Tumor Cell-released Autophagosome （TRAP） Induce Immunosuppressive PD-L1^hi IL-10⁺ Macrophage And Its Underlying Mechanisms

Tumor-associated macrophage（TAM）represents a major constituent of the leukocytes infiltrate in the tumor microenvironment where they mostly display tumor-promoting functions by facilitating tumor proliferation and survival,angiogenesis,metastasis,as well as immune suppression.Tumor-derived mediators such as cytokines（CSF-1,TGF-β,IL-10）,chemokines（CCL2,CCL5,CXCL12）,prostaglandin E2（PGE2）,metabolites（lactate）and exosomes likely contribute to the development of TAM with M2-like phenotype and tumor-promoting properties.However,the signals involved in communication between tumor and TAM are not completely understood.Our previous studies have shown that tumor cell-released autophagosome（TRAP）from supernatants of multiple murine tumor cell lines and malignant effusions or ascites of cancer patients was able to induce the differentiation of B cells into IL-10⁺regulatory B cells（Bregs）with immunosuppressive effect on T-cell proliferation via the TLR2-MyD88-NF-κB pathway.Moreover,treatment of human neutrophils with TRAP promoted the generation of reactive oxygen species（ROS）through macropinocytosis,contributing to the inhibition of T-cell activation and proliferation in a ROS-dependent manner.However,little is known about how TRAP affect macrophage polarization and function in vitro and in vivo.ObjectivesTo investigate whether TRAP could regulate macrophage polarization and the underlying molecular mechanisms involved in polarization;to ascertain the effects of TRAP-converted macrophage on immune responses and its mechanisms.Methods1.Investigation of TRAP-induced macrophage polarization and its mechanisms1.1 Investigation of TRAP-induced macrophage polarizationTRAP labeled with CFSE was incubated with BMDMs stained with PE-F4/80 antibody for 30 min,TRAP uptake was observed by confocal microscopy.BMDMs were stimulated with TRAP for 48 h,the expression of M1（CD86,MHC II）and M2（CD206）markers was determined by flow cytometry,the levels of IL-1β,IL-6,IL-10 and IL-12p70 in the supernatant were measured by ELISA,and the expression of co-inhibitory ligands including PD-L1,PD-L2,B7-H2,B7-H3,B7-H4,TIM-4 and VISTA was determined by flow cytometry.BMDMs were treated with TRAP for 6 h,mRNA expression of NOS2 and Arg1 in BMDMs was detected by qRT-PCR.BMDMs were treated with TRAP from EL4,B16F10,Hepa1-6 or 4T1 cells for48 h,the expression of PD-L1 and IL-10 was assessed by flow cytometry and ELISA,respectively.Mice were i.p.injected with four different doses of TRAP（0,10,30 and 100μg）at day 0 and the phenotype of peritoneal macrophages was analyzed at day 3.The expression of CD206 and PD-L1 on macrophages was determined by flow cytometry,and the mRNA expression levels of IL-10 and Arg1 were detected by qRT-PCR.1.2 Investigation of the mechanisms underlying TRAP-induced macrophage polarizationBMDMs derived from WT,TLR2^-/-,TLR4^-/-,or MyD88^-/-mice were incubated with TRAP for 48 h,the expression of PD-L1 and IL-10 was assessed by flow cytometry and ELISA,respectively.BMDMs were exposed to TRAP at indicated time points,and cell lysates were analyzed for p38,p-p38,STAT3 and p-STAT3 expression by western blot.BMDMs were pretreated with p38 inhibitor SB203580 for 1 h,and then co-incubated with TRAP for 4 h,the expression of STAT3 and p-STAT3 was detected by western blot.BMDMs were exposed to SB203580 or Stattic for 1 h,and followed by incubation with TRAP for 72 h,PD-L1 expression and IL-10 secretion was determined by flow cytometry and ELISA,respectively.TRAP was pretreated with Proteinase K,DNase I or