages/DC and T cell interactions and trafficking during inflammation and immune responses.The membrane bound CXCL16 also mediates the firm adhesion of CXCR6-expressing T cells.CXCL16 is not exclusively expressed on the cell surface.It is also found as soluble molecule that is constitutively generated by proteolytic cleavage of its transmembrane variant,a process called shedding.LPA,a bioactive lipid mediator has been indicated to regulate DC and epithelial functions during wound healing and inflammation responses.However,the direct link of CXCL16 expression with LPA has not been established.Using monocyte-derived macrophages/DC(MoDC),we investigated how LPA regulate CXCL16 production and shedding.Chapter three Chemokines secreted by DC are instrumental for DC to regulate their own migratory capacities and to recruit T lymphocytes during local tumor immune response.DC are the major producers of many chemokines including CCL2,CCL3, CCL4,CCL22.CCL2 may contribute to the initial migrating of monocyte derived DC(MoDC) into the inflammation and tumor sites,thereafter,chemokines secreted by MoDC themselves may influence DC further accumulation and T lymphocytes infiltrating.Recent experiments in mice showed that immature MoDC recruited into tumors weakly expressing MHC classâ…¡and not expressing activating costimulatory molecules B7,they were unable to activate the immune response,meanwhile these immature MoDC might recruit regulatory T cells(Tregs) into tumor sites and function as tolerogenic rather than immunogenic signals.Therefore,carefully and systemically monitoring MoDC chemokines expression profiles during their differentiation and maturation process will provide insight and overall guidance for DC vaccine development to improve the effectiveness of immunotherapy.Blood monocytes continuously repopulate DC populations to maintain homeostasis.We generated MoDC in vitro and monitored chemokines expression profiles during their differentiation and maturation.We found MoDC largely inherit a broad range of chemokines(CCL27,CXCL16,CXCL5,CCL24,CXCL3,CXCL8,CCL2,CCL4, CXCL7 and CCL18)from monocytes.Meanwhile,there was significant induction of CCL17,CCL22 and CCL23.Objective:To investigate the mechanisms which regulate the broad chemokines expression and functions.First,using siRNA targeting Notch signaling co-activator MAML1 to induce the functional CCL2 production regulated through the upregulation of TweakR in melanoma cells,this results in tumor cells to attract more innate immune cells into tumor microenvironment.Second,exploring the mechanisms of serum-borne LPA upregulating CXCL16 expression and shedding,this increased production of CXCL16 stimulated by LP A in macrophages/MoDC may involve multiple signaling transduction pathways,which would affect the chemotactic activity of macrophages/MoDC.Third,migration assays showed that spent culture supernatant from MoDC were chemotactic to activated T subtypes.Since the transcriptional regulation of CCL22 and CCL17 are similar to T helper chemokines like CCL3,CCL4,CCL5 and CXCL10 in several cell types,blocking antibodies or siRNA specifically targetting CCL17 and CCL22 would alter MoDC chemokines expression profiles as well as their abilities to recruit activated T subsets while reduce Tregs migration and infiltrating in vitro and in vivo,tipping the immune response toward the cytotoxic arm in tumor microenvironment and during DC vaccination.Methods and Results:Chapter one Induction of CCL2 by siMAMLl through upregulation of TweakR in melanoma cells1.SiMAML1 increased TweakR and CCL2 gene expression and protein production.Melanoma cell lines M624 were transfected with siMAMLl,along with control siRNA for 72 h,multiple genes expression analyzed by RT-PCR showed significant upregulation of TweakR and CCL2,with completely knocking down of siMAML1 and Hey1.TweakR ligand Tweak and CCL2 receptor CCR2 mRNA were both expressed at very low to no level and may not be significant in this cell system. MAML1 and Hey1 proteins were significantly decreased in siMAML1 transfected cells.The direct repressions of Hey1 on TweakR and CCL2 mRNA were verified using siHey1.Cell surface TweakR affected by siMAML1 and siHey1 were confirmed by FACS.CCL2 protein secretion regulated by siMAML1 and siHey1 were confirmed by ELISA.2.Exogenous Tweak upregualted CCL2 production.We thus examined the effect of recombinant Tweak on CCL2 production by M624 cells.Exogenous Tweak induced CCL2 production in a dose-and time-dependent manners,which was almost completely inhibited by blocking the Tweak/TweakR interaction with anti-TweakR antibody.3.SiMAML1 increased CCL2 dependent migration of monocytes.To study if CCL2 