Tumor Microparticles:The Underlying Mechanism Of Novel Tumor Vaccines Targeting Lysosomes Of Dendritic Cells

Objective:An effective tumor vaccine must have two conditions:one is to contain tumor antigens,and the other is to be able to activate appropriate natural immune signals.Previous laboratory studies have found that tumor cells derived microparticles(T-MP)-dependent dendritic cells(DCs)can induce tumor-specific T cell responses,suggesting its potential as a tumor vaccine.The research team further found that T-MP were localized to lysosomes after being taken up by DCs,which not only increased the p H of the lysosomal to facilitate the processing of tumor antigens,but also up-regulated the expression of CD80/CD86 expression,and effectively stimulates specific anti-tumor T cell responses.Therefore,this study will explore how T-MP regulate lysosome p H of DCs and the molecular mechanisms of CD80/CD86 up-regulation.Answering these basic questions is expected to reveal the specific mechanism of T-MP as tumor vaccine and lay a theoretical foundation for the development of new and efficient tumor vaccines.Methods:1.Detection of DC maturation:BMDCs were incubated with OVA-MP for a certain period of time,DC maturation was analyzed for expression of CD80/CD86,CCR7,MHC I/II,CD40,OX40L and PD-L1 by flow cytometry.2.OVA-MP uptake by DC and its location:BMDCs were incubated with PKH26/67-labeled MP for 24 hours,and then analyzed with mitochondria,ER,Golgi,early endosome,late endosome and lysosome Green/Red Trackers under two-photon confocal microscope.3.Detection of DC lysosomal p H:BMDCs were treated with OVA-MP at different time points and then incubated with 0.1?M Lyso-Sensor for 30 min,the BMDCs were observed by fluorescence microscope and the p H value of DCs was detected by microplate reader.BMDCs were treated with DPI or transfected with gp91^phox si RNAs respectively,and lysosomal p H value of BMDCs were analyzed.4.Detection of lysosomal related proteins:BMDCs were treated with or without OVA-MP for a certain period of time,and then the expression of LAMP1and LAMP2 was analyzed by real-time PCR,the expression of TFEB was analyzed by real-time PCR and Western blot,and the expression of V-ATPase subunits and NOX2 was analyzed by real-time PCR.5.Recruitment and activation of NOX2 on DC lysosomes:BMDCs were incubated with PKH26-labeled MP for a certain period of time,and then analyzed with gp91^phox,LAMP-1,and DAPI under two-photon confocal microscope.BMDCs were treated with or without OVA-MP at different time points and then the ROS production of BMDCs was detected by confocal microscope and flow cytometry.BMDCs were transfected with gp91^phox si RNAs or treated with DPI respectively,and then the ROS production of BMDCs were analyzed by flow cytometry.6.Detection of calcium ion channels and calcium ion concentration:BMDCs were treated with OVA-MP for a certain period of time,the expression of Mcoln1/2and Tpcn1/2 was analyzed by real-time PCR.OVA-MP loaded BMDCs were pre-treated with or without ER or mitochondria calcium release inhibitor ryanodine or CGP37157 for 1 hour and stained with Fluo-4AM for 1 hour.Then 200 n M ionomycin(Iono)was applied extracellularly at 30 s and the cytosolic calcium release was recorded by two-photon confocal microscope.7.Detection of TFEB localization,expression and its regulation of CD80/86in DCs:BMDCs were treated with OVA-MP for a certain period of time,the expression and location of TFEB were analyzed by two-photon confocal microscope and Western blot.TFEB si RNAs or control si RNAs(NC)were transfected into BMDCs.Twenty-four hours later,the expression of CD80/CD86was analyzed by flow cytometry.Chromatin Immunoprecipitation(CHIP)with anti-TFEB followed by PCR with CD80/CD86 promoter primers in BMDCs treated with or without OVA-MP.8.Detection of DC antigen presentation ability:Splenic CD8⁺T cells purified from OT-I mice were incubated with untreated BMDCs or OVA-MP-loaded BMDCs.T-cell proliferation was examined by CFSE dilution assay and the expression of H-2Kb-OVA(257-264)was detected by flow cytometry.BMDCs were transfected with gp91^phox si RNAs,treated with DPI or treated with NAC respectively,and then the T-cell proliferation was examined by CFSE dilution assay and the expression of H-2Kb-OVA(257-264)was detected by flow cytometry.Results:1.Flow cytometry results showed that BMDC maturation surface markers including CD80,CD86,CCR7,MHC-class I/II,CD40 and