The results from in vivo xenograft model suggested suppressed tumor growth and tumor weight under ASP treatment. ASP treatment also caused downregulated PD-L1 and Ki-67 levels in mice tumors. Moreover, the IFN-γ-caused PD-L1 accumulation was inhibited by ASP treatment. The administration of ASP decreased the expression of PD-L1 of OC cells in a coculture system with activated T cell or unstimulated PBMCs, along with decreased expression of PD-1 by activated T cells. ASP reversed PD-L1 expression caused by coculture with activated T cells and abolished the suppressed T cells activation and proliferation. Analysis on molecular mechanisms revealed that KAT5 bonded to the promoter region of PD-L1 and upregulated its expression via enhancing histone H3 lysine 27 acetylation (H3K27ac), whereas ASP downregulated KAT5 expression and blocked this phenomenon. Moreover, ASP enhanced the effect of antiPD-L1 therapy in the in vivo tumor model. Hence, we proposed that ASP decreased expression of PD-L1 protein via inhibiting the epigenetic regulation by KAT5 and suppressed the PD-1/PD-L1 signaling to attenuate tumor growth. ASP may be a promising adjuvant in OC immunotherapy.Ovarian cancer (OC) is a frequently occurred malignancy with high incidence and poor survival worldwide. In recent years, immune checkpoint inhibition that targets PD-1/PD-L1 axis has become an efficient and popular therapy for cancers. Aspirin (ASP), an anti-inflammatory drug, exhibits a wide spectrum of biological functions including anticancer property. However, the role of ASP treatment in ovarian cancer treatment remains unclear. In this work, we explored the role of ASP in modulating PD-L1 signaling during OC development. Notably,  Ovarian cancer (OC) is the primary cause of gynecologic cancer-related death globally . Though Aspirin (ASP), as an anti-inflammatory drug, has been widely used in treatment of various diseases in clinics . StudiesIn present research, we evaluated the functions of ASP in immunotherapy of OC and determined reduced level of PD-L1 in OC cells and decreased level of PD-1 in activated T cells. Further discussion on molecular mechanisms demonstrated that ASP epigenetically modulated the expression of PD-L1 in OC cells. Our findings provided novel evidence for the application of ASP in the immunotherapy of OC.2. For coculture, the cancer cells were seeded in the lower chamber of Transwell plates , and T cells were placed in the top chambers. The culture medium was collected and centrifuged to obtain conditioned medium. The conditioned medium was mixed with same volume of fresh medium for use.Human OC cellsSKOV-3 and TOV-21G and murine OC cell ID8 were obtained from American Type Culture Collection . All cells were maintained in DMEM  that contains 10% fetal bovine serum  at a 37°C incubator filled with 5% COSmall hairpin RNAs (shRNAs) that targets PD-L1 (shPD-L1) and KAT5 (shKAT5) were synthesized by GenePharma (China). For cell transfection, Lipofectamine 2000  was adopted following the manufacturer's instruction.Cell viability and proliferation were assessed by using cell counting kit-8 , colony formation, and 5-ethynyl-2′-deoxyuridine (EdU) assay. For CCK-8 assay, OC cells were placed in a 96-well plate  and cultured for indicated time. Then CCK-8 reagent was added to incubate for another 2 h. The absorbance values were measured at 450 nm in a microplate reader .For colony formation assay, the cells were suspended as single cells and seeded in a 12-well plate (500 cells/well) and cultured for 14 days. Next, the colonies were dyed with crystal violet  and captured by a digital camera .μM EDU reagent . The nuclei were stained with Hoechst 33342. The positive staining was captured under a fluorescence microscope .To conduct EDU assay, cells were fixed with 4% paraformaldehyde and permeabilized with Triton X-100 after treatment, followed by incubation with 50 To obtain T cells, we collected the peripheral blood mononuclear cells (PBMCs) from healthy donors using the Ficoll method and extracted the T cells using a Pan-T cell isolation kit  in line with the manufacturer's protocol. Next, the T cells were primed by using a T Cell Activation Kit . Briefly, T cells were incubated with MACS Beads  preconjugated with antiCD8, antiCD2, and antiCD3 antibodies and then loaded in FACS  for isolation.The determination of cell surface biomarkers was realized by flow cytometry. In brief, cells were suspended in PBS solution with 2% FBS and incubated with fluorescence-conjugated antibodies against CD4, CD8, PD-1, and PD-L1 for 30 minutes in dark. Next, the samples were washed with PBS and loaded in the FACS .6 cells/100 μL PBS) were subcutaneously inoculated to the left fat pad of each mouse. The mice were randomly divided into experimental groups when tumor size reached 100 mm3. For PD-L1 blockade, mice were intraperitoneally injected with anti-PD-L1 antibody  at a dose of 5 mg/kg for three times within one week after grouping. The ASP was administrated through gavage every day at a dose of 20 mg/kg. Mice in the control group were injected with Ig-G antibody and saline as control. The size of tumors and body weight were recorded every other two days. All animal experiments were authorized and conducted following the guideline of Animal Ethic Committee of our hospital.Five-week-old male C57BL/6 J mice were purchased from the Charles River Laboratory (USA). Murine ID8 cells  and captured under microscope .The tumor tissues were isolated, fixed, dehydrated, embedded by paraffin, and cut into 5-ΔΔCt− method. Relative quantification was conducted using β-Actin as the internal control.RNA was isolated from tumors and cells by using a TRIzol reagent . Transcription to cDNA was conducted using First-Strand Synthesis Kit . Gene levels were quantified using the SYBR Green manner following the 2ChIP assay was performed to evaluate the interaction between proteins and PD-L1 promoter region. In brief, OC cells were cross-linked in 1% formaldehyde, lysed, and sonicated to obtain chromatin fragments with length around 500 bp. Next, the fragments were incubated with antiKAT5, antiRNA polymerase II, antiH3K27Ac, or anti-IgG as control at 4°C overnight in rotation and then hatched with Dynabeads  at 4°C for another one hour. The samples were eluted with proteinase K  and quantified with qRT-PCR assay.t-test or one-way ANOVA. P < 0.05 was regarded as statistical significance.The data in this work were shown as mean ± SD of at least three independent experiments. GraphPad Prism software was adopted for statistical analysis. The comparison between two or more groups was conducted using Student Initially, we assessed the effect of aspirin on OC cells. Results from CCK-8 indicated that aspirin notably suppressed the viabilities of TOV-21G and SKOV-3 cells Figures . The colin vivo OC model. Tumorigenesis in nude mice showed that aspirin treatment attenuated the tumor growth and the tumor weight in the nude mouse xenograft model (Figures We next explored the effects of aspirin on  Figures . Moreove Figures .γ is a classic stimulator for PD-L1 expression. Here, we explored the function of aspirin in IFN-γ-induced PD-L1 enrichment. Flow cytometry analysis revealed that IFN-γ treatment notably stimulated the level of PD-L1 in OC cells (Figures γ-stimulated PD-L1 level in TOV-21G and SKOV-3 cells (Figures It is well-known that IFN- Figures . Neverth Figures . Moreove Figures .Subsequently, we explored the effects of aspirin on T cell function via using a cocultured system, that composed by activated or naive T cells in top chambers and OC cells in the bottom. Flow cytometry analysis revealed that treatment with aspirin suppressed PD-L1 level in TOV-21G cells that cocultured with activated T cell or naive PBMCs . BesidesWe next discussed the molecular mechanism of aspirin repressing PD-L1 level in OC cells. The qPCR analysis showed that aspirin treatment led to reduced KAT5 level in TOV-21G and SKOV-3 cells . The qPCin vivo model. Tumorigenesis in vivo demonstrated that monoclonal antibody against PD-L1 attenuated the tumorigenesis of murine OC ID8 cells, whereas the addition of aspirin significantly enhanced the inhibitory effect of antiPD-L1 antibody, manifested by decreased tumor growth, tumor weight, and expression of Ki-67 and PD-L1 (Figures Next, we determined the effect of aspirin as an adjuvant for antiPD-L1 therapy on OC cell growth using an  Figures .Ovarian cancer is the primary reason for women malignancies-related mortality with poor prognosis worldwide. Immune checkpoint inhibitors that target PD-1/PD-L1 axis have been regarded as promising treatments in cancer treatment. Aspirin is a classic anti-inflammatory agent recently found to be highly potential and effective for treatments of multiple cancers. Nevertheless, the effect of aspirin on OC remains unclear. In this work, we determined to evaluate whether aspirin targeted PD-L1 signaling and OC development.in vitro. Study on nude mouse model showed that aspirin treatment attenuated tumor growth and weight, along with decreased expression of Ki-67 and PD-L1. And the IFN-γ-stimulated PD-L1 expression in OC cells was suppressed by aspirin. Moreover, the PD-L1 and PD-1 expression of OC cells and activated T cells in the coculture system was inhibited under aspirin treatment. Aspirin treatment also recovered the proliferation and activation of T cells in the coculture system, as well as suppressed T cell apoptosis. Further study on in vivo mouse model proved the effect of antiPD-L1 therapy in OC. These results suggested the therapeutic effect of aspirin on OC development through targeting PD-1/PD-L1 signaling.Studies have demonstrated the potential anticancer effect of aspirin on OC development. Aspirin inhibits EGF-stimulated viability of OC cells in a COX-1-dependent manner . AspirinKAT5 is a lysine acetyltransferase that epigenetically modulates gene acetylation. Studies have reported the important function of KAT5 in cancer initiation and progression. For example, KAT5 stabilizes the expression of c-MYC to promote cell invasion and metastasis in ATC . FosB reIn conclusion, we discovered that aspirin suppressed PD-L1 expression through suppressing KAT5 and subsequently inhibited PD-1 and PD-L1 signaling to attenuate OC development. Aspirin may be an effective therapeutic drug for immunotherapy of OC.


