Diffuse large B-cell lymphoma (DLBCL) is an aggressive, heterogeneous non-Hodgkin B lymphoma, that 35% of cases produces relapsed/refractory (R/R) disease. At this point, the search of prognostic and predictive factors in DLBCL is indispensable through key biomarkers. Recently, we have found that the level of circulating MDSCs is a good marker of survival in a translational study based on the R2-GDP-GOTEL study (EudraCT Number: 2014-001620-29). Since Vitamin D is a known regulator of the immune system, we aimed to assess the evolution of circulating suppressor cells MDSCs, Tregs, and inhibited T cells in patients with deficit of vitamin D (<15 ng/mL) and sufficient levels of vitamin D (>15 ng/mL). We observed a reduction in blood suppressor cells in the group with normal vitamin D levels, but not in patients with vitamin D deficit, supporting that R2-GDP and vitamin D may have immunomodulatory functions that favors a better clinical evolution.The search of prognostic factors is a priority in diffuse large B-cell lymphoma (DLBCL) due to its aggressiveness. We have recently found that the level of circulating MDSCs is a good marker of survival in a translational study based on a trial (EudraCT Number: 2014-001620-29), using lenalidomide combined with R-GDP . Since Vitamin D is a known immunomodulator, we have studied blood levels of these cell populations comparing patients with deficit of vitamin D levels (<15 ng/mL with those with normal levels >15 ng/mL. Mann–Whitney U test was used to compare cells distributions between groups, Wilcoxon test to compare cells distribution at different times and Spearman test to measure the association between cell populations. Patients with vitamin D deficit maintained the increased level of immune suppressor cells, whereas we observed a depletion of all immune suppressor cells in patients with normal vitamin D levels. In conclusion, we have confirmed the importance of vitamin D in the response to treatment in R/R DLBCL, suggesting that vitamin D deficit may be involved in the immune deficit of these patients, and thus, vitamin D supplementation in these patients may help to obtain a better response, warranting further investigation. Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive non-Hodgkin lymphoma (NHL) subtype, representing between 30% and 40% of cases every year . One-thiDue to the aggressive behavior of the disease and its different clinical outcomes, some prognostic factors have been evaluated, including (but not limited to) vitamin D and the immune system . VitaminThe role of vitamin D in some biological processes is well known, like the bone metabolism, or the calcium homeostasis and regulation . However2D3 enhances innate immune responses by increasing the production of antimicrobial proteins and both chemotactic and phagocytotic responses of macrophages [In recent decades, a great interest has emerged in cancer concerning to the potential role of the immune system and its modulation by vitamin D, since vitamin D receptor (VDR), a member of the nuclear receptor superfamily, is expressed to a greater or lesser extent on both innate and adaptive immune cells, such as neutrophils, dendritic cells (DCs), or T and B lymphocytes . Vitaminrophages , and by rophages , found irophages .By contrast, VDR is highly expressed during the T cell activation and indirectly stimulates the polarization of T cells from Th1 and Th17 to Th2 phenotype via antigen-presenting cells (APCs) by inhibiting the expression of MHC-II or co-stimulatory molecules, and directly by inhibiting the production of IL-12, IFN-γ, or IL-17 and stimulating IL-4, again leading the polarization from Th1 and Th17 to Th2 cells, as well as allowing the development of regulatory T cells (Tregs) by increasing FOXP3 and IL-10 production . In cancIn this work, our purpose was to evaluate the evolution of different immune suppressor cells  throughout the R2-GDP treatment in R/R DLBCL patients with normal vitamin D (NVD) levels and vitamin D deficiency (VDD) , recruited in the R2-GDP-GOTEL study. In addition, we aimed to compare basal vitamin D levels of patients with those in healthy donors (HD) and to correlate initial vitamin D levels of patients according to OS at 24 months.From April 2015 to September 2018, the R2-GDP-GOTEL trial recruited 79 patients diagnosed of R/R DLBCL not candidates for ASCT from 18 Spanish hospitals. Major inclusion criteria included patients with R-CHOP-like treatments previously received and ECOG ≤ 1. They were treated with the R2-GDP schedule, based on lenalidomide, rituximab, gemcitabine, dexamethasone and cisplatin, and granulocyte colony-stimulating factor. Vitamin D levels could only be analyzed in 63 R/R DLBCL patients before treatment and in all HD (20). Data analyses in cycle 3 and EOI were lower due to adverse effects or death in some patients throughout the course of the treatment. A summary of the number of patients included in the data analysis is shown in p < 0.001), as shown in p = 0.010) compared with OS < 24 month patients, as shown in Serum vitamin D levels were significantly lower in R/R DLBCL patients before starting treatment compared with the age-matched HD (p = 0.047 (for M-MDSCs) and p = 0.020 (for G-MDSCs). Consequently, total MDSC levels were also remarkably reduced in the same group of patients (p = 0.002), as shown in p < 0.001 in both cases).Both M-MDSC and G-MDSC populations decreased in blood after treatment in the NVD group comparing with basal (p = 0.015), and between basal vitamin D and M-MDSCs after treatment , as shown in In addition, significant negative correlations were found between basal vitamin D levels and G-MDSCs before treatment . Statistical differences were also found in cycle 3 (p = 0.048) and EOI (p = 0.004) between NVD and VDD patients.Circulating Tregs were decreased in both vitamin D groups at the end of the induction with R2-GDP . Howeverp = 0.037) and after treatment , as shown in In addition, negative correlations were found between basal vitamin D levels and Tregs before , as shown in p = 0.037), CD8+ (p = 0.047) and total PD-1+OX40− T cells (p = 0.013).CD4+, CD8+, and total PD-1+OX40− T cell levels were significantly reduced after the R2-GDP schedule in NVD patients compared with basal determinations (p = 0.030).Negative correlations were found between initial vitamin D and EOI inhibited PD-1 T cells , but stap = 0.027 for CD4+, p = 0.014 for CD8+, and p = 0.002 for total CTLA-4+OX40− T cells), as shown in p = 0.003) and total CTLA-4+OX40− T cells (p = 0.021). Statistical differences between NVD and VDD groups were also identified at EOI in CD8+ (p = 0.015) and in both CD4+ and total CTLA-4 T cells (p < 0.001).In line with PD-1+OX40- T cells, all inhibited CTLA-4+OX40− T cell levels were also reduced in blood after treatment comparing with basal (p < 0.001), rS = −0.431 in CD8+ (p = 0.036), and rS = −0.735 in total exhausted CTLA-4+OX40− T cells (p < 0.001).Again, significant negative correlations were found between basal vitamin D levels and EOI CTLA-4 T cells, as show in 2D has been demonstrated to have anticarcinogenic properties [In recent years, there has been a great enthusiasm due to the promising role of vitamin D in the prevention and immunomodulation of cancer. The first observation of the inverse relationship between the exposure to sunlight and cancer mortality was made in 1941 by Apperly et al. . Since toperties . Becauseoperties  but its operties . In DLBCL, low concentrations of vitamin D have been found in patients at diagnosis and a deficiency of this vitamin type has been correlated with poorer clinical outcomes ,18,43,44Vitamin D has essential interactions with both innate and adaptive cells for normal immune functions, and impaired concentrations could produce dysregulated immune responses , thus coHere, we show the results from the same trial but focused on the concentration of serum vitamin D before starting R2-GDP schedule and its impact on immune suppressor cells in patients with deficit of vitamin D (<15 ng/mL) or normal levels of vitamin D (>15 ng/mL). We found increased baseline levels of circulating M-MDSCs, G-MDSCs, and total MDSCs independently of the vitamin D concentration. However, when we monitored MDSCs during the course of the treatment we observed a remarkably depletion of these cell populations in the group of patients that had vitamin D levels >15 ng/mL, while MDSC populations were broadly unchanged in the group with deficit of vitamin D. Lenalidomide has inhibited MDSC-mediated immunosuppression in multiple myeloma  and vitaThe role of vitamin D over Tregs also seems to be clear since this substance has been demonstrated to promote the differentiation of Tregs in many diseases ,54,55,56Apart from the regulatory subset of T cells, we also studied other T cell subpopulations, such as PD-1 and CTLA-4 T cells, mainly involved in different pathways as immune checkpoint in cancer ,50. We oWe studied circulating immune suppressor cells and vitamin D levels of patients from the R2-GDP-GOTEL study (EudraCT Number: 2014-001620-29), a phase II clinical trial that recruited a total of 79 patients diagnosed of R/R DLBCL who were not high-dose CT and ASCT candidates from 18 Spanish hospitals between April 2015 and September 2018. They received the R2-GDP schedule, based on lenalidomide + R-GDP . An age-matched group of 10 women and 10 men was also recruited as healthy donors in July 2019 from the Virgen Macarena University Hospital . Median age were 71.3 years old in R/R DLBCL patients and 68.2 years old in the healthy group.