Among the peptides, [WR]5, which contains alternate tryptophan (W) and arginine (R) residues, was found to be the most efficient in the delivery of siRNA by improving the delivery by more than 3-fold when compared to other synthesized cyclic peptides that were not efficient. The data also showed that co-formulation of [WR]5 with lipid DOPE significantly enhanced the efficiency of siRNA delivery by up to ~2-fold compared to peptide alone. Based on the data indicating the efficiency of [WR]5 in siRNA delivery, peptides containing arginine residues on the ring and tryptophan residues on the side chain, [R6K]W6 and [R5K]W5, were also evaluated, and demonstrated improved delivery of siRNA. The presence of DOPE again enhanced the siRNA delivery in most cases. [WR]5, [R5K]W5, and [R6K]W6 did not show any significant toxicity in MDA-MB-231, MDA-MB-468, and AU565 WT cells at N/P ratios of 20:1 or less, in the presence and absence of DOPE. Silencing of kinesin spindle protein (KSP) and Janus kinase 2 (JAK2) was evaluated in MDA-MB-231 cells in the presence of the peptides. The addition of DOPE significantly enhanced the silencing efficiency for all selected peptides. In conclusion, peptides containing tryptophan and arginine residues were found to enhance siRNA delivery and to generate silencing of targeted proteins in the presence of DOPE.A number of amphiphilic cyclic peptides—[FR] Post-transcriptional regulation of protein expression is an important intracellular mechanism that relies on the interference of non-coding RNAs (RNAi) and a protein complex containing Argonaute protein (AGO) and cofactors . Human AThroughout the last two decades, efficient non-viral siRNA delivery strategies have been pursued, which include different physical approaches . PhysicaN-acetylgalactosamine  to siRNA or incorporating it into the siRNA delivery system has been recently reported 4, and the corresponding peptide-capped gold nanoparticles were found to act as efficient molecular transporters of siRNA in human cervix adenocarcinoma (HeLa) cells. The mechanism of uptake for [WR]4 and [WR]5 was found to be endocytosis independent, since no significant difference in cellular uptake was observed in the presence of different endocytic inhibitors [5 and [WR]5-capped gold nanoparticles improved the intracellular uptake of siRNA by about 2- and 3.8-fold versus siRNA alone. We also found out that both delivery platforms were less toxic when compared to lipofectamine. Fluorescence microscopy data confirmed the localization of fluorescence-labeled siRNA in cytoplasm in the presence of [WR]5 and [WR]5-capped gold nanoparticles 4 and [WK]5. The rationale of this design is based on the preliminary data that cyclic peptide [WR]5 containing alternative tryptophan (W) and arginine (R) residues significantly enhanced the cellular of negatively charged phosphopeptides [5K]W5, and [R6K]W6. The peptides were used alone or in different ratios with siRNA for comparative studies in two triple-negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) for cyclic peptides and an additional breast cancer cell line (AU565) for [R5K]W5 and [R6K]W6.Herein, we investigated [WR]peptides  and siRNpeptides . FurtherFurthermore, we have evaluated the effect of addition of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) to the delivery system. Enhancing the level of interaction with lipophilic moieties of cell membrane by increasing the hydrophobic characteristics of the carrier has been a well-studied strategy in delivering nucleic acid to cytoplasm. We have previously reported using DOPE as an additional hydrophobic component for hybrid peptides. In 2017, we reported siRNA delivery to breast cancer cell lines using arginine and lysine-containing linear and cyclic peptides conjugated to a variety of fatty acids to enhance the hydrophobicity of the carrier. Our experiments showed a significant improvement in cell internalization and silencing efficiency of the siRNA after addition of DOPE to the delivery system . Finallyv/v) as the matrix that was mixed with the peptides before the measurement. Hanks’ Balanced Salt Solution (HBSS), fetal bovine serum (FBS), penicillin , and streptomycin (10 mg/mL), and SYBR Green II were provided by Life Technologies . iScriptTM Reverse transcription Supermix and iTaq Universal SYBR Green Supermix used in qPCR, as well as all western blot accessories  were obtained from Bio-Rad . 4′,6-Diamidino-2-phenylindole (DAPI) was provided by Vector Laboratories . Phosphate-buffered saline , Phalloidin, Sulforhodamine 101 (Texas Red), and other reagents were purchased from VWR . Scrambled negative control siRNA , 5′-carboxyfluorescein (FAM)-labeled negative control siRNA , and the siRNA targeting kinesin spindle protein  were purchased from Ambion . The siRNAs targeting Janus kinase 2 (JAK2) , mLST8  were purchased from Qiagen . Primers for KSP , and JAK2  were designed and ordered from Integrated DNA Technologies . Monoclonal antibody against JAK2  and anti-rabbit polyclonal secondary antibody HRP-linked (7074V) were supplied by Cell Signaling Technology, Inc. .All amino acids and resins were purchased from AAPPTec  for in-house peptide synthesis. All the other chemicals and reagents used in synthesis and purification of peptides, as well as TRIzol reagent and formaldehyde (37%) were purchased from Sigma-Aldrich . 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was obtained from Avanti Polar Lipids Inc. . The chemical structures of the final products were confirmed by high-resolution matrix-assisted laser desorption/ionization (MALDI) time-of-flight  from Bruker Inc. . Mass spectra were collected using a freshly prepared α-cyano-4-hydroxycinnamic acid (5 mg/mL) in water (0.1% TFA) and acetonitrile (0.1% TFA) in a 1:1 ration (® HTB-26™), MDA-MB-468 , and AU565 . All cell lines were incubated for the duration of experiments in 37 °C and 5% CO2 level. Dulbecco’s modified Eagle’s medium low glucose (DMEM), containing L-glutamine, sodium pyruvate, and glucose was used for MDA-MB-231 and MDA-MB-468 cells. RPMI 1640 medium was used for AU565. All media were supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were sub-cultured after reaching > 80% confluency and were discarded after ~40 passages.Three different breast cancer cell lines were included in this study to represent a variety of phenotypes: MDA-MB-231 -OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Lys(Dde)-OH as building block amino acids. The appropriate resin, H-Arg (Pbf)-2-chlorotrityl resin  was swelled in DMF under dry nitrogen for 15 min, three times. The solvent was filtered off. The next Fmoc protected amino acid (3 equiv.) was coupled to the N-terminal of the arginine in the presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (3 equiv.), and N,N-diisopropylethylamine (DIPEA) (6 equiv.) in DMF by agitating under dry nitrogen for 1.5 h. The reaction solution was filtered off after the completion of the coupling. The resin was washed with N,N-dimethylformamide . Fmoc deprotection was performed by using piperidine in DMF . The reaction solution was filtered off, and the resin was washed with DMF . The subsequent amino acids were coupled and deprotected in a similar manner. Fmoc deprotection was performed on the last amino acid. Capping reaction by acetic anhydride (3 equiv.) and (DIPEA) (6 equiv.) was performed on the last amino acid. The resin was washed with DMF  and methanol . The resin was dried under vacuum for 4 h. The resin cleavage cocktail, dichloromethane:trifluoroethanol:acetic acid  was freshly prepared. The cleavage cocktail was added to the resin, and the solution was mixed for 3 h. The filtrate was evaporated under low pressure. Hexane (2 × 20 mL) and DCM (2 × 15 mL) were added to the residue to remove the acetic acid from the mixture. The crude material was solidified as a white solid. The crude peptide was dried under vacuum overnight.Synthesis of [FR]4, [WR]5 , and [WK[WR]5 W5: MALDI-TOF (m/z): C93H124N32O12, calculated: 1881.0076, found: 1883.8647 [M+2H]+;[R6K]W6: MALDI-TOF (m/z): C110H146N38O14, calculated: 2223.1881, found: 2225.0676 [M+2H]+W5 is shown in 5K]W5 was performed by using Fmoc solid-phase peptide synthesis. The appropriate resin, H-Arg(Pbf)-2-chlorotrityl resin was used as the solid support. Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, and Dde-Lys(Boc)-OH were used as building block amino acids in the assembly of the peptide on the resin. After the protected linear peptide formation was confirmed by MALDI mass spectroscopy . However, while similar improved internalization was observed with the addition of DOPE to the delivery system, the trend among the different N/P ratios was different. Without DOPE, an N/P ratio of 40 showed the highest mean fluorescence, where after the addition of DOPE, an N/P ratio of 20 showed the highest efficiency in terms of mean fluorescence. There was no significant difference in the percentage of cells positive for fluorescence signal in MDA-MB-468 cells among [WR]5 study groups, which was partially due to overall high percentages (69–77%) observed for all groups.The study of siRNA intracellular delivery using cyclic and hybrid (positively charged ring and hydrophobic chain) peptides was performed using flow cytometry in two triple-negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) for cyclic peptides and an additional breast cancer cell line  for hybrid peptides. We investigated the efficiency of three N/P ratios for each peptide, and also determined the effect of an additional hydrophobic moiety to the carrier by adding DOPE to the formulation of each peptide/siRNA complex . Among cP ratios A,B. The 5, the addition of DOPE increased the mean fluorescence for both peptides . On the other hand, more variability was observed in AU565 cells, where the highest mean fluorescence was observed for N/P ratios of 80 and 20 for [R5K]W5 with and without DOPE, respectively, and N/P ratio of 80 for [R6K]W6 both with and without DOPE. Statistically significant differences were less frequent in terms of percentage of the cells positive for fluorescence signal, mostly due to high percentages observed for most study groups . This significant difference was not observed for the percentage of siRNA-positive cells . A higher N/P ratio is usually expected to create a higher interaction with the negatively-charged cell membrane. In fact, a positive overall charge (> + 5 mV) is usually recommended for effective delivery of nucleotides ,27,28. In = 3–5) 5. These CD spectra are distinct from a helical structure. In general, [WR]5 shows a distorted helical structure that may explain more efficient cellular permeability. This distorted helical structure is less obvious in [WK]5. However, neither of the two peptides demonstrated fully helical structure. We have conducted preliminary CD studies on several new hybrid peptides along with [R5K]W5 and [R6K]W6. For example, CD study on [R5K]W7 showed a negative peak at about between 222 to 227 nm that is significantly different than [WR]5. The peak at 222 nm is consistent with an α-helical protein, but no peak at 208 nm was observed to confirm the helical structure. It also showed positive band near 287 and 295 nm in near UV, suggesting the stacking or self-assembly of tryptophan residues. We also studied the effect of increasing the overall hydrophobicity of the delivery system by adding DOPE to the formulation. We have previously reported the enhanced cellular internalization of siRNA with increased hydrophobicity through conjugation of fatty acids to PEI 2.0 kilodaltons  ( 3–5) W5, and [R6K]W6 in a wide range of N/P ratios (5 (BR50 = 4.0). This could be explained by steric hindrance caused by the hydrophobic side chain on [R5K]W5 and [R6K]W6 peptides. [R6K]W6 showed a higher affinity than [R5K]W5 , which was also expected due to presence of six positively charged nitrogens in the structure of [R6K]W6 (compared to five positive charges for [R5K]W5).The affinity to bind to siRNA was determined for [WR]P ratios A. Comple5 and [R6K]W6 (~200 nm), particles formed with [R5K]W5 had a larger diameter (~310 nm). However, while all particles had a positive surface charge, the ξ-potential of particles formed with [R6K]W6 was significantly higher than the other peptides. This is probably due to the extra positive charge on the structure of this peptide.The