Sex steroid hormones like estrogens have a key role in the regulation of energy homeostasis and metabolism. In transwomen, gender-affirming hormone therapy like estradiol (in combination with antiandrogenic compounds) could affect metabolism as well. Given that the underlying pathophysiological mechanisms are not fully understood, this study assessed circulating estradiol-driven microRNAs (miRs) in transwomen and their regulation of genes involved in metabolism in mice.n = 20) and validation cohort (n = 30), we identified miR-224 and miR-452. Subsequent systemic silencing of these miRs in male C57Bl/6 J mice (n = 10) was followed by RNA-seq-based gene expression analysis of brown and white adipose tissue in conjunction with mechanistic studies in cultured adipocytes.Following plasma miR-sequencing (seq) in a transwomen discovery (Estradiol in transwomen lowered plasma miR-224 and -452 carried in extracellular vesicles (EVs) while their systemic silencing in mice and cultured adipocytes increased lipogenesis (white adipose) but reduced glucose uptake and mitochondrial respiration (brown adipose). In white and brown adipose tissue, differentially expressed (miR target) genes are associated with lipogenesis (white adipose) and mitochondrial respiration and glucose uptake (brown adipose).This study identified an estradiol-drive post-transcriptional network that could potentially offer a mechanistic understanding of metabolism following gender-affirming estradiol therapy. Sex hormones like estrogens have a key role in the regulation of energy homeostasis and metabolism . ParticuIt has become increasingly clear that post-transcriptional networks through an intricate interplay of non-coding RNAs such as microRNAs (miRs) and RNA binding proteins coordinate the expression of multiple sets of functionally related genes, that together shape the functional response of cells to a change in metabolic demand . Also inTherefore, to understand how trans-hormones (estradiol) associate with post-transcriptional regulation of functionally related genes in metabolism, this study assessed circulating estradiol-driven miRs in transwomen and their regulation of genes involved in metabolism in mice.A full description of the methods can be found in the Supplementary methods  and 4 mg estradiol valerate  or 100 µg/24 h transdermal estradiol  twice a week, as previously described  (Androcurescribed . Paired Plasma RNA from each patient sample was isolated from 200 μL EDTA-plasma by using the RNeasy Micro Kit (Qiagen).Plasma miR sequencing was performed by Exiqon according to protocol. Samples were sequenced on the Illumina NextSeq 500 system. Experiments were conducted at Exiqon Services, Denmark.Plasma EVs (70 nm) were isolated by applying 125 μL human plasma to a 3.64 mL Sepharose CL-2B size-exclusion chromatography (SEC) column, as previously described .Selected miRs were validated with quantitative PCR, using individual samples that comprised the pooled samples that were used for plasma miR sequencing. Taqman primers  were used according to the manufacturer’s protocols. Target gene mRNA primer sequences are listed in Supplementary Table 2.n = 10 per group, age = 8 weeks, Charles River Nederland) were randomized in three groups and received two s.c. injections of 25 mg/kg locked nucleic acid (LNA)-modified antisense miR-224 (antimiR-224), miR-452 (antimiR-452), or scrambled miR sequence (scramblemiR). Five days before injection, mice were individually housed in fully automated metabolic cages .Male C57Bl/6J mice .3H]oleate-labeled lipoprotein-like triglyceride (TG)-rich emulsion particles (80 nm) were prepared as previously described (14C]deoxyglucose ([14C]DG) was added in a 3H:14C = 4:1 ratio. After 6 h of fasting, mice were injected with 200 μL of emulsion particles (1 mg TG per mouse) via the tail vein. After 15 min, organs were harvested and dissolved overnight at 56°C in Solvable (Perkin Elmer).Glycerol trioleate uptake was observed in s.c.WAT(P  = 0.08), while antimiR-452 treatment of mice increased [3H]oleate uptake in s.c. white adipose tissue . AntimiR-224 treatment reduced [14C]DG uptake in subscapular brown adipose tissue (sBAT), while antimiR-452 treatment reduced [14C]DG in both interscapular brown adipose tissue (iBAT) and sBAT . In contrast, in WAT tissue from antimir-224 treated mice, AMP-activated protein kinase (AMPK) signaling (regulates adipocyte metabolism) and signal transducer and activator of transcription 3 (STAT3) (controls lipogenesis and adipocyte hypertrophy) were affected, while antimiR-452 treatment affected the HIPPO-pathway (adipocyte differentiation) and insulin receptor signaling. We subsequently used IPA to identify all potential miR-224/452 target genes that could serve as upstream regulators of differential top canonical pathways. When miR-224/452 target genes were plotted in heatmaps, many of them were upregulated in BAT and WAT , and some key metabolism-related target genes could be validated by qPCR   of WAT and BAT to identify metabolism-associated gene expression changes. Interestingly, using Ingenuity Pathway Analysis (IPA), we found that both miR-224/452 target genes are strongly enriched for genes that associate with metabolism, potentially indicating a coordinated regulation of multiple metabolic pathways by these miRs . By furt by qPCR  and C. S3 in BAT , which a3 in BAT , 21. Nr4ogenesis , antimiRis  , 32.FigPgc1α , CVON-RECONNECT (A J V Z), and CVON-GENIUS-2 (P C N R), the European Fund for the Study of Diabetes and Boehringer Ingelheimhttp://dx.doi.org/10.13039/100001003 (to A J V Z and R B), the Dutch Kidney Foundation (KOLLF grant 16OKG16 to R B) and the Dutch Diabetes Research Foundation .This study was supported by funding provided by the Netherlands B W F conducted experiments, acquired and analyzed data, and wrote the manuscript. M K, E N K, R W A L L, and S K acquired and analyzed data. J D, J L, G D T, A B, Y Y, and W S researched the data. R N, T J R, P C N R, A J V Z, and R B contributed to the discussion and reviewed and edited the manuscript. B W F, A J V Z, and R B are guarantors of this work and, as such, had full access to all the data in the study and responsibility for the integrity of the data and the accuracy of the data analyses. J M G J D and M K contributed equally. R B and A J v Z shared senior authorship.


R)-selective amination effectively is highly desirable in organic synthesis. Despite noticeable progress in the engineering of (R)-amine activity in pyridoxal-5’-phosphate-dependent transaminases of fold type IV, the specialization of the activity is still an intuitive task, as there is poor understanding of sequence-structure-function relationships. In this study, we analyzed this relationship in transaminase from Thermobaculum terrenum, distinguished by expanded substrate specificity and activity in reactions with L-amino acids and (R)-(+)-1-phenylethylamine using α-ketoglutarate and pyruvate as amino acceptors. We performed site-directed mutagenesis to create a panel of the enzyme variants, which differ in the active site residues from the parent enzyme to a putative transaminase specific to (R)-primary amines. The variants were examined in the overall transamination reactions and half-reaction with (R)-(+)-1-phenylethylamine. A structural analysis of the most prominent variants revealed a spatial reorganization in the active sites, which caused changes in activity. Although the specialization to (R)-amine transaminase was not implemented, we succeeded in understanding the role of the particular active site residues in expanding substrate specificity of the enzyme. We showed that the specificity for (R)-(+)-1-phenylethylamine in transaminase from T. terrenum arises without sacrificing the specificity for L-amino acids and α-ketoglutarate and in consensus with it.Creating biocatalysts for ( Transaminases  are pyridoxal-5’-phosphate (PLP)-dependent enzymes that catalyze the reversible stereoselective transfer of an amino group from an amino substrate to a keto acid or ketone, thereby producing a chiral amine or amino acid and a new keto compound . TransamR)-selective amine transaminases (R-TAs), and branched-chain L-amino acid transaminases (BCATs) (R)-selectivity or (S)-selectivity of the PLP-dependent TAs of fold type IV are the result of different amino acid compositions within similarly organized active sites . –12. R)-sve sites –16. Geneve sites , 17–19 -primary amines (that lack α-COOH group) using pyruvate as an amino acceptor [DAATs catalyze transamination between D-amino acids and a-ketoglutarate as the amino acceptor, thereby producing keto acids and D-glutamic acid , 21. In acceptor , 23. Theacceptor , 24. Theacceptor , 24, 25.R)-primary amines are poor substrates for BCATs (according to the CIP nomenclature (R)-primary amines result from L-amino acids by substituting the α-COOH group with an alkyl group) [BCATs catalyze transamination between L-branched-chain amino acids (BCAA) and α-ketoglutarate, producing keto acids and L-glutamic acid , 26, 27.l group) .R)-primary amines to keto acids, producing new D-amino acids and ketones [R)-primary amines to keto acids, producing new L-amino acids and ketones [Thermobaculum terrenum (TaTT) converts BCAA and L-aromatic amino acids at a high rate [R)-(+)-1-phenylethylamine (R-PEA) to α-ketoglutarate producing acetophenone and L-glutamic acid at a rate similar to that of canonical R-TAs -primary amines. Previously, Voss et al. succeeded in introducing R-TА-like activity into DAAT from Bacillus subtilis but failed to generate R-TA-like activity on the scaffold of canonical BCAT from Escherichia coli [TaTT as a transaminase with an expanded substrate specificity  can be changed more effectively than canonical BCATs or DAATs and chose a strategy to increase the R-TA-like activity of TaTT by changing residues in the active site. Although our results showed that the specialization of the generalist is not straightforward, the applied strategy allowed estimating the role of individual residues in the active site in achieving different types of activity. We found that the observed TaTT compatibility of the high specificity toward BCAA and α-ketoglutarate and