+CD11b+CD33+HLA-DRlow/−CD14+CD15−, G-MDSCs as CD45+CD11b+CD33+HLA-DRlow/−CD14−CD15+, Tregs as CD4+CD25highCD127low/−, inhibited PD-1+ T cells as CD3+CD4+PD-1+OX40− and CD3+CD8+PD-1+OX40−, and inhibited CTLA+ T cells as CD3+CD4+CTLA-4+OX40− and CD3+CD8+CTLA-4+OX40−. Absolute cell number was calculated by multiplying flow cytometry percentages with total leucocyte count from hematologic count . Total MDSCs were calculated as the sum of M-MDSC and G-MDSC counts, total PD-1 T cells as the sum of CD3+CD4+PD-1+OX40− and CD3+CD8+PD-1+OX40− T cell counts, and total CTLA-4 T cells as the sum of CD3+CD4+CTLA-4+OX40− and CD3+CD8+CTLA-4+OX40− T cell counts.Three peripheral blood analysis were carried out during the R2-GDP-GOTEL study: basal, cycle 3 and end of induction (EOI). MDSCs, Tregs, and inhibited PD-1+OX40− and CTLA-4+OX40− T cells were measured by flow cytometry using the FACSCanto II flow cytometry system  from EDTA-K3 tubes. Gates of cell populations are shown in Antibodies were obtained from Becton Dickinson Immunocytometry Systems  and were used at the manufacturer’s recommended concentrations.MDSCs: PerCP-Cy5.5 Mouse Anti-Human CD 45 (ref no. 564105), APC-Cy7 Rat Anti-CD11b (ref no. 557657), PE Mouse Anti-Human CD 33 (ref no. 555450), PE-Cy7 Mouse Anti-Human HLA-DR (ref no. 560651), FITC Mouse Anti-Human CD 14 (ref no. 555397), and APC Mouse Anti-Human CD 15 (ref no. 551376).Tregs: Human regulatory T cell cocktail (ref no. 560249), including PerCP Mouse Anti-Human CD4, PE Mouse Anti-Human CD127, and FITC Anti-Human CD25.PD-1 and CTLA-4 T cells: APC-Cy7 Mouse Anti-Human CD3 (ref no. 560176), PE-Cy7 Mouse Anti-Human CD4 (ref no. 557852), PerCP-Cy5.5 Mouse Anti-Human CD8 (ref no. 565310), APC Mouse Anti-Human PD-1 (CD279) (ref no. 558694), and PE Mouse Anti-Human CTLA-4 (CD152) (ref no. 557301).®  system.Basal vitamin D samples were collected in cryotubes from serum-separating tubes and stored at −80 °C. At the end of the R2-GDP-GOTEL study, all samples were measured in the same day by an automated chemiluminescence immunometric analysis using the LiaisonStatistical analysis and graphs were performed by GraphPad Prism 8.0.2 . Normal distribution of analyzed variables was checked by watching histogram, box plot, Q-Q plot, and the outcomes of normality tests of Shapiro–Wilk and Kolmogorov–Smirnov.p values ≤ 0.05 were considered for statistically significant differences.Non-parametric tests were used due to absence of normality. Mann–Whitney U test was used to compare basal cell distributions of all patients vs. HD, and NVD vs. VDD patients in every determination . The Wilcoxon test was used to compare cell distributions of patients in basal vs. EOI determinations. Data shown are median and 95% confidence intervals. Bivariate correlations among cell populations were carried out using Spearman coefficient. In last years, efforts in cancer research have been mainly focused on the targeting of immune suppressor cells to overcome tumor resistance. As we know, some emerging prognostic factors in DLBCL are the immune system and vitamin D. This is the reason why we decided to evaluate the evolution of immune response suppressor cells, such as MDSCs, Tregs, and both inhibited PD-1+OX40− and CTLA-4+OX40− T cells in R/R DLBCL patients with deficit and normal levels of vitamin D levels using R2-GDP as an immunomodulator in the R2-GDP-GOTEL study (EudraCT Number: 2014-001620-29). In B-cell lymphomas, blood vitamin D levels were shown to affect both the course and progression of the disease by different mechanism of action, such as a possible systemic effects, effects on pharmacokinetics or the activation of the NF-κB pathway , and the


Breast cancer (BC) is one of the major malignancies worldwide. Circular ribonucleic acids (circRNAs) are a class of conserved ribonucleic acid (RNA) molecules that play important roles in various diseases. Recently, circRNAs have been suggested to have diagnostic value and may function as potential diagnostic biomarkers for BC. Previously, hsa_circ_0043278 was found to be downregulated in human BC. However, its role in human BC has not yet been identified.in vitro. A xenograft experiment was conducted to validate the inhibitory effect of hsa_circ_0043278 on tumour growth. The interaction between hsa_circ_0043278 and miR-455-3p was confirmed by a dual-luciferase reporter assay. Mimics and inhibitors of miR-455-3p were designed to confirm the influence of hsa_circ_0043278 on the hsa_circ_0043278/miR-455-3p/etoposide-induced gene 24 (EI24) axis.The levels of hsa_circ_0043278 in BC cell lines were verified by quantitative reverse transcription–polymerase chain reaction (qRT–PCR). The overexpression vector and short hairpin RNA (shRNA) of hsa_circ_0043278 were transfected into MDA-MB-231 and MCF-7 cells, respectively. The effects of hsa_circ_0043278 on tumour cell growth, migration and invasion were measured by 3--2,5-diphenyl-tetrazolium bromide (MTT), colony formation, wound healing and Transwell assays in vitro and suppressed tumour growth in vivo. Downregulation of hsa_circ_0043278 led to the opposite results. Hsa_circ_0043278 expression was negatively correlated with that of miR-455-3p. In addition, mechanistic investigation proved that hsa_circ_0043278 directly bound to miR-455-3p and regulated EI24 and NF-κB expression in BC cells.Hsa_circ_0043278 was downregulated in BC cell lines. Furthermore, overexpression of hsa_circ_0043278 notably decreased BC cell viability and inhibited BC cell migration and invasion Hsa_circ_0043278 acts as a tumour suppressor gene in BC through the hsa_circ_0043278/miR-455-3p/EI24 axis and may be regarded as a new prognostic predictor or potential therapeutic target in BC.The online version contains supplementary material available at 10.1186/s12885-021-08989-w. Breast cancer (BC) is the second most common cancer worldwide and is a major cause of female death , 2. SomeCircular ribonucleic acids (circRNAs) are a class of stable, conserved ribonucleic acid (RNA) molecules , 9. ReceIn the present study, the low expression of hsa_circ_0043278 in BC cell lines was examined. Moreover, its target miRNA, miR-455-3p, has been considered an oncogene in BC owing to its inhibitory effect on etoposide-induced gene 24 (EI24) expression . EI24 haA total of 50 pairs of BC tissues and matched normal tissues were collected from BC patients at The First Affiliated Hospital of China Medical University (from January 2005 to December 2012). All patients were diagnosed with primary BC, female, between 27 and 81 years old, and did not receive chemotherapy or radiotherapy before surgery. Tissue specimens were obtained after surgical resection and stored at -80 °C in an ultralow-temperature freezer . The matched normal tissues were collected from regions more than 5 cm outside the edge of BC tissues. The study was approved by the Ethics Committee of The First Affiliated Hospital of China Medical University. All procedures performed in this study involving specimen collection and experiments were in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients.2).Human BC cell lines (MDA-MB-231 and MCF-7) and a normal mammary epithelial cell line (MCF-10A) were purchased from the Cell Bank of the Chinese Academy of Sciences. The MDA-MB-468, BT-549, SK-BR-3, T47D and HEK 293T cell lines were maintained in our laboratory. MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium  containing 10% foetal bovine serum . MCF-7 cells were cultured in minimum essential medium  containing 10% FBS and 0.01 mg/ml insulin from bovine pancreas . Michigan Cancer Foundation-10A (MCF-10A) cells were cultured in mammary epithelial basal medium . The other cell lines were cultured appropriately. All these cell lines were maintained at 37 °C in a humidified incubator with 5% carbon dioxide  using the SYBR Green method . β-Actin was used as the control for the normalization of circRNA and messenger RNA (mRNA) levels, while U6 was used for normalization of miRNA levels. Table -△CT method. All the experiments were repeated three times.Total RNA from tissues or cells was extracted with a simple Total RNA Kit  following the manufacturer’s instructions. The purity and concentration of the RNA were determined using a NanoDrop 2000 spectrophotometer . Complementary deoxyribonucleic acid (cDNA) was reverse transcribed from the total RNA using Moloney Murine Leukaemia Virus (M-MLV) Reverse Transcriptase (BioTeke). Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) was performed in an Exicyclerhttps://www.ncbi.nlm.nih.gov/). The miR-455-3p mimics and inhibitors were purchased from GenePharma . Vectors, mimics, and inhibitors were transiently transfected into BC cells with Lipofectamine 2000  following the manufacturer’s protocols. The sequences of the siRNAs and shRNAs are shown in Table To overexpress hsa_circ_0043278, the mature sequence of hsa_circ_0043278  was synthesized and then cloned into the pCD5-ciR vector  and was named “ov-circ”. A mock vector without the hsa_circ_0043278 sequence (ov-NC) served as the negative control. To knock down hsa_circ_0043278, two small interfering RNAs (siRNAs) targeting the back-splice junction site of hsa_circ_0043278 and a negative control siRNA (si-NC) were synthesized (GenScript). Consequently, qRT–PCR proved that siRNA-2 was the most effective siRNA, and it was used to construct the siRNA plasmid Fig. . The shRProteins from tissues and cells were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer  supplemented with 1 mM phenylmethylsulfonyl fluoride . A total of 40 μg (in 20 μl) of protein from each sample was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and was then transferred onto polyvinylidene difluoride (PVDF) membranes . Next, the membranes were incubated with 5% milk at room temperature for 1 h. Then, they were incubated with the appropriate primary antibody overnight at 4 °C and then with a horseradish peroxidase-conjugated secondary antibody . The specific primary antibodies included anti-EI24  and anti-NF-κB (P65)  antibodies. β-Actin and histone H3  served as the internal controls. Immune complexes were finally visualized with an enhanced chemiluminescence system (Beyotime).3 cells per well. MTT solution  was added to each well at the indicated time points , and the plates were incubated at 37 °C in 5% CO2 for 4 h. Next, the medium was removed, and 200 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The optical density at 570 nm was recorded using an automatic microplate reader .Cells were seeded in 96-well plates at a density of 3×102 for 2 weeks. The cells were fixed and stained with Giemsa solution . Colonies consisting of at least 50 cells were counted and photographed under a microscope.Briefly, cells in each group were seeded in 35-mm dishes at 500 cells/dish and