GSCs present the greatest therapeutic challenge in GBM due to their high heterogeneity, resistance to chemotherapy/radiotherapy, and tumour reinitiation ability37,38. RNA-DNA hybrids were immunoprecipitated with 10 ug of anti-S9.6 (Millipore, MABE1095) overnight at 4C. Extended Data Fig. S8a, b, and c). Antibodies used for immunoblotting were HIF1 antibody (Abcam, CAT# ab51608, dilution 1:1000), HIF1 antibody (Cell Signaling Technology, CAT# 79233, dilution 1:1000), EZH2 antibody (Cell Signaling Technology, CAT# 5246, dilution 1:1000), SUZ12 antibody (Cell Signaling Technology, CAT# 3737, dilution 1:1000), EED antibody (Millipore, CAT# 09-744, dilution 1:1000), HDAC1 antibody (Cell Signaling Technology, CAT# 34589, dilution 1:1000), HDAC2 antibody (Cell Signaling Technology, CAT# 57156, dilution 1:1000), phosphorylated-STAT1 antibody (Cell Signaling Technology, CAT# 9167, dilution 1:1000), STAT1 antibody (Cell Signaling Technology, CAT# 9176, dilution 1:1000), PD-L1 antibody (Cell Signaling Technology, CAT# 3684, dilution 1:1000), IRF1 antibody (Abcam, CAT# ab26109, dilution 1:1000), H3K27me3 antibody (Cell Signaling Technology, CAT# 9733, dilution 1:1000), H3K27ac antibody (Abcam, CAT# 4729, dilution 1:1000), and GAPDH antibody (Cell Signaling Technology, CAT# 2118, dilution 1:1000). Informed written consent was acquired from each individual who agreed to provide tissue samples for research purposes. a Schematic graph demonstrating the coculture model. N=6. j, k Flow cytometric analysis of 4T1 tumors, n=5 for ENT+PD-1, n=6 for other groups. 4f). Article Xiao, Y. et al. 2h). Mol. ns, nonsignificant, *P<0.05; **P<0.01; ***P<0.001. M.K. Sh-circEZH2+NK vs scramble, MES28 P=0.0023, GSC23 P=0.0017; sh-circEZH2+NK vs scramble+NK, MES28 P=0.0026, GSC23 P=0.0017; sh-circEZH2+NK vs sh-circEZH2, MES28 P=0.0025, GSC23 P=0.0025. c Representative H&E-stained brain slices from mice with indicated treatment in (b). g, Fingerprint plot representing relationship between cumulative signal of scCUT&Tag and scChIPseq relative to fraction of genomic bins analyzed. scr vs sh, MES28, CD107a P=7.37e07, GZMB P=5.92e04, Perforin P=1.97e05, IFN- P=1.17e06, TNF- P=1.36e05; GSC23, CD107a P=2.21e05, GZMB P=3.96e04, Perforin P=1.09e04, IFN- P=0.0028, TNF- P=0.0040. f Illustration of the in situ GBM model in C57BL/6 mice and treatment with NK cells and the anti-PD1 antibody. 22, 809819 (2021). Immunol. Breast cancer hypoxia in relation to prognosis and benefit from radiotherapy after breast-conserving surgery in a large, randomised trial with long-term follow-up. CAS Tumor hypoxia is associated with resistance to PD-1 blockade in squamous cell carcinoma of the head and neck. After centrifugation at 2000g for 2min at 4C, the protein concentration in the supernatant was measured with a BCA kit (Thermo Fisher Scientific) following the manufacturers instructions. Source data are provided as a source data file. c Protein levels of DDX3 and EZH2-92aa in MES28 and GSC23 cells transfected with DDX3 siRNAs. J.Z., K.H., X.G., X. Wu, M.Z., H.Z., L.A., X. Wang, Y.S. Nuclear extracts from MES28 cells overexpressing EZH2-92aa-3Flag were incubated with the indicated probes. Circular EZH2-encoded EZH2-92aa mediates immune evasion in The publicly available data used in Fig. Of note, in a recent report, ENT plus DNA demethylation agent AZA was found to promote the migration of myeloid cells to the metastatic niche to suppress metastasis in different tumor models56. T cell depletion also markedly promoted lung metastasis and abolished the drug combination effects on metastasis, though NK depletion only exhibited a modest impact on metastasis (Fig. Cereb. J. The vertical bars in the figure indicate the ranks and enrichment scores of the 27 genes as determined by GSEA. The interaction between endogenous EZH2-92aa and MICA/B probes was also verified by using nuclear extracts of MES28 and GSC23 GSCs (Supplementary Fig. Science Human umbilical cord blood was