hydrodynamic diameter and the ξ-potential of the peptide/siRNA particles were studied at N/P ratio of 20 to further characterize the peptide-siRNA interaction B. While 5K]W5 showed no significant effect on the viability of the cells in the N/P range studied. However, both [WR]5 and [R6K]W6 showed signs of toxicity in N/P ratios of 40 (except [WR]5 without DOPE) and 80. The drop in viability was most significant for cells exposed to scrambled siRNA/DOPE/peptide complexes at N/P ratio of 80 . The level of toxicity of [WR]5 was most significant in MDA-MB-468 cells, where N/P ratio of 80 caused 18.2 and 6.6% viability with and without DOPE .Safety of a delivery system is a key characteristic for the clinical applications of the carrier. We investigated the toxicity of the designed peptides after complex formation with siRNA, at different N/P ratios ranging from 1 to 80, in all breast cancer cell lines included in the study . In MDA-out DOPE B. In AU5io of 80 C. Overal5, [R5K]W5, and [R6K]W6 alone (without siRNA complexation) in healthy cells, cell viability assay was conducted in LLCPK  at the experimental concentration of 25 μM. None of the compounds showed any significant cytotoxicity at this concentration in the selected cell line (data not shown).To evaluate the cytotoxicity of [WR]6K]W6 showed the highest level of toxicity in all cell lines, which is consistent with the expectation due to the presence of six primary amine groups compared to five groups for the other two peptides. [R5K]W5 showed the least level of toxicity in all three cell lines, and in fact the cell viability was not significantly different at N/P ratio of 80 compared to the ‘No Treatment’ group in MDA-MB-231 cells. This indicates that [R5K]W5 is a more promising carrier compared to [WR]5 in both efficiency and safety aspects. No significant toxicity was detected at N/P ratio of 20 for any of the peptides, and therefore this ratio was selected for protein silencing experiments to avoid the interference of toxicity with the silencing effect .As emphasized before, a correlation between the efficacy of cellular internalization and cytotoxicity is usually expected for positively charged siRNA carriers. An increased cytotoxicity was in fact detected with increasing N/P ratio for all selected peptides in all three cell lines, and was most significant with N/P ratio of 80. W5/DOPE delivery system . A CCK assay was also used to investigate the viability of the MDA-MB-231 cells after targeting KSP. A very similar trend was observed in the viability pattern of the study groups with minor differences: scrambled siRNA delivered with [WR]5 demonstrated a slight decrease in viability of the exposed cells  that was statistically significant. Also, unlike the real-time PCR results, where [R6K]W6/DOPE showed more significant efficacy in downregulating KSP mRNA compared to [WR]5/DOPE study group, no significant difference was observed in the effect of the two delivery systems on the viability of the cells targeted with KSP siRNA. Again, targeting KSP by [R5K]W5/DOPE showed the most significant effect on cell viability (20.2%; 5K]W5 and [R6K]W6 after addition of DOPE to the delivery system.We targeted two model proteins as a proof of concept using the designed peptides that showed significant uptake in human breast cancer cells. The effect of targeting KSP was analyzed at mRNA level by real-time PCR and on cell viability using CCK assay . Peptidey 20.2%; B; howeve5K]W5 (6K]W6 showed the highest level of siRNA cellular internalization among the designed peptides in this cell line with N/P ratios of 40 and 80, the lower silencing efficiency of this peptide is not surprising due to the lower N/P ratio selected to avoid non-specific toxicity.KSP has been identified as a viable target for siRNA silencing as a therapeutic approach in cancer therapy. Delivering siRNA via a variety of delivery systems targeting KSP in different cancer types has shown effective apoptosis induction and tumor growth retardation in vitro and in vivo ,38,39,405K]W5 . This is5K]W5 and [R6K]W6. [R5K]W5/DOPE again showed the most significant drop in mRNA level (~80% silencing; A similar study design was used to investigate targeting JAK2 protein in the same cell line, and in addition to the real-time PCR, western blot was performed to confirm the effect of siRNA delivery on the protein expression level . The ana5K]W5/DOPE delivery system. In order to confirm the silencing efficiency at the protein level, we performed western blotting to determine inhibition of JAK2 expression by this delivery system, which further confirmed the silencing efficiency of the selected carriers, in correlation with our observations in uptake and PCR studies.In order to further confirm the silencing efficiency of peptide/DOPE/siRNA complexes, JAK2 was selected as a second model protein. JAK/STAT pathway is a crucial signaling cascade known to be involved in the proliferation and survival of different cancer cells . Silenci5 and [WR]4 and the corresponding peptide-capped gold nanoparticles improved the cellar delivery of siRNA of siRNA . Consist


Macrophages are highly versatile cells, which acquire, depending on their microenvironment, pro- (M1-like), or antiinflammatory (M2-like) phenotypes. Here, we studied the role of the G-protein coupled receptor G2A (GPR132), in chemotactic migration and polarization of macrophages, using the zymosan-model of acute inflammation. G2A-deficient mice showed a reduced zymosan-induced thermal hyperalgesia, which was reversed after macrophage depletion. Fittingly, the number of M1-like macrophages was reduced in the inflamed tissue in G2A-deficient mice. However, G2A activation was not sufficient to promote M1-polarization in bone marrow-derived macrophages. While the number of monocyte-derived macrophages in the inflamed paw was not altered, G2A-deficient mice had less macrophages in the direct vicinity of the origin of inflammation, an area marked by the presence of zymosan, neutrophil accumulation and proinflammatory cytokines. Fittingly neutrophil efferocytosis was decreased in G2A-deficient mice and several lipids, which are released by neutrophils and promote G2A-mediated chemotaxis, were increased in the inflamed tissue. Taken together, G2A is necessary to position macrophages in the proinflammatory microenvironment surrounding the center of inflammation. In absence of G2A the macrophages are localized in an antiinflammatory microenvironment and macrophage polarization is shifted toward M2-like macrophages. Once having crossed the endothelium and entered the inflamed tissue they differentiate to macrophages and polarize toward M1 or M2 phenotypes depending on their microenvironment (Macrophages are heterogeneous immune cells that exhibit a high plasticity and can be roughly classified by two polarized end states named M1 (proinflammatory) and M2 (antiinflammatory)  is essential for efferocytosis of apoptotic neutrophils –9. Lysop2 is esse−/− mice develop secondary lymphoid organ enlargement associated with increased T- and B-cell numbers, causing at an age of more than 1 year to a slowly progressive wasting syndrome, which is associated with lymphocytic infiltration into various tissues  mice . All experiments were performed according to the recommendations of the Guide of the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the local ethics committee .We used 8–12 weeks old C57BL/6N mice  and G2A-deficient , autotaxin , CD45 , CD80 , CD86 , CD206 BioRad, clone MR5D3 catalog number MCA2235GA, and Biolegend, clone C068C2 catalog number 141707), F4-80 , IL1β  11-7114-82, Ly6C , Ly6G , and TNFα .In all cases the experimenter was unaware of the treatments or the genotypes of the mice. 10 μl Zymosan  were administered subcutaneously into the plantar side of one hind paw. Thermal thresholds were determined using the Hargreaves test . BrieflyFACS analyses for inflamed paws and bone marrow-derived macrophages were performed as follows : The tisFor FACS sorting cells were prepared as described above with the exception that NaN3 was omitted from the buffers. The cells were stained with antibodies against F4-80, Ly6C, and either CD86 or CD206 in 50 μl PBS/1% FCS in for 30 min at 4°C. Then the cells were washed once with PBS and sorted using FACS Aria III (BD Biosciences) , 28.For generation of bone marrow-derived macropghages  femur anBone marrow-derived macrophages were serum starved overnight and then stained with 10 μM 5(6)-CFDA, SE  for 10 min. They were detached with accutase, washed and transferred into 5 μM polycarbonate membrane transwell inserts. Inserts were transferred to plates supplied with RPMI medium and incubated for 2 h in absence or presence of 10% serum or 9-HODE (1 μM). Migrated macrophages were lysed and fluorescence intensities were measured using an Infinite F220 .CT−ΔΔ.Bone marrow-derived macrophages were differentiated for 7 days using 20 ng/ml M-CSF. Monocytes were isolated from peripheral blood of naïve mice with F4-80 magnetic beads . Macrophages were isolated from inflamed paws 24 h after zymosan injection by FACS sorting. RNA was isolated using mirVana miRNA Isolation Kit  and transcribed using First Strand cDNA Synthese Kit . RT-PCR was performed for G2A (Mm02620285_s1) and GAPDH (Mm99999915_g1) with a 7500 Fast System and TaqMan Gene Expression Assay . Relative mRNA expression is shown as 2The MELC technology is an immunhistological imaging method that uses directly labeled antibodies to allow the sequential visualization of 20–40 proteins on the same sample , 29. Bri+/Ly6C+ and F4-80+/Ly6C− macrophages, first all cells positive for F4-80 were counted in the respective areas (for example 0–100 μm distance from the border of the zymosan covered area). Only cells outside of blood vessels, which could be clearly distinguished from neighboring cells were included in the analysis to avoid false positive or negative signals. Then coexpression of F4-80+ and Ly6C+ were determined as described above. The number of Ly6C-positive and -negative F4-80-expressing cells was finally calculated as percent of all F4-80-expressing cells present in a defined region.Images were analyzed for coexpression using an established protocol , 29. To Expression profiles were generated using the Image J function Plot Profiles. Three linear expression profiles were taken for each image. The distance was calculated for each pixel in regard to the border of the zymosan containing area (0 μm).Cytokines within the paw edemas were determined 24 h after zymosan injection using ELISA kits from R&D Systems. The edemas were isolated, homogenized and lysed for 30 min in Cell Lysis Buffer 2 (R&D Systems) before analyses. To normalize cytokine concentrations to the protein content, protein concentrations were determined using the Bradford protein assay.Tissue from inflamed paws was prepared at the indicated times and lysed in PBS by sonication. Lactate was measured using the Promega Lactate-Glo Assay  following the manufacturer's instructions.LC-MS/MS analysis of LPAs, LPCs, HETEs, and HODEs were deermined using a hybrid triple quadrupole-ion trap mass spectrometer QTrap 5500 , an Agilent 1260 HPLC binary pump, column oven, and degasser , and a HTC Pal autosampler . For details please see Supplemental Experimental Procedures.t-test were applied. p < 0.05 were considered statistically significant.All data are presented as mean±S.E.M. To determine statistically significant differences Two-way or One-way variants of ANOVA or Student's −/− mice 24 h after zymosan-injection  and monocyte-derived macrophages (F4-80+/Ly6C+/CD45+)   and M2-like (F4-80+/Ly6C+/CD206+) macrophages isolated from edemas 24 h after zymosan-injection -mediated paw inflammation is a common model to study mechanisms of acute tissue inflammation and inflammatory pain (nociception). Here, we compared the nociceptive response of wild type and G2A-deficient mice during paw inflammation induced by the TLR2-agonist zymosan. We observed a significantly decreased thermal hyperalgesia in G2An Figure , a time n Figure , 28. Sinn Figure , or clodn Figure  to deplen Figure . While Pe Figure . FACS and Figure  suggestid Figure  and FACS) Figure . However) Figure . Notablyn Figure . G2A mRNd Figure . Thus, s−/− mice had decreased numbers of M1-like macrophages, we studied whether or not G2A has a direct effect on