RNase,respectively,followed by incubation with BMDMs for 72 h.PD-L1 was evaluated by flow cytometry,and IL-10 was tested by ELISA.TRAP was preincubated withαHMGB1,αHSP60,αHSP70,αHSP90αandαHistones,respectively,and then treated with BMDMs for 72 h.The expression of PD-L1 and IL-10 was assessed by flow cytometry and ELISA,respectively.2.Investigation of the effects of TRAP-converted macrophage on immune responses and its mechanisms2.1 Investigation of the effects of TRAP-converted macrophage on T-cell proliferationCFSE-labeled purified CD3⁺T cells were activated by immobilized anti-CD3 plus soluble anti-CD28 mAb,and were either cultured alone or cocultured with BMDMs pretreated with or without TRAPs for 3 d,T cell proliferation was determined by flow cytometry.A transwell chamber was used to separate TRAP-converted BMDMs from T cells stimulated by immobilized anti-CD3 plus soluble anti-CD28 mAb for 3 d,T cell proliferation was determined by flow cytometry.BMDMs were left untreated or pretreated with B16F10 TRAP or B16F10-OVA TRAP for 2 d,were then incubated with CFSE-labeled OT-I splenocytes in the presence of OVA_257-264 peptide for 3 d at a ratio of 1:20,T cell proliferation was determined by flow cytometry.BMDMs derived from WT,TLR2^-/-,TLR4^-/-,or MyD88^-/-mice were left untreated or pretreated with B16F10 TRAP for 2 d,and cocultured with CFSE-labeled OT-I splenocytes for 3 d in the presence of OVA_257-264 peptide.The division of CD8⁺T cell was detected by flow cytometry.TRAP-converted BMDMs were cocultured with T cells stimulated by immobilized anti-CD3 plus soluble anti-CD28 mAb in the presence of anti-PD-L1 mAb,anti-IL-10 mAb or control IgG for 3 d,and T cell proliferation was determined by flow cytometry.2.2 Investigation of the effects of TRAP-converted macrophage on tumor growthB16F10 cells were mixed with WT or PD-L1^-/-BMDMs（2:1）treated with or without TRAP and then injected s.c.to C57BL/6 mice.Mice were randomly divided into five groups:B16F10,B16F10+WT-M?,B16F10+WT-M?（TRAP）,B16F10+PD-L1^-/--M?and B16F10+PD-L1^-/--M?（TRAP）,and tumor growth was monitored at the indicated days.2.3 Investigation of the roles of TRAP in TAM polarization and anti-tumor T-cell immune responses in vivoBeclin1-KD B16F10 and Ctrl-B16F10 cells were s.c.implanted to C57BL/6 mice,and tumor area was measured at the described days.Tumor bearing mice were euthanized around day 15,total TAMs were gated and analyzed for expression of CD206,PD-L1,CD86 and MHC II expression by flow cytometry.TILs were stimulated with Cell Stimulation Cocktail for 5 h,the frequency of CD4⁺IFN-γ⁺and CD8⁺IFN-γ⁺T cells was determined by intracellular staining.Single suspension cells form dLNs and spleens were stimulated with inactivated B16F10 cells at a ratio of 30:1 for 21 h,breferdin A plus monensin was added for the last 5 h of culture,and IFN-γproduction was determined by flow cytometry.T cells from TILs,dLNs and spleens were tested for Ki-67 expression by intracellular staining.3.Investigation of cancer patients derived TRAP-induced monocyte polarization and its functional effects on T-cell immune responsesThe correlation between concentration of TRAP and expression of PD-L1 on CD14⁺monocytes,total IL-10 in malignant pleural effusions or ascites of cancer patients was analyzed.CD163,PD-L1 and IL-10 expression on CD14⁺monocytes from peripheral blood of cancer patients and healthy donors was assessed by flow cytometry.Purified CD14⁺monocytes were treated with TRAP derived from cancer patients for 3 d,and the expression of CD163,PD-L1,CD86,HLA-DR and IL-10 was detected by flow cytometry and ELISA.LC3B⁺EVs and LC3B^-EVs were sorted from total