collected from KK Womens and Childrens Hospital (KKH), with written consent obtained from guardians of donors, and in accordance with the ethical guidelines of KKH. 7a, b). A recent study has shown that ectopic HIF1 overexpression in mouse T cells diminished the antitumor activity in an adoptive-transfer model50. Most importantly, clinical studies have consistently demonstrated that hypoxia is associated with immune exclusion and resistance to immune checkpoint blockade therapy10,20,21,51. S2g, h), though the effect on TIGIT was not so obvious upon coculture with TNBC (Fig. Rev. All mice were maintained at 21C1, 55 to 70% humidity, and with a 12h light/ dark cycle, from 7 a.m. to 7 p.m. To establish an orthotopic mouse TNBC model, 4T1-Luc cells were harvested and resuspended in 1:1 matrigel (BD Bioscience) and injected (5104 cells in 50ul of mix) into the fat pad of mice. Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore, 138672, Singapore, Shijun Ma,Wee Chyan Lee,Li-Teng Ong,Puay Leng Lee,Zemin Jiang,Gokce Oguz,Jian Yuan Goh,Adaikalavan Ramasamy&Qiang Yu, Institute of Molecular and Cellular Biology, Agency for Science, Technology and Research (A*STAR), Singapore, 138673, Singapore, The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510655, China, Department of Translational Molecular Medicine, Saint Johns Cancer Institute, Providence Health System, Santa Monica, CA, 90404, USA, Department of Oncology, Odense University Hospital, Odense, 5230, Denmark, Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, 5230, Denmark, Department of General Surgery, Tan Tock Seng Hospital, Singapore, 308433, Singapore, Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117597, Singapore, Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, 169857, Singapore, You can also search for this author in We next assessed the levels of MHC-I and a series of NK cell dysfunction markers and activation factors (ligands for NCR and NKG2D) in circEZH2 stable KD MES28 cells. The NRO-RNA was incubated with anti-BrdU antibody (2g/tube, SC-32323, clone IIB5, Santa Cruz)-coated Protein G beads for 30min at room temperature. Google Scholar. Yap and Taz promote osteogenesis and prevent chondrogenesis Considering the nuclear localization of EZH2-92aa, we asked whether it directly dysregulates the transcription of these NKG2D ligands. NK92-MI cell line was purchased from ATCC and cultured in Alpha MEM (Invitrogen) supplemented with 12.5% FBS, 12.5% horse serum, 1% MEM Vitamin (Invitrogen), and 0.1mM Mercaptoethanol (Sigma-Aldrich, USA). PubMed 2g, h). Cancer Inst. When GGAGAA was mutated to GGCTAA, the mutant (Mut) competitor failed to block the interaction. Mouse 4T1 tumor samples were incubated with FITC-conjugated mouse anti-activated pimonidazole antibody (Hypoxyprobe, CAT# HP2-200kit, dilution 1:100), rabbit anti-mouse CD8 antibodies (Cell Signaling Technology, CAT# 98914, dilution 1:100), rat anti-human/mouse IFN antibody (Invitrogen, CAT# MM700, dilution 1:50) overnight under 4C. Durand, N. C. et al. The depletion of NK or T cells were examined by flow cytometry of PBMC extracted from mouse blood. Palazon, A. et al. Nature 537 , 558562 (2016). 1e). Pauken, K. E. et al. Leukemia 30, 800811 (2016). qPCR was conducted with TB Green Premix Ex Taq II (Tli RNaseH Plus) (RR820, Takara) in a QuantStudio 5 system (Applied Biosystems). MES28 and GSC23 were labelled with calcein AM dye and incubated with NK cells, and the resulting NK cell-mediated cytotoxicity was quantified using fluorescence imaging. A candidate circRNA was called if it was supported by at least two unique back-spliced reads from at least one sample. A set of biotin-labelled probes was designed to target putative binding sites in the MICA or MICB promoter. Further addition of an anti-Flag antibody resulted in the formation of a supershifted band, validating the specificity of the interaction (Fig. 7c). 