incubated at 37 °C in 5% CO5 cells/well) and grown in 6-well plates for 48 h post transfection. Next, the cells were cultured in serum-free culture medium and treated with 1 μg/ml mitomycin C (Sigma-Aldrich) for 1 h. Wounds were created in the middle of the wells using 200-μL pipette tips. The cells were then washed and cultured in serum-free culture medium, and wound closure was monitored by imaging with a phase contrast microscope . After culture at 37 °C in 5% CO2 for 48 h, the widths of the wounds were measured again, and the migration rates were calculated in each group.Cells were seeded  in 200 μL of serum-free medium. An 800-μL volume of medium containing 20% FBS was added to the lower chambers. The pore size of the Transwell membrane was 8.0 μm. After incubation at 37 °C in 5% CO2 for 24 h, the cells that migrated to the bottom of the filter were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet  and were then photographed and counted under an inverted phase contrast microscope (Motic).For the migration assays, 2×104 cells in 200 μL of serum-free medium were seeded in the upper chambers of a Transwell plate with a Matrigel-coated membrane . An 800-μL volume of medium containing 20% FBS was added to the lower chambers. The pore size of the Transwell membrane was 8.0 μm. After incubation at 37 °C in 5% CO2 for 24 h, the cells in the lower compartment were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet (Amresco) and were then photographed and counted under an inverted phase contrast microscope (Motic).For the invasion assays, 2×107 cells/ml) of MDA-MB-231 cells stably transfected with the hsa_circ_0043278 overexpression vector or MCF-7 cells stably transfected with hsa_circ_0043278 shRNA was subcutaneously injected into the right axillae of the mice, and mice injected with mock vector or shRNA served as negative controls . The tumour sizes were measured once a week with a calliper. The tumour volumes were calculated using the following formula: 1/2 (length × width2). In brief, mice were sacrificed in the 4th week, and the tumours were then excised, photographed and weighed.A total of 18 two-month-old female BALB/c (nu/nu) mice were purchased from Wanleibio . The animal care and experimental procedures were approved by the Experimental Animal Ethics Committee of The First Affiliated Hospital of China Medical University. All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health . We made great attempts to reduce the pain experienced by the animals. Mice were adaptively fed for 1 week under specific pathogen-free conditions. To establish xenograft tumours, 0.2 mL (5 × 10The expression of NF-κB (P65) in the paraffin-embedded xenograft tissue was detected by immunohistochemistry. Tissues were incubated with the anti-NF-κB (P65)  primary antibody at 4 °C overnight and then with the secondary antibody at 37 °C for 60 min and an HRP-labelled streptavidin solution for 10 min. Next, tissues were stained by diaminobenzidine (DAB)  and observed under a microscope. The mean density of P65 in the tissues was calculated using Image-Pro software.The sequences of wild-type hsa_circ_0043278 or a mutant without miR-455-3p binding sites were synthesized and subcloned into the luciferase reporter vector pmirGLO (GenScript); the resulting vectors were named “circ-WT” and “circ-Mut”, respectively. HEK 293T cells were cotransfected with the vectors and miR-455-3p mimics or negative controls. Relative luciferase activity was measured with a Dual-Luciferase Assay Kit (KeyGEN BioTECH) in accordance with the manufacturer’s protocols after 48 h of incubation. Luciferase activity was measured using a Tecan Infinite M200 Pro luminometer .t test with the Wilcoxon signed-rank test. Comparisons of the means among multiple groups were performed by one-way analysis of variance (ANOVA) with the Kruskal–Wallis test. Pearson correlation coefficients were calculated for correlation analyses. Continuous data are presented as the mean ± standard deviation values (SD). A P value less than 0.05 was considered to indicate statistical significance. All experiments were repeated three times.Statistical analysis was performed using GraphPad Prism 7.0 software . The data for two groups were compared using a In our previous study, hsa_circ_0043278 was found to be downregulated 43-fold in BC tissues compared to matched normal tissues by microarray analysis. In addition, hsa_circ_0043278 was found to be downregulated in BC tissues compared to matched normal tissues by qRT–PCR . In thisP<0.01), whereas downregulation of hsa_circ_0043278 significantly increased the viability of MCF-7 cells  and was significantly increased in MCF-7 cells by downregulation of hsa_circ_0043278 . Conversely, the migration rate of MCF-7 cells was markedly increased by downregulation of hsa_circ_0043278 (P<0.01). Similarly, the results of the Transwell assays showed that upregulation of hsa_circ_0043278 significantly inhibited the migration  or mock vector (ov-NC) or MCF-7 cells transfected with hsa_circ_0043278 shRNA (sh-circ) or the negative control shRNA (sh-NC) into female nude mice. After 4 weeks, the tumours formed from ov-circ MDA-MB-231 cells were significantly smaller and lighter than the tumours formed in the control group (P<0.05), and the tumours formed from MCF-7 cells transfected with sh-circ were larger and heavier than the tumours formed in the sh-NC group . Conversely, downregulation of hsa_circ_0043278 significantly promoted tumour growth ; in contrast, downregulation of hsa_circ_0043278 significantly increased the level of P65 in xenograft tumour tissues , whereas its expression was significantly elevated after knockdown of hsa_circ_0043278 in MCF-7 cells . Moreover, the miR-455-3p mimics did not decrease the luciferase activity in either circ-Mut group  and that knockdown of hsa_circ_0043278 markedly decreased the level of EI24 in MCF-7 cells  and increased it in the cytosol in MDA-MB-231 cells (P<0.0001), while knockdown of hsa_circ_0043278 resulted in the opposite effects in MCF-7 cells  and MDA-MB-231 (with the lowest expression of hsa_circ_0043278) cells for subsequent research. Functional experiments suggested that overexpression of hsa_circ_0043278 significantly decreased the viability and the migration and invasion abilities of BC cells miRNAs have been reported to bind to the 3'UTR and inhibit the expression of their target mRNAs –31. CircFurthermore, EI24 has been reported to be a target gene of miR-455-3p . It is aAdditionally, we found that hsa_circ_0043278 expression was much lower in TNBC cell lines than in cell lines of other subtypes of BC. Thus, the relationship between TNBC and hsa_circ_0043278 needs to be verified in the future. The precise relationship between hsa_circ_0043278 and NF-κB also needs to be elucidated. Furthermore, the number of patient samples needs to be increased. Multicentre trials are also needed to investigate the roles of hsa_circ_0043278.This study identified downregulation of hsa_circ_0043278 in BC cells and tissues. Additionally, hsa_circ_0043278 was found to significantly inhibit BC progression by sponging miR-455-3p, thus influencing the level of EI24 and the activity of NF-κB. Hsa_circ_0043278 may be regarded as a novel prognostic predictor and a potential therapeutic target for BC. The hsa_circ_0043278/miR-455-3p/EI24 axis might be a crucial mechanism of BC cell progression.Additional file 1: Table S1. The sequences of primers in this studyAdditional file 2: Table S2. The sequences of siRNAs and shRNAsAdditional file 3: Table S3. miR-455-3p inhibitor sequenceAdditional file 4: Figure S1. Two siRNAs (siRNA-1 and siRNA-2) targeting the back-splice junction site of hsa_circ_0043278 were constructed, and the expression of hsa_circ_0043278 in MCF-7 cells was analysed by qRT–PCR. The data are presented as the mean ± standard deviation values (n = 3); **** P < 0.0001.Additional file 5: Figure S2. The expression of miR-103a-2-5p, miR-302b-3p, miR-302c-3p, and miR-520d-3p was measured by qRT–PCR after overexpression of hsa_circ_0043278 in MDA-MB-231 cells. The data are presented as the mean ± standard (n = 3).Additional file 6: Figure S3. Western blot bands of EI24 (A) and β-actin (B) after hsa_circ_0043278 overexpression in MDA-MB-231 cells.Additional file 7: Figure S4. Western blot bands of β-actin (A) and EI24 (B) after hsa_circ_0043278 downregulation in MCF-7 cells.Additional file 8: Figure S5. Western blot bands of NF-κB (P65) (A) and β-actin (B) in the cytosol after hsa_circ_0043278 overexpression in MDA-MB-231 cells.Additional file 9: Figure S6. Western blot bands of histone H3 (A) and NF-κB (P65) (B) in the nucleus after hsa_circ_0043278 overexpression in MDA-MB-231 cells.Additional file 10: Figure S7. Western blot bands of β-actin (A) and NF-κB (P65) (B) in the cytosol after silencing hsa_circ_0043278 in MCF-7 cells.Additional file 11: Figure S8. Western blot bands of histone H3 (A) and NF-κB (P65) (B) in the nucleus after silencing hsa_circ_0043278 in MCF-7 cells.Additional file 12: Figure S9. Western blot bands of EI24 (A) and β-actin (B) after hsa_circ_0043278 overexpression and/or transfection of the miR-455-3p mimic in MDA-MB-231 cells.Additional file 13: Figure S10. Western blot bands of β-actin (A) and EI24 (B) after hsa_circ_0043278 downregulation and/or transfection of the miR-455-3p inhibitor in MCF-7 cells.Additional file 14: Figure S11. Western blot bands of NF-κB (P65) (A) and β-actin (B) in the cytosol after hsa_circ_0043278 overexpression and/or transfection of the miR-455-3p mimic in MDA-MB-231 cells.Additional file 15: Figure S12. Western blot bands of histone H3 (A) and NF-κB (P65) (B) in the nucleus after hsa_circ_0043278 overexpression and/or transfection of the miR-455-3p mimic in MDA-MB-231 cells.Additional file 16: Figure S13. Western blot bands of NF-κB (P65) (A) and β-actin (B) in the cytosol after hsa_circ_0043278 downregulation and/or transfection of the miR-455-3p inhibitor in MCF-7 cells.Additional file 17: Figure S14. Western blot bands of histone H3 (A) and NF-κB (P65) (B) in the nucleus after hsa_circ_0043278 downregulation and/or transfection of the miR-455-3p inhibitor in MCF-7 cells.


