Journal: Nature Biomedical Engineering

Article Title: Engineered outer membrane vesicles enhance solid tumour CAR-T cell therapy

doi: 10.1038/s41551-025-01575-6

Figure Lengend Snippet: a , Representative IVIS images showing biodistribution of OMVs/POMVs in tumour-bearing mice. BALB/c mice were intravenously (i.v.) injected with 2.5 mg kg −1 DiR-labelled OMVs or POMVs. IVIS imaging was performed to detect DiR signals in tumour-bearing mice at 24 h post-injection. b , Representative IVIS images showing the biodistribution of OMVs or POMVs in tumour and major organs (H, heart; Lu, lung; Li, liver; S, spleen; K, kidney; I, intestine; T, tumour) at 6, 12, 24 and 48 h. c , Fluorescence intensity per gram of tumour tissue was quantified at indicated timepoints. d , Plots of all CD45⁺ immune cells by t -SNE. e , Stacked bar charts comparing the relative proportions of immune cell subsets within the total CD45⁺ compartment across samples. f , t -SNE plots showing monocyte–macrophage populations from mice treated with POMV or P-LNP, coloured by treatment group. g , Volcano plots showing upregulated and downregulated DEGs (on POMV treatment versus P-LNP treatment) in monocyte–macrophage populations. h , Enrichment of GO terms associated with DEGs derived from monocyte–macrophage populations comparing POMV and P-LNP treatments. i , Stacked bar charts showing the proportions of tumour-infiltrating T cell subclusters. j , Single-cell transcription levels of IFN-γ illustrated on t -SNE plots, highlighting differential expression across cell populations. For d – j , 4T1 tumour-bearing BALB/c mice received i.v. injections of 2.5 mg kg −1 P-LNP, POMVs or equal volume of PBS on days 7, 9, and 11 post tumour inoculation. Single-cell RNA sequencing was conducted to analyse the TME on day 13. k , 4T1-HER2 tumour-bearing BALB/c mice received i.v. injections of 2.5 mg kg −1 OMVs, OMVs + 3 mg kg −1 aPD-L1, POMVs or equal volume of PBS on days 7, 9 and 11 post tumour inoculation. FCM analysis was conducted to analyse the TME on day 13. l – s , FCM analysis of MDSCs ( l ), T reg ( m ), M2 macrophages ( n ), M1 macrophages ( o ), CD3 + CD8 + T cells ( p ), GZMB + CD8 + T cells ( q ), perforin + CD8 + T cells ( r ) and CD3 + CD8 + CD44 + CD62L − T effector memory cell (T EM ) ( s ) infiltrating in tumour tissues at day 13. n = 6 independent biological replicates per group for the data in c and l – s . The data and error bars represent the mean ± s.d. A statistical analysis was performed using a two-way ANOVA for c , followed by Šídák’s multiple comparisons test, one-way ANOVA for l – s , followed by a Tukey’s multiple comparisons test, two-sided Wilcoxon rank-sum test with Benjamini–Hochberg adjustment for g and one-sided hypergeometric test with Benjamini–Hochberg adjustment for h . P adj , adjusted P value.

Article Snippet: After coculture, the supernatants were collected for cytokine secretion analysis using mouse IL-2, TNF (CAS no. E-EL-M3063, Elabscience) and IFN-γ ELISA kits, following the manufacturer’s protocol.

Techniques: Injection, Imaging, Fluorescence, Derivative Assay, Single Cell, Quantitative Proteomics, RNA Sequencing

Journal: Nature Biomedical Engineering

Article Title: Engineered outer membrane vesicles enhance solid tumour CAR-T cell therapy

doi: 10.1038/s41551-025-01575-6

Figure Lengend Snippet: a , A heterogeneous population of 4T1 cells (50% HER2 positive, 50% wild type) was orthotopically implanted into the third mammary fat pad of BALB/c mice. The mice received intravenous (i.v.) injections of 2.5 mg kg −1 OMVs, OMVs + 3 mg kg −1 aPD-L1, POMVs or equal volume of PBS on days 7 and 12. CAR-T cells (5 × 10⁶) were intravenously administered on days 8 and 13, followed by weekly bioluminescence imaging. b , IVIS imaging of tumour growth at indicated timepoints. c , Tumour growth of individual mice and average tumour growth for each treatment group (PBS, CAR-T, POMVs, OMVs + CAR-T, OMVs + aPD-L1 + CAR-T and POMVs + CAR-T), with tumour volume measured up to day 21 post inoculation. d , The Kaplan–Meier survival curves are shown of orthotopic 4T1/4T1-HER2 mixture tumour-bearing mice after different treatments. e – h , Frequencies of M2 macrophages ( e ), MDSCs ( f ), T reg ( g ) and CD8 + T cells ( h ) infiltrated in tumour tissues at day 21. i – l , The frequency of CAR-T cells in CD3 + T cells ( i ) and frequencies of CAR-T cells expressing intracellular cytokines, including IFN-γ ( j ), perforin ( k ) and GZMB ( l ) in CAR-T cells at day 21. m , Percentages of HER2-positive 4T1 cells in tumour tissues after different treatments at day 21. n = 6 independent biological replicates per group for data in c – m . The data and error bars represent the mean ± s.d in e – m , and the data and error bars represent the mean ± s.e.m. in c . A statistical analysis was performed using a two-way ANOVA for c or one-way ANOVA for e – m , followed by a Tukey’s multiple comparisons test and log-rank (Mantel–Cox) test for d .

