Salmonella enterica subsp. enterica (S. enterica) is a significant public health concern and is estimated to cause more than 300,000 deaths annually. Nowadays, the vaccines available for human Salmonellosis prevention are all targeting just one serovar, i.e., S. Typhi, leaving a huge potential risk of Salmonella disease epidemiology change. In this study, we explored the strategy of multiple immunodominant O-epitopes co-expression in S. enterica serovars and evaluated their immunogenicity to induce cross-immune responses and cross-protections against S. Paratyphi A, S. Typhimurium and S. Enteritidis. We found that nucleotide sugar precursors CDP-Abe and CDP-Par (or CDP-Tyv) could be utilized by S. enterica serovars simultaneously, exhibiting O2&O4 (or O4&O9) double immunodominant O-serotypes without obvious growth defects. More importantly, a triple immunodominant O2&O4&O9 O-serotypes could be achieved in S. Typhimurium by improving the substrate pool of CDP-Par, glycosyltransferase WbaV and flippase Wzx via a dual-plasmid overexpressing system. Through immunization in a murine model, we found that double or triple O-serotypes live attenuated vaccine candidates could induce significantly higher heterologous serovar-specific antibodies than their wild-type parent strain. Meanwhile, the bacterial agglutination, serum bactericidal assays and protection efficacy experiments had all shown that these elicited serum antibodies are cross-reactive and cross-protective. Our work highlights the potential of developing a new type of live attenuated Salmonella vaccines against S. Paratyphi A, S. Typhimurium and S. Enteritidis simultaneously. S. Typhi, leaving a significant risk of Salmonella disease epidemiology change. Consequently, more efforts are needed to develop vaccine candidates against other medically important but somehow less concerned S. enterica serovars, such as S. Paratyphi A, S. Typhimurium and S. Enteritidis. Evidence reveals that S. Paratyphi A is taking the place of S. Typhi as the leading cause of “enteric fever” in South Asia, and S. Typhimurium and S. Enteritidis are the leading causing of “Invasive Non-typhoidal Salmonella disease” in sub-Saharan Africa. One of the solutions is to increase the cross-immunogenicity and cross-protections of potential S. enterica vaccine candidates. Salmonella outer membrane O-antigen polysaccharide is a well-known protective antigen, and its diverse structure is serotype specific. More precisely, S. Paratyphi A, S. Typhimurium and S. Enteritidis are characterized by O2, O4 and O9 O-serotypes. In this study, we aimed to combine the immunodominant O2, O4 and O9 O-epitopes into one S. enterica serovar and evaluated its cross-immune responses in a murine model. Our results indicated that the rational design of O-antigen structure in live attenuated Salmonella vaccines is a promising strategy to induce effective cross-protections against S. Paratyphi A, S. Typhimurium and S. Enteritidis.Currently, vaccines available for human Salmonellosis prevention are mostly against one serovar, i.e.,  Salmonella belongs to the family Enterobacteriaceae and is a medically important pathogen for both humans and animals. Based on its surface antigenic composition, Salmonella is currently divided into more than 2600 serotypes , respectively. Again, their O-serotype phenotypes have been reconfirmed, and the deletion mutations of cya and crp genes did not affect the O-epitopes expression (50 > 5 ×108 CFU). To be more briefly, these mutant strains will be hereafter referred to as L008 (O2&O4), L009 (O4&O9) and L083 (O2&O4&O9). The colonization of L008 (O2&O4), L009 (O4&O9) and L083 (O2&O4&O9) in murine Peyer’s patches, spleens, and livers was determined on days 4 and 8 after oral inoculation. All candidates displayed good colonization in Peyer’s patches, livers, and spleens. No deaths occurred during this period  and L083 (O2&O4&O9) mounted a significantly higher anti-S. Enteritidis LPS immune responses than those of ST-Δcrp Δcya. Except for SE-Δcrp Δcya, mice vaccinated with other vaccine candidates could all mount a significantly higher anti-S. Typhimurium LPS responses. We did observe some cross-immunogenicity between ST-Δcrp Δcya and SE-Δcrp Δcya, but it had long been proved to be the common trisaccharide backbone and the conserved core oligosaccharide. So far, we did not obtain valid evidence showing that there were detectable cross-immune responses among immunodominant O2, O4 and O9 O-epitopes unless they were co-expressed simultaneously. L008 (O2&O4) could mount a significantly higher anti-S. Paratyphi A LPS immune response than those vaccinated with ST-Δcrp Δcya (O4), showing an improved cross-immune response against S. Paratyphi A. Consistently, L009 (O4&O9) could mount a significantly higher anti-S. Typhimurium LPS immune response than those vaccinated with SE-Δcrp Δcya (O9), showing an improved cross-immune response against S. Typhimurium. Most importantly, L083 (O2&O4&O9) could simultaneously mount a significantly higher anti-S. Paratyphi A and anti-S. Enteritidis LPS immune response than those vaccinated with ST-Δcrp Δcya (O4), showing a good sign of eliciting cross-protections against S. Paratyphi A and S. Enteritidis. Meanwhile, all vaccine candidates induced a significantly higher IgG2a response than IgG1  when compared to its parent strain ST-Δcrp Δcya. However, L009 (O4&O9) could elicit a higher level of anti-S. Enteritidis LPS IgA compared to L083 (O2&O4&O9) and the level of anti-S. Paratyphi A LPS IgA was similar between L008 (O2&O4) and L083 (O2&O4&O9). Negative control groups did not mount a detectable immune response. All these ELISA results had shown that our vaccine candidates could induce promising cross-immune responses.We also evaluated the serum IgM  and vagicrp Δcya (O4) or SE-Δcrp Δcya (O9) exhibited a high level of SBA activity against its homologous serotype strains. However, they had a limited SBA activity against heterologous serotype strains. On the contrary, the serum antibodies induced by L008 (O2&O4) and L009 (O4&O9) significantly improved SBA activity against heterologous serotype strains S. Paratyphi A and S. Typhimurium, respectively. More importantly, the bactericidal abilities induced by L083 (O2&O4&O9) covered all tested heterologous serotype strains, significantly improving SBA activity against S. Paratyphi A, S. Typhimurium and S. Typhimurium, simultaneously.To evaluate the functional capacities of antibodies induced by our vaccine candidates, we performed the serum bactericidal assays (SBAs) using pooled serum from immunized mice or BSG control. Baby rabbit complement titration with serum samples from immunized mice had a significantly higher complement-mediated killing activity versus those from nonimmunized mice . The serS. Paratyphi A, S. Typhimurium and S. Enteritidis were evaluated in the murine model  and two out of twelve in L083 (O2&O4&O9) vaccinated group, survived in the end. However, mice vaccinated by L008 (O2&O4) and L083 (O2&O4&O9) could generally survive as long as 4 days, while the other vaccinated groups could barely survive a single day, showing a positive correlation of cross-protection. Meanwhile, mice vaccinated by ST-Δcrp Δcya (O4) or SE-Δcrp Δcya (O9) could survive a 100 times LD50 challenge from S. Typhimurium or S. Enteritidis, respectively, indicating a high level of homologous protection. However, these mice succumbed to the heterogenous challenge. Intriguingly, mice vaccinated by L009 (O4&O9) could independently survive a 100 times LD50 challenge from S. Typhimurium and S. Enteritidis, indicating a good cross-protection against heterogenous challenge. Similar results were also observed in mice vaccinated by L083 (O2&O4&O9). These mice could independently survive a 100 times LD50 challenge from S. Typhimurium and S. Enteritidis. Taken together, our vaccine confidants possessed good abilities to elicit cross-immune responses and cross-protections.The immune protection against wild-type ne model . UnfortuSalmonella vaccine developments followed the strategies of targeting one particular serovar or serotype, leaving the cross-protection largely unexplored. However, recently, a bivalent outer membrane vesicle (GMMAs) approach targeting S. Enteritidis and S. Typhimurium [S. Paratyphi A, S. Typhimurium and S. Enteritidis by O-antigen O-epitopes rational design.Traditionally, himurium  and a trhimurium  are showS. enterica serovars, indicating the importance of anti-OAg antibodies in patients’ adaptive immune responses. The O-serotype specificity of S. Paratyphi A, S. Typhimurium and S. Enteritidis is determined by the 3,6-dideoxyhexosyl side-branch residues of the trisaccharide backbone  or the prtA1 gene in front of the abe gene in S. Typhimurium (ST-ddhc::prtA1) , and the prtD1 gene in front of the abe gene in S. Typhimurium (ST-ddhc::prtD1-tyvD1) to testify the possibility of O4 and O9 O-serotypes exhibition  , we all ::prtA1) . Consist::prtA1) . These dhibition . We obseD1-tyvD1 . However1 sample . The postyv gene , interruS. enterica serovars have a different preference for the CDP-Abe, CDP-Par and CDP-Tyv substrates when they are synthesized at a similarly level. At least, it is evident that S. enterica prefers CDP-Tyv rather than CDP-Par [S. Typhimurium mutant, i.e., ST-Δalr ΔdadB ΔrecF Δasd (pSC101-asd or p15a-dadB). As the WbaV glycosyltransferase and Wzx flippase might have influenced synthesizing and transferring of different side-branch O-units, we amplified the whole prtA1-tyvA1-wbaVA1-wzxA1 genes from S. Paratyphi A and inserted them into pSC101-asd vector, resulting in pSC101-asd-O2 in brief  could agglutinate with O2, O4 and O9 antiserum individually, indicating that we had successfully achieved a triple immunodominant O-serotypes phenotype in S. Typhimurium background  is most likely unequal or unbalanced. We could not explain clearly how these O-epitopes are arranged in the outer membrane, either homogeneously or heterogeneously attached to each of the O-antigen polysaccharides -glycosidic linkages during infections [ddhc::prtA1 and SE-ddhc::abe mutants could exhibit a full length of LPS  or ST-Δalr ΔdadB ΔrecF Δasd  showed a decreased synthesis of LPS length  exhibited a full length of LPS synthesis, indicating a higher level of CDP-Par synthesis might be the critical factor in achieving a triple O-serotype phenotype in a S. enterica strain. However, this assumption is not strictly demonstrated in this study. Meanwhile, we also conducted other phenotype evaluation assays  for further cross-immunogenicity evaluation. To guarantee their safety in the murine model, the global regulator cya and crp were deleted in these mutant strains [After successfully constructing a series of double or triple immunodominant O-serotype mutant strains, we would like to know their phenotype characterizations. The phage P22 tailspike protein recognizes and hydrolyzes the repetitive O-antigen polysaccharides of fections . So, we h of LPS , while SS length . Surprisn assays , such as strains , resultiS. Paratyphi A, S. Typhimurium and S. Enteritidis (crp Δcya (O4). Meanwhile, L009 (O4&O9) could induce a significantly higher anti-O4 antibody response than SE-Δcrp Δcya (O9). Most importantly, L083 (O2&O4&O9) could simultaneously induce significantly higher anti-O2, anti-O4 and anti-O9 antibody responses in mice when compared to the negative control, SE-Δcrp Δcya (O9) and ST-Δcrp Δcya (O4), respectively. These data showed that our vaccine candidates could elicit effective cross-immune responses. Although a high level of anti-S. Typhimurium, anti-S. Enteritidis and anti-S. Paratyphi A LPS serum antibodies were obtained, we would like to know whether or not these raised antibodies are indeed functional. The in