macrophage polarization toward M1-like phenotypes. To ensure that G2A expression is sufficient to mediate biological activity, we first tested the G2A-agonist 9-HODE for its known ability of G2A to induce chemotaxis in macrophages  in relation to the distance from the border of the zymosan-covered region. Here, only cells outside of blood vessels, which could be clearly distinguished from neighboring cells were included in the analysis. We found that in wild type mice the percentage of F4-80+/Ly6C+/CD45+ macrophages of all F4-80+ cells dropped with increasing distance from the zymosan-containing area from 88% (0–100 μm) to around 50% (200-300 μm)  cells were preferentially located outside the zymosan-containing area in distances of >100 μm from the zymosan border , which sensitize peripheral sensory neurons  by G2A contributing to chemotherapy-induced painful neuropathies . However neurons –33. Howe neurons –33. The 2), and M2-like macrophages, which release mediators  that can counteract this sensitization. Since the status of the nociceptor sensitization, and therefore the pain response, depends on the balance of the pro- and antinociceptive mediators released within the inflamed tissue (Surprisingly, depletion of all macrophages by clodronate had no net effect on the nociceptive response. Clodronate depletes both, M1-like macrophages, which release pronociceptive mediators that sensitize nociceptors (i.e., IL1β, TNFα, PGEd tissue , 33, thed tissue .In summary, the data show that the G2A receptor mediates migration of macrophages along a proinflammatory axis toward sources of LPCs and, possibly, 5-HETE during zymosan-induced inflammation and thereby indirectly affects their polarization causing an antinociceptive effect. Thus, G2A seem to fulfill two opposing roles in macrophage polarization. On one side by promoting M2-like phenotypes through direct G2A activation and on the other side by promoting M1-like macrophage phenotypes by positioning macrophages in a proinflammatory microenvironment.in vivo experiments and performed FACS analyses. KK, MS, and SH performed RT-PCRs. GG and NF provided LC-MS/MS analysis data. AW performed FACS sorting. BB provided reagents. KS, TO, and SP performed MELC analysis. KS, KK, and SP designed experiments and wrote the paper. All authors read and approved the manuscript.KK, SS, NT, and JC did the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CD19-CAR-T therapy remarkably reduced the number of leukemia cells, and three patients achieved bone marrow remission . The efficacy of CD19-CAR-T therapy on B-ALL was positively correlated with the abundance of CAR and immune cell subpopulations, e.g., CD8+ T cells and natural killer (NK) cells, in the bone marrow. Additionally, CD19-CAR-T therapy mainly influenced the expression of genes linked to cell cycle and immune response pathways, including the NK cell mediated cytotoxicity and NOD-like receptor signaling pathways. The regulatory network analyses revealed that microRNAs , acting as oncogenes or tumor suppressors, could regulate the crosstalk between the genes encoding transcription factors  and histones  involved in CD19-CAR-T therapy. Furthermore, many long non-coding RNAs showed a high degree of co-expression with TFs or histones  and were associated with immune processes. These transcriptome analyses provided important clues for further understanding the gene expression and related mechanisms underlying the efficacy of CAR-T immunotherapy.Chimeric antigen receptor (CAR) T cell therapy has exhibited dramatic anti-tumor efficacy in clinical trials. In this study, we reported the  As one of the main types of leukemia, B cell acute lymphoblastic leukemia (B-ALL) is caused by the occurrence of genetic abnormalities in B cells, leading to the aberrant arrest of normal lymphoid maturation, evasion of apoptosis, and uncontrolled cell proliferation e.g., remission or non-remission) could provide insights into the underlying molecular mechanisms involved in CAR-T therapy.Most studies on CAR-T immunotherapy were focused on the clinical efficacy and new antigen development. However, few studies have investigated the alterations in gene expression and regulation of patients after CAR-T therapy. Transcriptome profiling has been widely used to investigate molecular mechanisms underlying the recurrence and therapy of cancers In this study, we investigated the clinical outcome and analyzed transcriptome profiles of bone marrow (BM) samples before and post CD19-CAR-T therapy from 4 adult patients with refractory B-ALL. Based on the analysis of differentially-expressed genes (DEGs), long non-coding RNAs (lncRNAs), and miRNAs, we proposed a schematic model of regulatory networks involved in the CD19-CAR-T therapy on B-ALL.Four patients approved for clinical trials of CD19-CAR-T therapy were selected in this study for further analysis . The worin vivo at 2 weeks post infusion ex vivo expansion  >1 for genes and transcripts per million reads mapped (TPM) >10 for miRNAs . Basic sTo investigate whether CAR-T therapy influenced the composition of T cell receptors (TCRs) in the neoplastic nidus, we examined the distribution of complementarity-determining region 3 (CDR3) sequences in RNA-seq data. A total of 685 CDR3 sequences were identified across samples. The R-D-D14 sample contained the highest number of CDR3 sequences, while the R-C-D14 sample had the lowest number. The number of CDR3 sequence varied among samples, and no dominant CDR3 sequence was found. The frequency of CDR3 sequences varied from 1% to 23% D, sugges+ T cells in the NR patient was notably different from the others  in leukemic pre-B cells were markedly decreased after CAR-T infusion in remissive patients  when comparing the gene expression levels before and after CD19-CAR-T infusion for each patient . Eighty ing DEGs B, but oning DEGs B and C, To further investigate biological functions of DEGs underlying CD19-CAR-T therapy, we performed the KEGG pathway and Gene Ontology (GO) enrichment analysis for all DEGs found in each patient. The top 20 enriched pathways and top 15 biological process terms are presented in P = 1.55E−9). Notably, 9 of 18 modules were highly correlated  with the apoptosis, cell cycle, and immune-related pathways, such as the NK cell mediated cytotoxicity, NOD-like receptor signaling pathway, and phagosome terms    to identify functional biological modules. In total, 18 co-expression modules were detected A, among me terms . The turctively) , which wctively) C. While ctively) . These rR > 0.9 and P < 1E−6)  . The lnc < 1E−6) . AlthougIST1H2BL .HIST1H4A and HIST2H4Ae.g., CEBPE and CHD4) and participates in the immune response pathways . Furthermore, the expression levels of tumor microenvironment related genes were dramatically changed after CAR-T infusion, such as immunostimulator/IL family/IFNR/GFR/chemokine families members, which may enhance the proliferation and activation of CAR-T cells and thus increase the anti-tumor activity Impressive results have been reported using CD19-CAR-T cells to treat patients with refractory B-ALL  patient B may be JUN or CEBPB and histone genes HIST1H4A, HIST1H4C, and HIST1H4EHIST1H4A and HIST2H4A is regulated by the TFs JUN and CEBPB as well HCG4P7. HCG4P7 is reportedly as an important immune regulatory molecule FOSDespite the differences in transcriptome profiles among these patients, most of the enriched DEG terms are related to the immune response and cell cycle . Our datAlthough biological replications are limited due to the restrictions of medical ethics, previous studies have shown that the cancer cells of leukemia are homogeneously dispersed in the BM compared with the solid cancers in vivo expansion of CAR-T cells in vivo expansion of CAR-T cells and the resulting alterations in immune cell population of CD19-CAR-T therapy on B-ALL. These could help to characterize clinically important features and develop treatments for patients with different conditions. Furthermore, our study suggests that the histone genes combined with their co-expressed lncRNAs and TFs, as well as the miRNA–TF–gene regulatory networks, may play vital roles in CD19-CAR-T therapy on B-ALL. These findings indicate an impact of these factors or modules for the CD19-CAR-T therapy on B-ALL, and may provide valuable clues for understanding the transcriptional and post-transcriptional regulatory mechanisms underlying CAR-T immunotherapy on cancers.Genetically-modified CAR-T cells act as “living drugs” to enable constant cytotoxic attacks on targeted malignant cells. The efficacy of CAR-T therapy depends on the tumor-specific antigens and further etc., were performed according to established standard operating procedures and protocols in the central laboratory at the Wuhan Union Hospital, China. Huazhong University of Science and Technology and the Wuhan Union Hospital ethics committees reviewed and approved this trial. All patients enrolled and treated in this trial gave written informed consent before participation. All clinical investigations were consistent with the Declaration of Helsinki. Only patients with relapsed or refractory B-ALL after standard therapies were deemed eligible for the CD19-CAR-T therapy.All procedures in this trial, including sample collection, processing, freezing, and laboratory analysis The CD19-CAR transgene comprises five parts: CD19 single-chain variable fragment, CD8 hinge, CD8-α transmembrane, 4-1BB costimulatory domain, and CD3 zeta chain . The tra2 fludarabine and 750 mg/m2 cetuximab) for 5 days to control the MRD level below 20%. Afterward, patients received a fractionated infusion of CD19-CAR-T cells .To improve the efficacy of CAR-T therapy, patients were pre-treated with a conditioning chemotherapy agent (30 mg/mex vivo expansion (D0-EV) and in the PB and BM samples collected from patients on D14 were determined with flow cytometry. The MRD level in BM samples was measured using flow cytometry  on D0 and D30. Patients with the MRD level <0.5% after 2 weeks were considered as CR. The presence of BCR-ABL fusion transcript in BM samples was detected with a real-time PCR system  with the primers (F: 5′-ACATCACGCCAGTCAACAG-3′ and R: 5′-GACGTAGAGCTTGCCATCAGA-3′). The tumor burden (TB) was calculated as the percentage of tumor cells among all karyocytes in BM samples on D0. Concentrations of cytokines in PB samples were determined with ELISA and with CRS grades (1–4) evaluated accordingly.The PB and BM samples were obtained from patients on D0, D14, and D30. The percentage of CAR-T cells and normal cells in the CAR-T cell culture after Total RNA was isolated from the BM samples of all four patients on D0 and D14 using the standard TRIzol protocol. The RNA quality was determined with the Agilent 2100 Bioanalyzer. Libraries for RNA-seq (Ribo-Zero) and small RNA sequencing were prepared according to Illumina’s TruSeq protocol. The libraries were sequenced on the Illumina Hiseq platform with the 2 × 150 bp paired-end strategy at BGI-Shenzhen . Base-calling was performed using the Illumina CASAVA v1.8.2 pipeline.RNA-seq reads containing <35 bp after adapter trimming or with poly-N or many low-quality bases  were removed. For small RNA sequencing reads, we filtered reads containing any N base or with a length >40 nt or <17 nt. The Q20, Q30, and GC content of the clean sequencing reads were calculated. All of the downstream analyses were based on the clean and high-quality sequencing reads.Sequencing data obtained from the BM at day 0 and 14 days after CAR-T infusion were analyzed using various bioinformatics tools. The detailed procedures of transcriptome profiling, such as gene/miRNAs expression analysis, immune cell proportion estimation, functional enrichment analysis, and co-expression regulatory network analysis are presented in File S1.CRA000746, which is publicly accessible at http://bigd.big.ac.cn/gsa.Sequencing data in this study have been deposited in the Genome Sequence Archive AYG, QZ, YW, and JY conceived the project. AYG, QZ, and WY supervised the study. WY and JY performed the clinical trial and biochemical examinations. HH, QZ, SC, FH, and CL performed the bioinformatics analysis. HH and QZ drafted the manuscript with the help of WY. AYG, QZ, and HH revised the manuscript. All authors read and approved the final manuscript.The authors declare that they have no competing interests.