OX40L were all upregulated upon 28-hour incubation with OVA-MP,but PD-L1 expression was not upregulated in the DCs,which indicates that OVA-MP can promote DC maturity.2.The colocalization results showed that OVA-MP were not found to be co-localized with mitochondria,endoplasmic reticulum(ER)or Golgi apparatus.However,OVA-MP were found to be co-localized with Rab5 positive early endosomes,Rab7 positive late endosomes and lysosomes,suggesting that OVA-MP are taken up via endocytosis and trafficked to the lysosomes of DCs.3.After OVA-MP treated BMDC for a certain time,qualitative and quantitative detection of lysosomal p H by confocal microscope and microplate reader showed that the lysosomal p H was markedly elevated and the lysosomal p H value actually increased to a peak of 8.5 within 24 hours.We used NADPH oxidase inhibitor diphenylene iodonium(DPI)or gp91^phoxsi RNA to block NOX2 activity.Immunofluorescence results showed that the above lysosomal p H value decreased to the level of untreated BMDCs.This suggested that endocytosed OVA-MP increase lysosomal p H via NOX2.4.After OVA-MP treated BMDC for a certain period of time,Real-time PCR and Western blot results revealed that the lysosomal-related genes LAMP1,LAMP2,and TFEB were up-regulated,indicating that endocytosed OVA-MP cause an increase lysosomal number in DCs.At the same time,The real-time PCR result did not show differential expression of subunit members of V-ATPase between OVA-MP-treated and untreated BMDCs,but show that endocytosed OVA-MP could upregulated the expression of gp91^phox.These data suggest that endocytosed OVA-MP increase lysosomal p H via the pathway of NOX2-mediated.5.Immunofluorescence colocalization results showed that gp91^phox was effectively recruited to lysosomes in MP-treated BMDCs.Consistently,ROS levels were elevated in OVA-MP-treated BMDCs,as evidenced by fluorescence microscopy and flow cytometry.We use DPI or gp91^phoxsi RNA to block NOX2activity,we found that the above ROS decreased to the levels of untreated BMDCs,suggesting that endocytosed OVA-MP increase lysosomal p H via NOX2-mediated ROS production.6.Real-time PCR results showed that the intracellular calcium levels were elevated in BMDCs upon uptake of OVA-MP.Notably,using ryanodine or CGP37157 to block calcium release from the endoplasmic reticulum or mitochondria didn't affect OVA-MP-mediated Ca²⁺increase in DCs,suggesting that it is lysosomes rather than ER or mitochondria that release Ca²⁺to the cytosol.7.Both immunostaining and Western blot showed that abundant TFEB was translocated into the nucleus in OVA-MP-treated DCs rather than in untreated BMDCs.To clarify whether TFEB regulates CD80/CD86 expression,we knocked down TFEB by the si RNA,which led to the downregulation of CD80/CD86expression.And then,we performed Chip-q PCR assay,and found that TFEB indeed bound the promoters of CD80/CD86 genes.Together,these data suggest that activation TFEB transcriptional activity by OVA-MP directly upregulates the expression of CD80/CD86.8.The results of flow cytometry showed that the MHC class I-OVA peptide complexes were confirmed be expressed on OVA-MP-loaded DC's membrane surface and markedly stimulated OVA-specific CD8⁺T cell proliferation,suggesting that BMDCs effectively process and present OVA antigen to T cells.We found that the DPI or si RNA treatment significantly inhibited T cell proliferation and the expression of MHC class I-OVA peptide complexes on the surface of those BMDCs was strikingly reduced.And then further used ROS scavenger NAC to treat OVA-MP-loaded BMDCs.The result showed that the inhibition of ROS production strikingly decreased OT-I T cell proliferation.Conclusion:DCs readily take up T-MP to lysosomes via endocytosis,where T-MP markedly increase lysosomal p H value via NOX2-catalyzed ROS production.This increased p H,coupled with T-MP-driven lysosomal centripetal migration,together promotes the formation of MHC class I-tumor antigen peptide complex.Meanwhile,endocytosis of T-MP results in the upregulation of CD80/CD86.T-MP-increased ROS activates lysosomal Ca²⁺channel Mcoln2,leading to Ca²⁺release.Released Ca²⁺activates TFEB,a lysosomal master regulator that directly binds to CD80/CD86 promoters,promoting gene expression.These findings elucidate a unique pathway through which DCs efficiently present tumor antigen from T-MP to CD8⁺T cells,potentiating T-MP as a novel tumor cell-free vaccine with clinical applications.