EVs of a lung cancer patient,and LC3B expression was determined by flow cytometry.Purified CD14⁺monocytes were treated for 3 d with total EVs,LC3B⁺EVs,LC3B^-EVs,respectively.Expression of CD163,HLA-DR,CD86,PD-L1 and IL-10 was detected by flow cytometry and ELISA.Monocytes were left untreated or pretreated with TRAP for 3 d,then incubated with CFSE-labeled autologous T cells for 5 d in the presence ofαCD3andαCD28,or monocytes and T cells were separated by a transwell chamber,T cell proliferation was determined by flow cytometry.Monocytes were left untreated or pretreated with TRAP from MDA-MB-231 cells（c-TRAP）and cancer patient（p-TRAP）for 3 d,then incubated with autologous T cells（1:3）for 20 h in the presence of immobilized anti-CD3.The percentage of CD25 and IFN-γexpressing T cells was examined by flow cytometry and total IFN-γsecretion was measured by ELISA.Results1.Investigation of TRAP-induced macrophage polarization and its mechanisms1.1 Investigation of TRAP-induced macrophage polarizationTRAP uptake was observed as early as 30 min and increased thereafter by confocal microscopy analysis.TRAP substantially increased CD206 and slightly reduced MHC-II expression,but failed to induce CD86.TRAP also upregulated the expression of PD-L1 but not other co-inhibitory ligands,including PD-L2,B7-H2,B7-H3,B7-H4,TIM-4,and VISTA,on macrophages.RT-PCR analysis showed that Arg1,but not NOS2,was increased.In addition,TRAP-treated macrophages secreted very high level of IL-10,low levels of IL-1βand IL-6,and no IL-12p70.TRAP from multiple murine tumor cell lines had similar effects on macrophages polarization.Mice that received TRAP had increased expression of CD206 and PD-L1,and elevated Il10 and Arg1 transcripts in peritoneal macrophages.1.2 Investigation of the mechanisms underlying TRAP-induced macrophage polarizationTRAP-induced PD-L1 expression was completely MyD88-dependent,and PD-L1upregulation was markedly diminished due to TLR4,but not TLR2 deficiency.IL-10 secretion was impaired in Tlr4^–/–or Myd88^–/–macrophages,although reduced IL-10 release was also observed in Tlr2^–/–macrophages.After stimulation of BMDMs with TRAP,phosphorylation of p38 was observed at 0.5 h,and declined rapidly after 1 h to the control level at 4 h.Phosphorylation of STAT3 was detectable at 2 h,and increased thereafter.Inhibition of p38activation by SB203580 repressed STAT3 phosphorylation and the induction of PD-L1 and IL-10.Pretreatment of macrophages with the STAT3 inhibitor Stattic also significantly diminished PD-L1 and IL-10 induction.Treatment of TRAP with proteinase K,but not DNase or RNase,significantly blocked the upregulation of PD-L1 and IL-10.Preincubation of TRAPs withαHMGB1,αHSP60,αHSP70,αHSP90αorαHistones had no effect on induction of PD-L1 on macrophages.2.Investigation of the effects of TRAP-converted macrophage on immune responses and its mechanisms2.1 Investigation of the effects of TRAP-converted macrophage on T-cell proliferationTRAP-treated macrophages,but not control macrophages,inhibited CD4⁺and CD8⁺T cell proliferation.Suppression was partially dependent upon cell contact,as TRAP-exposed macrophages had reduced suppressive activity on CD4⁺and CD8⁺T-cell proliferation when separated from T cells by transwell.TRAP-stimulated macrophages could also inhibit OVA_257-264 peptide specific CD8⁺T cell proliferation.Tlr4^–/–or Myd88^–/–,but not Tlr2^–/–macrophages treated with TRAP had diminished capability of inhibiting OT-I proliferation.PD-L1 blockade,and to a lesser extent,IL-10 neutralization,significantly restored T cell proliferation stimulated byαCD3/CD28 mAb.More importantly,dual PD-L1/IL-10 blockade