Tunicamycin induces both glycosylation depression and ubiquitination improvement of P-gp. However, the latter is not associated with large increases in molecular mass as evidence for polyubiquitination. Therefore, P-gp continues in maturation to an active membrane efflux pump rather than proteasomal degradation. P-gp-positive L1210 cells contain a higher quantity of ubiquitin associated with cell surface proteins than their P-gp-negative counterparts. Thus, P-gp-positive cells use ubiquitin signaling for correct protein folding to a higher extent than P-gp-negative cells. Elevation of protein ubiquitination after tunicamycin treatment in these cells leads to protein folding rather than protein degradation, resulting at least in the partial lack of cell sensitivity to tunicamycin in L1210 cells after P-gp expression. In contrast to tunicamycin, to understand why P-gp-positive cells are resistant to GalNAc-α-O-benzyl, further research is needed.Overexpression of P-glycoprotein  in neoplastic cells is the most frequently observed molecular cause of multidrug resistance. Here, we show that the overexpression of P-gp in L1210 cells leads to resistance to tunicamycin and benzyl 2-acetamido-2-deoxy-α- The development of a specific cell phenotype may include several alterations in the composition of sugars linked to proteins and lipids that form specific glycoms typical for this phenotype. Processes of tissue cell carcinogenesis are frequently associated with the strong remodelation of cell sugar composition ,2 detectMaackia amurensis agglutinin (MAA) and Sambucus nigra agglutinin (SNA) [Lycopersicon esculentum agglutinin (LEA) attached more effectively to the cell surface of P-gp-positive R and T cells than to P-gp-negative S cells [d-galactopyranoside , are lacking, reflecting the fact that N-glycosylation is a major posttranslational modification of P-glycoprotein [O-benzyl; (ii) to study the effects of both glycosylation inhibitors on binding of ConA and Galanthus nivalis agglutinin (GNA) to cell surface and membrane proteins; (iii) to study the effect of tunicamycin on P-gp ubiqutination in R and T cells.We have described several alterations in the binding of concanavalin A (ConA) to intracellular  or plasmin (SNA) . Changesin (SNA) . Alteratin (SNA) ,21. The in (SNA) . In a prin (SNA) . In cont S cells ,18, and  S cells ,16, did  S cells  or gluco S cells . Tunicam S cells . Data cooprotein . The inhoprotein . Howeveroprotein ,27. OnlyBoth R and T cells express large amounts of P-gp at the mRNA and protein levels as detected using RT-PCR or western blotting, respectively . The P-gO-benzyl and tunicamycin, respectively. S, R and T cells were passaged 1–3 times in the absence or presence of 0.1 mmol·dm−3 GalNAc-α-O-benzyl or 0.1 μmol·dm−3 tunicamycin in cultivation medium, and subsequently, the viability of the cells was assessed. After the first passage, tunicamycin induced a decrease in the number of viable cells to approximately 70% of the control value in all three variants of L1210 cells . In contrast, after the third passage, S cells could not be effectively passaged in the presence of either GalNAc-α-R and T cells contain massive protein bands with molecular masses in the 160–185 kDa region that can be immunodetected using a c219 mouse monoclonal antibody against P-gp a. In conO-benzyl did not alter the P-gp glycosylation status, and fully glycosylated P-gp variants with molecular masses in a similar range as unaffected control were observed. GNA was described to interact with high mannoses directly associated with P-gp through N-glycosylation [O-benzyl. However, R and T cells treated with tunicamycin did not contain protein bands in this molecular mass region that bind to GNA . Untreated and cells treated with tunicamycin or GalNAc-α-O-benzyl was altering the binding of ConA  bound to ConA more effectively as their P-gp-positive counterparts R and T cells b. More p of ConA b or GNA  of ConA c onto thTotal protein lysates isolated from R and T cells cultivated for four passages in the presence or absence of tunicamycin were immunoprecipitated using an anti-ubiquitin polyclonal antibody. The immunoprecipitate was further analyzed using western blotting with a c219 antimouse antibody a. Small O-benzyl and tunicamycin). Resistance of P-gp-positive cells to GalNAc-α-O-benzyl during repeated passages of cells in the presence of this inhibitor (O-benzyl improve pancreatic cancer cell sensitivity to 5-fluorouracil [O-benzyl.Both P-gp-positive variants of L1210 cells (R and T) are resistant to inhibitors of O- and N-glycosylation Tunicamycin may be a substrate for P-gp efflux and therefore could effectively be eliminated from the intracellular volume of P-gp-positive cells. While this idea has previously been discussed , direct (b)Tunicamycin induced the global inhibition of glycoprotein synthesis  associated with the elevation of immature proteins cell content leading to endoplasmic reticulum stress and apoptosis . P-gp-po(c)The application of tunicamycin increases the cellular level of UDP-N-acetylglucosamine, associated with different features of cell damage visible using electron microscopy, namely, in the membranes of the endoplasmic reticulum and nuclei . SignifiThe resistance of P-gp-positive leukemia cells to tunicamycin has recently been described for mouse L1210 and human SKM-1 or MOLM-13 cells ,27. The Tunicamycin also abolish specific P-gp N-glycosylation detectable with GNA either in R or T cells . GNA ligO-benzyl induced detectable changes in ConA or GNA binding to the surfaces of S, R and T cells measured using flow cytometry (Differences in cell binding capacity for ConA between S cells that bound larger amounts and R or T cells that bound lower amounts are shown in ytometry . Similarytometry .r 8500 kDa) can be covalently attached to lysines of target proteins either as a monomer or as a lysine-linked polymer. The ubiquitinated proteins showed an increase in molecular weight compared to the non-modified form, and the precise molecular weight of the modified proteins depends on the number of ubiquitins on the modified proteins [Several lines of evidence indicate that the lack of P-gp N-glycosylation induced by tunicamycin leads to the elevation of ubiquitination and subsequent proteasomal degradation of unglycosylated proteins . This efproteins . On a siproteins . Ubiquitproteins . Polyubiproteins . Taken t6 cells) were cultured in 4 cm3 RPMI 1640 media with l-glutamine (1 mg cm−3), 4% fetal bovine serum and 1 μg cm−3 gentamycin  in a humidified atmosphere with 5% CO2 and air at 37 °C for 48 h in the absence or presence of either tunicamycin (0.1 μmol·dm−3) or GalNAc-α-O-benzyl (0.1 mmol·dm−3). This procedure was termed as passage and was repeated three times. Numbers of viable cells after each passage were counted using a CASY Model TT Cell Counter . R cells were cultured for two passages without VCR prior to the experiments.The following three L1210 cell variants were used in this study: (i) S-drug-sensitive parental cells obtained from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH  ACC-123; (ii) R—P-gp-positive drug-resistant cells that overexpress P-gp after selection with vincristine  obtained−3) or GalNAc-α-O-benzyl (0.1 mmol·dm−3). Total cell proteins were fractionated to crude membrane fraction (CMF) and cytosolic fractions (CF) using the ProteomeExtract Subcellular Proteome Extraction Kit  according manufacturer’s protocol. Proteins from CMF (30 μg per line) were separated via sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) on polyacrylamide gradient gels (8–16%) using protocol published by Laemmli [P-gp and other membrane glycoproteins were detected by either western blot using specific antibody or Eastern blot using lectins (ConA and GNA) in crude membrane fractions isolated from S, R and T cells three times passaged in the absence or presence of either tunicamycin  were thermally denatured in 0.1 mol dm−3 phosphate buffer (pH 7.2) containing 20 mmol·dm−3 EDTA, 50 μmol·dm−3 SDS and 2.6 mm3 per dm3 β-mercaptoethanol for 15 min at 100 °C. Subsequently, the samples were cooled and 66 μmol·dm−3 of NP-40 (Sigma-Aldrich) were added. Either PNGase or Endo H  were added to the sample and incubated for 14 h at 37 °C. The samples were analyzed via Eastern blotting with GNA (see previous section).Membrane proteins 2 mg/cm−3) or GalNAc-α-O-benzyl (0.1 mmol·dm−3), the cells were harvested by centrifugation and washed three times with phosphate-buffered saline , resuspended in RPMI medium without fetal bovine serum (5 × 105 cells/cm3) and incubated for 60 min with FITC-labeled ConA or GNA (EY Laboratories Inc.) at a concentration of 1 mg/L in a humidified atmosphere supplemented with 5% CO2 at 37 °C [After three passages in the absence or presence of either tunicamycin (0.1 μmol·dmat 37 °C . After i−3). Subsequently, the cells were harvested, and whole-cell lysates were prepared by homogenization in SoluLyse  according to the manufacturer’s instructions. The protein concentration was determined using the Lowry assay and used for immunoprecipitation. The proteins (80–100 μg) from R and T cells were adjusted to a final volume of 300 μL using 50 mmol·dm−3 Tris-HCl (pH 7.0) and anti-ubiquitin antibody  was added. After 2 h of incubation at 4 °C, 20 μL of protein A/G PLUS-agarose (Santa Cruz Biotechnology) was added, and the mixture was incubated overnight at 4 °C. Subsequently, the precipitates were pelleted by centrifugation  and washed two times with 50 mmol/L Tris-HCl buffer. The precipitates were applied on 8% SDS-PAGE and subsequently the proteins were transferred by electroblotting onto nitrocellulose membrane, and the presence of P-gp in the immunoprecipitate was detected using the same antibodies as described for the Western blot procedure. Signal of rabbit IgG heavy chain originated from anti-ubiquitin antibody was detected as internal standard using goat anti-rabbit antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology).R and T cells were passaged four times in the presence or absence of tunicamycin  but with 5% of defatted BSA (Sigma Aldrich) and incubated for 240 min with anti-ubiquitin antibody (described in previous section) in a humidified atmosphere supplemented with 5% CO2 at 37 °C. After incubation, the cells were washed three times with RPMI medium containing 5% BSA and subsequently left to interact with secondary antibody . The labeled cells were either counted in the BD Accuri C6 flow cytometer or were evaluated using the InCell 2000 Analyzer .R and T cells were passaged four times in the presence of 0.1 μmol/dmO-benzyl. This resistance could be recognized