Anaplastic diffuse large B-cell lymphoma(A-DLBCL) is a rare morphological subtype characterized by the presence of polygonal, bizarre-shaped tumor cells. Our previous research found that A-DLBCL displays many genetic alterations and biological features that differ greatly from those of ordinary DLBCL. However, the status of tumor immune microenvironment components and checkpoint molecules in A-DLBCL remains unclear.Thirty A-DLBCL patients were enrolled to study tumor immune microenvironment components and checkpoint molecules and their associations with clinicopathological features and prognosis.vs 10%, P=0.004) than patients with ordinary DLBCL. FISH analysis showed that extra copies of PD-L1 were more frequent in A-DLBCL cases than in ordinary DLBCL cases . The numbers of PD-1+ TILs (tumor infiltrating lymphocytes) and CD8+T cells were significantly lower in A-DLBCL versus ordinary DLBCL. In contrast, the numbers of GATA3+ Th2 cells, FOXP3+ Tregs and CD33+ myeloid-derived suppressor cells (MDSCs) were significantly higher in A-DLBCL than in ordinary DLBCL. The associations between clinicopathological features and tumor immune microenvironment cell frequency were analyzed in A-DLBCL patients. Briefly, the number of PD-1+ TILs was lower and the number of CD33+ MDSCs was higher in patients with mutated TP53 compared to those with wild-type TP53. The number of FOXP3+ Tregs was much lower in patients with a noncomplete response (CR) to chemotherapy than in those with a complete response. The number of CD8+ T cells showed a decreasing trend in patients with high International Prognostic Index (IPI) scores and in those with concurrent MYC and BCL2 and/or BCL6 abnormalities. Univariate survival analysis showed that patients with PD-L1+, mPD-L1+ or mPD-L1+ status had a significantly poorer overall survival (OS) than those with PD-L1- status. An increase in the number of CD3+ T cells, FOXP3+ Treg cells and T-bet+ Th1 cells was significantly associated with prolonged OS in patients with A-DLBCL.Patients with A-DLBCL presented higher expression of PD-L1 (40% Our study suggests that A-DLBCL displays a distinct pattern of tumor immune microenvironment components and checkpoint molecules that distinguish it from ordinary DLBCL. The analysis of tumor immune microenvironment components and checkpoint molecules could help in predicting the prognosis of A-DLBCL patients and determining therapeutic strategies targeting the tumor immune microenvironment. Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma; it is more prevalent in elderly patients, with a median age in the 70s. However,it also occurs in young adults but rarely occurs in children. Clinically, most patients present with a rapidly growing tumor mass involving one or more lymph nodes and extranodal sites , 2. DLBCTP53 mutation, as well as concurrent abnormalities of MYC and BCL2 and/or BCL6, and most cases had a non-GCB immunophenotype. Patients with A-DLBCL follow an aggressive disease course and have poor prognosis  is a rare morphological type, representing approximately 3-4% of all DLBCL cases , 2, that+ T cells, allowing tumor cells to escape T-cell–mediated tumor-specific immunity, thereby promoting tumor development. Various studies have reported an association between increased expression of PD-L1 and poor prognosis in several cancers, including melanoma, lung cancer, ovarian cancer, and DLBCL  of B-cell lymphoma significantly contributes to tumor progression and immune evasion . RecentlWe analyzed 30 patients with A-DLBCL and 50 patients with ordinary DLBCL, both originally diagnosed, from June 2004 to April 2016. Four pathologists reviewed all cases according to the 2016World Health Organization classification of tumors of hematopoietic and lymphoid tissues . Sections were subjected to staining protocols (detailed information on the antibodies is listed in +) or in nonmalignant stromal cells . PD-L1 staining on tumor cells was considered positive in cases with moderate (2+) or strong (3+) cytoplasm reaction, and the percentage of the positive tumor cells was set as above 30%. Once reaching the 30% threshold, the PD-L1 positivity of nonmalignant stromal cells was ignored. Among PD-L1-negative DLBCL cases, in which PD-L1+nonmalignant microenvironment cells represented 20% or more of the total tissue, cellularity was defined as microenvironmental PD-L1(mPD-L1+) DLBCL . Quantification of cells was performed with the nuclear Kisight assay provided by the manufacturer (Applied Imaging) , CD8(cytotoxic T lymphocytes), T-bet(Th1 cells), GATA3(Th2 cells), FOXP3(regulatory T cells), CD68, CD163(M2-TAMs), and CD33. Germinal center B cell(GCB) and non-GCB subtypes of DLBCL were classified using the Hans  and Choi+) DLBCL . PD-1+ TImaging) .FISH analysis was performed using LSI probes for PD-L1 on a Thermobrite System  according to the manufacturer’s instructions. Images were collected using a workstation equipped with software . Areas with a minimum of 70% tumor cells were counted, and signals from 100 non overlapping nuclei were analyzed. Positivity was determined above and a 30% threshold for extra copies (defined as copy number ≥ 3/cell). The 9p24.1 gene (PD-L1 gene) was labeled with Spectrum Red, and centromeres were labeled with Spectrum Green as a control probe on 9p. After calculation of red and green in areas with a minimum of 70% tumor cells were counted, and signals from 100 non-overlapping nuclei, the numbers of red and green was calculated. 4 or more was considered as amplification. 3 was considered as copy gains . Pearson’s χThirty patients with A-DLBCL were enrolled, of whom 20 were male (66.7%) and 10 were female (33.3%), with a median age of 61.5 years. Only 1(3.3%) patient was classified as Ann Arbor stage I, 3(10%) as Ann Arbor stage II and 8(26.7%) as Ann Arbor stage III. The remaining 13(43.3%) patients were at Ann Arbor stage IV. Sixteen (53.3%) patients had elevated serum LDH, while 9(30%) had normal LDH levels. Clinical data are summarized in + A-DLBCL was identified based on double immunostaining for PD-L1 and PAX-5. Representative IHC images for PD-L1 are shown in +A-DLBCL was 40% (12 of 30), which was higher than that of ordinary DLBCL  according to our results.All A-DLBCL cases contained large bizarre-shaped tumor cells with abundant cytoplasm containing irregularly shaped nuclei. Membrane expression of PD-L1 by the tumor cells was observed in the investigated sections. PD-L1vs 4.0%, P=0.001).Amplifications targeting the PD-L1 locus were observed in 7 out of 30 A-DLBCL cases. Seven cases (23.3%) were amplifications, and 2(6.6%) were copy gains, and the other 21 cases (70%) were normal. Representative examples of cytogenetic alterations identified by FISH are presented in To evaluate the cellular composition of the TIME in A-DLBCL, we used markers targeting various cells of the TIME in IHC analysis. Representative immunostaining of A-DLBCL cases is shown in + large bizarre-shaped tumor cells, there were also cases with PD-L1+ nonmalignant stromal cells, and we defined them as mPD-L1+ (PD-L1+/PAX5-)  in A-DLBCL patients than patients with ordinary DLBCL.In addition to cases containing PD-L1vs 20.6 ± 4.1/HPF, represented by CD33, while M2 TAMs (CD163) and M1/M2 TAMs (CD68) showed no significant difference between A-DLBCL and ordinary DLBCL.We found that MDSCs were more abundant in A-DLBCL than in ordinary DLBCL(P=0.039), with 33.8 ± 4.2/HPF + T cells  and PD-1+ TILs  in A-DLBCL compared to ordinary DLBCL. Moreover, there seemed to be no significant difference in CD3+ T cells and Th1 cells between A-DLBCL and ordinary DLBCL cases.The distribution of TILs was diffuse in all cases. The difference between the numbers of cells expressing GATA3 and FOXP3 in A-DLBCL and DLBCL was significant after statistical analysis, indicating that there were more Th2 cells and Treg cells in the TME of A-DLBCL than in ordinary DLBCL. In contrast, staining of CD8 showed decreased numbers of CD8+ TILs (CD3) and MDSCs (CD33) in A-DLBCL cases. There seemed to be fewer PD-L1+ TILs in TP53 mutant cases than in TP53 wild type cases . For MDSCs, the result was the opposite . The comparison also showed that patients with fewer Treg cells  in the TIME(FOXP3) had a non-complete response(Non-CR) to chemotherapy, since high FOXP3 expression had a significant correlation with complete response. The number of CD8+ cells showed a decreasing trend in patients with high International Prognostic Index (IPI) scores . All results are shown in Same as in our previous study , the mut+/mPD-L1+ or mPD-L1+ had significantly poorer overall survival(OS) than those with PD-L1- status. In consistent with DLCBL, A-DLBCL patients with PD-L1+ has a poorer prognosis than PD-L1-. An increase in CD3+, FOXP3+ and T-bet+ cell numbers was significantly associated with prolonged OS in patients with A-DLBCL .+ and mPD-L1+ in A-DLBCL compared with ordinary DLBCL. In the TME, the numbers of PD-1+ TILs and CD8+ cells were significantly lower in A-DLBCL. In contrast, the numbers of GATA3+ cells, FOXP3+ cells and CD33+ cells were significantly higher in A-DLBCL patients. These differences between A-DLBCL and ordinary DLBCL were associated with poor prognosis in A-DLBCL.In this study, we demonstrated the molecular pathogenesis of primary A-DLBCL and its association with clinical characteristics. In brief, our findings showed high expression of PD-L1Traverse-Glehen et al.  divided + DLBCL is defined as 20% or more PD-L1+ nonmalignant cells among the total tissue cellularity in PD-L1– DLBCL. Studies have shown that the prevalence of mPD-L1+ DLBCL is approximately 15% in DLBCL  than those with PD-L1-in DLBCL. These results may help explain both tumor escape from host immune surveillance and inhibition of activated T-cells in A-DLBCL.PD-1 belongs to the B7/CD28 family and is expressed on the surface of activated T and B cells, follicular helper T cells, dendritic cells (DCs), and monocytes. The interaction between PD-1 and its ligand PD-L1 reduces T cell proliferation and cytokine release and inhibits survival proteins (e.g. bcl-xl), which results in apoptosis , 26, 27.in DLBCL , 37. Our+ tumor-infiltrating lymphocytes (TILs). Others have reported that the quantity of PD-1+ TILs is significantly positively correlated not only with PD-L1 expression in tumor cells but also with PD-L1 expression in tumor cells/macrophages. Moreover, the therapeutic response can be affected by several parameters, such as the mutational load of GATA3+ cells, FOXP3+ cells and CD33+ cells and the abundance of TILs in lung cancer or melanoma  (TIME cells include tumor-infiltrating lymphocytes (TILs), regulatory T cells, and myeloid-derived suppressor cells (MDSCs). Some studies have demonstrated that PD-L1-expressing tumor cells can induce apoptosis in PD-1melanoma –40. The melanoma  and is wn others –44. To bs(MDSCs) , 46 can s(MDSCs) , 48, it s(MDSCs) . In our Several studies have used CD163 as an M2 tumor-associated macrophage (TAM) marker and CD68 as a pan macrophage marker(M1+M2) –52. Emer+ T cells are generally thought to play a central role in the antitumor immune response, and the presence of CD8+has been reported as a prognostic factor in cancer , while the expression of Th2 markers in A-DLBCL was increased. According to existing reports, increased Th2 marker levels accelerate the secretion of interleukin(IL)-10 and IL-4 and inhibit cellular immune function, and the GATA3/T-bet(G/T) ratio of tumor-infiltrating lymphoid cells is an independent negative predictive marker for survival , 55. Oven cancer , 58. OurThe standard therapy for patients with DLBCL contains rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone(R-CHOP). By such regimen, approximately 60–70% of patients with DLBCL are cured of the disease. However, 30–40% of patients experience disease relapses or, in a small patient subset, are refractory to R-CHOP therapy. The great potential of our recent advances is based on the understanding of the molecular characteristics of DLBCL, and we hope this will be translated into the development of novel, highly effective therapies for patients with A-DLBCL. With the advent of novel targeting agents/regimens, one that specifically targets A-DLBCL will eventually be developed. In addition, our final cohort contains only 30 A-DLBCL cases, which make the results relatively preliminary. Our study requires more cases to make the conclusion closer to the real world.+, mPD-L1+, CD33+, CD163+ and GATA3+ cells and low levels of CD8+ and PD-1+cells in the TIME; these TIME features could highlight key molecular markers of the prognosis of A-DLBCL, which is poorer than that of ordinary DLBCL. A-DLBCL displays a distinct pattern of tumor immune microenvironment components and checkpoint molecules that distinguish it from ordinary DLBCL. These results could help in understanding the prognosis of A-DLBCL patients and determining therapeutic strategies that target the tumor immune microenvironment.In conclusion, an immunosuppressive state in A-DLBCL may be described by both high levels of PD-L1The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Medical Ethics Committee of the First Affiliated Hospital of the Air Force Medical University . The patients/participants provided their written informed consent to participate in this study.RL, ML and ZW contributed to the conception of the study; JX and KY performed the experiment; DZ, JM, JC, KW and YL contributed significantly to analysis and manuscript preparation; TX, JC and KW performed the data analyses and wrote the manuscript; QJ contributed greatly to the revised manuscript. YW, LF, QY, SG, GC, QC, HX, FL and CQ helped perform the analysis with constructive discussions. All authors contributed to the article and approved the submitted version.National Natural Science Foundation of China (81570180).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.


