Kupffer Cell Suppression Of CD8~+ T Cells In Human Hepatocellular Carcinoma Is Mediated By B7-H1/PD-1 Interactions

Partâ… .B7-H1 expression on antigen presenting cell subsetsin human HCC[Objective] Characterization of immunosuppressive phenotype in human HCC.[Methods] Cells were obtained from fresh HCC tumor or surrounding cirrhotic livertissues.The tissues were finely minced and further digested with 50 mL of HBSS(Life Technologies) containing 40 mg of collagenase,4 mg of DNaseâ… ,and 100 unitsof hyaluronidase (Sigma) for 2 h at room temperature.Tumor infiltrating leukocytes(TILs) or non-tumor infiltrating leukocytes (NILs) was isolated by Ficoll gradientcentrifugation and analyzed by fluorescence activated cell sorting (FACS).[Results] Significant quantities of APC subsets were noted in tumor infiltratingleukoctyes (TILs) as compared to surrounding hepatic parenchyma (non-tumorinfiltrating leukocytes,NILs).The proportion of HLA-DR⁺CD4⁺CD123⁺plasmacytoid DC (pDC) (1%±0.5%),HLA-DR⁺CD4⁺CD11c⁺ myeloid DC (mDC)(5%±2%),or HLA-DR⁺CD14⁺ Kupffer cells (KC) (20%±5%) in CD45⁺HLA-DR⁺leukocytes was not different between TILs and NILs.KC was the predominantpopulation which express B7-H1 and the percentage of KC in TILs expressing B7-H1was increased compared to NILs (19%±1.21% and 8%±3.37%,p＜0.05) in 15 HCCpatients.Using fluorescence microscopy,we confirmed that KC (n = 8) expressed B7-H1.Additionally,no significant cell surface B7-Hl levels were noted on pDC,mDC,or even HCC tumor cells.[Conclusion] The data indicate that KCs expressed high levels of B7-H1 in HCC. Partâ…¡.Tumor associated TNF-αand IL-10 induced B7-H1expression on KCs[Objective] Examination of the potential factors in the HCC microenvironmentcontributing to KC B7-H 1 up-regulation.[Methods] PBMC were obtained from fresh health donot blood then sorted for CD 14⁺cells.Hep G2 cells were obtained from American Type Culture Collection (Manassas,VA).The cells were grown in Dulbecco's modified complete Eagle's medium(DMEM).Six-well transwell chambers with a 0.4 um porous membrane (Corning-Costar) were used.Briefly,Hep G2 cells (2×10⁶/ml),recombinant human cytokines orantibodies as indicated were added in the upper chamber,and CD14⁺ cells(0.5×10⁶/ml) from blood of healthy donors) were plated to the lower chambers.After48 hours,CD14⁺ cells were harvested and B7-H1 expression was determined byFACS.Supernatants from transwell experiments and cultured cells were checked byhuman TNF-αand IL-10 ELISA kit (R&D systems,Minneapolis,MN)..[Results] Monocyte B7-H1 expression was significantly increased in the co-culturewith Hep G2 cells.High levels of TNF-αand IL-10 were detected in the co-cultures.We further explored the role of TNF-αand IL-10 in inducing monocyte B7-H1expression.Recombinant TNF-αand IL-10 moderately induced B7-H1 expression onmonocytes.Neutralizing antibody against TNF-αand IL-10 completely blockedmonocyte B7-H1 expression induced in the CD14⁺/Hep G2 co-culture.These resultssuggest that tumor associated TNF-αand IL-10 may contribute to increased KC B7-H1 expression in the HCC microenvironment.[Conclusion] To mimic the HCC microenvironment,monocytes were co-culturedwith Hep G2 cells in a transwell assay.High levels of TNF-αand IL-10 were found inthis co-culture system and contributed to B7-H1 up-regulation.Therefore,tumorconditioned KCs,are the likely mediator of B 7-H 1/PD- 1 interactions. Partâ…¢.Functional characteristics of tumor associated PD-1⁺CD8⁺ T cells in human HCC[Objective] Analysis of the functional characteristics of tumor associated PD-1⁺CD8⁺T cells in human HCC[Methods] Cells were obtained from fresh HCC tumor or surrounding cirrhotic livertissues and analyzed by FACS.PD-1 is the co-inhibitory signal receptor for B7-H1and CD8⁺ cytotoxic T cells are the main effector in anti-tumor immune responses,weexamined PD-1 expression on CD8⁺ T cells and the functional characteristics oftumor associated PD-1⁺CD8⁺ T cells in human HCC.To identify whether B7-H1⁺KCs interact with PD-1⁺CD8⁺ T cells in HCC,human HCC tumor specimens werefurther analyzed by IHC to determine their physical localization.