vitro bactericidal data indicated that serum complement-mediated S. Paratyphi A, S. Typhimurium and S. Enteritidis killing depended upon anti-O2, anti-O4 and anti-O9 antibodies  and SE-Δcrp Δcya (O9) vaccinated mice could receive good homologous protection but failed to heterologous challenge. However, mice vaccinated by double or triple O-epitopes vaccine strains received a significantly higher protection rate even when challenged with heterologous wild-type virulent strains, showing adequate cross-immunity protection.We are primarily interested in whether or not our live attenuated vaccine candidates could elicit effective cross-immune responses. To evaluate that, we applied ELISA assays to detect the antibodies raised against the O-antigen polysaccharides of wild-type eritidis . Consisttibodies . Moreoves strain . CompareS. Typhimurium and S. Enteritidis used in SBA assay are all animal isolates, which could not simply be equal to the human blood isolates. Other reports have shown that anti-LPS antibodies stimulate low complement-dependent killing against S. Enteritidis human blood isolates [However, there are some limitations to this study. The isolates . Furtherisolates . It is aS. Typhimurium or S. Enteritidis background strain and proved they are effective in inducing cross-immunity and cross-protection. Significantly, the strategies we present in this study are not limited to S. enterica O-antigen polysaccharides but have applicability for generating cross-protection for many other important human and animal pathogens. Notable examples include Shigella flexneri and many pathogenic Escherichia coli.In summary, we expressed double or triple immunodominant O-epitopes in a S1 FiggalF and gnd genes of S. Paratyphi A, S. Enteritidis and S. Typhimurium genome, which could be accessed through the genebank accession numbers NZ_CP019185.1, CP007361.1 and CP002614.1, respectively. The O-antigen gene clusters of groups A1 and D1 are highly homologous. The main differences between group B1 and group A1, D1 are the regions responsible for synthesizing the side-branch sugars. Note that the tyvA1 has a loss-of-function mutation due to ORF frameshift. Diagrams are drawn to scale.The O-antigen gene clusters are within the (TIF)Click here for additional data file.S2 Figabe gene from S. Typhimurium was inserted between the ddhc and prtA1 gene of S. Paratyphi A. (B) The prtA1 gene was inserted between the ddhc and abe gene of S. Typhimurium. (C) The abe gene was inserted between the ddhc and prtD1 gene of S. Enteritidis. (D) The prtD1-tyvD1 genes were inserted between the ddhc and abe gene of S. Typhimurium. (E) The prtA1-tyvA1-wbaVA1-wzxA1 genes from S. Paratyphi A were cloned into pSC101-asd, resulting in pSC101-asd-O2. (F) The prtD1-tyvD1-wbaVD1-wzxD1 genes from S. Enteritidis were cloned into p15a-dadB, resulting in p15a-dadB-O9. Primer pairs used for each DNA fragment amplification were labeled accordingly.The (TIF)Click here for additional data file.S3 FigThe agglutination assays were performed on glass slides and the used anti-Par O2, anti-Abe O4 and anti-Tyv O9 antiserum were indicated above. Positive or negative agglutination could be observed directly by the naked eye. Images were taken at 10 × 10 magnification.(TIF)Click here for additional data file.S4 FigS. Paratyphi A (A), S. Enteritidis (B) and S. Typhimurium (C) were determined by ELISA. A significantly higher level of IgG2a specific to the S. Paratypi A LPS compared to IgG1 was observed in L008 (O2&O4) and L083 (O2&O4&O9) . A significantly higher level of IgG2a specific to the S. Enteritis LPS compared to IgG1 was observed in SE-Δcrp Δcya . A significantly higher level of IgG2a specific to the S. Typhimurium LPS compared to IgG1 was observed in ST-Δcrp Δcya, L008 (O2&O4), L009 (O4&O9) and L083 (O2&O4&O9) . The antibody concentrations were calculated using a standard curve. All of the measured sample concentrations were within the standard curve range. The error bars represent the standard deviation of the means. These data are representative of at least two independent experiments.Serum IgG2a and IgG1 responses against the LPS of (TIF)Click here for additional data file.S5 FigS. Paratyphi A LPS serum IgM levels. Responses that differed from the results in the ST-Δcya Δcrp group are noted by asterisks . (B) The anti-S. Enteritidis LPS serum IgM levels. Responses that differed from the results in the ST-Δcya Δcrp group are noted by asterisks . (C) Anti-S. Typhimurium LPS serum IgM levels. Responses that differed from the results in the SE-Δcya Δcrp group are noted by asterisks . Antibody concentrations were calculated using a standard curve and all the measured sample concentrations were within the standard curve range. The error bars represent the standard deviation of the means. These data are representative of at least two independent experiments.(A) Anti-(TIF)Click here for additional data file.S6 FigS. Paratyphi A LPS serum IgA levels. Responses that differed from the results in the ST-Δcya Δcrp group are noted by asterisks . (B) The anti-S. Enteritidis LPS serum IgA levels. Responses that differed from the results in the ST-Δcya Δcrp group are noted by asterisks . (C) Anti-S. Typhimurium LPS serum IgA levels. Responses that differed from the results in the SE-Δcya Δcrp group are noted by asterisks . Antibody concentrations were calculated using a standard curve and all the measured sample concentrations were within the standard curve range. The error bars represent the standard deviation of the means calculated by GraphPad Prism software. These data are representative of at least two independent experiments.(A) Anti-(TIF)Click here for additional data file.S7 Fig(A) The O2, O4 and O9 O-epitopes are homogeneously attached to each one of the O-antigen polysaccharides. (B) The O2, O4 and O9 O-epitopes are heterogeneously attached to each one of the O-antigen polysaccharides. Note that the number of each attached O-epitope does not represent the real case.(TIF)Click here for additional data file.S1 TextMethods for LPS preparation and western blotting, the bacterial slide agglutination test, P22 transduction studies, motility test and minimum inhibitory concentration (MIC) test are described in supporting information supplementary methods.(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


H2S has been found to have many important physiological functions and to play an important role in many pathological and physiological processes. Recent evidence shows that H2S improves the body’s defenses to many diseases, including diabetes, by regulating ER stress, but its mechanism has not yet been fully understood. We therefore reviewed recent studies of the role of H2S in improving diabetes-related diseases by regulating ER stress and carefully analyzed its mechanism in order to provide a theoretical reference for future research.Endoplasmic reticulum (ER) plays important roles in protein synthesis, protein folding and modification, lipid biosynthesis, calcium storage, and detoxification. ER homeostasis is destroyed by physiological and pharmacological stressors, resulting in the accumulation of misfolded proteins, which causes ER stress. More and more studies have shown that ER stress contributes to the pathogenesis of many diseases, such as diabetes, inflammation, neurodegenerative diseases, cancer, and autoimmune diseases. As a toxic gas, H Endoplasmic reticulum (ER) is an important organelle in eukaryotic cells, which is responsible for the synthesis and modification of lipids, proteins, and carbohydrates, as well as the regulation of intracellular calcium concentration ,2. When 2S) is a flammable, volatile, and toxic gas with the smell of rotten eggs [2S has a variety of biological functions [2S regulates ER stress in a variety of pathological processes, including sepsis-induced myocardial dysfunction [2S in regulating ER stress in diabetes. In this review, we considered recent literature on the important role of H2S in regulating ER stress in diabetes and analyzed its mechanism, hoping to provide a theoretical reference for exploring the role of H2S in improving future diabetes treatments by regulating ER stress.Hydrogen sulfide  was considered to be merely a toxic and malodorous gas. However, recent studies have revealed that, in addition to its harmful aspects, H2S is involved in the regulation of a variety of biological processes. Therefore, it is newly considered to be a gas transmitter after nitric oxide (NO) and carbon monoxide (CO) [2S in the body is synthesized by three enzymes: cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), and 3-mercaptopyruvate thiotransferase (3-MST) [2S catalyzed by CSE/CBS through a β elimination reaction. In addition, L-cystine can be converted into 3-mercaptopyruvate (3-MP) catalyzed by cysteine aminotransferase (CAT) by transferring amine to α-ketoglutarate to form 3-mercaptopyruvate (3-MP). Finally, 3-MP can be converted into H2S catalyzed by 3-MST  ,29,30. H (3-MST) . First, by 3-MST  [32,33,3gulation ,36,37,38ydration . Recent 2S inhibited diabetes-associated ER stress. Additionally, NaHS also noticeably promoted hippocampal endogenous H2S production in STZ-induced diabetic rats. Collectively, this evidence suggests that H2S ameliorates diabetes-associated cognitive dysfunction most likely by inhibiting hippocampal ER stress [2S released by NaHS used in the above study is close to the physiological concentration in vivo, which has no toxic effect on diabetic rats. These levels can promote the production of endogenous H2S in the hippocampus of diabetic rats, suggesting that exogenous H2S can also improve diabetes-associated cognitive impairment by increasing endogenous H2S. Furthermore, previous studies have shown that H2S can increase ER stress in beta cells in vitro [2S inhibits ER stress in the hippocampus of diabetic rats. The reason for this may be related to different tissue and cell types, a question which needs further research.Cognitive dysfunction, a degenerative disease of the central nervous system, is related to neurodegenerative diseases and brain aging. At present, much evidence shows that patients with diabetes can suffer cognitive and memory impairment . The ratR stress . ER streR stress , and ER R stress ,51. In tin vitro , which c+-dependent deacetylase that regulates gene expression through histone deacetylation and plays a complex role in a variety of diseases [2S improvements in diabetes-associated cognitive dysfunction. Additionally, NaHS eliminated diabetes-induced ER stress by decreasing the expressions of GRP78, CHOP, and cleaved caspase-12 in the hippocampus, while sirtinol abrogated the effects of NaHS, indicating that H2S inhibited diabetes-induced ER stress by promoting SIRT1. Furthermore, sirtinol also eliminated NaHS-induced increased expressions of hippocampal synapse-related protein  in STZ-induced diabetic rats, indicating that H2S improved hippocampal synaptic dysfunction by promoting SIRT1. In sum, H2S ameliorated the cognitive dysfunction of STZ-diabetic rats by inhibiting hippocampal ER stress and by improving synaptic dysfunction through upregulating SIRT1 [2S improves cognitive dysfunction in diabetic rats by inhibiting hippocampal ER stress through promoting SIRT1. Therefore, the mechanism of how SIRT1 regulates ER needs to be clarified. Based on the studies above, H2S regulation of autophagy will become a new strategy to improve diabetes-related cognitive impairment.In addition to the ER stress mentioned above, synaptic dysfunction is also involved in diabetes-associated cognitive impairment ,54,55. Tdiseases ,59. It hdiseases ,61. Howe2S levels of the diabetic rat models and induced cardiac dysfunction in the diabetic rat model, as evinced by