Molecular details of field rabies virus (RABV) adaptation to cell culture replication are insufficiently understood. A better understanding of adaptation may not only reveal requirements for efficient RABV replication in cell lines, but may also provide novel insights into RABV biology and adaptation-related loss of virulence and pathogenicity. Using two recombinant field rabies virus clones (rRABV Dog and rRABV Fox), we performed virus passages in three different cell lines to identify cell culture adaptive mutations. Ten passages were sufficient for the acquisition of adaptive mutations in the glycoprotein G and in the C-terminus of phosphoprotein P. Apart from the insertion of a glycosylation sequon via the mutation D247N in either virus, both acquired additional and cell line-specific mutations after passages on BHK (K425N) and MDCK-II (R346S or R350G) cells. As determined by virus replication kinetics, complementation, and immunofluorescence analysis, the major bottleneck in cell culture replication was the intracellular accumulation of field virus G protein, which was overcome after the acquisition of the adaptive mutations. Our data indicate that limited release of extracellular infectious virus at the plasma membrane is a defined characteristic of highly virulent field rabies viruses and we hypothesize that the observed suboptimal release of infectious virions is due to the inverse correlation of virus release and virulence in vivo. Rabies virus (RABV) and related lyssaviruses are highly neurotropic rhabdoviruses, which cause an almost invariably fatal disease once neurological symptoms appear . The negThe RABV glycoprotein G is located at the surface of RABV particles and is responsible for receptor binding and ribonucleoprotein (RNP) release in the cytoplasm by pH-dependent membrane fusion. Although essential for cell entry and in vivo spread , G is diNeuronal cell adhesion molecule (NCAM1), p75 neurotrophin receptor (p75NTR), nicotinic acetylcholine receptor (nAChR), and metabotropic glutamate receptor subtype 2 (mGluR2) have been described as RABV entry receptors ,9,10. InAdaptation of field RABV isolates to more ubiquitous non-neuronal receptors may represent one key bottleneck to successful RABV cell culture replication. However, there is no experimental proof available that receptor usage differs between field and laboratory RABVs. By demonstrating infection of CNS-resident astrocytes by different field RABVs, a recent comparison of various field and lab-adapted RABV strains revealed an even broader host cell tropism of field RABVs in vivo . Beyond receptor usage, infection of glial cells by highly virulent field RABVs may also be determined by the replication potential and the capacity of the virus to antagonize the innate immune response in that specific cell type ,16. The Another receptor-independent factor is the glycosylation status of the surface glycoprotein G. Typically, in the course of rabies virus cell culture adaptation, the acquisition of additional N-glycosylation sites in the surface glycoprotein G leads to a more efficient G expression ,19,20,21The availability of recombinant field RABV clones  allowed Cells and viruses. BSR T7/5 baby hamster kidney (BHK) cells , murine Virus stock production and passages. After rescue of the recombinant RABVs (rRABVs) on the BHK cell clone BSR T7/5, rRABV Dog and rRABV Fox  stocks wGeneration of rRABV Dog-NTN and BSR T7/5-free rescue of rRABV Dog*. The full-length plasmid of rRABV Dog mutant rRABV Dog-NTN  was generated by sequential site-directed mutagenesis of the full length cDNA plasmid prRABV-DogA14 . After i2 wells and presence of the virus was monitored by indirect immunofluorescence with RABV N- and G-specific antibodies. Cell culture supernatants of positive samples were transferred to freshly seeded Na 42/13 cells for stock virus production. The absence of adaptive mutations in G was verified by Sanger sequencing of RT-PCR products.In order to avoid any BHK-specific adaptation, rRABV Dog* was directly rescued in Na 42/13 cells by co-transfection of prRABV-DogA14 with plasmids coding for RABV N, P, L , and cod5 cells were seeded in 3.5 cm dishes. After overnight incubation, the cell culture medium was replaced by 1 mL serum-free DMEM. To prepare the transfection mix, plasmid DNA and PEI were separately diluted in 400 µL DMEM each and then mixed after a 5 min incubation at room temperature. After an additional incubation of 20 min and 1 h after exchanging the cell culture medium for serum-free DMEM, the mix was added to the cell culture. The cell culture medium including the transfection mix was exchanged for fresh culture medium after 3.5 h incubation at 37 °C.DNA transfection. Plasmid DNA transfections into BSR T7/5 cells were performed with polyethyleneimine . Briefly, 2.5 to 5.0 × 106 BSR T7/5 in a T25 cell culture flask were infected at an MOI of 1. After 2 h incubation at 37 °C, the cells were trypsinized and transferred to 6-well cell culture plates. After an over-night incubation, the infection was monitored by GFP autofluorescence and the plasmid DNA was transfected. Two days later, the cell culture supernatants were collected, centrifuged , and the infectious virus titers in the supernatants were determined by titration on BSR T7/5 cells. In trans-complementation of G-deficient RABV. G gene-deleted SAD ΔG GFP was complemented by plasmid-expressed G protein as described previously . BrieflyTM Reagent (Invitrogen) according to the supplier’s instructions. Four hundred nanograms of RNA were used in a reverse transcription (RT) with 100 U RevertAid Premium Reverse Transcriptase (Thermo Scientific) according to manufacturer’s instructions. The 1.6 kb G-coding cDNAs of rRABV Dog variants passaged on BSR T7/5 cells were cloned in a plasmid vector by PCR amplification of the respective G-coding sequence with the primers 5‘-TAA TCA GAA TTC GGA AAG ATG GTT CCT CAG GTT CTT TTG-3‘ and 5‘-ATC TAT GCT AGC GAA GCG TCG AGA GGA TGA CC-3‘, and insertion of the EcoRI/NheI-digested PCR DNA fragment in an EcoRI/NheI-digested pCAGGS vector [RNA extraction, RT-PCR, and cloning of G gene cDNAs. RNA was prepared from infected cell monolayers with TRIzolS vector .Full length RABV sequencing. Total RNA was extracted from cell culture supernatant using peqGOLD TriFast . Full-length virus genome sequencing was done as described before . Brieflyg, the cell pellet was resuspended in 100 µL lysis buffer , 1.5 mM MgCl2, 1% (v/v) Triton-X-100, 20 µg micrococcal nuclease (NEB)). After incubation for 30 min at 30 °C, the RNA digest was stopped by the addition of TRIzolTM Reagent and the RNA was prepared. Amplicon sequencing and variant calling. For amplicon sequencing, viral RNA was prepared from infected BSR T7/5 cells . To this end, the cells were trypsinized and suspended in 5 mL cell culture medium. After a 5 min centrifugation step at 1500× By RT-PCR amplification with specific primers carrying a gene-specific and a sequencing platform-specific part , amplicoSince in the analyzed P/M region comprises a homopolymer sequence, the amplicons were generated to be compatible with Illumina technology. Here, for library preparation a two-step PCR protocol was employed. First, a set of gene-specific primers with overhang linker adapters were used for RT-PCR followed by a limited-cycle PCR with primers carrying Illumina specific dual index adapters. The amplicons were quantified with the KAPA Library Quantification Kit Illumina (Roche) and sequenced on an Illumina MiSeq instrument in combination with MiSeq reagent Kit v3 (Illumina).Subsequently, libraries were quality checked and adapter sequences were removed using 454 Sequencing Systems Software  prior to mapping with bowtie2 (v2.4.1) against the reference LN879480. Mapped reads were filtered for the specific gene regions and only reads covering primer to primer were selected for further analysis. A random subset of each 10,000 reads from both mapping directions was selected and analyzed for sub-consensus variants using LoFreq (v 2.1.3.1).Antibodies, sera, and chromatin stain. The monospecific rabbit sera N161-5 and P160-5 against RABV N and P proteins and the mouse monoclonal antibody E559 against RABV G protein have been described previously ,32. RabbIndirect immunofluorescence, confocal laser-scanning microscopy, and image processing. For indirect immunofluorescence detection, cells growing on glass cover slips were fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). After 30 min incubation at RT, the PFA was removed and the specimens were blocked by incubation with 0.025% skim milk powder in PBS for 20 min at RT. Immunostainings were performed by a two-hour incubation with primary antibody, two wash steps with PBS, and a subsequent 45 min incubation with Alexa Fluor-conjugated secondary antibodies. After two wash steps with PBS, the specimens were mounted on object slides using Kaiser’s glycerol gelatin.Images were acquired with a Leica DMI6000 TCS SP5 confocal laser scan microscope  with sequential acquisition of the fluorophores. Images were processed with the ImageJ software version 1.48b .4  and stained with uranyl acetate . After stepwise dehydration in ethanol, cells were cleared in propylene oxide, infiltrated with Glycid ether 100 , and polymerized at 60 °C for 3 days. Ultrathin sections were prepared with an ultramicrotome  and collected on 300 mesh EM grids . The grids were counterstained with uranyl acetate and lead citrate, and finally examined with a transmission electron microscope  at an accelerating voltage of 80 kV.Electron microscopy. For EM analysis, BSR T7/5 monolayer cells were infected at an MOI of 0.01 and fixed at 72 h post-infection for 120 min with 2.5% glutaraldehyde buffered in 0.1 M Na-cacodylate, pH 7.2, 300 mOsmol . Cell pellets were embedded in low melting agarose  and cut in small pieces. After that the samples were postfixed in 1.0% aq. OsOStatistical Analysis. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism (v8.1.0).Recombinant virus clones rRABV Dog and rRABV Fox were derived from field virus cDNA clones prRABV Dog and prRABV Fox, and serially passaged on murine Na 42/13 neuroblastoma, fibroblastoid baby hamster kidney (BHK-21 clone BSR T7/5), and epithelial Madin–Darby canine kidney cells (MDCK-II) . Prior t4 to 1 × 107 focus forming units (ffu) was observed over time. Also, a 30-fold titer increase was observed for rRABV FoxB-P10 (6 ffu/mL) strongly decreased for the P2 virus and then slowly increased again in later passages . Notably, the rRABV DogB-P10 titers were ~100-fold higher than those of the rRABV DogB-P1 from 16 hpi onwards a, confirTo assess whether the serial passaging of the viruses led to obvious alterations in the intracellular distribution of virus proteins, immunofluorescence analysis of BHK, Na 42/13, and MDCK-II cells infected with the rRABV DogB/N/M-P1 and -P10 viruses at an MOI of 0.01 were performed . Strong After the ten virus passages, all viruses exhibited a pronounced plasma membrane localization of the glycoprotein G, while the intracellular G accumulation was strongly reduced. These data indicated that the G protein of the field virus rRABV Dog accumulates within the infected cells and that serial cell culture passages led to an increased cell surface presentation of the viral glycoprotein. Co-localization studies of rRABV DogB-P1 G with the endoplasmatic reticulum (ER) marker calnexin further revealed that the G protein of non-BHK-adapted rRABV DogB-P1 accumulates in the ER . FollowiDeep sequence analysis of all six P10 viruses  was perfFor rRABV DogB-P10, N-P10 and M-P10, four, three, and five amino acid (aa) substitutions were identified, which exceeded a frequency of 50% of reads, respectively . All seqInterestingly, identical aa exchanges in G were identified in both passaged rRABV Dog and rRABV Fox viruses in a host cell-independent and -dependent manner. Of the rRABV DogB-P10 glycoprotein mutations D247N, A400T, and K425N, the D247N exchange was identified in all P10 viruses, whereas K425N specifically only appeared in BHK cell-passaged rRABV DogB-P10 and rRABV FoxB-P10, but not in Na 42/13 and MDCK-II cell-passaged viruses. These data indicated that D247N represents a general adaptive mutation, which independently appeared in the rRABV Dog and rRABV Fox viruses, either in the course of virus stock production on Na 42/13 cells or in subsequent cell passages. In contrast to D247N, independent selection of K425N in BHK cell-passaged rRABV DogB-P10 and rRABV FoxB-P10 without appearance in the other P10 viruses indicated its significance as a host cell-specific adaptation possibly required for increased virus replication in BHK cells. A400T only appeared in rRABV DogB-P10 and thus may represent a rRABV DogB-specific adaptive mutation. Other unique and abundant aa substitutions in G were identified in rRABV DogN-P10 (T187M), rRABV DogM-P10 (R264L and R346S), and rRABV FoxM-P10 (R350G and S379A). Similar to the K425N mutation in BHK cell-passaged viruses, arginine replacements R346S and R350G at positions in close proximity to each other in the glycoproteins of MDCK II-passaged rRABV DogM-P10 and rRABV FoxM-P10 may represent host cell-specific adaptations.Notably, beyond the G mutations, amino acid exchanges at an identical position in the C-terminal region of the phosphoprotein (R293C and R293H) were identified in two independent viruses, rRABV DogB-P10 and rRABV FoxM-P10, respectively, suggesting additional adaptation within the P gene. Whereas rRABV DogN-P10 and rDogM-P10 also exhibited one aa exchange in P at aa positions 83 and 277, no alterations were observed in the P gene of rRABV FoxB-P10 and rRABV FoxN-P10. Overall, the deep sequence analysis highlighted the selection of cell line-dependent and cell line-independent adaptive mutations, which may result in increased cell culture replication of the passaged viruses.High frequencies of 99.8%, 94.4%, and 99.5% for D247N, A400T, and K425N in rRABV DogB-P10  indicateIn trans-complementation of G gene-deleted and GFP reporter-expressing SAD ΔG GFP rabies virus with clones 1, 2, 3, 5, and 6 revealed that all tested G’s from rRABV-Dog-B-P10 led to increased infectious virus titers when compared to the wild-type dog virus G protein, although a significance level of * p ≤ 0.05 was only reached for clone 2 , A400T (DTK), and K425N (DAN) as well as different combinations thereof b. WhereaTo investigate the time course over which the mutation accumulated, genome regions comprising the phosphoprotein mutation R293C (382 kb cDNA fragment P/M), glycoprotein mutation D247N (308 bp cDNA fragment G-1), and A400T/K425N (391 kb cDNA fragment G-2) were amplified by RT-PCR from rRABV DogB virus passages P1, P3, P5, P7, and P10, and amplicon sequencing was performed.The amino acid exchange R293C in P , DogB waIn G, the amino acid exchange at position 247 (D247N) was already present in P1, as indicated by 99.8% frequency after the first passage. In amplicon G-1, additional non-silent single-nucleotide polymorphisms (SNPs) leading to amino acid substitutions S165P and T187M were detected , however, were not detectable anymore at P7 and P10. In the G-2 amplicon, both A400T and K425N were not detectable in P1 to P3. K425N was first detected in P5 at a frequency of 75% and then increased to 96% and 100% in P7 and P10, respectively. A400T first appeared in P7 at a frequency of 58% and rose to 96% in P10. An additional mutation at position 428 (S428L) was observed at low frequencies (8% to 1%) in P3 and P7, and a synonymous SNP appeared at 99–100% at nt position 4725 already in P1.In summary, these data revealed that D247N was already present in the rRABV DogB-P1 and that K425N was acquired between passages P3 and P5. A400T was acquired at a later time point between passages P5 and P7. Notably, once the mutations appeared, only few virus passages were required to replace almost the complete original sequence  and inclusion bodies . WhereasBy serial passaging of two recombinant field virus RABV clones of highly virulent fox and dog isolates , we idenin trans-complementation assays with G gene-deleted RABV [Similar to previous studies, where an increased glycosylation status of G was achieved by the D247N mutation, which was the result of neuroblastoma cell culture adaptation of the field virus strain 1088 ,21, hereted RABV , our comted RABV . ConsequWe also cannot exclude minor effects of the adaptive mutations on the susceptibility of the cells to virus infection. However, observed phenotypes shown for the G distribution shown in The selection of the D247N mutation in the two cell culture-passaged field viruses here and identification of the same mutation in passaged 1088 virus, SAD B19, RV-97, Nishigahara, and RC-HL vaccine strains  further Whereas D247N and other mutations lead to NxT/S glycosylation sequons in several RABV strains ,34,35,37In BHK cells, the combination of D247N, A400T, and K425N overcame either cell-specific intracellular retention or rapid re-internalization of non-adapted glycoprotein G, as demonstrated by the intracellular accumulation of non-mutated G protein at cytoplasmic inclusion bodies of rRABV Dog* and increased plasma membrane accumulation of G protein in rRABV DogB-NTN-/rRABV DogB-P10-infected BHK cells A. rRABV Rabies virus cytoplasmic inclusion bodies (IBs) are liquid organelles and sites of virus RNA synthesis ,39, whicAlthough a combination of all three G mutations  was identified in rRABV-DogB-P10, stepwise selection of K425N and A400T indicated a possible requirement of K425N for rapid selection of A400T  present at a frequency of 8.5% in rRABV DogB-P1 was lost in BHK-passaged rRABV DogB-P10, while it was established as a major amino acid replacement in neuroblastoma cell-passaged rRABV DogN-P10, indicating a positive selection of this mutation on Na 42/13 cells. In contrast to K425N, which appeared in both rRABV DogB-P10 and rRABV FoxB-P10, this mutation was not identified in rRABV FoxN-P10. Although, the wild-type G protein ectodomain sequences of the rRABV Fox and Dog field viruses differ at only three positions  may represent variations by chance, two amino acid exchanges in the C-terminal region of the phosphoprotein P were selected in two independent viruses on two different cell lines with P-R293C and P-R293H in rRABV DogB-P10 and rRABV FoxM-P10, respectively. Considering the low overall frequency of non-silent mutations observed , these dOur data suggest that improved virus release caused by only a few amino acid replacements in the ectodomain of G represents a major difference between cell culture-adapted and non-fixed field RABVs. This may not only be of interest for a better understanding of requirements for RABV cell culture adaptation, but also offers new possibilities to further investigate the impact of defined cell culture-adaptive mutations on virulence and pathogenicity in vivo. Since most fixed laboratory-adapted strains have acquired multiple mutations, with the progenitor isolates often not being available, the recombinant field virus clones with a defined set of cell line-specific adaptive mutations described here offer the possibility to address the question whether and to what extent improved virus release after cell culture adaptation influences RABV infection in the natural host. Whereas virus spread and disease development may possibly be sped up by the more efficient virus release, higher levels of antigen presentation and resultant stronger immune responses could result in an inverse correlation between virus release and virulence. The recombinant field viruses described here now allow the experimental dissection of adaptation, pathogenicity, and attenuation.