a significant activity toward R-PEA arose not only from the substitutions of residues having direct contact with the substrates but also as a result of a more subtle comprehensive structural adjustment of a functional dimer.In the current paper, we used a combination of bioinformatics approaches, site-directed mutagenesis, and kinetic and structural analyses to estimate the role of the residues in the hia coli . We suggTaTT variants were created through site-directed mutagenesis using the modified QuikChange protocol as described in [TaTT (WT TaTT) gene using the Tersus Plus PCR kit  and mutagenesis primers (E.coli Match I сеlls. Clones carrying target mutations were identified by a colony PCR assay performed using Taq DNA polymerase and a pair of primers—a check primer specified in The ribed in . Oligonu primers . AmplifiE. coli BL21(DE3)pLys cells  for the expression of TaTT variants fused at the N-terminus to a His6TEV-tag. The transformed cells were grown in LB media supplemented with ampicillin (100 μg/ml) until the OD595 reached 0.5, then were induced with 1 mM IPTG. After overnight incubation at 30 oC the cells were harvested by centrifugation at 5000 g for 20 min and resuspended in 50 mM Tris-HCl buffer, pH 8.0, containing 500 mM NaCl, 0.1% Triton, 20 mM imidazole, 1 mM PMSF, 0.2 μg/ml lysozyme and then lysed by sonication. The lysate was centrifuged at 28000 g for 30 min at 4 oC. Supernatant was filtered through a 0.2 μ filter  and applied to HisTrap HP column  equilibrated with 50 mM Tris-HCl buffer, pH 8.0, containing 500 mM NaCl, 20 mM imidazole and 0.02% (v/v) Triton X-100. The target proteins were eluted with a linear gradient from 40 to 500 mM imidazole in 50 mM Tris-HCl buffer, pH 8.0, containing 500 mM NaCl. The target proteins were incubated 1 h with 1 mM PLP at 30 oC and transferred into storage buffer  using PD-10 desalting columns (Cytiva). The purified proteins were concentrated up to 3–6 mg/ml with a 10 kDa cutoff centrifugal filter device  and stored in 50% glycerol at -20 oC. The WT TaTT production was described in [6TEV-tagged TaTT was expressed in E. coli BL21(DE3)pLys (Stratagene). The recombinant TaTT was isolated using subtractive Ni-affinity chromatography and gel filtration. The fractions showing the activity were stored in 50 mM Tris-HCl buffer, pH 8.0, containing 100 mM NaCl, 15 μM PLP, and 50% glycerol at -20°C.The plasmid constructs were transformed into TaTT mutants, after being isolated on the HisTrap HP column, were transferred into 50 mM Tris-HCl buffer, pH 8.0, supplemented with 100 mM NaCl, 1 mM EDTA, 5 mM β-mercaptoethanol, and (His)6-TEV protease (1 mg per 10 mg of the protein). The solution was incubated overnight at 4°C, dialyzed against 50 mM Tris-HCl buffer, pH 8.0, containing 500 mM NaCl and 20 mM imidazole, and applied to a HisTrap HP column. A (His)6-TEV protease and a cleaved (His)6-tag were absorbed on the column, and the flow-through was concentrated and applied to a Superdex 200 10/300 GL column (Cytiva) equilibrated in 15 mM Tris-HCl buffer, pH 8.0, supplemented with 50 mM NaCl and 20 μM PLP. Fractions of TaTT variants were concentrated up to 10–15 mg/ml and frozen at -70 oC. The protein purity was analyzed by SDS-PAGE (12%). The protein concentration was determined spectrophotometrically [For crystallization, the fractions of trically .TaTT and its variants at 0.5–1.5 μg/ml were measured in the overall transamination reaction with 5 mM L-leucine and 1 mM α-ketoglutarate in 50 mM Tris-HCl buffer, pH 8.0, supplemented with 50 mM NaCl and 60 μM PLP at 50°C. Samples (250 μl) were taken at several time points (0.5–4 min) and frozen to stop the reaction. The L-glutamate concentration was assayed in 50 mM Tris-HCl buffer, pH 9.0, supplemented with 1 mM NAD and 1.0–2.0 U GluDH . One unit (U) was defined as the formation of 1 μmol L-glutamate per minute. All measurements were performed at least twice. The direct acetophenone assay was applied according to [-1 cm-1) using an Evolution 300 UV-Vis spectrophotometer equipped with a Peltier accessory . One unit (U) was defined as the formation of 1 μmol acetophenone per minute. All measurements were performed in triplicates. The data were analyzed using Origin 8.0 .The activity assays were described in . Brieflyrding to . The conTaTT and its variants were obtained by incubating with the excess of both PLP and α-ketoglutarate overnight, followed by the transfer into the assay buffer using a Desalting column (Cytiva). The half-reactions of TaTT and its variants in PLP form (16–20 μM) with R-PEA were followed spectrophotometrically as described in [At is the absorbance at time t, ΔA is the difference between absorbance at t = 0 and t = ∞, A∞ is the final absorbance, and kobs is the observed rate constant. Linear parts of the obtained kinetic curves were analyzed with the following equation: 0 is the absorbance at t = 0. The vs. substrate concentration according to The first transamination half-reaction is a transition of the PLP form of TA into the PMP form accompanied by the deamination of the amino donor . Half-reribed in  using anPLP form 6–20 μM wAll measurements were performed in triplicates. The data were analyzed using Origin 8.0 software .TaTT and its homologs from PLP fold type IV enzymes was constructed using the recently described Mustguseal method [TaTT (chain A) was used as a query to run the protocol with default settings [To identify SSPs  a multipl method . The PDBsettings . The ressettings . The SSPTm) of the TaTT variants was determined using the Thermofluor ) assay. The samples diluted to 0.5 mg/ml in 50 mM sodium phosphate buffer, pH 8.0, containing 200 mM NaCl, 20 μM PLP and 1000× dilution of Sypro Orange dye (Sigma-Aldrich), were transferred to a 96-well PCR plates (Thermo Fischer Scientific) in the final volume 50 μl per well. Buffer mixed with Sypro dye was used as a negative control. Plates were placed in a real-time cycler , and the temperature was ramped up from 20 to 95°C in 1.0°C min-1. Relative fluorescence was measured using the FRET channel, and GraphPad Prism v8.0  was used to fit the collected data a sigmoidal curve and calculate Tm using Boltzmann model. Assays were run in triplicate, and Tm was reported as the average of the three runs.The apparent melting temperature (TaTT variants (mP3 and mP3O1) were crystallized by the "hanging drop" vapor diffusion method in 24-well VDX plates . 1.5 μl of protein was mixed with 1.5 μl of precipitant containing 100 mM HEPES, pH 7.5, 2.5–3.4 M NaCl , and set up over 500 μl of precipitant in a sealed reservoir. The crystallization plates were incubated for a week at 15 oC then inspected for crystal growth. The best crystals grew in 8–12 days and were rod shaped with a linear size of 200x200x100 μm. Before data collection, the crystals were briefly soaked in the mother liquor containing 25% glycerol as a cryoprotectant. They were then flash-cooled to 100 K in liquid nitrogen. The X-ray diffraction data for the TaTT variants were collected at the BL41XU beamline of a SPring8 synchrotron . The data were indexed, integrated, and scaled using Dials [The ng Dials . The proTaTT (PDB ID: 6GKR) as a starting model. The refinement of both structures was carried out using the REFMAC5 program of the CCP4 suite [The structures were solved by the molecular replacement method using the Molrep program , with thP4 suite . In bothP4 suite . The resThe visual inspection of the modeled structures and figure preparation were carried out by the COOT program  and the TaTT and homologous BCATs and R-TAs and identified subfamily-specific positions (SSPs) of BCATs and R-TAs  and substitutions in the bottom of the pocket . The effects of mutations were analyzed in a half-reaction with R-PEA and in the overall reactions.We compared sequences and structures of nd R-TAs , which and R-TAs  and the nd R-TAs . The genTaTT variants in the overall reaction is summarized in TaTT activity toward L-leucine and α-ketoglutarate (BCAT-like activity), R-PEA and α-ketoglutarate (observed uniquely for TaTT), and R-PEA and pyruvate (R-TA-like activity)  .TaTT activity in the overall reaction, with BCAT-like activity being the most susceptible to the changes. Only variants mP1and mP2 were comparable to WT TaTT in the reaction R-PEA + α-ketoglutarate. We observed an increase in R-TA-like activity at pH 7; interestingly, mP3O1 . The paTaTT activity. Mutations W32H and F39Y  destroyed the TaTT activity toward R-PEA as well.Any other changes in the O-pocket combining with the three mutations in the P-pocket were unsuccessful. The double mutation (Y166W + F39Y) was fatal for the -1M-1, which is 30 times lower than that at pH 9.0. For WT TaTT, the half-reaction analysis at pH 7.0 showed no saturation as well, with the specificity constant for R-PEA being (0.009 ± 0.001) s-1M-1. Apparently, the observed increase in the activity of mP3O1 in the overall reactions with R-PEA at pH 7.0  showed no saturation; the specificity constant for R-PEA was (2.6 ± 0.2) st pH 7.0  indicateTaTT are similar: pairwise RMSD between the Cα atoms of subunits did not exceed 0.3  or even the absence of the electron density for the side chain (mP3O1)  . When Y1 (mP3O1) . The forTaTT, the mP3 O-pocket loop lacks some stabilizing hydrogen bonds—between the backbone oxygen of S115* and the side chains of T197 and S173 and between the backbone oxygen of F114* and the side chain of Q170. These disturbances induced a shift of the loop, and thereby, the side chain of F114* shifted toward the cofactor and partially blocked the entrance into the active site , W32* has two conformations: one is similar to that in mP3, and the other is unique and occupies the WT position of the O-pocket loop main chain.There are large conformation changes in the O-pocket loop in the mP3 and mP3O1 variants ; moreoveive site . In WT TIn sum, the substitutions induced a rearrangement in the active site, including an enhancement of hydrophobicity and an increase in the O-pocket loop and P-pocket β-turn mobility.sequence-structure-function relationships [R)-selective amination as well. In 2010, Höhne et al. [sequence-function relationships encouraged the researchers to change the specificity of TAs by