The Immune Regulatory Role Of IL-17in Hepatocellular Carcinoma

Part I The role of IL-17in hepatocellular carcinoma developmentObjective: To investigate the role of IL-17in hepatocellular carcinoma (HCC)development.Methods: Three murine HCC models (subcutaneous HCC model, Orthotopic HCCmodel and DEN induced HCC model) were established. The HCC development wasmonitored by using IL-17-/-mice in three HCC models. CD8depletion antibody wasused to deplete the CD8+T cells in tumor bearing mice. RT-PCR and flow cytometrywere used to examine the IL-17R expression on Hepa1-6cells. MTT and Annexin-V/PImethods were used to evaluate the proliferation and apoptosis of tumor cells Hepa1-6.FACS analysis was used to examine the lymphocytes profiles (T cells subsets, memoryT cells, effector T cells, NK, NKT, Treg, DCs, M, neutrophils and MDSCs) of TILs intumor bearing mice. Intracellular staining were performed to examine the IFN-γexpression by CD4+T and CD8+T cells. Nonradioactive cytotoxicity assay wasperformed to evaluate the cytotoxic T cell activity. The serum levels of IFN-γ, IL-2andTNF-were measured by CBA assay. The proliferation and cytokines production ofCD8+T cells was measured by CFSE and flow cytometry.Results:(1) Three murine HCC models were successfully established.(2) Thetumor growth was markedly reduced in IL-17-/-mice compared with WT mice, whileadministration of rmIL-17intraperitoneally promoted tumor growth in subcutaneousHCC model. On days14after the implantation, IL-17-/-mice exhibited decreased size oftumor nodules when compared to WT mice, and WT mice treated with rmIL-17A exhibited increased tumor growth compared to PBS control in orthotopic implantationmodel. In DEN-induced HCC model, the maximum size of tumors and the number oftumor nodules were significantly decreased in IL-17-/-mice compared with WT mice.Treatment of WT mice with rmIL-17A enhanced the tumor growth compared with notreatment control.(3) Hepa1-6cells express low levels of IL-17R at both protein andmRNA levels. However, IL-17had no direct effect on tumor cell viability and apoptosis.(4) Compared with WT mice and mice treated with rmIL-17, the infiltration of CD8+Tcells was significantly increased in IL-17-/-mice. The CD8+T cells with memoryphenotypes also increased in IL-17-/-mice. Furthermore, the percentages oftumor-infiltrating IFN-γ+CD8+T cells were significantly higher in IL-17-/-mice thanWT and rmIL-17treated mice assessed by intracellular cytokine staining. However, thepercentages of CD4+T cells, Treg cells and IFN-γ+CD4+T cells showed no significantdifference among the three groups. The serum levels of IFN-γ, IL-2and TNF-werealso elevated in IL-17A-/-mice comparing to the WT mice and were further reduced bythe rmIL-17treatment. In a killing assay using Hepa1-6cells as targets, splenocytes andenriched CD8+T cells from tumor bearing IL-17-/-mice showed enhanced killingactivity, compared with those from WT mice. Tumor growth was significantly increasedto the similar levels by CD8+T-cell depletion in both WT and IL-17-/-mice.(5) Thedifferent concentrations of IL-17had no effect on either cytokine productions orproliferations of CD8+T cells.(6) the percentages of dendritic cells, macrophages, andneutrophils were not significantly changed in IL-17-/-mice. However, the percentages ofboth monocytic and granulocytic MDSCs were apparently reduced in IL-17-/-mice.Moreover, the administration of rmIL-17increased the tumor infiltrations of theMDSCs.Conclusion: IL-17promotes the development of HCC in murine models. IL-17promoted HCC development not by directly affecting tumor cell growth, but throughmodulating the anti-tumor immune responses, and possibly through promoting therecruitment of MDSCs. Part II The source of IL-17in the tumor microenvironmentObjective: To investigate the source of IL-17in the tumor microenvironment ofHCC.Methods: Orthotopic HCC