produced by M624 are functional we collected culture supernatant from siMAML1 transfected cells stimulated with exogenous Tweak,put it into the bottom wells of transwell system with monocytes in the top wells.We found that supernatant from siMAML1 transfected cells were much more chemoattractive to monocytes,and this chemoattraction increased with increasing doses of exogenous Tweak. Neutralizing CCL2 significantly blocked Tweak induced monocyte migration in siMAMLl transfected cells.Chapter two LPA enhances CXCL16 production stimulated by LPS from macrophages and regulates T cell migration1.Macrophages and MoDC express LPA receptors.Macrophages and MoDC were derived from CD14⁺monocytes from normal donor PBMC without age or gender discrimination.FACS confirmed the cell surface expression of LPA1 and LPA2,but not LPA3 on macrophages/MoDC,as well as their precursors CD14⁺monocytes. Similar results were observed when analyzing mRNA expression of LPA receptors by qualitative RT-PCR,where expression of both LPA1 and LPA2 mRNA were readily detectable.2.Macrophages and MoDC express and secrete CXCL16.FACS confirmed macrophages and MoDC expressed CXCL16 on the cell surface.A few monocytes expressed CXCL16 on their cell surface,almost all macrophages acquired CXCL16 after differentiation,MoDC expressed CXCL16 with the highest intensity on cell surface.ELISA showed both macrophages and MoDC secreted soluble CXCL16, while macrophages released more CXCL16 into their spent culture supernatants.3.LPA stimulates CXCL16 production from macrophages.To examine the effect of LPA on CXCL16 expression,we cultured macrophages,stimulated them with LPS to trigger chemokine release.We observed that LPA at 10μM significantly increased the production and release of CXCL16 from macrophages by more than 2 fold detected by ELISA.LPA did not affect CXCL16 cell surface presentation shown by FACS.Quantitative real time PCR showed LPA upregulated CXCL16 mRNA expressions in macrophages also.Control macrophages had basal CXCL16 mRNA (detectable at cycle 35),LPA increased CXCL16 mRNA(detectable at cycle 32),LPS induced CXCL16 mRNA(detectable at cycle 28),however,LPA and LPS synergized in CXCL16 mRNA transcription(detectable at cycle 22).By calculating the cycle threshold(CT)values of in each sample and ACT,we found that LPA enhanced CXCL16 mRNA transcription by 64 fold.4.Signaling pathways regulating CXCL16 production enhanced by LPA.By binding to specific LPA receptors,LPA activates complex cell signaling including PTx sensitive and-insensitive G proteins depending on cell types,triggering tyrosine phosphorylation and regulating rho-dependent actin reorganization.PTx,a specific inhibitor of G_i/oproteins,which has been shown to inhibit G_i/o-dependent LPA effects in different cell systems.Pretreatment of macrophages with 10 ng/ml of PTx for 16 hr before LPA suppressed CXCL16 protein production stimulated by LPS.In addition, pretreatment with 1μg/ml of exoC3,a specific inhibitor of Rho and 200μM of PDTC,an inhibitor of the NFκB-dependent pathway significantly decreased LPA enhanced CXCL16 protein production from macrophages,suggesting that the enhancement of LPA on CXCL16 production are G_i/o,Rho,and NFκB dependent. Similar results were also observed at the RNA level as detected by RT-PCR. y0,5.LPA enhances chemotactic activity of macrophages.Macrophages play an important role in immune cell trafficking into inflammation and tumor sites.To investigate CXCL16 production stimulated by LPA would functionally attract more T lymphocyte migration,chemotactic activity of spent culture supernatant collected from LPA/LPS treated macrophages was tested in trans-well system.Results showed that spent culture supernatant from LPA/LPS stimulated macrophages significantly increased CD3⁺T cells migration,and pre-incubation the spent culture supernatant with 10μg/ml blocking antibody against human CXCL16 significantly blocked CD3⁺cells migration.On the contrary,normal mouse IgG(10μg/ml)had no effects on CD3⁺cells migration,implying the enhanced chemotactic activity of macrophages stimulated by LPA was mediated through the increased production of soluble CXCL16.Consistent with the ELISA results of CXCL16 production regulated by LPA through several signaling transduction pathways above,the chemotactic activity of macrophages was also mediated through a G_i/o-,Rho-,and NF-κB-dependent pathways,since CD3⁺cells migration toward to spent culture supernatant collected from macrophages were blocked by pretreatment macrophages with PTx,exoC3,or PDTC.LPA and LPS alone had no significant enhancement on CD3⁺cells migration.Chapter three Specifically knocking down of CCL22 and