as a side effect of P-gp overexpression in these cells and may be independent of P-gp efflux activity. Tunicamycin inhibits P-gp specific N-glycosylation. In addition to this specific feature, both GalNAc-α-O-benzyl and tunicamycin did not induce detectable changes in the content of glycoprotein ConA and GNA ligands in either crude membrane fraction proteins or on the cell surface. However, ConA interacts with S cell glycoproteins in the crude membrane fraction or on the cell surface at a higher extent than in R and T cells. R and T cells contain higher amounts of ubiquitin linked with cell surface protein than S cells. Tunicamycin induced elevation of P-gp ubiquitination, but with no pronounced increase of molecular mass, excluding the likelihood of massive polyubiquitin labeling. Therefore, in these cells, P-gp is not sufficiently ubiquitinated for recognition in proteasomal degradation and continues maturation to a fully active membrane efflux pump. While the GalNAc-α-O-benzyl resistance of R and T cells is not understood, the resistance of these cell variants to tunicamycin could reflect alterations in ubiquitination mechanisms. The fact that the surface of R and T cells in the absence of tunicamycin contained higher amounts of ubiquitin indicated by the utilization of protein ubiquitination during cell maturation at a higher extent than in S cells. The acceleration of protein ubiquitination in these cells after tunicamycin treatment leads to protein folding rather than protein degradation.P-gp-positive variants of L1210 cells are also resistant to tunicamycin and GalNAc-α-


VisioProt-MSis highly compatible with many algorithms and software developed by thecommunity to integrate and deconvolute top-down and intact protein MS data. Itsdynamic and user-friendly features greatly facilitate analysis through severalgraphical representations dedicated to MS and tandem mass spectrometry (MS/MS)analysis of proteoforms in complex samples. Here, we will illustrate theimportance of LC-MS map visualization to optimize top-down acquisition/searchparameters and analyze intact protein MS data. We will go through the mainfeatures of VisioProt-MS using the human proteasomal 20S core particle as auser-case.The rise of intact protein analysis by mass spectrometry (MS) was accompanied byan increasing need for flexible tools allowing data visualization and analysis.These include inspection of the deconvoluted molecular weights of theproteoforms eluted alongside liquid chromatography (LC) through theirrepresentation in three-dimensional (3D) liquid chromatography coupled to massspectrometry (LC-MS) maps . With this aim, we developed a free andopen-source web application named VisioProt-MS ( Comment on: Locard-Paulet M, Parra J, Albigot R, et al. VisioProt-MS:interactive 2D maps from intact protein mass spectrometry.Bioinformatics. 2019;35(4):679-681. doi:10.1093/bioinformatics/bty680. PubMed PMID:30084957. PubMed CentralPMCID:PMC6378940. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378940/.1 and became quantitative,3 thereby allowing the in-depthcharacterization of proteoforms4 in complex samples.5 Such development was associated with the establishment of a specificlexicon,7dedicated databases (repository.topdownproteomics.org), and, needless to say, a panel ofbioinformatics tools.811Top-down proteomics consists in the analysis of intact proteins using liquidchromatography coupled to mass spectrometry (LC-MS), followed by theiridentification by tandem mass spectrometry (MS/MS). This informs on the proteincomposition of the analyzed sample, and their potential combinations ofpost-translational modifications, splicing events, and/or mutations. Intact proteinmass spectrometry (MS) recently increased in throughputx-axis representsretention time (RT), the y-axis represents deconvoluted MW, and thecolor represents the intensity of the MS signal. In addition, directly comparingthese maps reveals differences in proteoform footprints between samples and/orexperimental conditions.1216 Recently, we developed a freestandalone tool to facilitate this analytical step: VisioProt-MS.17 Here, we will illustrate its use through the analysis of the 20S coreparticle of the human proteasome.Typically, top-down and intact protein MS analysis relies on the measurement of thedeconvoluted molecular weights (MWs) of proteoforms after separation by liquidchromatography (LC). This can be facilitated by the graphical representation ofLC-MS three-dimensional (3D) maps, where the 18 It is vital to maintain protein homeostasis and the pool of free amino acidsavailable for protein synthesis. It also contributes to the immune response throughproduction of antigenic peptides. Its catalytic activity resides in the 20S coreparticle that is composed of 4 rings of 7 subunits each: two β-rings (β1-7)surrounded by two α-rings (α1-7). The names and theoretical MWs of these subunitsare presented in 19 The proteasome is highly studied in academia and industry in the context ofdrug development. The 20S core particle can be immunopurified using the anti-α2antibody MCP21,21 and as it is constituted of subunits of ~20 to ~30 kDa, it isspecifically suited for top-down and intact protein MS analysis.8 We present here the comparative top-down analysis of in-house-produced std20Sand commercial samples of purified std20S and i20S using VisioProt-MS.The proteasome is a ubiquitous macromolecular barrel-shaped complex of around 700 kDathat is responsible for protein degradation in eukaryotic cells.All reagents were provided by Sigma-Aldrich unless otherwise specified.Commercial standard and immuno-20S were purchased from Enzo Life Science.2. Cells were then washed twice withphosphate-buffered saline (PBS) and stored at ‒80°C. On the day of experiment,50 × 106 cells were lysed with 2 mL of lysis buffer , incubated for 15 minutes at 4°C, and sonicated. Non-soluble debris were removed by centrifugation(4000g for 15 minutes at 4°C) and the protein concentrationin the supernatant was determined by detergent-compatible (DC) assay (Bio-Rad).Aliquots were kept at ‒80°C until analysis. Immunopurification of the endogenous20S core proteasome was performed as described in Fabre et al.21Endogenous 20S was purified from Hek293-EBNA cells grown to 80% confluency inIscove’s Modified Dulbecco’s Medium  supplemented with 10%fetal bovine serum (FBS), 116 mg/mL L-Arginine, and 36 mg/mL L-asparagine (AcrosOrganics) at 37°C and 5% CONano-LC-MS and MS/MS analyses of commercial or immunopurified 20S were performedon a nanoRS UHPLC system (Dionex) coupled to an LTQ-Orbitrap Fusion Tribrid massspectrometer (Thermo Fisher Scientific). A total of 5 μL of sample at 0.3 µM wasloaded onto a reverse-phase C4-precolumn  at 20 μL/min in 2% acetonitrile (ACN) and 0.2% formic acid (FA).After 5 minutes of desalting, the precolumn was switched online with ananalytical C4 nanocolumn equilibrated in 95% solvent A  and 5% solvent B (0.2% FA inACN). Proteins were eluted using a binary gradient ranging from 5% to 40% (5minutes) and then 40% to 99% (33 minutes) of solvent B at a flow rate of 300nL/min. For the commercial 20S samples, the Fusion Tribrid (Thermo FisherScientific) was operated in single MS acquisition mode with the Xcalibursoftware (Thermo Fisher Scientific). The spray voltage was set to 1900 V, theion transfer tube temperature to 300°C, the RF lens to 60%, and the in-sourcedissociation to 50 V. The MS scans were acquired in the 700 to 2000 m/z rangewith the resolution set to 60 000 and using 10 µscans for averaging. For theintact protein and top-down MS analysis of the immunopurified std20s, the sprayvoltage was set to 1350 V, the ion transfer tube temperature to 270°C, the RFlens to 60%, and the in-source dissociation to 50 V. The MS and MS/MS scans wereacquired in the 400 to 2000 m/z range with a resolution of 120 000 and using 3µscans for averaging. Ions of interest were selected according to an inclusionlist of 14 precursor masses (corresponding to the 20S subunits) that wereanalysed by MS/MS with the option “DDA if parent mass list not found” unchecked.The isolation window was set to 5 Th with electron-transfer/higher-energycollision dissociation (EThcD) fragmentation (electron-transfer dissociation[ETD]: 20 ms and higher-energy collision dissociation [HCD]: 25%).8 and the proteoform footprints were visualized with VisioProt-MS v2.0.17 Top-down raw data were analyzed with Proteome Discoverer v2.2 (ThermoScientific) using the ProSight PD Top-Down Low/High node. Briefly, intactprotein spectra were deconvoluted with ReSpect .Tandem mass spectrometry spectra were deconvoluted with Xtract  and searched against acustom database including all human 20S subunits . The search was performed in absolute mass mode with afragment mass tolerance of 15 ppm and a precursor mass tolerance of 200 Da. TheMS/MS and proteoform-spectrum matches were visualized with VisioProt-MS. All thefigures were adapted from VisioProt-MS exports using Adobe Illustrator CS6v16.0.0.For MS traces, raw files were automatically deconvoluted with the rolling windowdeconvolution software RoWinPro22 using intact protein and top-down MS. VisioProt-MS allowed the visualizationof the most intense proteoforms based on their deconvoluted MWs  sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation coupled withanalytical techniques .VisioProt-MS is an easy solution to visualize and inspect intact protein and top-downMS data. It quickly provides an overview of all the detected MWs, reflecting dataquality and reproducibility regarding observed MWs, intensities, and RTs. It allowscomparison of not only multiple LC-MS runs (including from different deconvolutionsuites), but also LC-MS and LC-MS/MS runs of the same sample. Furthermore, itsdynamic features enable to pinpoint potential new proteoforms, quickly rejectwrongly assigned Proteoform Spectral Matches, and spot intense MS signals thatremain unassigned.8 Intact Protein Analysis ,DataAnalysis 4.2 (Bruker),9 TopFD (TopPIC Suite),10 and ProMex (Informed-Proteomics)11 for deconvoluted LC-MS data; and ProSight PD ,TopPIC (TopPIC Suite),10 and MSPathFinder (Informed-Proteomics)11 for the LC-MS/MS data. It is open source and has been developed to be easilyadaptable to other formats that may materialize from future technologicaldevelopment. Alternatively, it can be adapted and included in more complexworkflows. All the information concerning its features, compatibility, and usagescan be found in Locard-Paulet et al17 and on the associated online help (https://masstools.ipbs.fr/visioprothelp.html).Today, VisioProt-MS v2.0 is compatible with the following bioinformatics tools: RoWinPro,