model was used in this part of study. The IL-17production by different cell populations infiltrated in the tumor was screened byintracellular staining. Specific antibodies were used for phenotypic identification. TheVγ repertoire of the γδ T cells in the tumor infiltrating lymphocytes was examined byRT-PCR. The HCC growth was compared in TCRδ-/-and WT mice, and the IL-17levelin serum was measured by ELISA. in vivo Vγ4γδ T cells depletion and adoptivetransfer of WT or IL-17-/-Vγ4γδ T cells into TCRδ-/-mice were performed todemonstrate the critical role of IL-17producing Vγ4γδ T cells in tumor development.FACS analysis was used to examine the lymphocyte profiles (T cells subsets, memory Tcells, effector T cells, NK, NKT, Treg, DCs, M, neutrophils and MDSCs) of TILs intumor bearing mice. Intracellular stainings were performed to examine the IFN-γexpressions by CD4+T and CD8+T cells.Results:(1) CD3+T cells are the main source of IL-17，CD4+T cells, CD8+T cellsand γδT can all produce IL-17. However, γδ T cells exhibited the highest percentage ofIL-17+cells. Of all the IL-17producing cells, about60%were γδ T cells, while only20%were Th17cells, and the absolute numbers of γδ T cells were about4-fold higherthan that of Th17cells. Further analysis showed that the IL-17producing γδ T cellsexpressed RORγt, CCR6but did not express CD27.(2) The tumor infiltrating γδ T cellsexpressed all Vγ1, Vγ2, Vγ4, Vγ5, Vγ6and Vγ7genes.75%of IL-17-producing γδ Tcells were Vγ4γδ T cells.(3) TCRδ-/-mice exhibited reduced tumor growth comparingto the WT mice. The serum IL-17levels were also significantly decreased in TCRδ-/-tumor bearing mice. The percent of memory CD8+T cells and IFN-γ production by theCD8+T cells were also significantly increased in TCRδ-/-mice.(4) Depletion of Vγ4γδT cells resulted in significant reduction in tumor volumes in comparison with WT micetreated with the control Ab. Interestingly, Vγ4γδ T cell depletion in IL-17-/-mice didnot further suppress the tumor growth. FACS analysis showed that IL-17+γδ T cellswere significantly reduced in Vγ4γδ T cell depleted mice, while we observed similarlevels of Th17cells in both Vγ4γδ T cell depleted mice and WT mice. The infiltrationsof effector and memory CD8+T cells in tumors were significantly increased in Vγ4γδ T cell depleted mice. Moreover, the IFN-γ producing CD8+T cells were also significantlyincreased in Vγ4γδ T cells depleted mice compared with the mice treated with thecontrol Ab.(5) Transferring WT Vγ4γδ T cells in TCRδ-/-mice exhibited similar tumorgrowth as the WT mice. However, transferring IL-17-/-Vγ4γδ T cells showedsignificantly reduced tumor growth. Flow cytometry analysis showed increased memoryCD8+T cell infiltration in IL-17-/-Vγ4γδ T cell reconstituted mice, the percent of Tc1cells significantly increased in IL-17-/-Vγ4γδ T cell reconstituted mice comparing withthe WT mice.Conclusion: Vγ4γδ T cells not Th17cells are the main source of IL-17duringHCC development. Vγ4γδ T cells promote tumor growth through IL-17A production. Part III The mechanism of IL-17in promoting the development ofhepatocellular carcinomaObjective: To investigate the regulatory mechanisms of IL-17in promoting thedevelopment of HCC.Methods: RT-PCR and flow cytometry were used to examine the IL-17Rexpression on MDSCs. Transwell assay was performed to examine the role of IL-17onMDSCs migrations. The expression of CXCR2and CXCR4on MDSCs were examinedby flow cytometry. The expressions of CCL2, CCL5, CCL7, CCL20, CXCL12andCXCL5by Hepa1-6were measured by Realtime PCR. Transwell assay was performedto examine the effect of IL-17treated tumor supernatants on MDSCs migrations. Tumorbearing mice were treated with rmIL-17combined with the antagonist of CXCR2(SB265610) or CXCR4(AMD3100) to further examine the role of CXCL5/CXCR2interaction in IL-17promoted MDSCs migration and tumor growth. MDSCs and OT-ICD8+T