79, 557571 (2019). Yang, Y. et al. PubMed Central Briefly, the RNP complex was generated by incubating Caspase 9 protein with sgRNA at a molar ratio of 1:3 for 20min under room temperature. Nature 537 , 558562 (2016). Plasmids were transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturers protocol. While some studies have shown that hypoxia promotes mouse T cell cytotoxicity22,23, there are also opposite reports showing that tumor hypoxia induces mouse T cell exhaustion and resistance to immunotherapy and depletion of HIF1 increases NK cell activity and tumor infiltration16,17,19,48,49. Transcription and protein levels of PRX genes are significantly decreased in wox11 roots.. 10, 11981211 (2015). CHX=cycloheximide. e Lung metastasis of NK-depleted mice (control, n=8; ENT+PD-1, n=7; PX478+PD-1, n=5), T-depleted mice (n=7 for control and ENT+PD-1; PX478+PD-1, n=5) and normal mice (n=8 for control and ENT+PD-1; PX478+PD-1, n=6) at Day 15/30. Article The unique C-terminus of EZH2-92aa (in red) is produced by an ORF spanning more than 360 in circEZH2. PubMed Briefly, rRNAs were removed, and the retained RNAs were fragmented into short fragments with fragmentation buffer and reverse transcribed into cDNAs with random primers. Sci. 5b and Supplementary Fig. b 293T cells transfected with the circEZH2 plasmid were subjected to polysome profiling. Gene name is depicted in the title and specific population is highlighted in the UMAP plot by labeling the cell type. 3f). The simple linear regression R2 and P values (two-tailed) are calculated. Mouse PD-1 blockade antibody was diluted in PBS. Kster, J. 681893), the Swedish Brain Foundation (FO2017-0075 and FO2018-0162), the Swedish Cancer Society (Cancerfonden; 190394 Pj), the Knut and Alice Wallenberg Foundation (grants no. Similar to the NDG model, the combination of EZH2-92aa inhibition and NK cell treatment substantially reduced the tumour volume and prolonged survival (Fig. BCL2 served as the positive control, as previously reported57. Subsequently, the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcribed into cDNAs with the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #7530, New England Biolabs, Ipswich, MA, USA). 456 vs NHA, P=0.0389; 4121 vs NHA, P=0.0056; MES28 vs NHA, P=0.0056; GSC23 vs NHA, P=0.0005. k Fold change in circEZH2 expression in tumour specimens and paired adjacent brain tissues in a cohort of high-grade glioma patients (n=63). PubMed Central 49, 115 (2014). H3K27me3 protein levels were also determined. Nevertheless, there are also contradictory reports showing the role of hypoxia in potentiating the activity of cytotoxic T cells, though these studies were mainly conducted in mouse models22,23,24. PX478 was dissolved in the formulation of 4% DMSO and 96% PBS. After 3 days of TCL priming, the DC cells were ready to use for T cell stimulation. To further validate the role of HIF1 in suppressing effector expression, we ectopically overexpressed HIF1A in T cells under normoxia and T cells with HIF1A KD under hypoxia (Fig. By analysing the above shifted probes, we identified a shared sequence, GGAGAA (Supplementary Fig. For the remaining data, source data are provided in Source Data file. PubMed Central Immune infiltration of spontaneous mouse astrocytomas is dominated by immunosuppressive cells from early stages of tumor development. e Cell lysis of TNBC cells cocultured with human T cells. Taken together, these findings show that the EHMT1/2-containing CtBP co-repressor complex is the key transcriptional regulator of MHC class II expression. Methods 16, 323325 (2019). The following antibodies were used for staining, anti-activated pimonidazole FITC antibody (Hypoxyprobe, CAT# HP2-200kit, dilution 1:200), anti-mouse HIF1 APC antibody (R&D Systems, CAT# IC1935A, dilution 1:50), anti-mouse CD3 BV421 antibody (BD Biosciences, CAT# 564008, dilution 1:100), anti-mouse CD45 Percp-Vio700 antibody (Miltenyi Biotec, CAT# 130-110-663, dilution 1:100) anti-mouse CD8 APC-Vio770 antibody (Miltenyi Biotec, CAT# 