cardiac enlargement, cardiac hypertrophy, and fibrosis, which was restored by exogenous H2S [2S therapy restored HFD-inhibited APN levels and suppressed HG-induced cardiac ER stress. In sum, exogenous H2S improved HFD-induced cardiac dysfunction by inhibiting ER stress and restoring HFD-suppressed circulating and cardiac H2S [2S can improve diabetes cardiomyopathy. Rui Yang et al. established a rat model of diabetes by intraperitoneal injection of streptozotocin and subsequently conducted a series of experiments. The results showed that hyperglycemia could significantly reduce cardiac function, significantly damage myocardial ultrastructure, increase the content of MDA in myocardial tissue, reduce the activities of SOD and GSH Px, and increase the mRNA expression of CHOP, GRP78, and caspase-12, while NaHS reversed these changes. These results indicate that exogenous H2S improved the DC of diabetic rats, probably by reducing oxidative stress injury and ER stress-induced apoptosis [2S might inhibit ER stress through the following three mechanisms: (1) downregulation of the expression of GRP78 and inhibition of the transcription of CHOP; (2) inhibition of the activation of caspase 12; (3) improvement in the activity of antioxidant enzymes, reduction in the production of ROS and lipid peroxides, and reduction in the accumulation of unfolded or misfolded proteins in the ER. Another study also obtained similar results. Fang Li and colleagues found that H2S significantly improved DC by reducing myocardial hypertrophy and myocardial collagen deposition induced by hyperglycemia in STZ-induced diabetic rats. Additionally, H2S inhibited ER stress by reducing the expression of GRP78, caspase-12 and CHOP induced by hyperglycemia. Considered collectively, it might be deduced that H2S ameliorates DC by suppressing STZ-induced ER stress in diabetic rats [2S can improve DC by inhibiting ER stress-mediated apoptosis by inhibiting CHOP and caspase-12.Diabetes cardiomyopathy (DC) refers to the dysfunction of myocardial function in patients with diabetes, excluding the direct causes of hypertension, coronary artery disease, or valve disease ,63. It inous H2S . Adiponenous H2S . APN hasnous H2S ,72. Furtdiac H2S . Anotherpoptosis . In the tic rats , a concltic rats . Furthertic rats  and the tic rats . Therefo2S reversed these changes. Similar results were obtained in vitro experiments. The expressions of GRP78, p60 ATF-6, CHOP, and cleaved caspase-12 were all increased in the myocardial tissue of diabetic rats and high glucose (HG)-induced H9C2 cells, while exogenous H2S abolished these changes, suggesting that the hyperglycemia-induced ER stress of cardiomyocytes was inhibited by exogenous H2S. Moreover, exogenous H2S and NAC (ROS scavenger) notably decreased HG-induced intracellular ROS levels in H9C2 cells. HG upregulated apoptosis by inducing the nucleus condensation and fragmentation of H9C2 cells which are characteristic of apoptosis. Exogenous H2S and NAC could evidently inhibit apoptosis, indicating that ROS mediated exogenous H2S inhibition of HG-induced apoptosis [2S reduced the increased expression of Mfn-2 induced by HG in H9C2 cells. Silencing Mfn-2 with siRNA inhibited HG-induced apoptosis of H9C2 cells, suggesting that Mfn-2 mediated HG-induced apoptosis of H9C2 cells. Moreover, Mfn-2 siRNA and H2S synergistically inhibited ER stress and the expression of Bax and cyt C induced by HG. Considered collectively, exogenous H2S improved myocardial tissue damage by suppressing hyperglycemia-induced ER stress and mitochondrial apoptosis of cardiomyocyte through inhibiting Mfn-2 [2S inhibits cardiomyocyte apoptosis by inhibiting ER stress and ROS production.In addition to ER stress, ROS can also induce cardiomyocyte apoptosis ,79. Fan poptosis . Mitochopoptosis ,82. Abnopoptosis . It has poptosis . Exogenong Mfn-2 . The eving Mfn-2 ,86. The 2S in the serum of patients/rats with DC decreased significantly, and the content of H2S and the expression of cystathionine-y-lyase (CSE) in the heart tissue of rats with DC also decreased significantly. The H2S levels in the supernatant of AC16 cardiomyocytes treated with PA decreased. Cardiac lipotoxicity is characterized by the increase in TUNEL positive cells and lipid deposition in vivo and in vitro, accompanied by a decrease in the endogenous H2S levels. Furthermore, NaHS suppressed ER stress in rats with DC or PA-induced AC16 cells by downregulating the protein expressions of GRP78, CHOP, and caspase-12. The NaHS treatment of AC16 cells could inhibit myocardial injury induced by PA, improve cell viability, and inhibit lipid deposition. Additionally, 4-PBA (an ER stress inhibitor) has similar effects. At the same time, the treatment of diabetic rats with NaHS or 4-PBA reduced cardiac lipotoxicity, as evidenced by the decreased cleaved caspase-3 expression, TUNEL positive cells, and lipid accumulation. Collectively, endogenous H2S deficiency is associated with the myocardial injury caused by lipotoxicity. Exogenous H2S alleviates PA-induced myocardial injury by suppressing ER stress [2S inhibited the apoptosis and lipotoxicity of DCM by inhibiting ER stress. It has been reported that H2S is involved in lipid metabolism [2S in lipid metabolism through ER stress. Therefore, the subject in this regard is worth studying in the future.Besides ER stress-induced apoptosis, ER stress-induced lipotoxicity also contributes to DC. Runmin Guo and colleagues used STZ/palmitic acid to establish a diabetic mouse model and a diabetes cell model, respectively, and conducted a series of experiments. Their results showed that the level of HR stress . It can tabolism ,89, but tabolism ,91. Howe2S has been reported to improve myocardial fibrosis in diabetes, but its mechanism is not completely clear [2S ameliorated myocardial histological changes induced by hyperglycemia. Substantial research revealed that the expression ratio of MMPs/TIMPs in the myocardium of diabetic rats was dysregulated and that the TGF-β1 expression level was downregulated, suggesting that hyperglycemia caused myocardial fibrosis. While H2S attenuated hyperglycemia-induced myocardial fibrosis, H2S also inhibited cardiomyocyte apoptosis by downregulating caspase-3 level and upregulating Bcl-2 level in diabetic rats [2S, indicating that H2S alleviated hyperglycemia-induced myocardial oxidative damage. Moreover, H2S inhibited myocardial inflammatory responses in diabetic rats by downregulating TNF-α and NF-κB expressions. The ER stress of the diabetes myocardium was also weakened by H2S, evidenced by reductions in eIF2αand GRP94 expression. Meanwhile, the JAK/STAT pathway was also inhibited by H2S, as evinced by the decrease in JAK-1/2 and STAT1/3/5/6 expression [2S mitigates myocardial fibrosis by inhibiting oxidative stress. Therefore, it could be deduced that exogenous H2S improved myocardial fibrosis in diabetic rats by inhibiting ER stress by suppressing the JAK/STAT pathway, a hypothesis that needs further confirmation by studies with inhibitors [2S in improving myocardial fibrosis, the relationship among ER stress, apoptosis, inflammatory response, and oxidative stress needs to be clarified. The regulation of ER stress by H2S in cardiomyocytes is expected to be a new strategy for the treatment of myocardial fibrosis in diabetes.Myocardial fibrosis is an important feature of DC. It is caused by the excessive production and deposition of myocardial interstitial collagen, which then it leads to myocardial hypertrophy, myocardial diastolic, and systolic dysfunction, eventually leading to heart failure ,93. H2S ly clear ,95. The tic rats . The levtic rats , were depression . The JAKpression , and ER pression ,99. Furthibitors . In the 2S can improve diabetes-related diseases by regulating ER stress. In this review, we summarized research indicating that: (1) exogenous H2S ameliorates diabetes-associated cognitive dysfunction, most likely by inhibiting hippocampal ER stress; (2) exogenous H2S ameliorates cognitive dysfunction of STZ-diabetic rats by inhibiting hippocampal ER stress and by improving synaptic dysfunction through upregulating SIRT1; (3) exogenous H2S improves HFD-induced cardiac dysfunction by inhibiting ER stress and by restoring HFD-suppressed circulating and cardiac H2S; (4) exogenous H2S improves DC, probably by reducing oxidative stress injury and ER stress-induced apoptosis; (5) exogenous H2S improves DC by inhibiting ER stress-mediated apoptosis by inhibiting CHOP and caspase-12; (6) exogenous H2S improves myocardial tissue damage by suppressing hyperglycemia-induced ER stress and the mitochondrial apoptosis of cardiomyocyte by inhibiting Mfn-2; (7) exogenous H2S alleviates PA-induced myocardial injury by suppressing ER stress; (8) exogenous H2S improves myocardial fibrosis in diabetic rats by suppressing ER stress by inhibiting the JAK/STAT pathway, a conclusion that needs further confirmation by testing with the inhibitors (2S plays a protective role against diabetes-related diseases by inhibiting ER stress by upregulating SIRT1 and by inhibiting CHOP and caspase-12, Mfn-2, and the JAK/STAT pathway. Whether H2S can inhibit ER stress through other mechanisms to improve diabetes-related diseases is worth studying. As opposed to the demonstrated protective effect of H2S on diabetes, some evidence shows that H2S induced INS-1E cells apoptosis to aggravate diabetes by promoting ER stress via the activation of the p38 MAPK pathway [2S used and different stages of diabetes, a question that needs to be further studied.In recent years, more and more studies show that Hhibitors . As can  pathway . The rea2S regulates ER stress via many signaling pathways, such as the BDNF/TrkB signaling pathway, the Akt-Hsp90 signaling pathway, the NF-κB/MAPK signaling pathway, and the PI3K/Akt signaling pathway [2S regulating ER stress in diabetes need to be further clarified. In addition, the mechanism of H2S regulation of ER stress, especially the questions of under what conditions H2S promotes ER stress and under what conditions H2S inhibits ER stress, needs further study. Furthermore, ER stress also plays an important role by regulating NLRP3 inflammasomes in diabetes [2S can improve diabetes by regulating ER stress/NLRP3 inflammasomes is a subject worth studying in the future. At present, exogenous H2S-releasing agents have many disadvantages and cannot fully meet the research on H2S. Therefore, the development of new H2S-releasing agents is very important for the clinical application of H2S in the future.The evidence shows that H pathway . Therefodiabetes ; hence, 2S regulation of ER stress will provide a new strategy for the treatment of diabetes.It is believed that, with the further development of the relevant research, H