completely abrogated the suppressive function of TRAP-treated macrophages on T cell proliferation stimulated byαCD3/CD28 mAb.2.2 Investigation of the effects of TRAP-converted macrophage on tumor growthMice co-injected with Pdcd1l1^–/–BMDMs experienced slower tumor growth than WT BMDMs with or without TRAP pretreatment.Co-injection of TRAP-stimulated WT BMDMs significantly accelerated tumor growth compared to the co-injection of control WT BMDMs.TRAP treatment of WT but not Pdcd1l1^–/–BMDMs resulted in larger tumors as compared to the untreated group.2.3 Investigation of the roles of TRAP in TAM polarization and anti-tumor T-cell immune responses in vivoTAMs from mice bearing Beclin1 knockdown tumors had significantly decreased expression of CD206 and PD-L1,as well as slightly increased expression of CD86 and MHC-II whereas the effect did not achieve statistical significance.Furthermore,a higher frequency of IFN-γ-producing T cells was observed in Beclin1 knockdown tumors.Re-stimulation of T cells from spleens and dLNs also revealed a higher proportion of IFN-γ⁺CD4⁺and CD8⁺T cells in mice bearing Beclin1 knockdown tumors.Intratumoral and dLNs T cells in mice bearing Beclin1 knockdown tumors expressed higher level of the proliferation marker Ki-67.3.Investigation of cancer patients derived TRAP-induced monocyte polarization and its functional effects on T-cell immune responsesThere was a significant positive correlation between the concentration of LC3B⁺autophagosomes and the expression of PD-L1 and IL-10 in matched monocytes from effusions or ascites of cancer patients.Elevated levels of LC3B⁺EVs were observed in plasma of cancer patients compared with healthy donors.Moreover,elevation of CD163,PD-L1 and IL-10expression on CD14⁺monocytes from PBMCs of cancer patients was observed as compared to those from healthy donors.Peripheral blood CD14⁺monocytes treated with autophagosomes from cancer patients exhibited a significant increase in CD163 and PD-L1 expression and a decrease in HLA-DR expression.TRAP stimulation showed the trend of downregulating CD86expression,although the effect did not achieve statistical significance.Meanwhile,monocytes produced high level of IL-10 following TRAP stimulation.We also found that LC3B⁺EVs,isolated from malignant ascites of a lung cancer patient,were more potent than LC3B^-EVs to upregulate CD163,PD-L1 and IL-10 expression and downregulate HLA-DR expression of monocytes.TRAP-activated monocytes acquired the ability to suppress the proliferation of both CD4⁺and CD8⁺T cells.Meanwhile,TRAP-pretreated monocytes suppressed the expression of CD25 on T cells,and diminished the frequency of IFN-γ⁺T cells and IFN-γsecretion into the supernatant.Conclusions1.TRAP was sufficient to induce PD-L1^hiIL-10⁺macrophage.Protein components on TRAP mediated macrophage polarization through TLR4-MyD88-p38-STAT3 signaling pathway, however,the determinants still need to be fully investigated.2.TRAP-treated macrophages could suppress T-cell proliferation in vitro and promote tumor growth in vivo,the mechanisms appeared to predominately involve PD-L1/PD-1 signaling, with IL-10 playing a minor role.Interference with autophagosome formation in tumor cells blocked M2-like macrophage polarization,enhanced anti-tumor T cell responses,and inhibited tumor growth.3.The levels of LC3B⁺autophagosomes were correlated with the expression of PD-L1 and IL-10 in matched monocytes from cancer patients.TRAP from cancer patients was capable of transforming human monocytes into a HLA-DR^loCD163^hiPD-L1^hiIL-10⁺phenotype that significantly suppress the proliferation,activation and IFN-γsecretion of T cells.