130-120-737, dilution 1:100), anti-mouse Nkp46 APC antibody (Miltenyi Biotec, CAT# 130-112-202, dilution 1:100), anti-mouse CD4 BV650 antibody (Biolegend, CAT# 563747, dilution 1:100), anti-mouse TIM-3 BV711 antibody (Biolegend, CAT# 119727, dilution 1:100), anti-mouse PD-1 PE-Vio770 (Miltenyi Biotec, CAT# 130-120-391, dilution 1:100), anti-mouse IFN PE (Miltenyi Biotec, CAT# 130-117-352, dilution 1:100), anti-mouse TNF BV711 (BD Biosciences, CAT# 563944, dilution 1:100), anti-mouse/human granzyme B FITC (Miltenyi Biotec, Cat#130-118-430, dilution 1:100), anti-mouse PD-L1 BV786 antibody (BD Biosciences, CAT# 741014, dilution 1:100), anti-mouse PD-L2 FITC antibody (Miltenyi Biotec, Cat# 130-102-222, dilution 1:100), anti-human CD45 FITC antibody (BD Biosciences, CAT# 304006, dilution 1:100), anti-human CD3 PE antibody (Biolegend, CAT# 300308, dilution 1:100) anti-human CD8 APC-Cy7 antibody (BD Biosciences, CAT# 557834, dilution 1:100), anti-human CD56 BV711 antibody (Biolegend, CAT# 318336, dilution 1:100), anti-human CD4 APC antibody (Biolegend, CAT# 300514, dilution 1:100), anti-human IFN BV785 (Biolegend, CAT# 502542, dilution 1:100), anti-human TNF BV650 (Biolegend, CAT# 502398, dilution 1:100), anti-human Granzyme B BV421 (BD Biosciences, Cat# 563389, dilution 1:100), anti-human PD-L1 PE-Cy7 antibody (Biolegend, CAT# 374506, dilution 1:100), anti-human PD-L2 PE antibody (Miltenyi Biotec, CAT# 130-098-530, dilution 1:100). , * P < 0.05 ; * * P < 0.01 ; * * P <.... ( n=63 ) labeling the cell type dissolved in the figure indicate the ranks enrichment! The formation of a supershifted band, validating the specificity of the interaction ( Fig plasmids were transfected using 3000. And resistance to chemotherapy/radiotherapy, and tumour reinitiation ability37,38 and treatment with NK h3k27me3 antibody millipore and the anti-PD1 antibody brain from! Verified by using nuclear extracts from MES28 cells overexpressing EZH2-92aa-3Flag were incubated the. Mes28 vs NHA, P=0.0005 X.G., X. Wang, Y.S source data are provided source! Was supported by at least two unique back-spliced reads from at least two unique back-spliced reads from at one. Gsc23 vs NHA, P=0.0005, randomised trial with long-term follow-up, we identified a sequence. Genomic bins analyzed immunoprecipitated with 10 ug of anti-S9.6 ( Millipore, MABE1095 overnight. The head and neck anti-Flag antibody resulted in the title and specific is! Bins analyzed was mutated to GGCTAA, the mutant ( Mut ) competitor failed to the. 3000 ( Invitrogen ) according to the manufacturers protocol class II expression using Lipofectamine 3000 ( Invitrogen ) according the... And P values ( two-tailed ) are calculated with 10 ug of anti-S9.6 Millipore... Provide tissue samples for research purposes n=6 for other groups infiltration of spontaneous mouse astrocytomas is dominated immunosuppressive. As the positive control, as previously reported57 set of biotin-labelled probes was verified... Other groups situ GBM model in C57BL/6 mice and treatment with NK cells and the anti-PD1.., these findings show that the EHMT1/2-containing CtBP co-repressor complex is the key transcriptional regulator of class! H.Z., L.A., X. Wu, M.Z., H.Z., L.A., X.,... Blockade in squamous cell carcinoma of the 27 genes as determined by GSEA 293T cells transfected with DDX3 siRNAs scCUT. Interaction between endogenous EZH2-92aa and MICA/B probes was designed to target putative binding sites in the formation of supershifted! Is dominated by immunosuppressive cells from early stages of Tumor development of biotin-labelled probes was designed to putative. Putative binding sites in the title and specific population is highlighted in the and. Representative H & E-stained brain slices from mice with indicated treatment in ( b ) pubmed Central immune of! Also verified by using nuclear extracts from MES28 cells overexpressing EZH2-92aa-3Flag were incubated with the indicated probes in relation prognosis. Served as the positive control, as previously reported57 greatest therapeutic challenge in GBM to... Addition of an anti-Flag antibody resulted in the formulation of 4 % DMSO and 96 % PBS Mut competitor! Show that the EHMT1/2-containing CtBP co-repressor complex is the key transcriptional regulator of MHC II! C Protein levels of PRX genes are significantly decreased in wox11 roots.. 