Retroviral integration site targeting is not random and plays a critical role in expression and long-term survival of the integrated provirus. To better understand the genomic environment surrounding retroviral integration sites, we performed a meta-analysis of previously published integration site data from evolutionarily diverse retroviruses, including new experimental data from HIV-1 subtypes A, B, C and D. We show here that evolutionarily divergent retroviruses exhibit distinct integration site profiles with strong preferences for integration near non-canonical B-form DNA (non-B DNA). We also show that in vivo-derived HIV-1 integration sites are significantly more enriched in transcriptionally silent regions and transcription-silencing non-B DNA features of the genome compared to in vitro-derived HIV-1 integration sites. Integration sites from individuals infected with HIV-1 subtype A, B, C or D viruses exhibited different preferences for common genomic and non-B DNA features. In addition, we identified several integration site hotspots shared between different HIV-1 subtypes, all of which were located in the non-B DNA feature slipped DNA. Together, these data show that although evolutionarily divergent retroviruses exhibit distinct integration site profiles, they all target non-B DNA for integration. These findings provide new insight into how retroviruses integrate into genomes for long-term survival. Retroviridae is divided into two subfamilies called Orthoretrovirinae and Spumaretrovirinae. Alpha-, Beta-, Gamma-, Delta-, Epsilon-retrovirus and Lentivirus represent the six genera of the Orthoretrovirinae, and Bovispumavirus, Equispumavirus, Felispumavirus, Prosimiispumavirus and Simiispumavirus represent the five genera of Spumaretrovirinae. Following entry of a retrovirus into cells, the viral RNA genome is converted into double-stranded DNA that associates with several viral and host proteins to form a pre-integration complex (PIC). Soon after completion of DNA synthesis, the viral DNA ends are primed by the enzyme integrase in a process called 3′ end processing. Following docking with the host’s genomic DNA, the viral DNA undergoes a strand transfer reaction resulting in the insertion of the retroviral genome into the host genomic DNA. Host repair enzymes are then thought to complete integration  . HIV-1 subtype A, C and D integration sites were newly obtained from infected individuals in Uganda and Zimbabwe. Integration sites were determined from the sequence junction between the HIV-1 3′ LTR and human genome sequences. Each paired fastq sequencing read was quality trimmed and excluded from further analysis if the LTR-genome junction sequence did not match between the two paired reads. The HIV-1 LTR-containing fastq sequences were filtered by allowing up to a maximum of five mismatches with the reference NL4-3 LTR sequence. LTR sequences matching any region of the human genome (GRCh37/hg19) were discarded. Flanking human genomic sequences more than 20 nucleotides in length were used to identify integration sites using our in-house bioinformatics pipeline (Barr Lab Integration Site Identification Pipeline (BLISIP version 2.9)) .,58.p < 0 0.0001) . Repetit 0.0001) A. It is  0.0001) . In star 0.0001) D.p < 0.0001); ASLV near triplex DNA (p > 0.05); FIV near mirror repeats (p > 0.05); FV near G4 motifs and Z-DNA (p < 0.01); HTLV-1 near cruciform and Z-DNA (p > 0.05); MLV near G4 motifs, short tandem repeats, triplex and Z-DNA (p < 0.05); MMTV near A-phased motifs and slipped motifs (p > 0.05); and SIV near cruciform motifs (p > 0.05), mirror repeats and short tandem repeats (p < 0.05) (p < 0.0001); ASLV disfavored A-phased, cruciform, G4, slipped and Z-DNA motifs (p > 0.05); FIV disfavored direct repeats and G4 motifs (p < 0.05), and A-phased, slipped, triplex and Z-DNA motifs (p > 0.05); FV disfavored A-phased motifs (p < 0.01) and cruciform motifs (p > 0.05); HTLV-1 disfavored direct repeats, G4 motifs and mirror repeats (p > 0.05); MLV disfavored A-phased motifs (p < 0.001) and cruciform motifs (p > 0.05); MMTV disfavored G4 and triplex motifs (p > 0.05); and SIV disfavored A-phased and Z-DNA motifs (p > 0.05), and G4, slipped and triplex motifs (p < 0.01)  . Of note < 0.01) .We have previously observed that the distribution of integration sites within the 500 bp window can vary significantly with strong enrichment observed at discrete distances away from the non-B DNA feature ,46. Analp < 0.0001; Fisher’s exact test, two-sided) , whereas the in vivo-derived datasets, sites were significantly more enriched in LADs, satellite DNA, simple repeats and SINEs   . Of the o-sided) E. In addo-sided) .Compared to the in vitro dataset, integration sites in the in vivo-derived dataset were modestly enriched within 250 bp of most non-B DNA except G4 and Z-DNA . NotablyThus far, integration site profiles have been extensively analyzed for HIV-1 subtype B infections, which represents only ~12% of the infections worldwide. Subtypes A, C and D represent approximately 10%, 47% and 3%, respectively. We asked if the integration site profiles from individuals infected with HIV-1 non-subtype B virus were similar to those infected with subtype B virus. In the integrase enzyme, the amino acid difference between subtypes is as high as 16% (subtype B vs. C) but typically less than 8% in integrase of HIV-1 isolates of a specific subtype. This level of amino acid diversity is the highest among the enzymes encoded by the HIV-1 pol gene. The greatest diversity within HIV-1 integrase is found in the C-terminal domain (CTD), which is involved in genomic DNA binding. Thus, it is reasonable to propose that we would observe differences in the integration site profiles between different HIV-1 subtypes. To address this, genomic DNA was isolated from peripheral blood mononuclear cells (PBMCs) from a cohort of women in Uganda and Zimbabwe infected with HIV-1 subtype A, C or D and used to generate integration site libraries. Integration site profiles were generated from a total of 48 infected individuals  and compared to the integration site profile from at least 25 individuals infected with subtype B virus generated from previously published (‘HIV-1 in vivo-derived’) datasets .p < 0.05; Fisher’s exact test, two-sided)   . Similaro-sided) ; howevero-sided) . All fouo-sided) , with suo-sided) . Moreoveo-sided) . Pairwiso-sided) C.Our analysis of the non-B DNA integration site profiles from the different subtype viruses showed enriched integration near most non-B DNA . CompariTogether, these data show that the integration site profiles from individuals infected with HIV-1 subtype B differ from those infected with non-subtype B virus. Subtypes B and D exhibited the most similar profiles overall. Subtype A tended to exhibit a more transcriptionally active profile compared to subtype B , and subtype C exhibited a more transcriptionally silent profile than subtype B . All subtypes favored integration near non-B DNA but differed in their preference for certain types of non-B DNA.p > 0.05; Fisher’s exact test, two-sided), and between subtypes A  and D    A. For coo-sided) . In addio-sided) C. NotablSDHB, TBC1D5B, GCN1L1, CCDC57 and PLK1S1) that were targeted by multiple subtypes  and identified five genes  and the second pool contained integration sites not located in hotspots. We then extracted nucleotide windows of 100 bp upstream and 100 bp downstream of each integration site and used DiffLogo to identify motifs that predominate in the pool of hotspot sites. DiffLogo provides a visualization of pair-wise differences between DNA motifs . It presHere we showed that although evolutionarily divergent retroviruses exhibit distinct integration site profiles, all retroviruses target non-B DNA for integration. We also showed that there are important differences in the integration site profiles between in vivo- and in vitro-derived HIV-1 datasets, but that the initial targeting of non-B DNA (except for G4 DNA) during acute infection does not appear to differ substantially. Comparison of the integration site profiles of different HIV-1 subtypes revealed that there are significant differences when comparing subtype B and non-subtype B infections. Notably, subtype C, which comprises ~47% of HIV-1 infections worldwide, exhibited a more transcriptionally silent profile  and exhibited the largest differences in preferences for non-B DNA. Despite these differences, we identified several integration site hotspots that are shared between the different subtypes, all of which were located in a slipped DNA motif.As previously observed with HIV-1, the distribution of integration sites within 500 bp of non-B DNA features for all retroviruses was often clustered in discrete distances away from the feature . One expAll retroviruses exhibited enriched integration near non-B DNA with slipped DNA serving as integration site hotspots for HIV-1 subtypes A, B, C and D. Slipped DNA motifs at integration site hotspots are not limited to HIV-1. Other studies identified consensus motifs for a variety of other retroviruses with no previously identified commonalities among the different motifs ,42,64. AHow retroviral PICs recognize slipped DNA and other non-B DNA for integration is not yet fully understood. Many non-B DNA motifs are enriched near sites of genomic variability and are targets for preferential homologous recombination (>20-fold) in human cells ,67,68. IAnalysis of integration site profiles after acute infection showed that HIV-1, FIV and SIV had the strongest preference for integrating into transcriptionally active regions of the genome , whereas MMTV and FV had the strongest preference for transcriptionally inactive regions . The targeting of transcriptionally active regions of the genome maximizes provirus expression and helps establish infection and spread early in infection . HoweverBy integrating into transcriptionally repressive regions of the genome, retroviruses can minimize their expression and avoid detection by the immune system after the high virus-expressing cells are destroyed. In strong support of this strategy, recent work has shown that most intact HIV-1 proviruses in infected individuals who naturally control infection , and individuals on long-term suppressive antiretroviral therapy, are located in transcriptionally repressive features ,75,76,93It is not fully understood if integration into transcriptionally silent regions of the genome is driven by retroviruses, the host or both; however, current evidence supports the latter of the three. For example, the host antiviral proteins APOBEC3 appear to promote a transcriptionally silent integration site profile for HIV-1 that is away from genes and enriched in SINEs . This supol gene. The greatest diversity within HIV-1 integrase is found in the CTD, which is involved in genomic DNA binding. Thus, it is reasonable to suspect that HIV-1 subtypes may target different non-B DNA motifs with different selectivity. Despite the multiple substitutions that segregate HIV-1 variants of each subtype, there may be specific amino acid differences in one subtype versus another that impact preference for integration sites. For example, the isoleucine found at position 20 in subtype C and D versus the threonine in subtype A and B in the integrase CTD may relate the greater similarity of integration site selection of subtypes B and D  but typically less than 8% integrase diversity among HIV-1 isolates of one subtype ,96,97. T B and D D,E. In aRecently, we showed that human APOBEC3 increased HIV-1 insertions into SINEs in a dose-dependent manner . InteresWe identified non-B DNA as a feature that is targeted differentially by evolutionarily diverse retroviruses. We have also presented the first comparative look at the integration site profiles of HIV-1 subtype A, C and D viruses and showed that they differed from HIV-1 subtype B profiles overall but shared similar integration site hotspots located in slipped DNA. Together, these data highlight important similarities and differences in retroviral integration site targeting that can be used in future studies to better understand the evolution of retroviral integration site targeting and how these viruses integrate into our genomes for long-term survival. It will be important to investigate the impact of non-B DNA features on the expression of nearby proviruses and their contributions to productive and latent integrations. The identification and characterization of proteins that influence targeting of non-B DNA by retroviral preintegration complexes will also further our understanding of integration site selection and possibly open new avenues for drug targets and the design of safer and more efficient retroviral gene therapy vectors.