Moraxella catarrhalis is a human-adapted pathogen, and a major cause of otitis media (OM) and exacerbations of chronic obstructive pulmonary disease. The species is comprised of two main phylogenetic lineages, RB1 and RB2/3. Restriction–modification (R-M) systems are among the few lineage-associated genes identified in other bacterial genera and have multiple functions including defense against foreign invading DNA, maintenance of speciation, and epigenetic regulation of gene expression. Here, we define the repertoire of R-M systems in 51 publicly available M. catarrhalis genomes and report their distribution among M. catarrhalis phylogenetic lineages. An association with phylogenetic lineage (RB1 or RB2/3) was observed for six R-M systems, which may contribute to the evolution of the lineages by restricting DNA transformation. In addition, we observed a relationship between a mutually exclusive Type I R-M system and a Type III R-M system at a single locus conserved throughout a geographically and clinically diverse set of M. catarrhalis isolates. The Type III R-M system at this locus contains the phase-variable Type III DNA methyltransferase, modM, which controls a phasevarion (phase-variable regulon). We observed an association between modM presence and OM-associated middle ear isolates, indicating a potential role for ModM-mediated epigenetic regulation in OM pathobiology. Moraxella catarrhalis is a Gram-negative bacterial colonizer of the human respiratory tract. Although often carried asymptomatically in the nasopharynx  in infants and children, and exacerbations of chronic obstructive pulmonary disease (COPD) in adults. It is one of the three most prevalent bacterial causes of OM, along with Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi), and is detected in middle ear fluid in up to 56% of OM cases by PCR  systems are among the few genes associated with distinct phylogenetic lineages or virulent versus avirulent subpopulations . R-M sysngitidis , Neisserorrhoeae , H. inflfluenzae , S. pneueumoniae , and Helr pylori . R-M sysr pylori . Howeverr pylori , DNA repr pylori , and epir pylori , reviewer pylori . Four mar pylori . Type I r pylori , site-spr pylori  or epigeM. catarrhalis: Mca25239IIP (Type I)  that vary in their TRDs  and M.Mca195I (modM3) in a panel of middle ear and nasopharyngeal carriage isolates, and observed a significant association of the modM3 allele with middle ear isolates from patients with OM  ; Mca2523(Type I) ; Mca2523(Type I) ; and Mca(Type I) . We preveir TRDs . We haveeir TRDs . For exa with OM . HoweverM. catarrhalis genomes , and by literature search  using Geneious version 10.1.3  was present, regardless of missing subunits or inactivating mutations. R-M systems were considered to be the same system if >70% nucleotide sequence identity to the reference strain was observed in the DNA methyltransferase and/or restriction endonuclease genes, when present. Each predicted nclature  in the cstructed .http://www.geneious.com; last accessed October 23, 2018) .General sequence alignments and pairwise identity calculations were performed using ClustalW and Geneious version 10.1.3  , with th3, 2018) . AlignmeM. catarrhalis (species ID: 1232) phylogenetic dendrogram was sourced from NCBI GenBank  agar  at 37 °C with 5% CO2. Multiplex PCR reactions were performed using GoTaq Flexi DNA polymerase  as per the manufacturer’s instructions  with 1 ng genomic DNA or 1 µl of boiled cell lysate as template. The eight multiplex PCR primers used are detailed in modM1 (M.Mca23246IP): 400 bp, modM2 (M.Mca25239I): 300 bp, modM3 (M.Mca195I): 500 bp,  relative to RB1 and RB2/3 lineage strains (41.6%)  , and altbspecies . Nineteebspecies  and 2. Sbspecies  and 2. Lbspecies  and 2. Abspecies . None ofM. catarrhalis strains was generated, with strains ordered based on phylogeny (P = 0.0014) whereas the mutually exclusive Type I R-M system is associated with the RB2/3 lineage . At locus 2, four putative Type II R-M systems  are found that are mutually exclusive and differentially distributed between strains (P < 0.0001), whereas McaA9IIP is associated with the RB2/3 lineage . Locus 3 contains a putative Type II R-M system McaCO72IP (P = 0.4329), the associated restriction endonuclease gene (R.McaCO72IP) is only present in one RB1 strain (CO72) compared with 11/19 RB2/3 strains (P < 0.0001). The putative Type IV R-M system Mca25239MrrP at locus 4 is present in all 26 RB1 lineage strains, whereas it is absent from all 19 RB2/3 strains (P < 0.0001) and three short open reading frames (ORFs) encoding hypothetical proteins are present instead (P < 0.0001).A presence/absence matrix of R-M systems in 51 hylogeny . Comparihylogeny , table 1 strains ; Mca2523caCO72IP . Althoug instead . Convers instead  is assocM. catarrhalis RB1 and RB2/3 strains compared with strains of the divergent group. Two R-M systems, McaZ7547IP at locus 2 and Mca23246IIP at locus 11 (P < 0.00001). Conversely, two putative Type II R-M systems, McaCO72IP at locus 3 (P < 0.00001). The putative Type III R-M system Mca25239ORF1708P was present in strains from all lineages, however it was found at locus 5 in RB1 and RB2/3 strains but at locus 12 in divergent strains. Differences in R-M system presence were also observed between strains within the divergent group. Five to six R-M systems are present in strains Z7542, Z7546, Z7547, and Z7574, whereas strains 304_MCAT and 324_MCAT harbored only one R-M system, McaA9IIP at locus 2  with no proximal hsdR or hsdM genes identified  (hsdS2 and hsdR genes (R.Mca25240ORF1547P) are absent (hsdR and hsdM genes were well conserved within their respective loci (88–99% nucleotide identity); however, when comparing hsdR and hsdM genes between loci 1 and 8 only 44% nucleotide identity was observed, suggesting that these are distinct R-M systems at loci 1 and 8.Type I R-M systems are a complex of three subunits: Restriction endonuclease (HsdR), DNA methyltransferase (HsdM), and a specificity subunit (HsdS) that is comprised of two TRDs that recognize bipartite sequences and together determine the recognition sequence of the Type I R-M system complex . Three pentified a. Intere“hsdS2”) a, in alle absent . SeparathsdS genes across the three loci were aligned and although the TRDs were highly variable, two hsdS homology groups were identified based on the presence of homologous regions that are unique to each homology group, adjacent to the variable TRDs  in different strains , indicating that recombination between has taken place , suggesting domain movement between the two homology groups is unlikely.Nucleotide sequences of all four ble TRDs b. The hsen place c. HowevehsdS gene in RB1  and RB2/3  lineage strains, suggesting recombination occurs between M. catarrhalis phylogenetic lineages at the whole hsdS gene as well  were found in strains from multiple phylogenetic lineages alongside lineage specific TRDs, suggesting recombination between lineages has occurred at the TRD level. In addition, TRDs A and B were also found co-located within the same  as well  online.modM) undergoes phase variation, mediated by a 5′-(CAAC)n-3′ tetranucleotide repeat tract in its ORF, and is an epigenetic regulator of a phasevarion  (modM2 (mca25239Imod) (modM3 (mca195Imod) , modM5 (McaS11IIPmod), and modM6 (mcaN1IPmod). Nucleotide sequence alignments of modM1-6 showed that the conserved N and C-terminal regions share 91–97% nucleotide identity, whereas the TRD shares only 36–50% identity between alleles  while modM3 is more commonly associated with the RB2/3 lineage . The rare alleles modM4, modM5, and modM6 were found exclusively in RB2/3 strains, whereas modM1 was found in only one strain, Z7574, belonging to the divergent group.Type III R-M systems consist of a restriction endonuclease (Res) and a DNA methyltransferase (Mod) that contains the TRD and determines the recognition sequence of the system. Four putative Type III R-M systems were identified in this study: Mca25239IP at locus 1, McaC031IP at locus 10, Mca25239IIP at locus 11, and the previously discussed Mca25239ORF1708P at both locus 5 and 12 . We prevsevarion . Three d46IPmod) , modM2 (239Imod) , and mod195Imod)  that dif195Imod) . In this alleles a. In addmca23246IIPmod (modN) that also contains a 5′-(CAAC)n-3′ tetranucleotide repeat tract in its ORF was previously identified in M. catarrhalis 23246  and was thought to exist solely in this strain  was identified that differs in its central TRD  (modO gene was found in 19 strains and has a 5′-(CAACG)n-3′ pentanucleotide repeat tract upstream of its annotated ORF. Six alleles of modO were found, which have 99% nucleotide identity in the N and C-terminal regions and 35–54% nucleotide identity in the TRD: modO1 (mcaA9IPmod), modO2 (mcaC10IPmod), modO3 (mcaF21IIPmod), modO4 (mcaN1IPmod), modO5 (mcaN6IPmod), and modO6 (mcaZ7542IPmod)  . The mod42IPmod) c. NucleoM. catarrhalis TRD sequences, we searched for hsdS and mod TRD homologs in other Moraxella species and distantly related genera. Homologues of hsdS TRDs with a high e-value (1e–100 or greater) in conjunction with high amino acid sequence identity (>90%) were found for TRDs A, C, E, G, and S in the closely related Moraxella sp. HMSC061H09, and for TRDs E, I and N in Moraxella lacunata, a commensal Moraxella species that occupies the same niche (mod TRDs were found in other Moraxella species for modM1 (Moraxella cuniculi), modM4 (Moraxella sp. RCAD0137 and Moraxella bovoculi), and modM6 (Moraxella equi and Moraxella lacunata) (hsdS TRD G in Cyanobacterium sp. IPPASB-1200 (89%), hsdS TRD H in Weeksella sp. HMSC059D05 (96%), and modM3 in Bibersteinia trehalosi (99%)  were also observed, and these potentially constitute novel TRDs in other species (In order to investigate the origin of me niche  online. acunata)  online. si (99%)  online.  