replacing key residues within similar structural scaffolds. Voss et al. [B. subtilis to R-TA-like activity. The mutated DAAT showed 0.33 U/mg activity in converting R-PEA but lost its native activity toward D-amino acids  as well as R-TA-like activity with R-PEA and α-ketoglutarate/pyruvate, producing acetophenone and L-glutamate/L-alanine (Vmax is 0.33 U/mg). Earlier, we suggested that the expanded substrate specificity of TaTT resulted from the changes in the specificity-determining sequence motifs . Thccessful . In bothe motifs . These ce motifs , thus faTaTT), polarized by the neighboring conserved arginine residue, the other is formed by the backbone atoms of the b-turn polarized by the other conserved arginine residue from the βX-strand (R43 in TaTT) [TaTT, and no severe change in R-TA-like activity was observed  destroyed the binding of the α-COOH group in TaTT and led to the loss of both BCAT-like and R-TA-like activities. However, triple mutation improved the affinity to R-PEA more than 60 times.In canonical BCATs, P-pocket contains two α-COOH group binding sites: one is formed by the phenyl group of the tyrosine residue from the βY-strand (Y101 in in TaTT) , 28, 29 observed . The douTaTT as well. The improvement can be attributed to the incorporation of a positive charge in the O-pocket, which appears to adjust R-PEA coordination by fixing the aromatic moiety away from the charged side chain of R115. These changes imitated the key residues in the active site of natural R-TAs and appeared to favor the R-PEA binding. Further substituting the unique TaTT residues W32H and F39Y for R-TA ones at the bottom of the O-pocket  decreased the TaTT affinity to R-PEA. Double substitution Y166W + F39Y induced the loss of both TaTT activities. The structural and SSP analysis of BCATs showed that the tyrosine residue (Y166 in TaTT) is coupled with phenylalanine residue (F39 in TaTT), and this couple is essential for function and PLP-coordination; the latter in TaTT occur similarly to canonical BCATs  underpinned the specificity of PLP coordination. At the structural level, the inactivation of TaTT seems to result from the reduced mobility of the introduced tryptophan and tyrosine residues compared to their analogs in native R-TAs. In R-TAs, the tryptophan residue  was not achieved. The HPLC analysis of the products of transamination between R-PEA and pyruvate catalyzed by mP3O1 revealed only L-alanine , and between R-PEA and α-ketoglutarate producing ketone and L-glutamate (R-TA-like activity). The latter is a unique reaction because canonical R-TAs are inactive with α-ketoglutarate and catalyze the amino group transfer between R-PEA and pyruvate, producing acetophenone and D-alanine. The specificity for R-PEA arises without sacrificing the specificity for L-Leu and α-ketoglutarate and in consensus with it: TaTT demonstrates one of the highest rates of α-ketoglutarate conversion. Incorporating key residues of canonical R-TAs in the TaTT active site, we succeeded in enhancing the affinity to R-PEA; however, we destroyed the activity in the overall transamination reactions due to the lack of a proper binding of substrates with the α-COOH group. We found that the active site of TaTT is characterized by the relaxation of some conserved for BCATs interactions, which favored the specificity for R-PEA and enhanced the specificity for α-ketoglutarate.PLP-dependent TAs of fold type IV are informative objects in studying S1 FigTaTT.Unique reactions catalyzed by (PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.


Irreversible inhibitors of Bruton tyrosine kinase (BTK), pioneered by ibrutinib, have become breakthrough drugs in the treatment of leukemias and lymphomas. Resistance variants (mutations) occur, but in contrast to those identified for many other tyrosine kinase inhibitors, they affect less frequently the “gatekeeper” residue in the catalytic domain. In this study we carried out variation scanning by creating 11 substitutions at the gatekeeper amino acid, threonine 474 (T474). These variants were subsequently combined with replacement of the cysteine 481 residue to which irreversible inhibitors, such as ibrutinib, acalabrutinib and zanubrutinib, bind. We found that certain double mutants, such as threonine 474 to isoleucine (T474I) or methionine (T474M) combined with catalytically active cysteine 481 to serine (C481S), are insensitive to ≥16-fold the pharmacological serum concentration, and therefore defined as super-resistant to irreversible inhibitors. Conversely, reversible inhibitors showed a variable pattern, from resistance to no resistance, collectively demonstrating the structural constraints for different classes of inhibitors, which may affect their clinical application. Bruton Tyrosine Kinase (BTK) belongs to the TEC-family of non-receptor tyrosine kinases and is an important component of the B-Cell Receptor (BCR) signaling pathway , 2. BTK ®) is the most studied BTK inhibitor and the first in this new class approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA)  and FBS (2%), whereas B7.10 cells were activated with H2O2 (4 mM) and mouse anti-chicken IgM .Cells were collected after starvation-inhibition and then activated for 5 min at room temperature. Washout experiments were performed three times in serum-free medium prior to activation. COS-7 and HEK-293T cells were activated with pervanadate [0.02% HWhole-cell lysate was obtained by using modified RIPA buffer  containing a phosphatase inhibitor cocktail . Cell lysates were preheated for 5 min at 65 °C with sample buffer . Immunoblotting was performed as previously described . The folBTK-inhibitor co-crystal structures were obtained for ibrutinib (Protein Data Bank id 5p9j) , CGI-174In order to study the efficacy of both covalent and non-covalent BTK inhibitors in the context of resistant CLL cells, we created BTK variants by substituting the gatekeeper T474 residue, known to be associated with drug resistance , 53. SinWe further investigated the potential influence of gatekeeper residue substitutions when combined with replacement of C481. For that purpose, we generated five double BTK variants: T474A/C481S, T474I/C481S, T474M/C481S, T474M/C481T, and T474S/C481S , and almost complete BTK inhibition was obtained at 3 μM  complex, 95% carrying the double-variant E545K/E726K [Gatekeeper variations have been detected in treated CLL patients with a frequency of 4% and in the presence of the resistant C481S variation . The rol5K/E726K .Upon substitution of the gatekeeper residue, we found that most, but not all, variants are expressed at normal levels and are catalytically active. BTK was inhibited by ibrutinib in all the single variants with the exception of T474M, which does not explain the enrichment of T474I clones in a subset of treated patients . VariantWe generated five double variants to investigate effects on inhibitor binding. T474I/C481S, T474M/C481S and T474M/C481T variants are super-resistant to ibrutinib, while T474A/C481S and T474S/C481S are sensitive and partially sensitive, respectively, at the clinically relevant 0.5 μM concentration. We hypothesize that super-resistance appears when ibrutinib cannot bind covalently to C481S, it acts instead as non-covalent inhibitor and forms hydrogen bonds with E475 and M477 similar to in the wild-type . The repThe sensitivity of T474A/C481S and T474S/C481S variants to ibrutinib is compatible with explanation that substitutions carrying smaller side chains are less likely to clash with other residues. In T474S/C481S the polar character of the side chain is also retained. The single variants T474A and T474S showed slightly reduced enzymatic activity, as previously reported for T474A , but sinThe non-covalent BTK inhibitors RN486 and CGI-1746 are highly selective for BTK in in vitro and in vivo models, implying a potential for treatment of patients with C481S resistant variations , 41, 58.Supplementary data


Dear Editor,1 one of the latest ones was identified in Southern California in October 2020 and was subsequently detected in 26 other states in the United States as well as other countries as of January 2021. This strain, derived from coronavirus D614G mutation, is characterized by three genetic variations leading to three novel amino acid substitutions  in the spike (S) protein  in infectivity compared to the wild type in 293T-ACE2-TMPRSS2 . Figure n = 6).For vaccinated monkey sera, when compared with D614G pseudovirus, both CAL.20C and L452R-D614G pseudotyped viruses showed markedly reduced neutralization with a median of 2.4- and 2.3-fold, respectively Fig. . There wn = 11) and from vaccine recipients (Pfizer-BioNTech) (n = 8). For convalescent sera, a significant loss of neutralization activity was observed against CAL.20C and the L452R-D614G when compared to D614G, with a median reduction of 1.5-fold for both comparisons  is playing in the world’s fastest-growing surge of COVID-19 cases in India. Close monitoring of these variants containing L452R may guide us to the need of developing an additional COVID-19 vaccine with L452R in the RBD. The same implication can be applied to the development of therapeutic neutralizing antibodies.Supplementory