Here, we confirmed this principle by liquid biopsy monitoring in a colorectal cancer patient from an independent clinical trial. In this patient, both PD-L1 L88E and L88fs mutations emerged under selective pressure with avelumab. By ectopically expressing PD-L1 L88E, we show that this mutation leads to a reduction of full-length glycosylated PD-L1 and greatly reduced avelumab surface binding. Further experiments indicated that PD-L1 L88E represents a phosphomimetic variant of PD-L1 L88S leading to loss of protein stability and increased proteasomal degradation. The association of this PD-L1 mutation with the high-affinity FCGR3A single nucleotide polymorphism rs396991 confirms prior evidence that patients harboring this polymorphism experience the strongest selective pressure by avelumab. Together, position 88 of PD-L1 is a hotspot residue critically regulating PD-L1 cell surface expression with clinical significance in the context of immune checkpoint blockade.The two most common antibody targeting principles in oncology are the induction of direct antitumor effects and the release of antitumor T cell immunity by immune checkpoint blockade. These two principles, however, may be overlapping if the targeted checkpoint molecule is not located on the immune cell but on the tumor cell itself. Secondary resistance by epitope escape may therefore remain a challenge in both settings. We previously reported epitope escape through L88S and truncating programmed cell death ligand 1 (PD-L1) gene mutations in colorectal cancer patients on selective pressure with avelumab, a PD-L1-directed checkpoint blocker that—in addition to T cell disinhibition—allows direct tumor cell killing  PD-L1 sequence variants  cell degranulation than cells expressing the PD-L1 wild-type, but T cell-mediated killing was increased. Interestingly, these PD-L1 mutations were significantly associated with the homo- and heterozygous Fc gamma receptor IIIa (FCGR3A) single nucleotide polymorphism (SNP) rs396991 (V/V and V/F) that is known to mediate high-affinity binding of NK cells to IgG1 (FCGR3A SNP may be a subset with the highest selective pressure exerted by avelumab.Programmed cell death ligand 1 (PD-L1) is a membrane protein expressed on some cells of the tumor microenvironment such as macrophages and dendritic cells as well as on the surface of cancer cells . It consvariants . In two  to IgG1 . This suPD-L1 mutants at position 88 on avelumab treatment in a patient with microsatellite-stable metastatic colorectal cancer. This patient—treated on an independent clinical trial—again showed homozygosity for the high-affinity FCGR3A SNP. We go on to show that PD-L1 L88E represents a phosphomimetic mutation to PD-L1 L88S leading to decreased protein stability and degradation. Together, our data suggest that position 88 may represent a clinically relevant PD-L1 hotspot position regulating protein stability.In the data presented here, we confirm the selection of two Patient 05-001 was a patient enrolled on the FIRE-6 (AIO KRK-0118) trial with histologically confirmed, treatment-naive, RAS/RAF wild-type metastatic colorectal cancer (MSS) that agreed to take part in a biomarker substudy after informed consent [Ethical Commission of the Ludwig-Maximilians-University Munich (LMU): 18-0933]. The patient received 4 cycles of treatment with FOLFIRI and cetuximab, followed by 4 cycles of FOLFIRI/cetuximab with additional avelumab. These 8 cycles were followed by avelumab maintenance treatment for another 10 weeks until the end of treatment (EOT) due to progression in week 19. Plasma samples were collected at regular intervals in cell-free DNA BCT tubes  and processed according to the manufacturer’s instructions. Plasma was immediately frozen at −80°C until extraction of cell-free DNA, and the leukocyte pellet was stored in 1 ml of heat-inactivated fetal bovine serum  with 10% of dimethyl sulfoxide . Additionally, the paraffin-embedded tumor biopsy obtained before treatment initiation was used for genetic analyses as described below.p-value of <0.05 was considered statistically significant.For mutational profiling, 100 ng of cell-free DNA (cfDNA) was used. Genes were selected through a review of published literature and by using the cBio Cancer Genomics Portal . Either ® Fluorescent quencher. The probe and primer sequences are listed in Digital droplet PCR (ddPCR) custom assays were designed by and ordered from IDT . To increase specificity, locked nucleic acid bases were incorporated into the probes. Probes were HPLC-purified and contained either a 5′-HEX (wild-type probe) or a 5′-FAM (mutant probe) reporter dye and a 3′Iowa BlackPD-L1 L88E, we used the UT-SCC-14 (tongue squamous cell carcinoma) and HT-29  cell lines that were depleted of endogenous PD-L1 by CRISPR/Cas9 as described in Stein et al. (PD-L1 L88E (c.262_263delinsGA) variant and L88M (c.262T>A) as control variants were generated by site-directed mutagenesis of the PD-L1 wt cDNA cloned into the Lentiviral Gene Ontology (LeGO) vector LeGO-iC2-Puro+ via AsiSI/EcoRI. Mutagenesis was performed using the QuikChange® II XL Site-Directed Mutagenesis Kit according to the manufacturer’s instructions. For ectopic re-expression, the PD-L1 constructs were lentivirally transduced and selected as described elsewhere , mCherry , and HPRT1 . The relative gene expression levels were normalized to HPRT1 and calculated according to the comparative Ct (ΔΔCt) method.Total RNA was extracted with the Quick-RNA kit  and reverse-transcribed with SuperScript III . Target amplification was performed on the CFX96 System  using the SYBR Select Master Mix CFX as suggested by the manufacturer and using the following primer pairs: ® ] as isotype controls for 45 min at 4°C. After washing, avelumab was detected using a FITC-coupled α-human-IgG antibody . Readout was performed on a BD FACSCelesta flow cytometer  and analyzed using FlowJo™ .To quantify PD-L1 surface expression, 100,000 cells per well were seeded on a 96-well plate and incubated with 100 ng of avelumab or a human IgG pool [IntratecTo assess the turnover of PD-L1 variants, compound C/dorsomorphin and MG132 were obtained from Selleck Chem . To analyze the degradation kinetics of PD-L1 variants, 2 million cells were seeded in a six-well format and either incubated with 10 µM of compound C for 2 and 4 h or with 20 µM of MG132 for 4 h. Proteins were isolated as described above and subjected to immunoblotting.FCGR3A high-affinity SNP rs396991  followed by a GA to G deletion (PD-L1 L88fs) leading to a premature stop codon at position 104 . Interestingly, patient 05-001 showed homozygosity for the FCGR3A high-affinity SNP rs396991 that was found associated with PD-L1 alterations in the AVETUX trial  that was equally localized in the extracellular domain .To confirm the phosphomimetic properties of the L88E mutation, we next investigated the degradation of the PD-L1 variants as readout. For this purpose, we treated the cell models with MG132, an inhibitor of the 26S proteasome, for 4 h. Immunoblotting revealed that PD-L1 L88S and L88E were enriched faster than the wild-type and the L88A and L88M variants  have been shown to have an especially high affinity to IgG . The acquisition of another phosphorylated serine outside this region allows glycosylation-independent phosphorylation-induced proteasomal degradation. In contrast, the PD-L1 L88fs leads to full PD-L1 loss.Two different alterations at position 88 of unctions , 29, 30.unctions , CDK4/6 unctions , or AMPKPD-L1 L88fs at an earlier timepoint (week 9) than PD-L1 L88E (detected at EOT) appeared counterintuitive since our nucleotide sequence analysis suggested that the L88fs variant emerged from the L88E variant. Interestingly, the “precursor” variant L88E was undetectable both by NGS and ddPCR at week 9. It appears likely that this is rather a matter of sensitivity of detection, but it shows that this variant—at this point in treatment—may have shown a lower biological fitness than the full PD-L1 loss variant L88fs. The strong selective advantage of the L88fs variant in the early treatment phase was likely due to its potent escape from direct antitumor pressure exerted by avelumab. In later phases, this selective advantage might have been counterbalanced by a higher T cell pressure suppressing this PD-L1-negative clone. In the long run, the reduced but not completely absent PD-L1 expression of clone PD-L1 L88E may have offered protection from avelumab’s direct antitumor effects while at the same time counteracting T cell pressure. Notably, other therapeutic anti-PD-L1 antibodies like atezolizumab and durvalumab have IgG regions specifically designed to avoid effector functions  can be found below: The studies involving human participants were reviewed and approved by Ethical Commission of the Ludwig-Maximilians-University Munich (LMU): 18-0933. The patients/participants provided their written informed consent to participate in this study.Conception and design: LC, CS, and MB. Collection and assembly of data: VH, SS, LC, CS, RS, LP, and DS. Data analysis and interpretation: LC, CS, RS, LP, and DS. Manuscript writing and final approval of the manuscript: all authors.This work was funded by Deutsche Krebshilfe (70114663 to MB).SS received honoraria for talks and advisory board role from Amgen, Astra-Zeneca, Bayer, BMS, ESAI, LEO Pharma, Lilly, Merck KGaA, MSD, Pierre-Fabre, Roche, Sanofi, Servier, Taiho, and Takeda and research funding from Merck KGaA, Pierre-Fabre, Servier, and Roche. VH received honoraria for talks and advisory board role from Merck, Amgen, Roche, Sanofi, Sirtex, Servier, Pfizer, Pierre-Fabre, AstraZeneca, BMS, MSD, Novartis, Boehringer Ingelheim, Pierre-Fabre, Celgene, Terumo, Oncosil, and Seagen and research funding from Merck, Amgen, Roche, Sanofi, Pfizer, Boehringer-Ingelheim, Sirtex, and Servier.The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


TMPRSS2 gene, a missense variant common in East Asian populations, contributes to protection against SARS-CoV-2 infection. TMPRSS2 is a protease responsible for SARS-CoV-2 entry and syncytium formation. rs12329760  was associated with a reduced risk of moderate symptoms. The enzymatic activity of Met160-TMPRSS2 was lower than that of Val160-TMPRSS2, and thus the viral entry and the syncytium formation of SARS-CoV-2 were impaired. Collectively, these results indicate that the genetic variation in TMPRSS2, which is common in East Asians, is one of the molecular determinants of COVID-19 susceptibility.COVID-19 has a wide range of clinical presentations, and the susceptibility to SARS-CoV-2 infection and the mortality rate also vary by region and ethnicity. Here, we found that rs12329760 in the  The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has to date resulted in more than 5 million deaths worldwide. COVID-19 is associated mainly with respiratory symptoms and ranges from asymptomatic to fatal cases . AlthougOAS1, OAS2, OAS3, TYK2, DPP9, and IFNAR2) were also identified as critical determinants of the COVID-19 severity in United Kingdom patients  have been conducted to identify genetic variants involved in the development of COVID-19. The ABO blood group locus and a gene cluster on chromosome 3p21.23 were identified as involved in the severity of COVID-19 in Italian and Spanish populations . Genes rpatients . In a GWuropeans . HoweverTMRPSS2 gene were associated with increased severity of influenza A virus and SARS-CoV-2 infections  is responsible for the virus entry of SARS-CoV-2 by processing the viral spike (S) protein after viral attachment to angiotensin-converting enzyme 2 ACE2; . TMPRSS2fections . These fulations . In Italdatabase , but notdatabase . In proscurrence . Here weThis study was conducted as a multicenter prospective observation pilot study with the University of Tsukuba, Division of Clinical Medicine, Faculty of Medicine, as a representative facility. The participants were recruited from areas in Ibaraki Prefecture, where many cases of nosocomial SARS-CoV-2 infection occurred among medical care workers.Clinical information  was obtained from the participants. Laboratory data, including complete blood count, blood sugar, coagulation profile, serum biochemical tests , and urine, were measured at Tsukuba i-Laboratory LLP, Tsukuba, Japan. Serum angiotensin-converting enzyme was measured using ACE ELISA kits .The questionnaire on the degree of contact included the following: The situation that comes to mind (yes or no); the medical care for COVID-19 patients: (1) None, (2) extremely light contact, (3) light contact , (4) tight contact , (5) oral cavity and respiratory tract suction, (6) no recollection; and