cells were cocultured with SIINFEKL stimulation in the presence of rmIL-17oranti-IL-17. CFSE and3H-TdR assay were performed to evaluate the effect of IL-17onthe suppressive functions of MDSCs. To determine whether MDSCs were essential forIL-17A mediated immune suppression in vivo, mice were treated with rmIL-17A followed by anti-Gr-1antibody (clone RB6-8C5) or gemcitabine to deplete MDSCs. Toexamine the role of MDSCs in IL-17producing γδ T cell differentiation, sorted γδ Tcells were cocultured with or without MDSCs, respectively, in some experiments,anti-IL-1β, anti-IL-23antibodies or rmIL-17and anti-IL-17were added.Results:(1) MDSCs showed the highest IL-17R expression and almost all theMDSCs expressed IL-17R with expressions of IL-17RA and IL-17RC at thetranscriptional level. However, IL-17has no direct effect on MDSCs migrations.(2)Tumor infiltrating MDSCs expressed both CXCR2and CXCR4. IL-17treated tumorcells up-regulated a group of chemokine expressions in a dose dependent manner,especially CXCL5and CXCL12, but in the tumor microenvironment only CXCL5waselevated in WT tumors compared with IL-17A-/-tumors. IL-17treated tumorsupernatants induce the migration of MDSCs significantly in a dose dependent manner.The increase of MDSCs migration was completely blocked when neutralizinganti-IL-17antibody or the antagonist of CXCR2(SB265610) was added during theIL-17A treatment.(3) Mice treated with CXCR2antagonist together with rmIL-17showed a significant reduction in tumor growth comparing with rmIL-17treatmentalone. CXCR2antagonist treatment decreased the MDSCs at the tumor sites andincreased the percentages of memory and IFN-γ producing CD8+T cells.(4) IL-17Asignificantly enhanced the suppressive function of MDSCs in terms of inhibiting T cellproliferation. Different concentrations of IL-17A also promoted the suppressive effectsof MDSCs in terms of inhibiting IFN-γ and TNF-productions by the CD8+T cells in adose dependent manner. Furthermore, the suppressive function was mainly mediated bythe monocytic MDSCs and IL-17treatment could further enhance it.(5) Depletion ofMDSCs by Gr-1antibody or gemcitabine in the rmIL-17A treated mice led to thereduced tumor growth in comparison with rmIL-17A and control mAb treated mice. Theinfiltration of MDSCs at the tumor sites was significantly reduced. Memory and IFN-γproducing CD8+T cells were also increased after MDSCs depletion.(6) The IL-17producing cells, both Th17cells and γδT cells were also decreased in the absence ofMDSCs.(7) Although MDSCs significantly promoted the IL-17production by γδ T IL-1β and IL-23induction. This induction of IL-17production by γδ T cells by MDSCscould be partially blocked by anti-IL-1β or anti-IL-23antibodies. The combinedanti-IL-1β and anti-IL-23antibody treatment further reduced the IL-17production by γδT cells. However, granulocytic MDSCs could induce slightly higher percentage ofIL-17-producing γδT cells and higher level of IL-17production.(8) IL-17couldsignificantly increase the percent of IL-17producing γδ T cells in a dose-dependentmanner. Adding neutralizing anti-IL-17antibody could block this increase of induction.The IL-1β and IL-23expressions of MDSC cells were significantly elevated withrmIL-17treatment.Conclusion: IL-17promoted MDSCs infiltration to the tumor sites in a CXCR2dependent manner through the up-regulation of CXCL5production by the tumor cells.IL-17A impaired the anti-tumor immune responses through promoting the migrationsand suppressive activities of the MDSCs at the tumor sites. IL-17could also increaseIL-1β and IL-23productions by the MDSCs to further promote the IL-17producing γδT cell differentiation. The interactions among γδ T cells, MDSCs and tumor cells form apositive feed-back loop through IL-17, IL-1β and IL-23as well as CXCL5in the tumormicroenvironment to suppress the anti-tumor immune responses and promote tumorgrowth.