Article Snippet: After coculture, the supernatants were collected for cytokine secretion analysis using mouse IL-2, TNF (CAS no. E-EL-M3063, Elabscience) and IFN-γ ELISA kits, following the manufacturer’s protocol.

Techniques: Imaging, Expressing

Journal: Nature Biomedical Engineering

Article Title: Engineered outer membrane vesicles enhance solid tumour CAR-T cell therapy

doi: 10.1038/s41551-025-01575-6

Figure Lengend Snippet: a , A schematic representation of the BROAD-CAR platform: POMVs specifically target tumour cells, delivering a CAR-targeting plasmid that induces the expression of HER2 ECDs on the tumour cell surface, thereby facilitating effective CAR-T cell activation. b , c , Fluorescence images ( b ) and FCM analysis ( c ) showing the expression of HER2 on 4T1 cells at different timepoints after treatment with 10 μg ml −1 plasmid-loaded OMVs (OMV@H) or BROAD-CAR. 4T1 cells were treated for 4 h at 37 °C, and fluorescence images and FCM histograms were acquired at different timepoints post treatment to quantify HER2 expression. Scale bar, 100 μm. d – f , CAR-T cells were cocultured with 4T1-HER2 cells (E:T of 1:2) and treated with 10 μg ml −1 OMV@H, BROAD-CAR or equal volume of PBS for 48 h, followed by FCM ( d,f ) and ELISA ( e ) analysis: frequencies of CD69 and CD107 surface expression on CAR + CD8 + T cells ( d ); cytokine levels of IL-2, TNF and IFN-γ evaluated by ELISA ( e ); frequencies of CAR + CD8 + T cell subsets, including stem cell memory (T SCM ), central memory (T CM ), effector memory (T EM ) and effector T cells (T eff ) ( f ). g , Dose-dependent cytotoxicity of CAR-T cells (E:T of 1:2) against 4T1 cells treated with BROAD-CAR (0–48 μg ml −1 ) for 48 h, evaluated by the LDH assay. h , Cytotoxicity of CAR-T cells against 4T1 cells at the indicated E:T ratios after 48 h coculture with 10 μg ml −1 OMV@H, BROAD-CAR or equal volume of PBS. n = 6 independent biological replicates per group for data in c – h . The data and error bars represent the mean ± s.d. A statistical analysis was performed using two-way ANOVA followed by Šídák’s multiple comparisons test for c or a Tukey’s multiple comparisons test for d – f and h and one-way ANOVA for g , followed by a Tukey’s multiple comparisons test.

Article Snippet: After coculture, the supernatants were collected for cytokine secretion analysis using mouse IL-2, TNF (CAS no. E-EL-M3063, Elabscience) and IFN-γ ELISA kits, following the manufacturer’s protocol.

Techniques: Plasmid Preparation, Expressing, Activation Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay

Journal: Nature Biomedical Engineering

Article Title: Engineered outer membrane vesicles enhance solid tumour CAR-T cell therapy

doi: 10.1038/s41551-025-01575-6

Figure Lengend Snippet: a , C57BL/6J mice were orthotopically implanted with Hepa1-6 tumour cells and received intravenous (i.v.) administrations of 2.5 mg kg −1 POMVs, BROAD-CAR or an equivalent volume of PBS on days 7, 9 and 11, followed by CAR-T cell infusions (5 × 10 6 cells) on days 12 and 17. Bioluminescence imaging was conducted weekly to monitor tumour progression. b , In vivo bioluminescence imaging of tumour progression following different treatments. c , Image of the livers after different treatments. d , H&E staining of mouse liver after different treatments. Scale bar, 1,000 μm. The dashed black circles delineate the tumor boundary. e , The Kaplan–Meier survival curves are shown of orthotopic Hepa1-6 tumour-bearing mice after different treatments. f , g , FCM analysis of CD8 + ( f ) and CAR + ( g ) in CD3 + T cells after different treatments at day 21. h–k , Frequencies of CAR-T cells expressing intracellular cytokines, including, including TNF ( h ), perforin ( i ), GZMB ( j ) and IFN-γ ( k ) at day 21. l , HER2 expression levels in Hepa1-6 tumour tissues and other organs after BROAD-CAR-T treatment on day 21. n = 6 independent biological replicates per group for data in e – l . The data and error bars represent the mean ± s.d. A statistical analysis was performed using a log-rank (Mantel–Cox) test for e and one-way ANOVA for f – l , followed by a Tukey’s multiple comparisons test.