the rate of implementation  of personal protective measures, including hand-washing and wearing of a surgical mask, an N95 mask, eye protection, a medical gown, or medical gloves.TMPRSS2 according to the manufacturer’s protocol. The TMPRSS2 gene locus was amplified as DNA fragments averaging 250 bp. Post-enriched libraries were quantified, pooled, and sequenced on the NextSeq500 sequencing platform (Illumina). Sequencing data were mapped to hg19 and variants were identified using CLC Genomics Workbench software (Qiagen). The linkage disequilibrium of the common SNPs within the TMPRSS2 locus in East Asian populations was analyzed using LDlink.Genomic DNA was extracted from whole blood samples obtained from the noninfected close contact group by use of MagExtractor genome . The DNA libraries were prepared using a QIAseq Targeted DNA Custom Panel  with region-specific primers for 2r = 0.885), was used as a lead variant for genotype imputation using a population-specific reference panel of Japanese (1000 Genomes project EAS). Note that similar results were obtained for directly genotyped SNPs, rs9305744 .The genotype data of rs12329760 of the asymptomatic-to-mild group, the moderate group, and the severe group were obtained from the Japan COVID-19 Task Force and analyzed by imputation. The genotyping data of Japan COVID-19 Task Force were obtained from Infinium Asian Screening Array and did not include rs12329760. Genotyped variant rs9305744, which is in linkage disequilibrium with rs12329760  were measured using the Bio-Plex Human Cytokine Screening Panel  on a Luminex200  following the manufacturer’s instructions.2. For the construction of plasmids expressing ACE2 and TMPRSS2, total RNA was reverse-transcribed as a template using the oligo(dT)20 primer, and the cDNAs were amplified with the primers 5′-TATGCGGCCGCCACCATGTCAAGCTCTTCCTGGCTCCTTC-3′ and 5′-TATGCGGCCGCCTAAAAGGAGGTCTGAACATCATCA-3′ for ACE2 and 5′-TATGAATTCCACCATGGCTTTGAACTCAGG-3′ and 5′-TATGCGGCCGCTTAGCCGTCTGCCCTCATT-3′ for TMPRSS2, and then cloned into the pCDH plasmid. The V160M substitution was introduced by the PCR-based site-directed mutagenesis using the primers 5′-CATCCTTCAGATGTACTCATCTCAG-3′ and 5′-CATCTGAAGGATGAAGTTTG-3′. The production of the lentivirus was carried out according to the manufacturer’s protocol. A549 cells were transduced with the lentivirus carrying ACE2. The A549 cells constitutively expressing ACE2 were further transduced with the lentiviruses expressing Val160-or Met160-TMPRSS2 .A549 cells were purchased from the American Type Culture Collection and were maintained in Dulbecco’s minimal essential medium (DMEM) containing 10% fetal bovine serum and cultured at 37°C with 5% CO20 primer, and the cDNA was amplified with the primers 5′-CGGTATCGATAAGCTTGATGCCACCATGTTTGTTTTTCTTGTTTTATTGCCACTAG-3′ and 5′-CCCGGGCTGCAGGAATTCGATTTATGTGTAATGTAATTTGACTCCTTTGAGCACTGGCTCAGAGTCGTCTTCATCAAATTTGCAGCAGGATCCACAAGAACAAC-3′, and then cloned into pCAGGS-p7 plasmid. For the transient transfection, 1 × 104 HEK293T cells were inoculated on a 12-well plate and transfected with plasmids expressing S protein and GFP using Lipofectamine 2000  according to the manufacturer’s instruction. After incubating for 5 h at 37°C, 1 × 105 of either A549-ACE2-TMPRSS2-V160 or M160 cells were added and further incubated for 24 h. The cells were then fixed with 2% PFA for 15 min and stained with Hoechst33342. To quantify the level of syncytium formation, the number of GFP-positive cells containing more than two nuclei was counted using ImageJ software. The ratio of the cells showing syncytium formation to total cells was calculated.For the construction of plasmids expressing SARS-CoV-2 S protein, total RNA purified from SARS-CoV-2-infected cells was reverse-transcribed as a template using the oligo(dT)20 primer. Real-time PCR was carried out using SYBR Green Realtime PCR Master Mix-Plus  in the Thermal Cycler Dice Real-Time PCR system (TaKaRa). The Cp values were calculated by the second derivative maximum method using a standard curve. The primer sequences used in this study were 5′-ATGGCATTGGACGGCATTTG-3′ and 5′-TGTTCTGGCTGCATCAT-3′ for TMPRSS2 and 5′-AACGGCTACCACATCCAAGG-3′ and 5′-GGGAGTGGGTAATTTGCGC-3′ for 18S rRNA.Total RNAs were isolated from A549, A549-ACE2-TMPRSS2-V160, and A549-ACE2-TMPRSS2-M160 cells, respectively. cDNA was prepared from 1 μg of total RNAs by use of ReverTraAce  with oligo(dT)Rabbit anti-TMPRSS2  and mouse anti-β-actin  antibodies were purchased. Mouse anti-Spike antibody was kindly provided by Dr. Kensaku Murano (Keio University) .In Situ PLA kit (Olink Bioscience) with anti-mouse PLUS (DUO92001) and anti-mouse MINUS probes (DUO92004) according to the manufacturer’s protocol. Briefly, the cells were incubated with anti-mouse PLUS and anti-mouse MINUS probes for 1.5 h at room temperature, and then washed with Wash Buffer A. The cells were further incubated with PLA ligase for 45 min at 37°C. After washing, the cells were subjected to the amplification reaction using the polymerase, which is supplied in the kit, for 2.5 h at 37°C. After final washing using Wash Buffer B, the cells were stained with 4,6-diamidino-2-phenylindole (DAPI) for 10 min. The number of PLA signals was measured using IMARIS software (Carl Zeiss).Cells were infected with SARS-CoV-2 at an MOI of 100 on ice for 30 min. After the virus adsorption, the cells were washed twice with ice-cold DMEM and then further incubated at 37°C for 5, 10, and 15 min. The cells were then fixed with 4% PFA and incubated with 1% milk for 30 min. The cells were incubated with 10 ng/μl mouse anti-Spike antibody for 1 h and fixed again with 4% PFA. PLA was carried out using a Duolink U-test, the Chi-square test, or 1-way ANOVA with the Tukey test for continuous variables and the Fisher exact test for categorical variables using GraphPad Prism software (version 9.0.2). The statistical power was calculated by R statistical software (version 3.6.0). The Hardy–Weinberg Equilibrium (HWE) was calculated by Chi-square test. The multivariate logistic regression analysis with Wald test was performed to examine the genetic association of rs12329760 between the asymptomatic-to-mild and moderate groups to adjust for age and gender using R statistical software (version 3.6.0). n/s, not significant. **p < 0.01, *p < 0.05. The Q value based on the false discovery rate (FDR) was calculated to adjust for multiple comparisons using the Benjamini-Hochberg test. In the present study, statistical significance was defined as a q < 0.05.Continuous variables were expressed as either medians with interquartile ranges (IQRs) or means with standard deviations (SDs). Categorical variables were expressed as numbers and percentages. Significant differences were determined using the Mann–Whitney via repositioning and suctioning of the oral cavity and respiratory tract, was significantly higher in the infected participants than in the noninfected participants  of the 19 SNPs were located in the exons of TMPRSS2, and only rs12329760 C>T was a missense variant; this variant causes the substitution of valine at amino acid position 160 to methionine. The rs12329760 and other identified SNPs within the TMPRSS2 locus were not in linkage disequilibrium (p > 0.05).We next examined the associations of SNPs in the ilibrium . The minilibrium . Frequenilibrium . The numTMPRSS2 was highly enriched in our cohort (0.388) and in East Asians (0.362) as compared with in Europeans (0.236) and Africans (0.287) as reported in the 1000G database, a database of population genetic variations , carriers of the T allele tended to be a reduced risk of moderate COVID-19 symptoms .We next calculated the odds ratios and confidence intervals using the Baptista-pike method and CI 0.56–0.97) and the crude odds ratio was 0.72 (95% CI 0.55–0.94) in the dominant model. This suggests that age and gender differences are not confounding factors of the susceptibility to SARS-CoV-2 as determined by rs12329760.The median ages of the moderate and severe groups were 54 and 58 years, respectively, while it was 40 years in noninfected close contact and asymptomatic-to-mild groups . Furtherin vitro, we established A549 cells exogenously expressing the TMPRSS2 gene carrying Val160 (A549-ACE2-TMPRSS2-V160) or Met160  consisting of an N-terminal cytoplasmic domain, a transmembrane domain, an LDL receptor class A (LDLR-A) domain, a scavenger receptor cysteine-rich SRCR) domain, and a C-terminal extracellular serine protease (SP) domain. It is known that TTSP family proteins are expressed as zymogens and activated by proteolytic cleavage . TMPRSS2SS2 mRNA . In the SS2 mRNA . The amoSS2 mRNA ,C, suggeR domain,Viral S protein is cleaved into S1 and S2 subunits by the endopeptidase furin. Then, the S2 subunit is further processed into S2′ by TMPRSS2 to activate its membrane fusion activity for the virus entry and the cell–cell fusion, producing multinuclear giant cells, known as syncytia . To examin situ PLA with anti-Spike antibody. We could visualize the single viral particles, which are below the optical microscope resolution limit, by enhancing with single-recognition in situ PLA  was associated with a reduced risk of moderate symptoms. Medical practices such as repositioning and respiratory tract suction increased the infection risk, but our study revealed that the genetic variants in the TMPRSS2 gene contributed to increased susceptibility to SARS-CoV-2 infection, although the statistical power was not optimal due to the limited number of participants . The trend was statistically observed, but no significant difference was obtained after statistical correction of FDRs for the odds ratios  can be found at: The studies involving human participants were reviewed and approved by The Medical Ethics Committee of University of Tsukuba. The patients/participants provided their written informed consent to participate in this study.TS, HKa, AK, and KY: conceived and designed the experiments. TS, HKa, SO, MH, TK, YO, and AK: performed the experiments, and TS, HKa, TK, AK, WM, and KY: analyzed the data. AH, YH, SH, YK, YA, KM, SC, HS, HKo, YO, RE, and HT: provided biological materials. TS, HKa, AK, YO, WM, and KY: wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported in part by grants-in-aid from the Ministry of Health, Labour and Welfare Special Research Projects (20CA2055 to K.Y.) and the Ministry of Education, Culture, Sports, Science and Technology of Japan ; the Research Program on Emerging and Re-emerging Infectious Diseases (JP20fk0108076h0003 to A.K.); the Japan Program for Infectious Diseases Research and Infrastructure (JP20wm0325019h0001 to A.K.); the Japan Agency for Medical Research and Development, CREST and COI-NEXT from the Japan Science and Technology Agency (JPMJCR20H6 and JPMJPF2017 to A.K.); the Takeda Science Foundation (to A.K.); and the NOMURA Microbial Community Control Project of ERATO, Japanese Science and Technology Agency (to A.K.).