CAR T-cell therapies targeting the B-cell maturation antigen eliminate tumors in relapsed/refractory multiple myeloma patients, however durable remissions remain difficult to attain. Transforming growth factor beta (TGF-β) is a multifunctional cytokine abundantly expressed in the multiple myeloma bone marrow niche, where it promotes an immunosuppressive tumor microenvironment. We hypothesized that BCMA CAR T-cells armored to resist the suppressive effects of TGF-β will provide an advantage in treating multiple myeloma. The armored B2ARM CAR T cells, co-expressing BCMA targeting CAR with TGF-β dominant-negative receptor II, were generated by lentiviral transduction of primary human CD4+ and CD8+ T cells. The B2ARM CAR T cells eliminated MM.1S multiple myeloma targets in long-term cytotoxicity assays, even under TGF-β-high conditions, whereas cytotoxic function of the non-armored B2 CAR -T cells was inhibited by TGF-β. Concordantly, after long-term exposure to targets in the presence of TGF-β, the B2ARM CAR T cells were enriched for Granzyme B, CD107a, Ki67 and polyfunctional cells T-cells , as determined by flow cytometry. In addition, the B2ARM CAR T-cells, but not the conventional B2 CAR T-cells, resisted the TGF-β-mediated suppression of activation (CD25), exhaustion , and differentiation to T effectors (CD45RA+ CD45RO-CD62L-). In NSG mice bearing RPMI-8226 tumors overexpressing TGF-β, the B2ARM CAR mediated 100% tumor rejection and survival, superior infiltration of tumors on day 7 post CAR T treatment (%CD3+CAR+), and greater expression of IFN-γ, TNF-α, Ki67, Granzyme B, and PD-1, as compared to tumor-infiltrating non-armored B2 CAR T-cells. In NSG RPMI-8226 xenograft model in which tumors were additionally supplemented with TGF-β injections on days -1 through 11 of CAR T treatment, the B2ARM CAR T cells rejected tumors faster than the non-armored B2 CARs, and showed greater numbers of CD3+ and CD3+CAR+, central memory (CD45RO+CD62L+) and effector memory (CD45RO+CD62L-) T cells in the peripheral blood 18 days after treatment. In summary, the armored B2ARM CAR T cells mediate superior persistence, proliferation, multi-functionality, effector differentiation and anti-tumor function in pre-clinical models of multiple myeloma, while abrogating TGF-β-mediated suppression. Despite the recent surge in the development of novel therapeutics, multiple myeloma (MM), a hematologic disorder characterized by malignant proliferation of plasma cells, remains incurable. The advent of immunotherapies, including CAR T-cell therapy, has improved the prognosis of patients with relapsed/refractory multiple myeloma (RRMM) . HoweverMM cells predominantly localize in the bone marrow (BM), and the development and accumulation of MM in the BM is supported by a BM microenvironment replete in factors that specifically promote the expansion of MM cells and facilitate escape from immunosurveillance –12. Of pCip1, and p27Kip1, as well as through downregulation of IL-2 production from T-cells  sequences  of MM patients. To create an armored BCMA CAR that would remain resistant to the inhibitory effects of TGF-β, we co-expressed the B2 CAR construct with the dominant negative (DN) TGFBRII armor, that lacks the kinase domain of the receptor . We confMM cells localize in the BM where there are a number of potential sources of TGF-β, including BM stromal cells and the MM cells , 42. We We further examined the phenotypic characteristics of the B2ARM and B2 CAR T-cells following long-term exposure to target cells in the presence of TGF-β. We evaluated CAR proliferation capacity, degranulation status, and apoptotic potential in TGF-β-exposed long term co-cultures of B2 and B2ARM CARs, by assessing the expression of the specific markers Ki67, Granzyme B, CD107a, and annexin V, respectively, at the end of the long term co-culture with MM.1S cells Figure 4The percentage of B2 T cells expressing Ki67 was significantly decreased in the presence of TGF-β, as compared to B2 in the absence of TGF-β  mice with established tumors of the RPMI-8226 cell line, which produces the latent form of TGF-β  and CAR T-cell (CD3+CAR+) numbers in the blood of the mice that received the armored B2ARM CAR T-cells compared to the non-armored B2 CARs  supplemented with 30 U/ml of recombinant human IL-2 . After 18-24 hours, the T-cells were transduced with lentiviral vectors containing the CAR constructs. The T-cells were incubated with the lentiviral vectors for 2 days, and the cultures were subsequently washed and re-suspended in fresh TexMACS media with IL-2 and maintained at a density of 0.5 x 106 cells/ml. On day 6 or 7 after the start of T-cell culture, the cell surface expression of the CARs was assessed by flow cytometry.Primary CD4 and CD8 T-cells were activated with TransAct  according to the manufacturer’s protocol. The cells were cultured overnight at a density of 1 x 106 CAR T-cells were resuspended in FACS buffer (Miltenyi Biotec’s AutoMACS Rinsing Solution + MACS BSA Stock Solution) and incubated with 0.5 ug of recombinant human BCMA Fc Chimera Protein  for 20 mins at 4°C. Cells were washed twice and re-suspended with FACS buffer and incubated for 20 mins at 4°C with anti-Fc-Alexa Fluor 647 at 1 to 200 dilution. Cells were again washed twice and resuspended in FACS buffer and incubated for 20 mins at 4°C with anti-CD4-VioBlue or anti-CD8-VioGreen  at 1:50 dilution and 7AAD at 1:20 dilution. Cells were subsequently washed and analyzed using the MACSQuant Analyzer 10 flow cytometer . The TGF-β receptor II was detected by antibody clone REA903 (Miltenyi Biotec). For exhaustion marker staining, CAR T-cells were resuspended in FACs buffer and incubated with anti-PD-1-PE-Vio 770  and anti-LAG-3-APC  at 1 to 30 dilution. Memory markers were stained by incubating CAR T-cells with anti-CD45RO-PE-Vio 770, anti-CD45RA-APC, and CD62L-PE  at 1 to 30 dilution. For both exhaustion and memory staining panels, cells were additionally stained with CD8-VioGreen, CD3-VioBlue, and 7AAD. Cells were incubated with the antibodies for 20 mins at 4°C, and subsequently washed then acquired using the MACSQuant Analyzer 10 flow cytometer.To assess the cell surface expression of BCMA CARs, 0.5 – 1 x 10For intracellular cytokine staining, T-cells were incubated with target cells for 5-6 hours at 37°C in the presence of Brefeldin A . Cells were subsequently stained as previously described with cell surface markers CD8-VioGreen and CD3-VioBlue. After cell surface staining, cells were fixed and permeabilized with Fixation/Permeabilization Solution Kit  according to the manufacturer’s protocol. Cells were then stained with anti-IFN-γ-APC, anti-TNF-α-APC-Vio 770, and IL-2-PE at dilutions suggested by the manufacturer. Similarly, for additional intracellular T cell marker staining, fixed and permeabilized T cells were stained with antibodies to Granzyme B, or KI67, as indicated. For cell surface staining, non-fixed or permeabilized T cells were stained with antibody to CD107a, or annexin V-FITC binding reagent . All antibodies were from Miltenyi Biotec (Auburn CA), unless otherwise noted. After staining, cells were analyzed using the MACSQuant Analyzer 10 flow cytometer.For the long term co-culture experiment, CAR T-cells were co-cultured with target cells, MM.1S expressing GFP, at an ETT ratio of 0.1 – 0.3. The cells were cultured in 6-well plates with TexMACS media that was either treated with 10 ng/ml of human recombinant TGF-β  or remained untreated. The co-culture was fed by adding TGF-β-treated media or untreated media every 2-3 days. The absolute counts of T-cells and target cells at different time points during the long-term co-culture was assessed by quantifying the number of CD3+ cells and GFP+ cells using flow cytometry. The absolute counts were determined by normalizing the number of acquired cells in a specific volume using CountBright Absolute Counting Beads . When less than 15% of the target cells remained, cells from the co-culture were added to fresh target cells to initiate the subsequent round of co-culture. Additional rounds of co-culture were done until the T-cells no longer proliferated.Combinatorial gating function in Flow Jo software and SPICE analysis were utilized to determine the ability of individual cells to produce multiple cytokines during long-term co-culture with MM.1S cells. Each pie chart represents one treatment group. Slices within the chart represent fractions of the total T cell population organized based on the number of cytokines, which T cells contained in that fraction produced in response to treatment . Arcs on the outside of the pie chart signify which cytokine(s) were produced by T cells denoted within the corresponding slice of the pie chart .3 or 50 x 103 cells per well in a 96-well plate and cultured for 4 days in TexMACS media. The supernatants were collected and either remained untreated or treated with 1 M HCL for 15 mins to activate latent TGF-β. The Human TGF-β 1 Quantikine ELISA Kit  was used to detect active TGF-β in the supernatants. The ELISA was performed according to the manufacturer’s protocol.RPMI-8226 and MM.1S target cell lines were seeded at 25 x 10scid Il2rgtm1WjlPrkdc/SzJ) Stock No: 005557, Jackson Laboratory  were intradermally injected on the abdomen with 8 x 106 RPMI-8226 tumor cells. The CAR T-cells were intravenously injected after the tumors were allowed to engraft for 18-20 days and have reached volume sizes of > 60 mm3 as measured via caliper. Tumor volumes were calculated as: V = (L)*(W2)/2, where V-volume, W-tumor width, L- tumor length. For groups receiving CAR T-cells, 5 x 106 - 2 x 106 CAR T-positive cells, normalized per CAR expression percentage, were infused as indicated in each study. The number of T-cells that was infused in the UTD group was the mean of the total T-cells that was injected in the CAR T-cell groups. On day 6-7 after T-cell infusion, where indicated, 3-5 mice from each group (except the untreated group) were sacrificed for tumor harvest, while the rest were monitored for tumor progression and survival. Tumor sizes and body weights were measured every 2-3 days. Mice with tumor sizes reaching > 2000 mm were sacrificed. Where indicated, percentage of body weight change was calculated relative to day 0 of CAR T-cell administration.Female 7 to 8-week old NSG mice (NOD.Cg-The development of various treatment options, including CAR T-cell therapy, has greatly improved the prognosis of MM patients. Nevertheless, most patients eventually relapse, and MM remains an incurable disease. Some of the shortcomings of CAR T-cell therapy in MM have been attributed not only to antigen escape but also to failed expansion and limited persistence of CAR T-cells . While tin vivo, B2 CAR T-cells mediated greater tumor rejection as compared to B1 CAR T-cells.The B1 and B2 CAR T cells incorporate fully-human scFv sequences. In comparison to B1, B2 CAR T-cells exhibited high transduction efficiency and potent cytotoxicity in overnight killing assays against target MM cell lines, MM.1S and RPMI-8226. Moreover, the B2 CAR exhibited superior functionality compared to B1 CAR in long-term co-culture with MM.1S cells. B2 CAR retained potent cytotoxic capability