CCL17 expression by siRNA during DC differentiation and maturation affect T subsets recruitment in vitro and in vivo1.Chemokine expression of MoDC.MoDC generated from monocytes exhibited typical DC morphologies acquired CD11c and lost CD14 antigen,these MoDC also expressed high intensity of CD83,CD86,CD40 and HLA-DR after maturation with LPS.MoDC or fresh purified monocytes were stimulated with LPS and spent culture supernatant were collected and a sensitive chemokine protein arrays were performed. The resulting spots that corresponded to each chemokine were analyzed by densitometry and compared to assay-specific positive controls and relative expression of each chemokine was analyzed.Monocytes constitutively express CCL27,CXCL16, CXCL5,CCL24,CXCL3,CXCL8,CCL2,CCL4,CXCL7,CCL18,and low levels of CXCL1 and CCL3.MoDC continue to express the above chemokines with significant upregulation of CCL17,CCL22 and CCL23 by quantified by ELISA.Real time PCR confirmed the upregulations of CCL17,CCL22 and CL23 were reflected at RNA levels compared with the constitutively expressed CCL2.2.Chemotaxis of MoDC to activated T cells.Phenotypes of naive and activated CD3⁺T cells were confirmed by FACS.CD4⁺to CD8⁺ratios were about 2 in these healthy donors tested.Spent culture supernatant from monocytes and MoDC were placed in the bottom wells of transwell and CD3⁺T cells were placed on the top wells. Spent culture supernatant from monocytes had basal chemotatic abilities to both naive and activated CD3⁺T cells compared to media control.Meanwhile,spent culture supernatant from MoDC exhibited significantly higher chemotatic abilities to activated CD4⁺and CD8⁺cells.There is no significant difference between the chemotactic recruitment of na(?)ve T cells between spent culture supernatant from MoDC and monocytes.Spent culture supernatant from monocytes had equal chemotatic abilities to both CD4⁺and CD8⁺T cells.However,spent culture supernatant from MoDC attracted significantly more activated CD4⁺than CD8⁺, meanwhile,fewer na(?)ve CD4⁺than CD8⁺T cells.There was no significant difference in the ratios of CD4⁺to CD8⁺in na(?)ve and activated CD3⁺T cells before they were loaded into the transwells.To investigate the expressions of chemokine receptors on CD3⁺T cells which would bind to those significantly upregulated chemokines(CCL17,CCL22,CCL23)during DC differentiation and maturations,CCR1(receptor for CCL23),CCR4(receptor for CCL17 and CCL22),and CCR8(receptor for CCL17)in comparison with CCR2 (receptor for CCL2)and CXCR6(receptor for CXCL16)expressions were analyzed on activated CD4⁺and CD8⁺cells.Result showed that higher levels of CCR4 and CCR8 expression on CD4⁺cells.CD8⁺cells mainly expressed CCR2 and CXCR6. These observations suggest that more activated CD4 T cells may migrate toward CCL17 and CCL22 gradients depend on their CCR4 and CCR8 expression,while CD8⁺T cells may travel according to CCL2 and CXCL16 gradients from MoDC during DC differentiation and maturation.3.MoDC are highly chemptactic to Tregs Activated CD3⁺T cells transwell assays were set up as above and activated CD4⁺cells migrated were stained for CD25 and Foxp3.Compared to spent culture supernatant from monocytes,spent culture supernatant from MoDC attracted significantly higher numbers of CD25⁺Foxp3⁺CD4⁺Tregs.This similar chemotactic ability was seen by MoDC to recruit natural Tregs.4.Blocking CCL22 and CCL17 can regulate the ratios of CD4⁺to CD8⁺as well as frequency of Tregs recruited by MoDC Spent culture supernatant from MoDC was pretreated with anti-CCL2,anti-CCL17,anti-CCL22,anti-CCL23,or isotype control,then were placed in the bottom of transwell and CD3⁺T cells were placed on the top wells.CD4⁺cells migrated to the bottom wells were analyzed.Blocking CCL2 had very little effect on the ratio of CD4⁺to CD8⁺and the frequency of Tregs migrated.While blocking CCL17 and CCL22 significantly decreased the ratio of CD4⁺to CD8⁺and the frequency of Tregs migrated.Surprisingly,blocking CCL23 increased the ratio of CD4⁺to CD8⁺and the frequency of Tregs migrated.5.Specifically knock-down CCL22 and CCL17 expression by siRNA can regulate the ratios of CD4⁺to CD8⁺as well as the frequency of Tregs recruited by MoDC We applied siRNA specific for CCL17 and CCL22 and evaluated the effect of silencing gene expression of CCL17 and CCL22 post-transcriptionally.Compare to the non-specific siRNA control with over 90%transfection efficiency,siRNA specifically knocked down either CCL22(＞80%)or CCL17(＞70%)at the mRNA levels during MoDC differentiation and maturation.Chemokine protein array showed that either CCL22 or CCL17 expression was significantly decreased after separate transfections with siRNA