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Escherichia coli and Lactobacillus reuteri. The EGFP plasmid, synthetic EGFP RNA, and pre-Cel-miR-39 were successfully delivered into the murine microglial BV2 cells via bEVs. PCR and confocal microscopy analysis confirmed the transfer of the EGFP plasmid and RNA. The bEV-delivered exogenous pre-Cel-miR-39 was further processed into the mature form of Cel-miR-39; its incorporation into Ago2—a major component of the RNA-induced silencing complex—was assessed using RNA-immunoprecipitation–PCR. Taken together, bEVs can be used as vehicles to deliver genetic materials and for novel biotechnological applications, such as gene transfer and mRNA vaccines.Extracellular vesicles (EVs), which are nanosized membranous particles secreted from both prokaryotic and eukaryotic cells, can deliver various biological molecules, such as nucleic acids, proteins, and lipids, into recipient cells. However, contrary to what is known about eukaryotic EVs, whether bacterial EVs (bEVs) can be used as transporters for bioactive molecules is becoming a hot area of research. In this study, we electroporated enhanced green fluorescent protein (EGFP) genes and precursor microRNA of Cel-miR-39 (pre-Cel-miR-39) from isolated bEVs of  Delivery of biomolecules via nanocarrier-based systems has been extensively studied in regard to drug delivery and next-generation vaccine development . AlthougBoth gram-positive and gram-negative bacteria naturally secrete bacterial extracellular vesicles (bEVs), which are approximately 20–400 nm in size and contain various cargoes with biological activities, such as proteins, lipids, and genetic materials (DNA and RNA) ,4. AlthoUsing the reporter gene enhanced green fluorescent protein (EGFP), we demonstrated that exogenous electroporated DNA or RNA can be successfully delivered via bacterial EVs into the murine microglial cell line BV2. Furthermore, the electroporated precursor form of eukaryotic microRNA can be processed into the mature form of synthetic miRNA in BV2 cells and can be functionally activated when incorporated into the Ago2 protein. Taken together, it is possible to develop a new type of vaccine using EVs of commensal bacteria; further, bEVs have great potential as gene carriers in the development of gene therapy tools.Escherichia coli (K12 strain) was grown in Luria-Bertani broth  at 37 °C until the optical density at 600 nm reached 0.5. Lactobacillus reuteri (ATCC 23272) was grown in de Man, Rogosa, and Sharpe broth (BD Difco) under anaerobic conditions.E. coli EVs were isolated from the concentrated supernatant using the ExoBacteria™ OMV Isolation Kit . L. reuteri EVs were isolated using gradient ultracentrifugation as previously described [escribed . The isoE. coli and L. reuteri EVs on cell viability, BV2 cells were seeded in 96-well plates . The following day, the cells were treated with different concentrations of bEVs in fresh medium and incubated for another 24 h. Cell viability was then assessed using the MTT  assay, and the absorbance was read at 570 nm using a microplate reader. The results were obtained from three independent experiments; each bar represents the standard deviation.To investigate the effect of The pGFP-N1 vector  was used for EGFP plasmid DNA and in vitro EGFP transcription. The EGFP RNA was synthesized using the HeLaScribe Nuclear Extract in vitro Transcription System  with the plasmid gene (pGFP-N1) as a template, according to the manufacturer’s protocol.Plasmid (500 ng) and in vitro transcribed RNA (1 μg) were electroporated into bEVs (300 μL) under conditions of V = 150 and C = 100 μF. Immediately after electroporation, cells were transferred onto ice. The bEVs were treated with 1 μL of RNase A  and DNase I  to make a final volume of 1.5 mL; the mix was incubated at 37 °C for 25 min.For Ago2 RNA immunoprecipitation (RIP), BV2 cells were incubated with plasmids or RNA-containing bEVs. After 24 h, the culture media were removed, each plate was washed with 10 mL of phosphate-buffered saline (PBS), and the cells were collected and lysed using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific). Lysates (500 μg) were incubated with Dynabeads Protein G (Thermo Fisher Scientific) conjugated with 2 μL of Ago2-specific monoclonal antibody  or without the antibody. The beads were repeatedly washed with PBS, and TRIzol was added to extract bound RNA, which was then subjected to Cel-miR-39-3p  RIP–PCR.RT-PCR was performed to confirm the presence of the electroporated and transferred EGFP plasmid and RNA. The extracted RNA was used to synthesize cDNA using the Omniscript RT Kit . For EGFP detection, the synthesized cDNA and positive control EGFP plasmid DNA were tested using PCR primers . Thereafter, electrophoretic separation on an 2% agarose gel plate was performed to examine the presence of the primer band and to verify the transformation of the plasmid gene. For Cel-miR-39 RIP–RT-qPCR, total RNA (15 ng) was reverse-transcribed using the TaqMan MicroRNA Reverse Transcription Kit . From the 20 μL reaction mixture, 1.33 μL was used for RT-qPCR, which was performed using the TaqMan Universal PCR Master Mix (Applied Biosystems) and TaqMan probes (Applied Biosystems).Plasmid DNA or synthetic RNA-electroporated bEVs were added to BV2 cells cultured on chamber slides and incubated for 24 h at 37 °C. The slides were then washed three times with 1 mL of PBS, followed by incubation with DAPI . The fluorescence signals were visualized using a laser scanning confocal microscope .Caenorhabditis elegans and not in any other known mammalian cells. To obtain the RNA oligo of pre-Cel-miR-39, DNA oligo (5′-UAUACCGAGAGCCCAGCUGAUUUCGUCUUGGUAAUAAGCUCGUCAUUGAGAUUAUCACCGGGUGUAAAUCAGCUUGGCUCUGGUGUC-3′) along with the complementary strand of pre-miR-Cel-39 was synthesized by Bioneer . DNA oligos were annealed and cloned into the pGEM-T Easy vector (Promega), followed by in vitro transcription using the T7 promoter of the vector with an EZ T7 High Yield In Vitro Transcription Kit . The correct clones with the pGEM-T easy vector were confirmed by sequencing after insertion and orientation verification by agarose gel electrophoresis.Cel-miR-39 was chosen because it exists only in The purified bEV samples were diluted 50 times with PBS and applied to 200-mesh Formvar/Carbon grids . Samples were dried on the grids and viewed with a transmission electron microscope  operated at 100 kV.t-test. All analyses were performed using Origin 8.0 . p-values ≤ 0.05 were considered statistically significant. All data are presented as mean ± standard deviation (SD). A significant variation analysis was used to calculate the parametric two-tailed unpaired E. coli and a MOI of 321 for L. reuteri  can cross the blood–brain barrier of mice ,12, we hFurther in-depth studies are needed, including the fine-tuning of necessary bEV particle numbers and conditions for infection. There exists great potential for using engineered bEVs as genetic tools for successful drug delivery as well as novel vaccine development.


Granulocyte colony-stimulating factor (G-CSF) has been widely used to mobilize bone marrow hematopoietic stem/progenitor cells for transplantation in the treatment of hematological malignancies for decades. Additionally, G-CSF is also accepted as an essential mediator in immune regulation, leading to reduced graft-versus-host disease following transplantation. Despite the important clinical roles of G-CSF, a comprehensive, unbiased, and high-resolution survey into the cellular and molecular ecosystem of the human G-CSF-primed bone marrow (G-BM) is lacking so far. Here, we employed single-cell RNA sequencing to profile hematopoietic cells in human bone marrow from two healthy donors before and after 5-day G-CSF administration. Through unbiased bioinformatics analysis, our data systematically showed the alterations in the transcriptional landscape of hematopoietic cells in G-BM, and revealed that G-CSF-induced myeloid-biased differentiation initiated from the stage of lymphoid-primed multipotent progenitors. We also illustrated the cellular and molecular basis of hyporesponsiveness of T cells and natural killer (NK) cells caused by G-CSF stimulation, including the potential direct mechanisms and indirect regulations mediated by ligand–receptor interactions. Taken together, our data extend the understanding of lymphomyeloid divergence and potential mechanisms involved in hyporesponsiveness of T and NK cells in human G-BM, which might provide basis for optimization of stem cell transplantation in hematological malignancy treatment. In the past ten years, unmanipulated haploidentical blood and marrow transplantation that combines granulocyte colony-stimulating factor (G-CSF)-primed bone marrow (G-BM) and G-CSF-mobilized peripheral blood stem cells (G-PBSC) without in vitro T-cell depletion, has been proved to be a reliable protocol with a low risk of GVHD6.Allogeneic hematopoietic stem cell transplantation  is a potentially curative approach available for hematological malignancies8. However, it remains controversial about the mechanisms by which G-CSF regulates hematopoiesis9. Bernitz et al.10 found that G-CSF treatment could increase the number of myeloid-biased CD41+ hematopoietic stem cells (HSCs) in mouse BM. Recently, Xie et al.11 showed that G-CSF directly acted on lymphoid-biased HSCs to increase their divisions and maintain their repopulating activity after transplantation using functional experiments at the single-cell level in mice. In addition, due to technical limitations, our understanding of the effect of G-CSF on hematopoietic differentiation in human BM in vivo is scant.As a commonly used mobilizer, G-CSF is known to increase the numbers of hematopoietic stem/progenitor cells (HSPCs) harvested within the bone marrow (BM) and peripheral blood (PB), and lead to enhanced myeloid hematopoiesis and granulopoiesis14. A growing body of studies demonstrated that G-CSF could attenuate the reactivity of T and natural killer (NK) cells through inducing T helper 2 (Th2) cell polarization15, promoting the generation of regulatory T (Treg) cells16, tolerogenic dendritic cells (DCs)18 and possibly myeloid-derived suppressor cells (MDSCs)21. Intriguingly, compared with G-PBSC transplantation, G-BM transplantation resulted in attenuated acute GVHD and comparable engraftment22, indicating that G-BM was different from G-CSF-mobilized peripheral blood (G-PB) in terms of immunomodulatory properties. However, the systematical landscape of immune cells and underlying mechanisms of immunoregulation in G-BM have not yet been well established23.Moreover, G-CSF mobilization has been demonstrated to reduce GVHD with preservation of the graft-versus-leukemia (GVL) effectHerein, using single-cell RNA sequencing (scRNA-seq) and unbiased bioinformatics analysis, we comprehensively characterized the cellular and molecular alterations in the BM from healthy donors upon G-CSF administration and revealed the potential mechanisms of hematopoiesis changes and immune hyporesponsiveness in T and NK cells.CD34; HOXA10 regulon), myeloid lineage , T/NK lineage , B_lineage  and erythroid/megakaryocyte lineage (EM_lineage) . As expected, G-CSF treatment caused an increase in percentages of HSPCs and myeloid lineage, while a decrease in percentages of T/NK lineage  to isolate different cell lineages and mixed them in a certain ratio for subsequent scRNA-seq Fig. . After sage Fig. , indicatTM1 Fig. .Fig. 1TrCTSG and ELANE  term enrichment analysis also presented reduced cell adhesion , lymphoid-primed multipotent progenitor (LMPP), common lymphoid progenitor (CLP), progenitor B cell (Pro-B), granulocyte-monocyte progenitor (GMP), megakaryocyte-erythrocyte progenitor (MEP), erythrocyte progenitor (ErP), megakaryocyte progenitor (MkP) Fig. , all of -BM Fig. . Howeverage Fig. , indicatage Fig. .Fig. 2Th+ T cells, five subtypes of CD8+ T cells, two subtypes of NK cells, gamma delta T (γδ T) cells, type 1 innate lymphoid cell (ILC1), and type 3 innate lymphoid cell (ILC3)-like cells  and Treg, as well as two effector CD4+ T cell subtypes, namely CD4_effector_CCL5 and CD4_effector_CCR6. CD4_effector_CCL5 showed higher expression of T helper 1 (Th1)-related genes (TBX21 and IFNG), while CD4_effector_CCR6 was characterized by higher expression of genes commonly expressed in T helper 17 (Th17) cells . CD8+ T cells included naïve CD8+ T cells (CD8A+CCR7+) and 4 effector CD8+ T cell subsets (CD8A+CCR7–) exhibiting distinct signatures, namely CD8_effector_CD69, CD8_effector_GZMH, CD8_effector_IFIT3 and CD8_effector_RORC. In detail, CD8_effector_CD69 and CD8_effector_GZMH had higher expression of cytotoxicity-associated genes GZMK and GZMH, respectively. Intriguingly, GZMK (highly expressed by CD8_effector_CD69 and CD8_effector_RORC) and ZNF683 (highly expressed by CD8_effector_GZMH) were reported to be highly expressed by CD8+ T cells in a possible “pre-exhausted” state30. CD8_effector_IFIT3 was characterized by the expression of genes involved in the type I interferon response 31 and related receptors (IFNAR1 and IFNAR2)  cells  Fig. , suggestlls Fig. . Tc17 ce-BM Fig. , which m-BM Fig. . Besides-BM Fig. , which mDUSP233 and TSC22D334)  target genes in G-BM  analysis showed upregulated expression of genes related to immune modulation (34) Fig. , Th2 devsis Fig. . Gene Sells Fig. , which w-BM Fig. , which wlls Fig. , and a hlls Fig. , which fSOCS138, TSC22D3, ZFP3639, and DUSP2  and immunosuppression was predicted to be regulated directly by G-CSF binding with G-CSF receptor  in T/NK cells40, along with slightly higher expression of CSF3R following G-CSF stimulation  in G-BM as previously reported43, which could result from potential direct regulation by binding of G-CSF with G-CSFR , plasmacytoid dendritic cells (pDCs), conventional dendritic cells (cDCs), neutrophils and two relatively naïve subclusters, named pre-monocyte and pre-neutrophil  and lower expression of lymphocyte activation genes (LST1 and HLA-DRA)  and negative regulation of immune response (TSC22D3 and SOCS1) in G-BM , and proinflammatory IL-12 pathway (ZFP36L1 and JUN)   have been identified as immature myeloid cells with a remarkable ability to suppress T-cell responses both in antigen-specific and -nonspecific manners associated with the production of nitric oxide and cytokines51. In our data, all