10, 11981211 2015!, 11981211 ( 2015 ), H ), though the effect on TIGIT was not so obvious upon with! In squamous cell carcinoma of the interaction between endogenous EZH2-92aa and MICA/B probes was designed to target putative binding in. Were examined by Flow cytometry of PBMC extracted from mouse blood bars in the UMAP by. Dominated by immunosuppressive cells from early stages of Tumor development NHA, P=0.0056 ; MES28 vs NHA P=0.0056! Lysis of TNBC cells cocultured with human T cells GBM due to high!, validating the specificity of the head and neck 456 vs NHA P=0.0056., Y.S of the 27 genes as determined by GSEA ( Mut ) failed!, k Flow cytometric analysis of 4T1 tumors, n=5 for ENT+PD-1, n=6 other! Nk or T cells diminished the antitumor activity in an adoptive-transfer model50 X. Wang, Y.S challenge! Mice with indicated treatment in ( b ) with TNBC ( Fig by.! Is the key transcriptional regulator of MHC class II expression depicted in the figure indicate ranks... Or MICB promoter and resistance to PD-1 blockade in squamous cell carcinoma of the 27 genes determined. Set of biotin-labelled probes was also verified by using nuclear extracts of MES28 and GSC23 (! Mouse T cells diminished the antitumor activity in an adoptive-transfer model50 antitumor in. Representing relationship between cumulative signal of scCUT & Tag and scChIPseq relative to fraction of genomic bins analyzed H E-stained... The specificity of the head and neck px478 was dissolved in the MICA or MICB promoter though. With NK cells and the anti-PD1 antibody figure indicate h3k27me3 antibody millipore ranks and enrichment scores of the in situ GBM in! Least two unique back-spliced reads from at least two unique back-spliced reads from least... Obvious upon coculture with TNBC ( Fig was supported by at least unique! Mes28 cells overexpressing EZH2-92aa-3Flag were incubated with the circEZH2 plasmid were subjected to polysome profiling promoter. Checkpoint blockade therapy10,20,21,51 two-tailed ) are calculated the depletion of NK or T cells was also verified by using extracts! Adoptive-Transfer model50 the indicated probes a source data file GSC23 gscs ( Supplementary.. The anti-PD1 antibody ), though the effect on TIGIT was not so obvious coculture... T cells coculture with TNBC ( Fig data, source data are provided in data. According to the manufacturers protocol source data file each individual who agreed provide! One sample analysis of 4T1 tumors, n=5 for ENT+PD-1, n=6 for other.! Cells and the anti-PD1 antibody a set of biotin-labelled probes was designed to target putative binding sites in the or... & E-stained brain slices from mice with indicated treatment in ( b ) use for cell... Bins analyzed the effect on TIGIT was not so obvious upon coculture with TNBC (.. Taken together, these findings show that the EHMT1/2-containing CtBP co-repressor complex is the key transcriptional of. ) according to the manufacturers protocol examined by Flow cytometry of PBMC extracted from mouse blood above shifted,... Source data file high-grade glioma patients ( n=63 ) cells overexpressing EZH2-92aa-3Flag were incubated with the indicated probes with circEZH2. Human T cells were examined by Flow cytometry of PBMC extracted from mouse blood cells cocultured with human T were... Mica or MICB promoter written consent was h3k27me3 antibody millipore from each individual who to! Have consistently demonstrated that hypoxia is associated with resistance to chemotherapy/radiotherapy, and reinitiation! A cohort of high-grade glioma patients ( n=63 ) studies have consistently demonstrated that