Article Snippet: After coculture, the supernatants were collected for cytokine secretion analysis using mouse IL-2, TNF (CAS no. E-EL-M3063, Elabscience) and IFN-γ ELISA kits, following the manufacturer’s protocol.

Techniques: Imaging, In Vivo, Staining, Expressing

Journal: bioRxiv

Article Title: T cells use TNF and IFNγ for paracrine killing with target discrimination programmed by pathogen-derived factors

doi: 10.64898/2025.12.23.696308

Figure Lengend Snippet: (A) WT and Prf1-/- anti-B7H3 CAR-T cells were co-cultured with B16 melanoma cells in a 96W plate in triplicates. An E:T ratio of 1:1 was used. Control IgG or neutralizing anti-TNF and anti-IFNγ (50 ug/mL) were added to the wells. Target viability was assessed by YOYO-3 staining and live-cell imaging for 72 h using the IncuCyte S3 instrument. Confluency of the cells in each well were also quantified. Data is presented as YOYO-3 counts normalized to confluency in each well in arbitrary units. Values are triplicate mean ± SD. Data from one representative experiment is shown. (B) The experiment in (A) was independently performed 3 times. The mean confluency at 60 h of 9 wells from the 3 biological replicates is shown in the bar chart. ( C) B16 cells were stimulated in triplicates with 100 ng/mL TNF, IFNγ, or both, and imaged in the IncuCyte for 48 h in the presence of YOYO-3. Values are triplicate mean ± SD. Data from one representative experiment is shown. (D) The experiment in (C) was performed 3 times. The mean confluency of 9 wells at 60 h from the 3 biological replicates is shown in the bar chart. ( E) WT, FADD KO or STAT1 KO B16 cells were treated with a combination of 100 ng/mL TNF and IFNγ. Cell death was analyzed for 48h in the IncuCyte as in (A). Representative data from one experiment is shown. (F) WT, FADD KO or STAT1 KO B16 cells were co-cultured with Prf1 -/- CAR-T cells against B7H3 at an E:T ratio of 1:1. Target killing was analyzed as in (A). ( G ) Western blot analysis of the indicated proteins in B16 cells stimulated for 24 h with TNF and IFNγ as indicated. (H) B16 cells were co-cultured with Prf1-/- CAR-T cells at 1:1 ratio in the presence of control IgG or antibodies against TNF and IFNγ (50 ug/mL). Data in (G, H) are representative of 3 independent experiments. p values from Mann-Whitney tests are shown for data in (B, D).

Article Snippet: Antagonist antibodies used were from following sources: murine-TNF (clone XT3.11, Bio X cell BE0058) murine IFNγ (clone XMG1.2, Bio X Cell BE0055), human-TNF (Bio X Cell SIM0001) and human IFNγ (Bio X Cell BE0235).

Techniques: Cell Culture, Control, Staining, Live Cell Imaging, Western Blot, MANN-WHITNEY

Journal: bioRxiv

Article Title: T cells use TNF and IFNγ for paracrine killing with target discrimination programmed by pathogen-derived factors

doi: 10.64898/2025.12.23.696308

Figure Lengend Snippet: (A) Nuclear extracts from B16 cells treated for 24 h with 10 ng/mL TNF, IFNγ, or both were blotted for canonical and non-canonical NFκB and STAT1. HDAC1 was used as a loading control. (B) Biological triplicate samples of RNA isolated from B16 cells treated as in (A) were analyzed using the Nanostring nCounter Host Response panel. Heatmap shows a group of genes from unsupervised clustering whose expression was synergistically induced by TNF and IFNγ. (C) Supernatants from B16 cells treated as in (A) were analyzed for level of CCL2 and CXCL9 by ELISA. Values are the mean ± SD from three independent experiments, each performed in technical triplicates. (D) Schematic of paracrine killing assay. B16 WT cells were stably transduced with nuclear green fluorescent protein (GFP) and B16 B7H3 KO cells with nuclear raspberry fluorescent protein (RFP). The two population of targets were mixed and co-cultured with CAR-T cells against B7H3. (E) Mixed GFP⁺ WT and RFP⁺ B7H3 KO B16 cells (1:3 ratio) were co-cultured with Prf1+/+ CAR-T cells at E:T ratio of 1:1. Control IgG or anti-TNF/IFNγ (50 ug/mL) was also added. GFP and RFP counts in each well was quantified by IncuCyte live-cell imaging for 60 h. Values are mean ± SD from triplicate well. Data from one representative experiment is shown. (F) Experiment in (E) was performed three times. Bar graphs represent mean GFP and RFP counts in the wells at endpoint from the three biological replicates. (G) RFP+ B16 B7H3 KO cells were co-cultured with media, Prf1+/+ or Prf1-/- CAR-T cells against B7H3 at an E:T of 1:1 and imaged for 48 h. (H) Mean confluency of wells at endpoint from three independent experiments in (G). (I) 1:3 mixture of B16-cOVA and RFP⁺ B16 WT cells were co-cultured with OT-1 T cells at an E:T ratio of 1:2. RFP counts in each well was quantified by IncuCyte imaging for 60 h. Values are triplicate mean ± SD. (J) Experiment in (I) was performed three times. Bar graphs represent mean RFP counts in the wells at endpoint from three biological replicates. p values from Mann-Whitney tests are shown for data in (C, F, H and J).