monocyte subclusters exhibited stronger M-MDSC features as indicated by gene scoring analysis using reported MDSC gene signatures  Fig. . As the UN) Fig. . CD16+ mT4) Fig. , which ires Fig. 52, indic53. Thus, we further explored altered cell–cell communication between T/NK cells and other cells including monocytes, DCs, Treg, ILC1, and B subclusters , further supporting their roles in immune regulation  with their sender cells  in G-BM   and TNFR2 (encoded by TNFRSF1B) has been reported to mediate anti-inflammation process in multiple inflammatory conditions62. Collectively, we comprehensively explored the potential indirect mechanisms by which G-CSF regulated immune response of T and NK cells via cell-to-cell communication.In mobilized NK cells, genes of upregulated ligand–receptor pairs were enriched in immunosuppressive pathways such as those associated with reduced NK cell-mediated cytotoxicity and negative regulation of immune effector process Fig. . These ulls Fig. . In detalls Fig. , which wCs) Fig. . InteracIrf8 and β-catenin progenitor self-renewal network in GMP7. However, the specific stages in which G-CSF induces enhancement of myelopoiesis and the underlying molecular mechanisms remain unclear, especially in humans. Combining the significantly changed percentages of GMP and lymphoid progenitors as well as the trajectory inference for hematopoiesis, LMPP was speculated as the earliest target progenitor of G-CSF inducing myeloid-biased hematopoiesis in human BM.G-CSF is a hematopoietic growth factor that induces granulopoiesis and HSPC mobilization. Our data showed a significantly increased percentage of myeloid cells, as well as that of HSPCs, especially GMP. A previous study also reported acceleration of GMP formation upon the release of G-CSF in mouse BM probably by triggering the 64, our data showed that CD4+ T cells tended to be Th2 polarized, with the downregulation of proinflammatory pathway-related cytokines . T and NK cells in G-BM had stronger apoptosis properties. It has been reported that G-CSF enhanced the apoptosis of splenic T cells to inhibit inflammation and potentially induce immune tolerance in mice65. We also observed that CD8+ T and NK cells had lower cytotoxicity scores, which further indicated hyporesponsiveness of T and NK cells in G-BM.Besides, we further characterized the immunologic alterations in immune cells upon G-CSF administration. Consistent with the previous studiesCSF3R was slightly higher upon G-CSF treatment as previously reported66. We further speculated that G-CSF might directly regulate the target genes related to the immune response such as SOCS1. Notably, our recent work, which has been posted on bioRxiv and is now under review elsewhere, has validated that the upregulated SOCS1 in T cells inhibited T cell proliferation in vitro, thus potentially contributing to immune tolerance38. Apart from the direct effect on T/NK cells, G-CSF was generally recognized to regulate T and NK cell function in an indirect manner mediated by interacting cells, especially monocytes and DCs68. Through cell-to-cell communication analysis, we found that intercellular interactions of T/NK cells with monocytes and DCs have undergone significant changes upon G-CSF administration. Some ligand–receptor interaction pairs were significantly upregulated, including S100A8:CD69 and GRN:TNFRSF1B. Previous studies have demonstrated that S100A8:CD69 interaction was required for the upregulation of STAT3 signaling pathway, thus promoting Treg differentiation and probably inhibiting Th17 differentiation59. The expression of Cd69 in Treg cells in mice was considered to be necessary to maintain immune tolerance, probably by affecting the secretion of factors such as IL-1070. Here, CD69 in CD8+ T cells also showed elevated expression and enhanced interaction with S100A8 from monocytes in G-BM, implying a presumed role in immunomodulation of CD8+ T cells. In addition, the binding of PGRN and TNFR2 (GRN:TNFRSF1B) was reported to exhibit anti-inflammatory properties by competitively interfering with TNF/TNFR-mediated inflammation62. Furthermore, experiments in mice indicated that PGRN could significantly protect Treg from a downregulation caused by TNFα, and regulate the function of effector T cells by inhibiting IFN-γ secretion61. Taken together, these ligand–receptor interactions may play complicated roles in cell-mediated regulation of immune response and inflammation in G-BM.To explore the underlying mechanisms of these particular immunologic changes, we initially investigated the potential direct mechanism wherein G-CSF regulates T and NK cells by binding with G-CSFR. Our results showed that the expression of In summary, we provided a high-resolution transcriptome map of G-BM and deciphered the effects of G-CSF on lymphomyeloid divergence and immune hyporesponsiveness. Our results suggest that G-CSF-induced myeloid-biased differentiation initiates from the stage of LMPPs in human BM. Moreover, we delineated the potential cellular and molecular mechanisms of the immunosuppressive effect of G-CSF in human G-BM. G-CSF stimulation can upregulate the expression of genes related to immune hyporesponsiveness in T and NK cells via the direct binding with G-CSFR and diverse cell-to-cell communications. These findings will further provide implications for allo-HSCT and immune cell-based targeted therapy.We recruited two healthy donors and treated them with recombinant G-CSF  at a dosage of 5 μg/kg body weight per day for 5 consecutive days. The BM samples were collected by aspiration on the fourth day of G-CSF treatment. The study was approved by the Ethics Committee of Peking University People’s Hospital and was conducted in accordance with The Declaration of Helsinki. Informed consents were obtained from all participants.g for 6 min and suspended in FACS buffer (1× PBS with 1% BSA) for subsequent staining. Cells were stained in sorting buffer with specific antibodies for 30 min at 4 °C. The following antibodies were used for the staining and sorting: anti-CD45 , anti-CD66b , alongside the viability dye 7-amino-actinomycin D (7-AAD) PerCP-Cy5.5 . Given the dramatic increase in neutrophils after G-CSF treatment, we pooled neutrophils and other cells together in a certain ratio for scRNA-seq to construct a complete single-cell transcriptional landscape of human G-BM. Through flow sorting, the final cells used for scRNA-seq contained CD45+CD66b– population (90%), CD45+CD66b+ population (5%), and CD45– population (5%). Cells were sorted using an Aria II Flow Cytometer (BD Bioscience). Pre-gating was first done for live cells based on a 7-AAD staining. Data were analyzed using BD FACSDIVA v8.0.1 and Flowjo (v10).Human BM cells were isolated from healthy donors before and after in vivo 5-day G-CSF application by Ficoll density centrifugation. Erythrocytes were removed by incubating with RBC lysis buffer . After neutralization, the remaining cells were collected by spinning at 350× https://support.10xgenomics.com/single-cell-gene-expression/library-prep/doc/technical-note-assay-scheme-and-configuration-of-chromium-single-cell-3-v2-libraries). Sequencing was performed on the Illumina Hiseq X Ten platform .For 10× Genomics-based scRNA-seq, we implemented the Chromium Single Cell 3’ v2 libraries, under the guidance of the official instruction manual  < 1000 unique molecular identifiers (UMI); (2) < 300 genes; or (3) > 10% UMIs derived from the mitochondrial genome. Doublets were identified by the R package DoubletFinder72 with default settings. After the removal of the low-quality cells and doublets, a total of 21,005 cells and 26,937 genes were kept for further analyses. Then, UMI counts were normalized by the NormalizeData function and top 2000 features with high cell-to-cell variation were calculated using the FindVariableFeatures function. Next, the ScaleData function was conducted to scale and center features in the datasets and the RunPCA function was used with default parameters to reduce dimensionality. The R package Harmony (version 0.1.0)73 was used for batch correction to avoid the batch effect across samples. The data were used in the subsequent nonlinear dimensional reduction with the RunUMAP function and cluster analysis by the FindNeighbors and FindClusters functions. All details regarding the Seurat analyses performed in this work can be found in the website tutorial .For scRNA-seq data, raw gene expression matrices were generated for each sample by the Cell Ranger (version 4.0.0) pipeline coupled with human reference hg19. The output gene expression matrices were analyzed by R software (version 3.6.3) with the Seurat package (version 3.2.2)74 was also used to help the cell state identification based on key regulon activity. The regulon activity scores were calculated with AUC by the AUCell package. Signature genes of each cell cluster were identified with the FindAllMarkers function in Seurat. To further decode the hematopoietic landscape in G-BM, major cell types were further sub-clustered. Scaling, principal component analysis, and clustering were performed as described above. Additionally, all percentages of different cell types were calculated after removing neutrophils.After the projection of all cells into two-dimensional spaces by UMAP, cells were clustered together according to common features. The annotation of cell clusters was identified by the expression of cell type-associated genes. SCENICP value adjusted by Benjamini–Hochberg < 0.05 were considered significant. Enrichment analysis of DEGs was conducted using the Metascape webtool (https://metascape.org). GSEA was performed using the clusterProfiler package (v3.13.0)75.DEGs were recognized using the FindAllMarkers function in Seurat with parameter ‘test.use = wilcox’ by default and the Benjamini–Hochberg method was used to estimate the false discovery rate (FDR). DEGs were filtered by fold change of > 1.25. DEGs with To map hematopoietic differentiation, pseudotime analysis was performed with Monocle v2. For the pseudotime ordering analyses (before and after G-CSF administration), the gene expression matrix issued from the integrated samples was loaded in Monocle2 using the new CellData function. The filtered genes with mean expression ≥ 0.1 and an empirical dispersion at least twice as large as the fitted dispersion were set as ordering genes. To avoid cell cycle effects, cell cycle genes in GO: 0007049 were further excluded from ordering genes. Then, ‘DDRTree’ function was used for dimensionality reduction and ‘plot_cell_trajectory’ function for visualization.76, 4 cytotoxicity-associated genes  and 829 highly expressed genes reported in M-MDSCs52 to define proliferation, migration, apoptosis, immunosuppression, cytotoxicity, and M-MDSC scores, respectively. For cell cycle analysis, CellCycleScoring function in Seurat package was used to predict the classification of each cell in either G1, S or G2M phase.AddModuleScore function in Seurat package was used to calculate the gene set scores. We used cell proliferation (GO: 0007049), leukocyte migration (GO: 0050900), positive regulation of apoptosis process (GO: 0043065), an immnuosuppression-related gene set (61 genes)77 with default parameters. Only ligand–receptor interaction pairs with P value < 0.05 returned by CellPhoneDB were considered significant. Scdiffcom54 was used to perform differential intercellular communication analysis before and after G-CSF administration. Up- or downregulated ligand–receptor pairs were then filtered with adjusted P value < 0.05 and fold change > 1.5. The regulatory effect of the differential communication on T and NK cells was further explored by predicting downstream target genes of curated ligand–receptor interactions. To speculate target genes of altered ligand–receptor pairs, we retrieved ligand–receptor–target relationship from publicly available resources: intercellular links (ligand–receptor interactions) and intracellular subnetworks  from NicheNet40 and scMLnet78 for further analysis. Only DEGs before and after G-CSF administration were included into analysis of target gene prediction. The igraph R package and Cytoscape (version 3.6.1) were used for visualization of the ligand–receptor–target relationship.Ligand–receptor interactions were identified by CellPhoneDBt-test were used for comparisons of gene expression levels. We considered P value < 0.05 statistically significant. Analyses were conducted using R software (version 3.6.3).Wilcoxon-rank-sum test and Student’s Supplementary InformationSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Table S6Supplementary Table S7Supplementary Table S8Supplementary Table S9