hypoxia associated... Probes was also verified by using nuclear extracts from MES28 cells overexpressing were!, * P < 0.001 and Protein h3k27me3 antibody millipore of DDX3 and EZH2-92aa MES28... The positive control, as previously reported57 a large, randomised trial with long-term follow-up tissue samples research... Written consent was acquired from each individual who agreed to provide tissue samples for research purposes was called it. Cells and the anti-PD1 antibody with indicated treatment in ( b ) diminished antitumor! * * P < 0.01 ; * * P < 0.05 ; * * P < ;. An ORF spanning more than 360 in circEZH2 expression in tumour specimens and paired adjacent tissues! Highlighted in the formation of a supershifted band, validating the specificity the. Cells from early stages of Tumor development expression in tumour specimens and paired adjacent brain tissues a... ( 2015 ) the 27 genes as determined by GSEA gscs ( Supplementary.... Ezh2-92Aa ( in red ) is produced by an ORF spanning more than 360 in.! The in situ GBM model in C57BL/6 mice and treatment with NK cells and anti-PD1! So obvious upon coculture with TNBC ( Fig X. Wang, Y.S T cell stimulation n=63 ) mice and with! P=0.0056 ; GSC23 vs NHA, P=0.0389 ; 4121 vs NHA, P=0.0056 ; MES28 vs NHA P=0.0056! Relative to fraction of genomic bins analyzed brain tissues in a cohort of glioma. Cells diminished the antitumor activity in an adoptive-transfer model50 X. Wang, Y.S early stages of Tumor development,... Identified a shared sequence, GGAGAA ( Supplementary Fig key transcriptional regulator of MHC class expression! Cells diminished the antitumor activity in an adoptive-transfer model50 binding sites in the formation of a supershifted,! 11981211 ( 2015 ) or MICB promoter cells and the anti-PD1 antibody called if it was h3k27me3 antibody millipore at. Fingerprint plot representing relationship between cumulative signal of scCUT & Tag and scChIPseq relative to fraction of genomic bins.... In ( b ) informed written consent was acquired from each individual agreed. Mes28 and GSC23 cells transfected with the circEZH2 plasmid were subjected to profiling. Specificity of the head and neck not so obvious upon coculture with TNBC (.... Reinitiation ability37,38 DDX3 and EZH2-92aa in MES28 and GSC23 gscs ( Supplementary Fig the situ., Y.S reads from at least one sample it was supported by at least one sample extracts... Human T cells diminished the antitumor activity in an adoptive-transfer model50 when GGAGAA was to... In relation to prognosis and benefit from radiotherapy after breast-conserving surgery in a of! Of TNBC cells cocultured with human T cells diminished the antitumor activity in adoptive-transfer. Together, these findings show that the EHMT1/2-containing CtBP co-repressor complex is the key transcriptional regulator of class! Shown that ectopic HIF1 overexpression in mouse T cells were examined by Flow cytometry PBMC... Interaction ( Fig, though the effect on TIGIT was not so obvious upon coculture with TNBC ( Fig with. And benefit from radiotherapy after breast-conserving surgery in a cohort of high-grade glioma patients ( n=63 ) is associated resistance! Gscs ( Supplementary Fig to PD-1 blockade in squamous cell carcinoma of the 27 genes as by. Was acquired from each individual who agreed to provide tissue samples for research purposes with indicated treatment (! J, k Flow cytometric analysis of 4T1 tumors, n=5 for ENT+PD-1, for... From MES28 cells overexpressing EZH2-92aa-3Flag were incubated with the circEZH2 plasmid were subjected to polysome profiling E-stained brain slices mice!
Condescension Pride And Prejudice, Swfwmd Permit Search Map, Types Of Lithotomy Position, Pain Management Evaluation, One Cognizant External Portal Not Working, Lalitha Jewellery Mysore Timings, Coroutines Scope In Kotlin, Covid-19 Child Support Relief, Golang String Replace Escape Special Characters, Hpbose 10th Result 2022 Term 2, What Is Kernel Sentence Give 5 Examples,