species  online.modM2 and modM3 alleles in a collection of 81 nasopharyngeal carriage and OM-associated middle ear isolates from children and found a statistically significant association of modM3 with OM isolates  and 76% (416/549) of strains, respectively. A statistically significant difference in the presence of the Type I or Type III R-M system was observed between child nasopharyngeal carriage and OM-associated middle ear isolates. The Type I and Type III R-M systems were present in 24% (91/378) and 76% (287/378) of child nasopharyngeal carriage isolates, respectively. Interestingly, middle ear isolates from children with OM (32/32) always contain an allele of the Type III ModM R-M system, with the Type I system never found (P = 0.0005) (P = 0.0038) (P = 0.0685).We previously investigated the distribution of the isolates . This stisolates , fig. 6amodM2 was the most prevalent allele, present in 82% of isolates overall , followed by modM3 which was present in 18% of total isolates . The modM1, modM4, modM5, and modM6 alleles were rare and not found in any strains in the six panels. Child nasopharyngeal carriage and OM-associated middle ear isolates were distributed between four panels of isolates; the biOMe panel   compared with nasopharyngeal carriage isolates  was observed (P = 0.0262) (P = 0.0024). However, no statistically significant association was found between either modM2 or modM3 and nasopharyngeal carriage or OM-associated middle ear isolates in the biOMe panel (P = 0.6625) or the GROMIT panel (P = 0.3380). No significant difference was observed in the distribution of R-M systems or modM alleles in child nasopharyngeal isolates regardless of the patient’s prior/current OM status . Similarly, no difference was observed between strains isolated from COPD patient sputum samples during an exacerbation versus periods of stable colonization (P > 0.05).In the 416 strains containing the Type III R-M system, stralia) , the GROstralia) , the Kalstralia) , and thestralia) . When al 0.0262) b. ConsisN. meningitidis , which may further increase the complexity of the M. catarrhalis methylome and impact M. catarrhalis pathobiology and vaccine development.R-M systems are ubiquitous in bacteria, where they have fundamentally been associated with defense of the cell from incoming DNA, such as from bacteriophages . The prengitidis , Staphyls aureus , and Burdomallei . Furtherdomallei . All curdomallei . Epigenedomallei . In thisM. catarrhalis species is comprised of two closely related phylogenetic lineages, RB1 and RB2/3 . Consistent with our results, a prior study by drg gene in N. meningitidis, which encodes a modification-dependent restriction endonuclease and replaces the dam methylase in the ST-1, ST-4, ST-5, ST-32, and ST-41/44 lineages  and it was shown that ModM2 and ModM3 methylate different target sequences, 5′-GARm6AC-3′ and 5′-ACm6ATC-3′, respectively  in this study that contain different TRDs than the previously characterized alleles and are also predicated to methylate different target sequences. A second phase variable Type III DNA methyltransferase, modN, was previously identified in an atypical strain more closely related to Moraxella canis than M. catarrhalis  which was also found in atypical strains of M. catarrhalis. In addition, a novel Type III DNA methyltransferase (ModO), with six alleles (modO1-6) was identified in this study and is part of the locus 10 R-M system McaC031IP. Unlike modM and modN, that contain simple sequence repeats in their ORF that mediate phase variation at the translational level, modO contains a 5′-CAACG(n)-3′ repeat upstream of its ORF, which hypothesize will mediate phase variation at the transcriptional level. We predict that ModM1-6, ModN1-2, and ModO1-6 will each methylate different target sequences and regulate a different set of genes, that is, control different phasevarions, but this requires experimental confirmation. The presence of up to three independently switching phasevarions in individual M. catarrhalis strains is reminiscent of some Neisseria strains, that also contain up to three independently phase-variable methyltransferases  demonstrated that the Type III R-M system (modM) was the most common across the six panels, occurring in 76% of isolates. Interestingly, all OM-associated middle ear isolates contained a Type III R-M system modM gene at locus 1 with the Type I system never occurring in these isolates. This suggests that strains containing a nonphase-variable Type I R-M system may be attenuated in their ability to infect or persist in the middle ear when compared with strains containing a phase variable Type III modM gene. This may be reflective of phylogenetic lineage as genome strains containing the Type I R-M system are associated with the less virulent RB2/3 lineage. Analysis of the distribution of modM alleles in these 549 isolates revealed that modM2 was the most common allele, present in 82% of isolates, while modM3 was found in the remaining 18% of isolates. When isolates were split by clinical manifestation, modM3 was found in child OM-associated middle ear isolates at a significantly higher frequency than child nasopharyngeal carriage isolates. Although consistent with our previous study, wherein modM3 was associated with child OM-associated middle ear isolates versus nasopharyngeal carriage isolates in the BRPRU panel   , this asio, USA)  or GROMIio, USA)  panels. M. catarrhalis, and that many of these show associations with the phylogeny of the species. From these data, it is tempting to speculate that the acquisition of lineage associated R-M systems has shaped the M. catarrhalis population structure, potentially contributing to the differing genomic content, and by extension, the pathogenic potential of the M. catarrhalis phylogenetic lineages. In addition, a number of highly variable R-M systems that exhibit allelic variation, and are potentially phase variable were identified in M. catarrhalis. These novel R-M systems may contribute to pathogenesis if they facilitate epigenetic regulation in a manner analogous to the previously described ModM2 phasevarion (The data presented in this study show that a diverse range of systems exist in sevarion , but addSupplementary DataClick here for additional data file.


H/CD8H) in 45, type II (PD-L1L/CD8L) in 106, type III (PD-L1H/CD8L) in 8, and type IV (PD-L1L/CD8H) in 82 cases. The type I TME was significantly associated with high TIL (P = 3.0E-11) and EBV+ status (P = 8.55E-08). In conclusion, QIA results correlated well with the mRNA expression levels and classified TME of gastric cancers.The use of four groups of tumor immune microenvironments (TME) based on PD-L1 and tumor-infiltrating T lymphocytes (TIL) is a reliable biomarker for anti-PD-1/PD-L1 inhibitor therapy. We classified the TME in 241 gastric cancers which were subdivided according to 40 EBV+, 76 microsatellite instability-high (MSI-H), and 125 EBV-/microsatellite-stable (MSS) subtypes by quantitative image analysis (QIA) and correlated the results with mRNA expression levels. The mean PD-L1 ratio and CD8 ratio in EBV+, MSI-H, and EBV−/MSS GCs were significantly different (P < 0.006). The PD-L1 ratio and CD8 ratio obtained by QIA correlated well with the RNA levels of PD-L1 (r = 0.63) and CD8 (r = 0.67), respectively. The TME were type I (PD-L1 This variability among pathologists, coupled with the inherent heterogeneity seen among cancers, suggests that a more objective and truly quantitative strategy is the true gold standard toward which the biomedical community should strive2. Therefore, the advent of high-throughput acquisition technologies, such as automated slide scanners and computerized analysis of tissue images, is highly desirable. Studies have shown that quantitative software can detect changes in disease states that are missed by visual inspection3.Precision cancer treatment depends not only on next-generation sequencing (NGS), but also on visual assessment of protein biomarker expression seen on immunohistochemistry (IHC) slides. Pathologists interpret IHC results, a difficult and time-consuming task. However, the final IHC results are subjective and qualitative in nature. There is inter-observer variability, and this method is not well suited to the evolving landscape of biomedical research4. The use of four TME groups based on PD-L1 and tumor-infiltrating lymphocytes (TIL) status is a reliable biomarker for anti-PD-1/PD-L1 inhibitor therapy. Immunohistochemistry (IHC) is currently used to detect PD-L1 on tumor cells and immune cells. However, positivity and clinical significance vary due to the type of specimen used , type of PD-L1 primary antibodies, and pathological interpretation method7. The addition of immune cellular markers to PD-L1 IHC would provide deeper insight into the understanding of the complex TME8.The tumor immune microenvironment (TME) is increasingly recognized as a key factor in multiple stages of disease progression, particularly local resistance, immune-escaping, and distant metastasis. The TME substantially impacts the future development of frontline interventions in clinical oncology9. We hypothesized that if a full-section quantitative image analysis (QIA) method can classify TME subtypes, it can be used as a predictive biomarker for anti-PD-1/PD-L1 inhibitor therapy with high reproducibility. For this purpose, we performed IHC for PD-L1 and CD8+ T cells in three distinct subtypes of gastric cancer (GC) , interpreted them with QIAl and correlated the results with mRNA expression levels.Recently, immunotherapy has become a promising approach to treat GCIn QIA of whole slide imaging, representative images of PD-L1 Fig.  and CD8 The QIA results regarding PD-L1 ratio, PD-L1 count, CD8 ratio, and CD8 count in 40 EBV+, 76 MSI-H, and 125 EBV(−)/MSS GCs are described in Table Interestingly, although the PD-L1 ratio and count were significantly higher in both the EBV+ and MSI-H GC groups, the CD8 ratio and CD8 count were significantly higher in the EBV+ GC group, but not in the MSI-H GC group, compared to the MSS GC group. This result suggests that CD8+ cells heavily infiltrate both EBV+ and MSI-H GC, but there is a lower degree of infiltration in MSI-H GC compared to EBV+ GC.Direct comparison of mRNA expression levels and QIA results showed a higher correlation between PD-L1 mRNA expression level and PD-L1 ratio (r = 0.63) than with PD-L1 count r = 0.22). CD8 mRNA was more highly correlated with CD8 ratio (r = 0.67) than CD8 count (r = 0.34) , followed by CD8 count (r = 0.54), EBV status, and host inflammatory response (r = 0.46) Fig. .Figure 310. Of all 241 patients, the high PD-L1 ratio  group was comprised of 53 cases, and the low PD-L1 ratio  group was composed of 188 cases. A CD8 ratio of > 0.1636454 was defined as CD8-high (CD8H), and 127 patients were identified as CD8H. There were 30 (69.8%), 54 (68.4%), and 28 (22.4%) PD-L1H cases of EBV+, MSI-H, and EBV−/MSS GC, respectively.Because PD-L1 and CD8 ratios by QIA showed high correlation with PD-L1 combined positive score (CPS) and significant survival effects compared to IHC-positive cell counts, we used PD-L1 ratio and CD8 ratio in further analyses. A cut-off value was set with the Maxstat package to divide PD-L1 and CD8 ratio status into two groups: high and lowIn survival analyses, EBV+ status (P = 0.007) was significantly associated with longer overall survival (OS), while EBV+ (P = 0.0025) and MSI-H P < 0.001) status were significantly