The high-activity adenine base editors (ABEs), engineered with the recently-developed tRNA adenosine deaminases (TadA8e and TadA9), show robust base editing activity but raise concerns about off-target effects.In this study, we perform a comprehensive evaluation of ABE8e- and ABE9-induced DNA and RNA mutations in Oryza sativa. Whole-genome sequencing analysis of plants transformed with four ABEs, including SpCas9n-TadA8e, SpCas9n-TadA9, SpCas9n-NG-TadA8e, and SpCas9n-NG-TadA9, reveal that ABEs harboring TadA9 lead to a higher number of off-target A-to-G (A>G) single-nucleotide variants (SNVs), and that those harboring CRISPR/SpCas9n-NG lead to a higher total number of off-target SNVs in the rice genome. An analysis of the T-DNAs carrying the ABEs indicates that the on-target mutations could be introduced before and/or after T-DNA integration into plant genomes, with more off-target A>G SNVs forming after the ABEs had integrated into the genome. Furthermore, we detect off-target A>G RNA mutations in plants with high expression of ABEs but not in plants with low expression of ABEs. The off-target A>G RNA mutations tend to cluster, while off-target A>G DNA mutations rarely clustered.Our findings that Cas proteins, TadA variants, temporal expression of ABEs, and expression levels of ABEs contribute to ABE specificity in rice provide insight into the specificity of ABEs and suggest alternative ways to increase ABE specificity besides engineering TadA variants.The online version contains supplementary material available at 10.1186/s13059-022-02618-w. Single-nucleotide variants (SNVs), a universal feature of plant, animal, and human genomes, have been widely identified in association with agronomic traits and human diseases –3. VarioThe pioneer ABE7s, which are composed of a tRNA adenosine deaminase TadA7.10) and CRISPR/Cas systems, perform remarkably clean and efficient A•T to G•C conversions in the genomes of a variety of species, including human, mouse, and rice, without inducing obvious genome-wide off-target DNA mutations  and CRIS. HoweverThe tRNA adenosine deaminase TadA, a key component of ABEs, induces site-specific inosine formation on RNAs . RecentlThe relatively small genome (~ 0.4 Gb) of self-pollinated rice and the absence of endogenous ADAR family make rice an ideal model organism to examine the DNA and RNA specificity of gene editing tools. Here, we investigated the off-target DNA and RNA mutations induced by ABE8es and ABE9s in rice through whole-genome sequencing (WGS) and transcriptome sequencing.Agrobacterium tumefaciens-mediated transformation, we obtained three independent transgenic plants for each construct except 46bM and 49bM, which had three plants from two independent transformation events , we identified around 200–400 SNVs and around 250–350 indels from each plant , we chose four ABEs that are composed of different variants of tRNA adenosine deaminase and CRISPR/Cas systems with different PAM compatibility: rBE46b (SpCas9n-TadA8e), rBE49b (SpCas9n-TadA9), rBE50 (SpCas9n-NG-TadA8e), and rBE53 (SpCas9n-NG-TadA9) Fig. a. For eaons Fig. : Fig. S3Agrobacterium infection, or ABEs. The induced mutations in the two alleles are two independent events following binomial distribution, so the probability of the homozygous mutations is p2, the probability of being wild type (WT) is (1-p)2, and the probability of the heterozygous mutations is 2 * p * (1-p), assuming that the induced mutation ratio for each allele was p and the ratio of the WT allele was 1-p. A binomial test for all loci of homozygous SNVs or indels revealed that these loci did not follow a binomial distribution  is -p2, and Agrobacterium infection  and right border (RB) and identified four plants with a partial T-DNA insertion characterized by the missing TadA8e, TadA9, or SpCas9n-NG fragment  –38, resuUsing deeply sequenced genomes and transcriptomes, we systematically studied ABE-induced RNA mutations. ABEs induce RNA A>G mutations in one-third of plants with high ABE expression but do not induce mutations in two thirds of plants with low ABE expression. When ABEs segregated out, RNA mutations diminished. In addition, T-DNA integration analysis suggested that stable ABEs induce more off-target SNVs than those whose T-DNA has not been integrated into the genome. Together, these data highlight the importance of controlling the expression of ABEs in future applications, such as using inducible or photoactivatable transcription systems, ribonucleoprotein-based delivery in clinic gene therapy , 40, andWithout the noise from A-to-I mutations mediated by ADAR proteins, we were able to obtain a clean set of ABE-induced RNA mutations and discovered that ABEs induced clustered A>G mutations, which provided useful information for defining and characterizing true ABE RNA targets. Furthermore, given the existence of common and unique mutations in plants regenerated from the same callus, we provide robust experimental evidence that plants with different on-target editing could be derived from the same T-DNA insertion event with a shared set of off-target SNVs. Therefore, we highly recommend using two independent transgenic lines from separated calli (with two different T-DNA insertion sites and two sets of non-overlapping SNVs) in gene function studies.The properties of the small genome, self-pollination, and the absence of ADAR proteins make rice a model organism to employ large-scale sequencing approaches to evaluate ABEs’ off-target activity. The pioneering comprehensive analysis of ABE-induced DNA and RNA mutations using whole-genome and transcriptome sequencing in rice sheds light on defining and characterizing ABEs’ specificity. The discovery that Cas proteins, TadA variants, transient expression, and the expression level of ABEs contribute to ABEs’ specificity in rice points out alternative ways improving ABEs’ specificity including combinatorial optimization of Cas/deaminase (SpCas9n-TadA8e) and temporal control of ABEs’ expression besides the traditional protein engineering of deaminases.Oryza sativa) genomic loci  and four rice genomic loci  were targeted by rBE46b and rBE49b, respectively. Three genes  were targeted by both rBE50 and rBE53. Plant IDs and their corresponding information are described in Additional file In this study, five rice , the resistant callus lines were transferred onto RM plates to generate transgenic rice seedlings. All information on target gene mutations of each seedling examined in this study is given in Additional file The genome editing constructs were individually introduced into the Agrobacterium infection (namely C1) and regenerated from calli co-cultured with the empty EH105 strain (namely C2). Also, seedlings were regenerated from calli infected with EH105 strains harboring SpCas9 only (namely Cas). All rice materials were grown in the greenhouse under a 16-h-light/8-h-dark photoperiod, 28/25 °C temperature cycle, and 75% humidity.To eliminate background mutations, 10 individual Kitaake plants grown from seeds were used directly. Seedlings were regenerated from rice calli without Genomic DNA of 4-week-old rice plants was extracted using the CTAB method . Approximately 200 mg of fresh rice leaves was collected in a 2-ml centrifuge tube containing disposable metal balls. After being quickly frozen in liquid nitrogen, samples were ground to a fine powder using a tissue grinding apparatus . Following chloroform extraction, isopropanol precipitation, and 70% EtOH washing, genomic DNAs were eluted with 50 μL of double-distilled water supplemented with 1 μL of 10 U/μL RNase I  and stored at − 80 °C for later experiments.RNA was extracted with TRIzol reagent  according to the manufacturer’s instructions. Briefly, 100 mg of fresh rice leaves was sampled, quickly frozen in liquid nitrogen, and ground to a powder with a tissue grinding apparatus. Then, 1 ml of TRIzol reagent was added to the sample followed by chloroform and isopropanol treatment. Finally, RNA pellets were dissolved in 50 μL of RNase-free water (0.1% DEPC-treated) and stored at − 80 °C for later experiments.The on-target genomic regions were amplified using Phanta Max Super-Fidelity DNA Polymerase  and locus-specific primers  from each sample were used to construct the DNA libraries using a NEBNext Ultra DNA Library Prep Kit for Illumina  following the manufacturer’s instructions. DNA libraries were sequenced on the Illumina platform in the 150-nt paired-end mode with an average coverage depth of 40×  via BWA [The clean reads were mapped to the Kitaake genome V3 from Phytozome . The coverage of T-DNAs between the left border (LB) and right border (RB) was assessed using the R library ShortRead. The expression of ABEs was quantified as the average raw read number of Cas proteins and TadA variants normalized by the total read number in millions. Since we used T0 plants, the copy number of T-DNA integration was calculated as the relative T-DNA coverage versus half coverage of the rice genome.DNA-free RNAs (0.2 μg) were used to construct the RNA-seq libraries using a NEB Next Ultra RNA Library Prep Kit for Illumina  following the manufacturer’s instructions. RNA-seq libraries were sequenced on the Illumina platform in the 150-nt paired-end mode  [The clean reads were mapped to the Kitaake V3 genome and annotation from Phytozome via STAR aligner with a maximum of eight mismatches per paired-end read . GATK wane.com/) .We combined all A>G off-target SNVs obtained from plants with RNA off-target activities. For each A>G SNV, we calculated the number of reads with nucleotide A, T, G, and C separately in the 5′ and 3′ 30-bp region with a read coverage larger than 10. The genetic change ratio was calculated as the number of Gs divided by the total number of As and Gs if the reference is A. The genetic change ratio was calculated as the number of Cs divided by the total number of Cs and Ts if the reference is T. Positions with an A>G mutation ratio of higher than 0.05 were used as the numerator, while positions of A/T with a read coverage larger than 10 were used as the denominator. Similarly, we combined all A>G off-target SNVs obtained from plants through WGS and calculated the percentage of A>G mutations at the 5′ and 3′ 30-bp flanking regions.quartiles (Q3), respectively. The upper whisker is min(max(x), Q3 + 1.5 × IQR), and the lower whisker is max(min(x), Q1 − 1.5 × IQR). IQR (interquartile range) = Q3 − Q1. Black dots located outsides the whiskers are outliers.The horizontal line in the box represents the median value, and the bottom and top of the box are the lower (Q1) and upper Additional file 1: Supplementary tables. Table S1, Summary of plants with ABEs. Table S2, Mapping statistics of whole-genome sequencing. Table S3, Summary of sgRNA-dependent on-target and off-target loci. Table S4, Summary of all the homozygous SNVs. Table S5, Summary of all the homozygous indels. Table S6, Summary of genomic SNVs detected through WGS. Table S7, Summary of the overlapping SNVs between each of the plants with whole-genome sequencing. Table S8, Mapping statistics of whole-transcriptome sequencing. Table S9, Summary of all the transcriptomic SNVs. Table S10, Summary of clustered A>G DNA SNVs. Table S11, Primers used to verify DNA SNVs by Sanger sequencing. Table S12, Primers used to verify RNA SNVs by Sanger sequencing.Additional file 2: Supplementary figures. Fig. S1. Sanger sequencing chromatograms of on-target mutations in plants harboring rBE46b and rBE49b. Fig. S2. Sanger sequencing chromatograms of on-target mutations in plants harboring rBE50 and rBE53. Fig. S3. IGV browser views showing the on-target mutations for 36 plants harboring ABEs. Fig. S4. Analysis of SNVs and indels identified by whole-genome sequencing. Fig. S5. Analysis of the remaining background homozygous DNA mutations. Fig. S6. Characterization of ABE-induced genomic mutations. Fig. S7. Distribution of six types of SNVs. Fig. S8. Distribution of SNVs at given regions of the genome. Fig. S9. Chromosomal distribution of SNVs. Fig. S10. On-target and off-target mutations in plants from the same calli. Fig. S11. Off-target SNVs in plants with incomplete T-DNA insertions. Fig. S12. Distribution of SNVs with different copy numbers of T-DNA insertions. Fig. S13. Transcriptome-wide distribution of ABE-induced off-target mutations. Fig. S14. Heatmap demonstrating A>G mutations in transcriptomes with more than 5 A>G SNVs detected. Fig. S15. The 5′ and 3′ flanking A>G mutations in transcriptomes with ABEs containing A>G RNA SNVs and in transcriptomes with SpCas9 only lacking A>G RNA SNVs. Fig. S16. The 5′ and 3′ flanking A>G mutations in transcriptomes with ABEs but without A>G RNA SNVs. Fig. S17. IGV genome browser views showing the off-target RNA mutations. Fig. S18. IGV genome browser views showing A>G mutations with flanking A>G SNVs in genome sequencing data. Fig. S19. Sanger sequencing chromatograms of off-target A>G DNA mutations. Fig. S20. Sanger sequencing chromatograms of off-target A>G RNA mutations.Additional file 3: Review history