despite repeated and prolonged exposure to MM cells, whereas B1 CAR showed reduced killing of target cells after prolonged co-culture with MM cells. The ability of the B2 CAR T- cells to maintain a robust proliferative capacity after prolonged exposure to MM cells is crucial for clinical success, as high expansion of CAR T-cells in the blood has been associated with better clinical responses for patients treated with other BCMA CAR T-cells , 9. MoreTGF-β plays an important role in the etiology on MM, and the MM bone marrow tumor microenvironment is TGF-β-rich, owing to both production of this cytokine by the MM cells, and by stromal cells, fibroblasts and osteoblasts in the resorpted bone marrow –17. In oHere, we show that the armored B2ARM CAR T-cells, but not the original B2 CAR T-cells, retained their functional potency in the presence of the suppressive TGF-β. In the long term co-culture with MM cell line MM.1S, the B2ARM CAR T-cells exhibited robust proliferation and cytotoxicity, despite prolonged treatment with exogenous TGF-β. The cytotoxic capability of the B2ARM CAR T-cells during exposure to TGF-β remained similar to its untreated counterpart, while the B2 CAR without DN TGFBRII failed to clear the tumor cells with TGF-β treatment. Taken together, these results demonstrate that the B2ARM CAR T-cells are resistant to TGF-β. Additionally, since TGF-β has been documented to induce apoptosis through the intrinsic and extrinsic pathway , 51, we TGF-β is known to suppress the effector functions of T-cells by inhibiting granzyme B and cytokine expression , 28. Conin vivo, we used three separate xenograft models of intradermal implantation of tumor cells into NSG mice. In the first model, the parental RPMI-8226 MM cells, expressing the latent form of TGF-β, were implanted. In the second model, a modified RPMI-TGFB cell line, engineered to secrete the active form of TGF-β, was implanted. In a third model, RPMI-TGFB cells were implanted and tumors were injected with the active form of TGF-β every three days for a total of 5 injections in the course of CAR T treatment.To evaluate the effectiveness of B2ARM CAR T-cells in vivo models with or without TGF-β injection, the armored B2ARM CAR T-cells eradicated tumors as well as B2 CAR T-cells. Additionally, the B2ARM CAR T-cells treatment resulted in a modest survival benefit for mice that were implanted with the RPMI-TGFB cell line. In addition, B2ARM CAR T cells were present in tumors in greater numbers as compared to the non-armored B2 CARs, and T cells extracted from tumor tissue of B2ARM CAR T – treated mice demonstrated greater cytokine and granzyme B production, in support of the functional superiority of the B2ARM CAR T-cells in vivo.In all three models, including the parental RPMI-8226 tumor cells, and RPMI-TGFB in vitro observation that the armored B2ARM CAR T-cells are able to rapidly differentiate to memory T-cell populations despite prolonged exposure to TGF-β, while the non-armored B2 CAR T-cells had decreased differentiation capacity.In the RPMI-TGFB model with the added exogenous injection of TGF-β we observed increased presence of memory T-cell populations following treatment, in the peripheral blood of the mice that received the armored B2ARM CAR T-cells compared to the mice that received the B2 CARs. These findings show the capacity of the armored B2ARM CAR T-cells to differentiate to memory T-cell populations. These data also confirm the in vitro model in the presence of continuously high TGF-β levels may better represent the true TGF-β-suppressive BM tumor microenvironment in the patients. Indeed, under TGF-β-high conditions, we were able to demonstrate the superiority of the armored B2ARM CAR T-cells as compared to the non-armored B2 CAR in vitro. Taken together, these results underscore the functional advantage of the TGFβ-armor in the B2ARM CAR T-cells in TGF-β-rich environments.It should be noted that the MM intradermal models have their limitations in fully replicating the full pathology of MM. In patients, MM cells predominantly localize in the TGF-β-enriched bone marrow, where various cells including bone marrow stromal cells, osteoblasts, and MM cells are all high sources of TGF-β . Therefoin vivo models which better recapitulate the complex TGF-β-rich immunosuppressive microenvironment of the MM BM.While the dearth of suitable MM tumor microenvironment models which accurately represent the MM milieu in patients currently remains a limitation for CAR T assessment, future studies will aim to develop ex-vivo or in vitro and in vivo. The armored B2ARM CAR T-cell design may therefore help overcome the limitations of the current BCMA CAR T-cell therapies, and prevail in the suppressive tumor microenvironment of multiple myeloma.The elevated levels of TGF-β in the BM of MM patients has had consequential repercussions, including immunosuppression and the development of myeloma bone disease . While tThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by LabCorp Drug Development, Ann Arbor, MI. All procedures were conducted in compliance with the applicable laws, regulations, and guidelines of the National Institutes of Health and with the approval of LabCopr Drug Development’s Animal Care and Use Committee. LabCorp Drug Development is an AAALAC accredited facility.DS, LA, BW, BD, and ZZ contributed to conception and design of the manuscript. LA, BW, YX, BV, and DW performed experiments and acquired results. LA, YX, PH, BV, DW, ZZ, and DS analyzed and interpreted results. LA, DS, BW, PH, and YX drafted and revised manuscript. DS, LA, BW, PH, YX, ZZ, BD, and PD critically reviewed manuscript. All authors have approved the final version of the manuscript.DS, LA, YX, ZZ, BV, DW, PH, PD are employees of Lentigen Technology, a Miltenyi Biotec Company. BW is an employee of Miltenyi Biotec.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.


Chimeric antigen receptor T (CAR-T) cell therapy has limited effects in the treatment of solid tumors. Sulforaphane (SFN) is known to play an important role in inhibiting tumor growth, but its effect on CAR-T cells remains unclear. The goal of the current study was to determine whether combined CAR-T cells and SFN could provide antitumor efficacy against solid tumors.The effect of combined SFN and CAR-T cells was determined in vitro using a co-culture system and in vivo using a xenograft mouse model. We further validated the effects of combination therapy in patients with cancer.In vitro, the combination of SFN and CAR-T cells resulted in enhanced cytotoxicity and increased lysis of tumor cells. We found that SFN suppressed programmed cell death 1 (PD-1) expression in CAR-T cells and potentiated antitumor functions in vitro and in vivo. As a ligand of PD-1, programmed cell death ligand 1 (PD-L1) expression was also decreased in tumor cells after SFN treatment. In addition, β-TrCP was increased by SFN, resulting in higher activation of ubiquitination-mediated proteolysis of PD-L1, which induced PD-L1 degradation. The combination of SFN and CAR-T cell therapy acted synergistically to promote better immune responses in vivo compared with monotherapy. In clinical treatments, PD-1 expression was lower, and proinflammatory cytokine levels were higher in patients with various cancers who received CAR-T cells and took SFN orally than that in the control group.SFN improves the cytotoxicity of CAR-T cells by modulating the PD-1/PD-L1 pathway, which may provide a promising strategy for the combination of SFN with CAR-T cells for cancer immunotherapy.The online version contains supplementary material available at 10.1186/s12916-021-02161-8. Chimeric antigen receptor T (CAR-T) cells have been successfully used to treat hematological malignancies . HoweverSulforaphane (SFN) is an isothiocyanate that is enriched in cruciferous vegetables . Since SAlthough there are extensive studies, whether and how SFN modulates the tumor microenvironment remains unknown, and whether SFN is available for cancer therapy in the clinic has not been well explored. The exhaustion of CAR-T cells in the tumor microenvironment hinders their antitumor capacity. One of the reasons for this was PD-1/PD-L1 engagement. In this study, we report that SFN downregulated PD-1 expression in CAR-T cells by inhibiting the PI3K/AKT pathway, while SFN promoted PD-L1 degradation in tumor cells by activating the ubiquitination-mediated proteolysis pathway. Furthermore, SFN enhanced the antitumor ability of CAR-T cells both in vitro and in vivo. Our findings revealed the effect of SFN on the antitumor response, which may provide a potential strategy for immunotherapy.2 incubator.The human lung cancer cell lines A549, H322, and the human embryonic kidney cell line 293 T were purchased from the cell bank of the Chinese Academy of Sciences . All cell lines were cultured in DMEM high glucose  containing 10% FBS , 100 U/mL penicillin, and 100 μg/mL streptomycin without mycoplasma at 37 °C in a humidified 5% CO+ T cells were positively selected using human CD3 microbeads  according to the manufacturer’s instructions. The selected CD3+ T cells were incubated in RPMI 1640 medium  containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 200 U/mL IL-2 .Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using density gradient centrifugation. CD3+ T cells treated with or without SFN  were transduced with lentivirus particles encoding meso CAR and cultured with 200 U/mL IL-2 and with or without 15 μM SFN.The meso single-chain fragment variable (scFv) was synthesized by Sangon Biotech  and cloned into the pCDH lentivirus vector, containing CD3ζ and CD28 activation domains . After aFACS Canto II flow cytometer  was used for flow cytometry analysis. Surface marker staining was conducted with fluorescence-conjugated antibodies for 30 min in the dark at 4 °C. For intracellular staining, the cells were stained with antibodies against intracellular cytokines after surface antibody staining, fixation, and permeabilization. All antibodies were purchased from BioLegend : APC/Cyanine7 anti-human CD8 (SK1), PE anti-human CD107a (H4A3), PE anti-human PD-1 (EH12.2H7), APC anti-human Tim-3 (F38-2E2), PE anti-human CD69 (FN50), PE anti-human PD-L1 (29E.2A3), APC anti-human IFN-γ (4S.B3), PE anti-human IL-2 (MQ1-17H12), APC anti-human Perforin (dG9), and PE anti-human/mouse Granzyme B Recombinant (QA16A02), except PE anti-human mesothelin .Tumor cells were incubated with CAR-T cells at different effector to target (E:T) ratios for 6 h. Tumor cells were collected and stained with Annexin-V  in Annexin-V Binding Buffer  for 15 min in the dark. Flow cytometry was used for analysis after the addition of propidium iodide .PD-1 , phospho-AKT (Ser473) , phospho-mTOR (Ser2448) , phospho-S6 ribosomal protein (Ser235/236) , AKT (pan) , mTOR , S6 Ribosomal Protein , β-TrCP , and β-Actin , except anti-PD-L1 antibody [EPR19759] .To collect whole-cell lysates, cells were lysed in RIPA lysis buffer supplemented with a Protease Inhibitor Cocktail , and the cell lysates were centrifuged at 12,000 rpm. The supernatant was collected, mixed with loading buffer, and boiled for 15 min to disengage the protein secondary structure. The mixture was resolved on SDS-PAGE gels  and then transferred onto nitrocellulose membranes . After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies overnight at 4 °C. The bands were probed with the appropriate secondary antibodies and detected by enhanced chemiluminescence. The following primary antibodies were purchased from Cell Signaling Technology : We first purified and enriched the RNA from samples. For mRNA enrichment, RNA with a polyA tail was enriched using magnetic beads with Oligo-dT. For RNA purification, DNA probes were used to hybridize the rRNA. The hybridized products were subsequently degraded by RNase H and DNase I. Next, the RNA was shredded into fragments and was reverse-transcribed to cDNA using random N6 primers. The ends of the synthesized double-stranded DNA were filled, the 5 'ends were phosphorylated, and the ligation products were amplified by PCR with specific primers. After thermal denaturation into a single strand, the PCR products were circularized with a bridge primer to obtain a single-stranded circular DNA library. Finally, the BGISEQ-500 platform was used for high-throughput sequencing.Whole gene counts from RNA-seq data were selected for the GSEA enrichment analysis . “c2.cp.Total RNA was extracted with TRIzol , and the quality and concentration were detected using a NanoDrop 2000 spectrophotometer . cDNA was synthesized using the Primescript RT Reagent Kit . Quantitative real-time PCR (qRT-PCR) analysis was performed using the SYBR Green Master Mix . The primers used are shown in Additional file 5) were seeded in 6-well plates. Three BTRC short interfering RNA (siRNA) or scramble (siScr) sequences  were transfected into cells using jetPRIME transfection reagent  according to the manufacturer’s protocol. After 48 h, transfection efficacy was tested by qRT-PCR and western blot.A549 or H322 cells (1.5 × 105) were injected subcutaneously (s.c.) into mice. Five days later, mice received 5 × 106 meso CAR-T cells intravenously injection (i.v.) with SFN  or anti-PD-1  treatment. Tumor growth was measured by bioluminescent imaging using a Xenogen IVIS-200 Spectrum camera and Living Image version 4.4  for image acquisition and analysis. For the analysis of tumor- and spleen-infiltrating meso CAR-T cells, 7 days after meso CAR-T cell injection, cells from fresh tumor tissues were harvested using a tumor dissociation kit  according to the manufacturer’s instructions, while the spleens were ground directly for cell collection. Phenotypes and cytokine secretion were evaluated using flow cytometry. Mice were sacrificed when the values of total flux were over 2 × 1011.All mouse experiments were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University. Female 5-week-old NOD-SCID mice were purchased from Beijing Vital River Laboratory Animal Technology Company and were fed in the Henan Key Laboratory for Pharmacology of Liver Diseases. Luciferase-expressing H322 (H322-luc) cells ) overnight at 4 °C, followed by incubation with horseradish peroxidase-linked secondary antibody at room temperature for 30 min. After stained with the DAB solution, the samples were counterstained with hematoxylin, dehydrated, and sealed with neutral gum.Tissue sections from xenograft tumor were first deparaffinized. The sections were placed in 0.01 M sodium citrate buffer for antigen retrieval. Then, 3% HPD-L1 expression was evaluated by the immunohistochemical scoring (IHS). The proportion of positive cells was scored as follows: 0 (no positive cells), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%).The staining intensity was scored on a 0 to 3 scale . The final score was based on the addition of these two scores: negative , weakly positive , moderately positive , and strongly positive .Peripheral blood was obtained from patients with cancer who voluntarily participated in a clinical trial approved by the Institutional Review Board of the First Affiliated Hospital of Zhengzhou University . Written informed consent was obtained from all the participants. Four patients received sulforaphane from broccoli sprout extract , two capsules per day for 30 days at the beginning of CAR-T therapy, and another four patients only received CAR-T therapy as a control group. The clinical characteristics of the patients are presented in Additional file t test was used for comparison of two groups. Both methods were achieved by the “Analyze” function in GraphPad Prism. Survival curves were determined by Kaplan-Meier analysis using the R packages “Survival” and “Survminer.” All boxplot and barplot images are presented as the mean ± SEM. Statistical significance is shown as *(P < 0.05), **(P < 0.01), ***(P < 0.001), and **** (P < 0.0001), and ns meant not significant (P > 0.05).Data were analyzed using GraphPad Prism 8.2.1 and R language (version 3.6). Student’s + T cells with a second-generation anti-mesothelin CAR construct containing the CD3ζ and CD28 activation domains , including untreated group (control), IFN-γ-treated group (IFN- γ) and IFN- γ together with SFN-treated group (IFN- γ + SFN). Interestingly, PD-L1 mRNA expression was not consistent with the protein level, which showed no significant difference between the IFN-γ and IFN-γ + SFN groups. Furthermore, this result was confirmed in our samples   of SFN only (SFN only), or meso CAR-T cell infusion with exogenous SFN administration (CAR-T + SFN), or anti-PD-1 treatment (CAR-T + anti-PD-1) Fig. A. Althouing Fig. K, L. The+ CAR-T cells in the blood drawn at different time points (Fig. To determine whether SFN could promote CAR-T cell cytotoxicity in patients with cancer who received adoptive immunotherapy, we simultaneously treated patients with SFN orally and examined PD-1 expression and cytokine secretion of CD8nts Fig. A. We obsnts Fig. B–E, whicSFN has been shown to directly suppress tumor growth . In this+ tumor cells, whereas the effector functions of CAR-T cells were reversed by PD-1 blockade [+ T cells in patients by downregulating PD-1 expression [A major limitation of CAR-T cells is the dysfunction and exhaustion of the infused T cells , 34. PD-blockade . Our preblockade . In addipression . Herein,Upregulation of PD-L1 on tumor cells suppresses CAR-T cell antitumor capacity after chronic antigen stimulation . IFN-γ pPTMs, including ubiquitination, phosphorylation, glycosylation, methylation, and acetylation, play key roles in regulating protein biological processes, such as activity, degradation, and interaction . Accumul+ T cell in mice model [In Liang’s review, SFN inhibited T cell activation by increasing ROS level and decreasing GSH . Accordice model . Thejassce model . In our Since SFN displays dramatic outcomes in preclinical models, whether and how SFN affects human bodies should be taken into account. To date, several clinical trials have evaluated the effect of SFN in both healthy donors and patients with cancer . A randoIn our study, we found that SFN both decreased PD-1 expression on CAR-T cells and PD-L1 on tumor cells. Furthermore, CAR-T cell infusion combined with SFN administration showed a stronger ability against tumor growth in mice model, suggesting that this combination therapy may provide a strategy for the clinical application of immunotherapy. However, there are still several limitations in this study. Although we observed effect of SFN on patients with different type of cancer, the sample size we used was small with a relatively short observation time. The therapeutic effect of SFN for other tumors needs further verification. In addition, the antitumor mechanism of SFN in vivo remains unclear. For further study, we will expand the sample size of patients with solid tumor for observation of the safety and efficacy. Future in vivo study will be also needed to investigate the underlying mechanisms of SFN against tumor.In conclusion, we reported that SFN not only potentiated CAR-T cell function by downregulating PD-1 expression, but also reduced PD-L1 expression by activating the ubiquitination-mediated proteolysis pathway, which indicated the double benefit of combination therapy in inducing tumor remission.In this study, we found that SFN regulated immune response through the PD-1/PD-L1 pathway. Further study showed that SFN downregulated PD-1 expression on CAR-T cells by inhibiting PI3K/AKT signaling pathway, and PD-L1 degradation was induced by SFN in a ubiquitination-dependent manner. In addition, SFN in combination with CAR-T cell treatment enhanced antitumor response both in mice and patients. Hence, SFN has a potential therapeutic effect in the design of combination therapies with CAR-T cells.Additional file 1: Table S1. Primers of genes.Additional file 2: Table S2. Clinical characteristics of the enrolled patients.Additional file 3: Figure S1. The quantitative analysis of Western blot data. A Relative expression of PD-1 protein level in control and SFN group. The quantitative analysis of PD-1, p-AKT, p-mTOR, p-S6, t-AKT, t-mTOR, t-S6 protein level in control group, SFN group, and PI3K/AKT inhibitor group. B Relative expression of PD-L1 protein level of control, IFN- γ and IFN- γ + SFN group in A549 and H322. C The quantitative analysis of β-TrCP protein level of control, IFN- γ and IFN- γ + SFN group in A549 and H322. D The quantitative analysis of β-TrCP protein level of siRNA transfection efficacy in A549 and H322 cells. E Relative expression of PD-L1 protein level was detected in the presence of IFN- γ or SFN after si-Scr or si-BTRC by western blot in A549 and H322 cells