[Results] The percentage of PD-1⁺CD8⁺ T cells was higher (63±20.31%) in tumortissues than that in adjacent tissues (11.5±6.03%,P＜0.05).The expression of PD-1on CD8⁺ T cells was confirmed by fluorescence immunohistochemistry (IHC).Theseresults indicate that most of CD8⁺ T cells express PD-1 in human HCC.We initiallycompared the in vivo proliferation status of CD8⁺ T cells in tumor tissues versusadjacent tissues,and of PD-1⁺CD8⁺ T cells versus PD-1^-CD8⁺ T cells in human HCCusing Ki67,a proliferative marker for cells in mitosis.The levels of Ki67⁺CD8⁺ Tcells were lower in tumor tissues than adjacent tissues.Furthermore,PD-1⁺CD8⁺ Tcells were less proliferative than PD-1^-CD8⁺ T cells in the same individual humanHCC.The percentage of TNFα⁺IFNγ⁺ and Granzyme B⁺/Perforin⁺ T cells wassignificantly reduced in PD-1⁺CD8⁺ T cells than PD-1^-CD8⁺ T cells.A significantinfiltration ofB7-H1⁺ and PD-1⁺ cells was detected in the peri-tumor stroma.[Conclusion] These results indicate that PD-1⁺CD8⁺ T cells in HCC patients arefunctionally impaired.B7-H1⁺ KCs and PD-1⁺CD8⁺ T cells were co-localized in HCCand appeared to be in close contact with each other,providing the possibility for directphysical interaction. Partâ…£.B7-H1/PD-1 interactions mediate impaired effectorT cell function in human HCC[Objective] To evaluate whether disruption of the B7-H1/PD-1 interaction wouldrestoreeffector function to CD8⁺ T cells[Methods] CD8⁺ T cells (5×10⁵/ml) were added to 96-well round-bottom clusterplates and stimulated with anti-human CD3 (2.5 ug/mL,clone:UCHT1,BDBiosciences) and anti-human CD28 (1.2 ug/mL,clone:CD28.2,BD Biosciences) inthe presence of HCC associated KCs (1×10⁵/ml) from the same tissue for 5 days.Neutralizing monoclonal antibody against human PD-1 (clone:M3,5ug/ml) and B7-H1 (clone:5H1,5ug/ml) (17) or isotype controls were used in co-culture as indicated.After co-culture,cells were subjected to FACS analysis or proliferation assay.ForELISPOT,all the cells were transferred to MultiScreen filtration plates and incubatedfor another 48 h in the presence of HCC tumor cells lysate (from 1 x 10⁴ per well) at37℃,5% CO₂.After standard ELISPOT procedure,spots were counted using theImmunoSpot analyzer (Cellular Technology,Ltd.).Data are reported as an averagenumber of spots per 1×10⁵ responders±SEM of triplicate samples.[Results] Blockade of B7-H1 or PD-1 with specific monoclonal antibodies resulted inenhanced T cell proliferation as shown by [³H]-thymidine incorporation and Ki67expression,T cell effector cytokine expression,granzyme B,and perforin expression.To evaluate whether B7-H 1/PD- 1 interactions affect tumor antigen-specific responses,we further performed HCC-specific IFNγELISPOT analysis using HCC-derived KCsloaded with HCC lysates to stimulate autologous HCC CD8⁺ T cells.Blocking B7-H1or PD-1 using monoclonal antibodies increased the antigen-specific IFNγ-secretingspots,suggesting that B7-H1⁺ KCs interact with PD-1⁺CD8⁺ T cells and contribute todysfunction of effector T cells in HCC.[Conclusion] In HCC T cell function could be rescued by blocking B7-H1/PD-1interactions.Targeted therapies against this pathway are a promising approach toenhance T cell immunity to human HCC.