The development of nanocarriers (NC) for biomedical applications has gained large interest due to their potential to co-deliver drugs in a cell-type-targeting manner. However, depending on their surface characteristics, NC accumulate serum factors, termed protein corona, which may affect their cellular binding. We have previously shown that NC coated with carbohydrates to enable biocompatibility triggered the lectin-dependent complement pathway, resulting in enhanced binding to B cells via complement receptor (CR)1/2. Here we show that such NC also engaged all types of splenic leukocytes known to express CR3 at a high rate when NC were pre-incubated with native mouse serum resulting in complement opsonization. By focusing on dendritic cells (DC) as an important antigen-presenting cell type, we show that CR3 was essential for binding/uptake of complement-opsonized NC, whereas CR4, which in mouse is specifically expressed by DC, played no role. Further, a minor B cell subpopulation (B-1), which is important for first-line pathogen responses, and co-expressed CR1/2 and CR3, in general, engaged NC to a much higher extent than normal B cells. Here, we identified CR-1/2 as necessary for binding of complement-opsonized NC, whereas CR3 was dispensable. Interestingly, the binding of complement-opsonized NC to both DC and B-1 cells affected the expression of activation markers. Our findings may have important implications for the design of nano-vaccines against infectious diseases, which codeliver pathogen-specific protein antigen and adjuvant, aimed to induce a broad adaptive cellular and humoral immune response by inducing cytotoxic T lymphocytes that kill infected cells and pathogen-neutralizing antibodies, respectively. Decoration of nano-vaccines either with carbohydrates to trigger complement activation in vivo or with active complement may result in concomitant targeting of DC and B cells and thereby may strongly enhance the extent of dual cellular/humoral immune responses. In the field of biomedical applications, nanocarriers (NC) have gained increasing interest based on their capacity to codeliver different drugs and biologicals in a targeting manner to distinct cell types . TargeteCR3 is commonly expressed by myeloid cells, comprising monocytes/macrophages (MAC), PMN and conventional dendritic cells type 2 (cDC2), enabling efficient binding and internalization of complement-opsonized pathogens as well as of immune complexes . InteresCR3 also binds numerous serum factors , has alsGiven the important role of DC for the initiation and shaping of immune responses, we wanted to delineate the relative importance of CR3, as expressed by cDC2 only, and of CR4 as expressed by all (murine) DC, for the binding/uptake of complement-opsonized NC. Furthermore, due to the general finding of strong accumulation of most types of NC in the liver, including DEX-coated NC ,23, we a−/− DC as well as CD11b-blocking antibodies, we show that CR3, but not CR4 was critical for binding/uptake of complement-opsonized NC. Finally, within B cells, we identified the B-1 subpopulation as highly active in NC binding, with CR-1/2 playing a major role. In the case of DC and B-1 cells, NC engagement attenuated the expression of activation markers after subsequent LPS-mediated stimulation. Our results suggest that nano-vaccines, which codeliver antigen and native protein adjuvant and address DC and B cells in a complement-dependent manner, may be capable of inducing profound cellular and humoral immune responses.We show here that within liver DC, MAC and liver sinusoidal endothelial cells (LSEC) bound complement-opsonized NC to an increased extent. However, similar results were obtained using serum devoid of complement activity, suggesting the involvement of other receptors. SR-A, which have been frequently discussed in this regard ,12,13, dTo delineate the role of activated complement as a constituent of the protein corona of an NC for its binding to immune cells, we comparatively assessed the binding characteristics of fluorescence-labeled NC coated with the carbohydrates dextran (DEX), solid iron oxide core (FeO-DEX) and bionized nanoferrite (BNF-DEX) and starch (BNF-starch), and included albumin-based NC as an internal control .All myeloid cell types, comprising neutrophils , macrophages, and  DC, as well as NK cells and B cells, engaged carbohydrate-coated NC to a higher extent when these were pretreated with native mouse serum to enable adsorption of active complement on the NC surface . This efIn the case of albumin NC, only B cells displayed considerably higher binding in the case of pretreatment with native serum . All splFor numerous types of NC, considerable accumulation in the liver has been noted . BesidesAs shown in + cells, which thereby express CR4 (CD11c/CD18). Splenic DC largely comprise CD11b-positive cDC2, which co-express CR3 and CR4, and CD11b-negative DC that constitute either cDC1 or plasmacytoid (p) DC. As exemplified for FeO-DEX, cDC2 engaged this type of NC to a higher extent than cDC1/pDC at either condition . The minor fraction of CD11b+CD11c− (CR3+) cells is considered as MAC.To delineate specifically the relative contribution of CR3 and CR4 for the binding and uptake of complement-opsonized NC by DC, we set up cultures in which bone marrow (BM) progenitor cells were differentiated using GM-CSF . After o+ BMDC, which thereby co-express CR3 and CR4, bind all types of carbohydrate-coated NC to a higher extent when these were complement-opsonized due to pre-incubation with native mouse serum as compared to NC left untreated or pre-incubated with h.i. serum (+CD11c−) within the same cultures displayed similar binding patterns . Interestingly, CD11b−/− DC displayed lower FeO-DEX engagement not only in the case of NC pretreatment with native but also h.i. serum, which may suggest the involvement of CR3 in binding of FeO-DEX with a complement-deficient protein corona  blocking antibody. Blockade of CD11b, but not the application of a corresponding isotype control antibody, significantly reduced binding of FeO-DEX pretreated with a native mouse  free culture medium or in normal culture medium displayed only minor cell binding A, which n corona B. To rulve mouse C. Heparive mouse D. This f−/− BMDC  confirmed that complement-opsonized FeO-DEX were internalized to a higher extent by WT than by CD11b−/− BMDC . When h.Engagement of DC by immune complexes via CR3 has been shown to affect their activation state . To deli+ B cells constituted B-1 as deduced from co-expression of CR3 (+ B-1a and CD5− B-1b cells. Both B-1 subpopulations readily engaged FeO-DEX as assessed after over-night incubation (right panel). B-1 cells bound directly applied FeO-DEX at a much higher extent than B-2 cells, both in terms of frequency  and display a different ontogeny [B-1 cells reside predominantly in the peritoneum and pleural cavities . As an eontogeny . In our ontogeny  and exerontogeny  but alsoontogeny ,57. Altoontogeny  and paraontogeny  infectioontogeny .Conventional antigen-specific B-2 cells play an important role in the later course of infection by producing antibodies that opsonize pathogens  and bindTo date, most NC-focused immunotherapeutic strategies aimed to induce adaptive immune responses to target DC , which aAltogether, our results confirm that the design of NC for biomedical purposes needs to take into account the possibility of recognition of surface structures like carbohydrates by the complement system, resulting in complement opsonization and predominant interaction with CR-expressing leukocytes. Above, in some cases, an NC may trigger the alternative complement pathway due to its “foreign” surface as recently demonstrated for silica particles , and theAside from complement activation also other constituents of the protein corona may yield targeting, e.g., of CR3, like denatured fibrinogen , and of In general, our study confirms the necessity to include serum in in vitro studies on cellular interaction of NC to mimic somewhat more closely the behavior of NC in vivo on a cellular level, as shown by us  and othe+ Th [+ T cells [+ T cells [With regard to a translational perspective, our results suggest that complement-opsonized NC could be a suitable vaccine platform to induce a broad cellular and humoral immune response, similar to pathogens that are recognized by the complement system . As depi+ Th  and CD8+ T cells . The lat T cells . On the  T cells . In the  T cells  and trig T cells  of CR1/2 T cells . Alterna T cells , would b T cells  or antib T cells . A nano- T cells ,84. Targ T cells . In addi T cells ,28 and m T cells , which a T cells  and B ce T cells  that eng T cells  and B ce T cells .Follicular dendritic cells (FDC) contribute to affinity maturation and memory B cell induction and survival  by long-Fluorescent-labeled types of NP were obtained from micromod  or from Miltenyi Biotech . The characteristics of the various types of NP are listed in −/− mice on a C57BL/6 background (B6.129S4-Itgamtm1Myd/J) were bred and maintained in the Central Animal Facility of the Johannes Gutenberg-University Mainz under specific pathogen-free conditions on a standard diet according to the guidelines of the regional animal care committee. Female mice (6–12 weeks) were used throughout all experiments. The “Principles of Laboratory Animal Care”  were followed. Mice were sacrificed for organ retrieval according to § 4(3) TierSchG.C57BL/6 mice and CD11b2+ and Mg2+). The cell suspension was mixed with a double volume of freshly prepared 30% HistoDenZ (Sigma-Aldrich) and overlaid with 1 mL of cold HBSS buffer. After centrifugation , the cell containing interphase was retrieved and washed. Erythrocytes were lysed using a hypotonic buffer.Liver nonparenchymal cells (NPC) were isolated using a liver dissociation kit and the gentle MACS dissociator (both Miltenyi Biotec) as recommended by the manufacturer with some modifications. In brief, after liver dissociation, cells were resuspended in 1 mL cold HBSS buffer (w/o CaSpleen cells were isolated using a 40 µM cell strainer (Greiner Bio-One) to obtain a single-cell suspension. In parallel settings, spleen cells were resuspended in medium , and 50 µM ß-mercaptoethanol ) containing 5% FCS (PAN).5/mL) were seeded in 12-well cell cluster plates (Greiner Bio-One) in an IMDM-based culture medium (see above) supplemented with 10 ng/mL recombinant murine GM-CSF . Culture media was replenished on days 3 and 6 of culture.Bone marrow cells (2 × 106/500 µL). Three hours after the onset of incubation, samples were harvested and subjected to flow cytometric analysis. In some cases, cells were harvested after over-night incubation. In some cases, samples were stimulated overnight with LPS . The relevance of distinct CR for NC binding was assessed using CR-blocking antibodies and a corresponding isotype control antibody  applied 45 min prior to administration of NC.Aliquots of the different NP were pre-incubated in parallel with equal volumes of native or heat-inactivated mouse serum for 1 h at 37 °C in low protein binding 0.5 mL reaction tubes (Greiner Bio-One). Afterward, untreated and differentially pretreated NP were applied at concentrations evaluated in corresponding pre-experiments see  to BMDC The contribution of class A scavenger receptors for binding of FeO-DEX was tested using various pharmacological inhibitors. To this end, cells were pre-incubated with fucoidan (25 µg/mL), dextran sulfate and chondroitin sulfate (each 25 µg/mL), and polyinosinic acid and polycytidylic acid (each 12.5 µg/mL) for 1 h prior to application of differentially pretreated FeO-DEX.After incubation with NP, cells were washed in staining buffer (PBS/2% FCS), and Fcγ receptors were blocked by applying rat anti-mouse CD16/CD32 antibody . Afterward, samples were incubated  with distinct sets of fluorescence-labeled antibodies as indicated in BMDC (d8 of culture) were incubated with FeO-DEX as indicated, washed with FACS buffer , and transferred onto chamber slides . Cells were incubated with anti-CD11c-PE/Cy7 antibody and DAPI for nuclear staining. Confocal microscope: LSM510-UV .p < 0.05.Statistical analysis was performed using GraphPad Prism Software v5.0 . Results were expressed as mean ± standard error of the mean (SEM). Differences among groups were tested by one-way ANOVA, followed by post hoc Tukey’s test, assuming significance at