for CCL22 or CCL17,which were confirmed by ELISA. Spent culture supernatant from MoDC with specifically knocking down either CCL17 or CCL22 attracted fewer CD4⁺while more CD8⁺T cells and significantly lower numbers of Tregs.6.Intratumoral injections of MoDC transfected with siCCL22 and siCCL17 recruited fewer Tregs but more CD8⁺T cells When human breast cancers were established in athymic nude mice,control MoDC or MoDC transfected with siRNA for CCL22 and CCL17 during the last 2 days of differentiation and maturation were injected into the tumors followed by mixture of sorted Tregs and activated CD8⁺T cells.Tumors were dissected and digested into single cell suspensions,infiltrating lymphocytes were analyzed by FACS.Compared to the control MoDC,MoDC developed with siCCL22 and siCCL17 transfections significantly recruited more CD8⁺T cells(10.5%vs 3.56%)and fewer Tregs(0.24%vs 4.59%).In these siRNA transfected MoDC,CCL22 and CCL17 were almost completely suppressed for 5-7 days analyzed by real time RT-PCR and ELISA of sorted human intratumor CD11⁺ cells.IHC performed on these tumor samples confirmed the localization CD11c⁺ MoDC in tumors and the reversed colocalizations of CD8⁺T cells and Tregs.Conclusion:Chapter one Our study found the transcription and activation of transcriptional repressor Hey1,resulted from the constitutive activation of Notch signaling might actively repress TweakR protein and cell surface expression which suppressed CCL2 expression and secretion,therefore might reduce immune cells normal recruitment and immune surveillance in melanoma tumor microenvironment,indicating Notch as one of the molecular mechanisms that mediate tumor escape from natural immune surveillance.Unlike gamma-secretase inhibitors,siMAML1 specifically block Notch transcriptional signaling,amplified Tweak-TweakR interaction to induce CCL2 production in melanoma cells,therefore increas the innate immune cells infiltration. Therefore,like small-molecule drugs,siMAML1 targeting Notch signaling should be explored as a therapeutic strategy for cancer immunotherapy.Chapter two LPA has been indicated to promote early T cell migration to tissue sites of immune responses and regulate T cell proliferation and secretion of numerous cytokines.LPA has also been reported to regulate the differentiation and activation of mononcytes/macrophages and their further interactions with endothelial cells.Here, we demonstrated that LPA increases CXCL16 production and shedding stimulated by LPS in addition to other inflammatory cytokines,therefore directing more activated T cells to the inflammation and tumor sites to develop functional immune responses.LPA enhanced CXCL16 production stimulated by LPS through activation of NF-κB,G_i/o-and Rho-signaling pathways.Since most of CXCL16 binding CXCR6 expressing cells in blood are IFN-γsecreting helper T cells,cytotoxic T cells,or CD56⁺T cells that are enriched for effector functions,thus upregulation of CXCL16 by LPA in macrophages and MoDC most likely leads to the(T helper 1)Th1 and cytotoxic effector function in inflammation and tumor sites.We hope this mechanic link between LPA and chemokines would advance our knowledge in lipid mediated immune responses.Chapter three As professional antigen presenting cells,DC stimulate T cell-mediated adaptive immune response,which largely rely on DCs maturation and activation status.Chemokines secreted by DC are instrumental to recruit T cells during local inflammation and tumor immune response.Tregs play a central role for the maintenance of peripheral tolerance by active suppression of T effector functions and for the immunosuppressive role in tumor microenvironment.Our results showed that MoDC at different differentiation stages from monocytes produce relative different levels of immune stimulating and suppressive chemokines.MoDC produce both T effector chemotatic chemokines(high CCL2,CCL4,CXCL16)and Tregs chemotatic chemokines(high CCL22,CCL17),therefore they might recruit both Tregs and CD8⁺T effectors.By regulating the relative expression levels of CCL22 and CCL17 using siRNA might significantly reduce the frequency of Tregs while selectively increase CD8⁺T effectors migrated into tumor site to perform cytotolytic activities during DC priming and DC vaccination.In general,using siRNA or LPA to regulate multiple chemokines(CCL2,CXCL16, CCL22/CCL17,et al.)from different cell types in tumor microenvironment,may successfully increase both inate and adaptive T immune cells recruitment to local inflammation or tumor microenvironment,therefore enhance local immune responses. This study shall lead to a new strategy to improve the effectiveness of tumor immunotherapy.