Red blood cell (RBC)-mimicking nanoparticles (NPs) offer a promising platform for drug delivery because of their prolonged circulation time, reduced immunogenicity and specific targeting ability. Herein, we report the design and preparation of RBC membrane-bound NPs (M@AP), for tumoral photodynamic-immunotherapy. The M@AP is formed by self-assembly of the positively charged aggregation-induced emission luminogen (AIEgen) (named P2-PPh3) and the negatively charged polyinosinic : polycytidylic acid (Poly(I : C)), followed by RBC membrane encapsulation. P2-PPh3 is an AIE-active conjugated polyelectrolyte with additional photosensitizing ability for photodynamic therapy (PDT), while Poly(I : C) serves as an immune-stimulant to stimulate both tumor and immune cells to activate immunity, and thus reduces tumor cell viability. When applied in tumor-bearing mice, the M@AP NPs are enriched in both the tumor region as a result of an enhanced permeability and retention (EPR) effect, and the spleen because of the homing effect of the RBC-mimicking shell. Upon light irradiation, P2-PPh3 promotes strong ROS generation in tumor cells, inducing the release of tumor antigens (TA). The anti-tumor immunity is further enhanced by the presence of Poly(I : C) in M@AP. Thus, this strategy combines the PDT properties of the AIE-active polyelectrolyte and immunotherapy properties of Poly(I : C) to achieve synergistic activation of the immune system for anti-tumor activity, providing a novel strategy for tumor treatment. Immunotherapy has great potential, but the response of some tumors to immunotherapy is not high. Herein, we developed a biomimetic red cell membrane loaded with aggregation-induced emission luminogen and polyinosinic:polycytidylic acid nanoparticles to enhance the efficacy of tumor immunotherapy. It has shown great clinical success against a wide variety of malignancies in recent years . Currentimmunity . Additioimmunity . Polyinoimmunity . TLR3 isimmunity . As a TLimmunity ,11. The immunity ,13. Howeimmunity ,15. Furtimmunity , greatlyet al. demonstrated that local PDT not only inhibits the growth of primary tumors, but also suppresses the off-target distant tumors because of the infiltration of immune cells [Photodynamic therapy (PDT) is a novel non-invasive treatment option that has several advantages, including strong efficacy, target ability and negligible side effects, which has been used for treatment of tumors and other diseases ,18. PDT ne cells . Therefone cells ,24, the ne cells . Their eet al. demonstrated that nanoerythrosomes derived from RBC membranes promote TA delivery to the spleen to enhance cancer immunotherapy [To achieve tumor selectivity, nanoparticles (NPs) can be functionalized with naturally derived bio-membranes to construct biomimetic systems . Red blootherapy . Therefoin vivo results showed that the majority of the M@AP NPs are enriched in tumors, which induces tumor cell death upon PDT and promotes the release of TA to activate anti-tumor immunity. Furthermore, some of the M@AP NPs target the spleen because of the homing effect and directly activate the anti-tumor immune response. Therefore, we have demonstrated that M@AP NPs, as a combined PDT and immunotherapy agent, exhibit a direct inhibitory effect on tumors and evoke an anti-tumor immune response, representing a novel combination strategy for effective cancer treatment.Based on these features of RBC membranes, we envisioned that RBC membrane-camouflaged NPs loaded with photosensitizing AIEgen and immunologic adjuvant Poly(I : C) would strongly enhance anti-tumor immunity. In this study, we constructed such multi-functional NPs (M@AP NPs) by self-assembly of a positively charged AIE PS (i.e. P2-PPh3) and negatively charged Poly(I : C) in a poly (lactic-co-glycolic acid) (PLGA) matrix, which were then further encapsulated in an RBC membrane Fig. a. Our inP2-PPh3  was synthesized according to our previous report . P2-PPh3v/v) than in pure DMSO solution (7.3%). However, the quantum yields of M@AP in the DMSO/THF mixture and pure DMSO solution are very similar, 27.9% and 28.6%, respectively. Zeta potential measurement showed that P2-PPh3 has a large number of positive charges and that the zeta potential is lower in the resultant M@AP after assembly with Poly(I : C) and RBC membrane  was performed to verify the protein load on the NPs, confirming that M@AP indeed contains proteins from the RBC membrane, while Poly(I : C) and P2-PPh3 are protein-free Fig. c. UV-Visane Fig. e. The avane Fig. f and S4.ane Fig. g. Comparane Fig. h and S5.ane Fig. i and S6.–2) for 3 min, and a strong green fluorescence from ROS-activated DCFH-DA and a red fluorescence from PI were observed in M@AP-treated cells, indicating that M@AP produces ROS and effectively kills cells under such conditions . B16-F10 cells (a mouse melanoma cell line) incubated with M@AP show red emission from the M@AP, indicating successful cell uptake of the NPs . After co-incubation with DCFH-DA for 20 min, the B16-F10 cells were irradiated with white light  in the TA-treated group were significantly higher than those in PBS-treated group, but there was no significant difference in CD19 (B lymphocytes)- and CD49b  cells)-positive cells  in M@AP NPs, we first constructed M@AP-FITC NPs using FITC-tagged Poly(I : C) . After 8 h incubation with M@AP-FITC, fluorescent signals from both Poly(I : C)-FITC and P2-PPh3 were detected in the cells . Then, we prepared M@A NPs using the same procedure but without adding Poly(I : C). The effects of different NPs (without light irradiation) on the cell viability of B16-F10 cells were determined using Cell Counting Kit-8 (CCK-8) tests, from which results revealed that only M@AP NPs can induce significantly lower cell viability . The pharmacokinetics of M@AP and AP (NPs without the RBC membrane) are shown in Fig. in vivo, the biodistributions of AP and M@AP in the tumors and organs were detected using an in vivo imaging system (IVIS®)  Fig. b and c. in vivo, unilateral B16-F10 tumor-bearing mice with tumor volumes of ∼100 mm3 were randomly divided into five groups: PBS (Light −), M@A (Light −), M@AP (Light −), M@A (Light +) and M@AP (Light +). As shown in Fig. To investigate the anti-tumor efficacy of the NPs P > 0.05), indicating that the immune effect induced by PDT is insufficient to inhibit growth of the adjacent tumor. Interestingly, the non-treatment side tumor in the M@AP (Light −) group is significantly inhibited (P < 0.05), and the treatment effect for the non-treatment side tumor in the M@AP (Light +) group is the highest (P < 0.001)  Fig. h. We att01) Fig. h. Simila01) Fig. i, which To further explore the anti-tumor mechanism of the NPs, we examined the histopathology of the tumor tissues using H&E staining. The results reveal that the tumor tissues in the PBS and M@A (Light −) groups are compact, whereas those in the M@AP (Light −), M@A (Light +) and M@AP (Light +) groups are sparse with a large number of vacuolar necrotic cells Fig. j. Furthein vivo. T-lymphocytes are key mediators of tumor destruction and their specificity for tumor-expressed antigens is of paramount importance [+ T-cells are the preferred tool for investigating anti-tumor activities as they perform tumorous antigen presentations, and CD4+ T-cells are also necessary to support the normal functioning of CD8+ T-cells [+ T-cells and CD8+ T-cells in the peripheral blood from mice are significantly higher in the M@AP (Light −), M@A (Light +) and M@AP (Light +) groups compared with the control group, with the M@AP (Light +) group showing the most obvious increase. Furthermore, as analyzed by CLSM imaging  promotes tumor-specific T-cell responses. In addition, tumor-associated macrophages (TAMs) are the major infiltrating leukocytes of the tumor microenvironment, among which M1 macrophages are reported to phagocytose tumor cells [+, CD11b+ and CD86+) are significantly increased in tumor sections from M@AP-treated groups, especially the M@AP (Light +) group , suggesting that M@AP promotes the differentiation of macrophages into the M1 type. In addition, we also investigated the population of regulatory T (Treg) cells (FOXP3+ and CD4+), which play important roles in suppressing anticancer immunity. As shown in Fig. S26, the absolute number of Treg cells in the tumor tissues is relatively low in all groups  and M@AP (+Light) groups). But the proportions of Treg cells in the M@AP, M@A (+Light) and M@AP (+Light) groups are decreased, among which the degree of decline in the M@AP (+Light) group is the most obvious.We then investigated the mechanism by which M@AP NPs activate the immune system against tumors portance . CD8+ T- T-cells . As showor cells . Our resFurthermore, aged or damaged RBCs are eliminated by scavenger cells such as macrophages and dendritic cells in the spleen . PolyI d. Next, 3, randomized mice were treated with PBS (Light −), M@A (Light −), M@AP (Light −), M@A (Light +) or M@AP (Light +). The interval of drug injection and light irradiation is shown in Fig. ® imaging, observation with the naked eye and H&E staining . Quantitative analysis revealed that M@AP NPs simultaneously kill local cancer cells directly and stimulate the immune cells to release cytokines that present cytotoxicity against tumor cells. We further applied the system in a lung metastasis mouse model and demonstrated the excellent anti-metastasis capability of our approach, which provides a potential treatment strategy for preventing tumor recurrence and metastasis.In this study, we developed a multi-functional nanoplatform for delivering immunologic adjuvants for synergistic anti-tumor photodynamic immunotherapy. The M@AP nanoplatform consists of a complex comprising an AIE-conjugated polymer as a photosensitizer and Poly(I : C) as an immunologic adjuvant, a PLGA matrix and an RBC membrane shell. nwab039_Supplemental_FileClick here for additional data file.