associated with longer disease-free survival (DFS) in univariate analysis  in 45 (18.7%) cases, type II (PD-L1L/CD8L) in 106 (44.0%) cases, type III (PD-L1H/CD8L) in 8 (3.3%) cases, and type IV (PD-L1L/CD8H) in 82 (34.0%) cases. The clinicopathological characteristics of the four TME types are summarized in Table Based on previously reported melanoma casesTME type I was most significantly associated with LELC and CLR histology (P = 3.0E-11) and EBV+ status (P = 8.55E-08). We also found that TME type IV included 10 cases with LELC histology consisting of two EBV+, five MSI-H, and three EBV−/MSS GC cases. Although EBV+ GC cases were mostly present in type I TME, it is noteworthy that MSI-H GC cases were somewhat scattered throughout diverse TME subtypes; 17.1% were type I, 42.1% were type II, 4% were type III, and 36.8% were type IV. In survival analyses, patients with type I TME showed a longer OS compared to patients with type IV TME (P = 0.0294).In this study, we examined archived tumor specimens obtained from a large cohort of patients with stage IB-IV GC for IHC expression of PD-L1 and CD8 in three distinct subtypes of GC (EBV+/MSI-H/EBV− and MSS) by QIA, and correlated them with mRNA expression level and manual interpretation. Both PD-L1 ratio and CD8 ratio correlated well with their respective mRNA levels and manual interpretation results. The PD-L1 ratio correlated well with CD8+ immune cells and TME subtype by QIA, and also correlated well with host inflammatory response and EBV+ GC.13. In GC, ~50% of EBV+ and ~60% of MSI subgroups show high levels of PD-L1 expression, highlighting the molecularly defined patient population most likely to derive benefit from immune checkpoint blockade16. Recent meta-analyses have shown GCs with high PD-L1 expression had deeper tumor infiltration (pT) and were more positive for lymph node metastasis (pN), suggesting that PD-L1 is expressed through oncogenic stimulation and is associated with poorer prognosis17. Given that high PD-L1 expression is closely associated with EBV+ and MSI-H GC, PD-L1 may also be expressed through adaptive immune resistance, which occurs due to the many cytotoxic T-cells and neoantigens present within tumor cells (secondary to EBV itself and the many frameshift mutations seen in EBV+ and MSI-H cancers) and is associated with favorable prognosis. These contradictory results suggest that PD-L1 expression in GC is caused either by oncogenic stimulation resulting in poor prognosis and by adaptive immune resistance affecting favorable prognosis bringing favorable prognosis, and those results are consistent with previous observations in melanoma18.Therapies blocking the PD1/PD-L1 axis have resulted in different rates of tumor response for a variety of cancer types11. In the present study, we performed PD-L1 IHC with SP142, an FDA-approved antibody, in whole sections of 241 surgically resected GCs. The cases included many EBV+ (N = 40) and MSI-H (N = 76) GCs, and the study measured PD-L1 with QIA to assess its clinical significance and possible utility in pathology. In our previous study on TME classified by manual interpretation of PD-L1 and CD8 using the same IHC-stained slides, type I (PD-L1H and CD8H) showed the best survival and was enriched with EBV+ and MSI-H GC cases. The classification of TME groups in The Cancer Genome Atlas (TCGA) data based on PD-L1 and TIL status, the type I TME was associated with oncogenic viral infection19. In the present study, we found similar results to these TCGA results and our previous analyses: EBV+ GC cases are mostly type I TME, while MSI-H GC cases are somewhat scattered, with a diversity of TME subtypes19. Various expression patterns of PD-L1 and their prognostic significance have also been reported21. We recently found that all EBV+ GCs with PD-L1 expression responded dramatically to pembrolizumab; however, not all MSI-H GCs express PD-L1, and only patients with PD-L1-expressing GC responded well to pembrolizumab. PD-L1 expression was the most useful of the predictive markers used in that study22.Classifying GC based on PD-L1 expression and TIL may be more complex than the initial morphological studies performed in melanoma using IHC analyses suggest, and will likely require quantitative and special determination to be used as highly predictive tools to define optimal therapy for patients with advanced cancersIn this study, we introduced QIA to interpret the IHC results of PD-L1 and CD8, and the results were directly compared with manual interpretation results and mRNA expression levels. Given that manual interpretation has limitations, such as low reproducibility and high inter-observer variation, analysis by QIA can reduce inter-observer variation, provide more objective and unbiased assessments, and improve the reproducibility of interpretation. Moreover, it can facilitate the collection of large amounts of data for statistical analysis and increase the efficiency of pathological interpretation by assessing numerous samples in a short time. In the present study, QIA of IHC-stained, whole-slide images correlated very well with microscopic interpretation of PD-L1 and mRNA levels. Moreover, the CD8 ratio significantly correlated with CD8 mRNA levels. Our results highlight the positive aspects of QIA for digital images. Future work should focus on deep assessment of IHC-stained images with integrated regional information.8. To overcome these limitations, we are planning future stratification studies on GC patients with known responses to immunotherapy.Although this study was the first to analyze PD-L1 and CD8 expression status by QIA in a large cohort of GC cases, it has some limitations. First, selection bias is possible because this is a retrospective study, and no patients were treated with immunotherapy. Second, although SP142 is an FDA-approved antibody, this clone was developed for atezolizumab biomarker assays for urothelial carcinoma and non-small cell lung cancersIn conclusion, QIA correlated well with mRNA levels and the manual interpretation results of IHC. We successfully classified four TME groups of GC based on the QIA results of whole digitalized slide images for PD-L1 and CD8 expression status. These results demonstrate that QIA can be used as a diagnostic tool to classify TME subtype.12. No patient had received preoperative chemotherapy and/or radiation therapy or had any other uncontrolled cancer at the time of GC diagnosis. The mean follow-up period was 48.4 ± 20.1 months. Four patients developed other organ cancers  during the follow-up period. Clinical data, including demographic features, pathological characteristics, and treatment outcomes, were obtained by reviewing medical records, and information on deaths was provided by the national statistical office. Tumor stage was defined according to TNM classification as described in the 8th edition of the AJCC cancer staging manual23. Informed consent was obtained from all patients and the Institutional Review Board of the Samsung Medical Center approved this study. This study was carried out in accordance with the approved guidelines.Patients who underwent surgery for primary gastric carcinoma from September 2004 to May 2012 at the Samsung Medical Center were eligible for this study. Among them, 40 EBV− positive cases, 76 MSI-H cases, and 125 EBV(−)/MSS cases were selected, for a total of 241 patients with GC, as previously describedIHC was performed on each representative whole section from all 241 formalin-fixed paraffin-embedded (FFPE) GC samples. Staining for PD-L1 was conducted using a Food and Drug Administration (FDA)-approved rabbit anti-human PD-L1 monoclonal antibody . The percentages of tumor cells and peritumoral immune cells that stained positive for PD-L1 (combined positive score: CPS) were analyzed independently by two pathologists (J.C. and K.M.K.). Staining for CD8 in FFPE tissue sections was conducted using a CONFIRM-anti-CD8 (SP57) rabbit monoclonal primary antibody without dilution with Ventana BenchMark XT via the OptiView DAB IHC Detection Kit .24 was used to separate the pure DAB and hematoxylin-stained areas, leaving a complimentary image. Hematoxylin-stained images were then used for cell segmentation based on our segmentation algorithm25. The chromogenic intensity in DAB-stained images was then quantified . In the quantitative image analysis, single cell-based information, including pixel intensity and cell size measurements , was visualized using a bivariate scatter plot  were determined based on the distribution of scatter plots, and the same value was used across all samples. The PD-L1/CD8 positive cell ratio was defined as the absolute number of positive cells per total number of cells.All IHC-stained slides were digitally scanned at 20x magnification using a ScanScope Aperio AT Turbo slide scanner (Leica Microsystems). The computational whole slide digital image workflow encompasses three steps: 1) image preprocessing, (2) nuclei segmentation, and (3) quantitative image analysis. In image preprocessing, a color deconvolution technique image pr28, and part of the results were published previously29.Total RNA extracted from the available GC FFPE specimens was used to measure PD-L1 and CD8 gene expression with nCounter® Gene Expression Human Immunology Panel containing 494 human immune signature genes . For all selected cases, archival tissue was available for RNA extraction, with an estimated tumor cell percentage over 60% after dissection under microscopy. Total RNA (200 ng) was extracted from three to four 4-μm-thick tissue sections of representative primary tumor blocks using a High Pure RNA Paraffin kit . RNAs were hybridized to target sequence-specific capture probes and fluorescent-labeled reporter probes. The mRNA-probe complexes were washed, immobilized, and quantified by fluorescence imaging as previously describedThe clinicopathological characteristics of patients, such as age, sex, pTNM stage (AJCC 7th edition), disease-free survival (DFS), and overall survival (OS), were analyzed. The SPSS 18.0 statistical software program  was used for statistical analyses. PD-L1 expression status, CD8 expression status, and clinicopathological variables were compared using Pearson’s chi-squared test, and the results were further compared using a linear-by-linear association. The mean values of PD-L1 ratio and CD8 ratio were compared using a Kruskal-Wallis test. The Kaplan-Meier method was used to estimate DFS and OS. To evaluate the associations between clinicopathological factors and survival, a Cox proportional hazard model was used. P-values less than 0.05 were considered statistically significant.Figure S1