Article Snippet: Antagonist antibodies used were from following sources: murine-TNF (clone XT3.11, Bio X cell BE0058) murine IFNγ (clone XMG1.2, Bio X Cell BE0055), human-TNF (Bio X Cell SIM0001) and human IFNγ (Bio X Cell BE0235).

Techniques: Control, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Stable Transfection, Transduction, Cell Culture, Live Cell Imaging, Imaging, MANN-WHITNEY

Journal: bioRxiv

Article Title: T cells use TNF and IFNγ for paracrine killing with target discrimination programmed by pathogen-derived factors

doi: 10.64898/2025.12.23.696308

Figure Lengend Snippet: (A) Prf1-/- CAR-T cells were co-cultured at varying E:T ratios with B16 targets that are WT, TAK1 KO, TBK1/IKKε DKO and HOIP KO, and analyzed in the IncuCyte for 48 h. Cell death data for E:T of 1:16 is shown. (B) The experiment in (A) was independently performed 3 times. The mean confluency at 60 h of 9 wells from the 3 biological replicates is shown in the bar chart. (C) The indicated B16 targets were co-cultured with media or with Prf1-/- CAR-T cells at varying E:T ratios with control IgG or anti-TNF/IFNγ (50 ug/mL). Target killing was analyzed in the IncuCyte for 48 h and data for E:T of 1:16 is shown. (D) RFP-labeled B7H3 KO targets of the indicated genotypes were mixed with 10% unlabeled B7H3⁺ B16 WT cells and co-cultured with Prf1 -/- CAR-T cells at varying E:T ratios. Paracrine killing was quantified by RFP counts of the B7H3-negative targets in the IncuCyte. Data from E:T ratios of 1:16 and 1:256 are shown. (E) 2.5 x 10 5 B7H3 KO or B7H3/TBK1/IKKε TKO B16 cells that were unmixed or mixed with 10% WT B16 cells were implanted into B6 hosts. 5 days later, mice were administered Prf1-/- CAR-T cells against B7H3 via tail vein infusion and tumor growth measured. Spider plots shown were compiled from two independent experiments, each with N=5 per group. Thick red line indicates average tumor volume in each group of mice. Bar chart shows mean tumor volume ± SD for each group at day 23. p values from Mann-Whitney tests are shown for data in (B and E).

Article Snippet: Antagonist antibodies used were from following sources: murine-TNF (clone XT3.11, Bio X cell BE0058) murine IFNγ (clone XMG1.2, Bio X Cell BE0055), human-TNF (Bio X Cell SIM0001) and human IFNγ (Bio X Cell BE0235).

Techniques: Cell Culture, Control, Labeling, MANN-WHITNEY

Journal: bioRxiv

Article Title: T cells use TNF and IFNγ for paracrine killing with target discrimination programmed by pathogen-derived factors

doi: 10.64898/2025.12.23.696308

Figure Lengend Snippet: (A) B16 cells were transiently transfected with expression plasmids encoding FLAG-VP35-T2A-ZSG, FLAG-YOPJ-T2A-ZSG or hCD19-T2A-ZSG as negative control. The next day, cells were stimulated with 10 ng/mL TNF and IFNγ for 24 hours followed by flow cytometry analysis. Dot plots are FSC versus SSC for each sample with an overlay of ZSG fluorescence in green. Representative dot plot from one experiment is shown. Bar chart shows the percentage loss in ZSG signal in cytokine-treated samples relative to media for each transfected DNA from four experiments. Values are mean ± S.D. (B) RFP-labeled WT or TAK1 KO HeLa cells were mixed with 10% unlabeled BCMA-transfected HeLa cells and co-cultured with healthy donor human T cells at varying E:T ratios in the presence of the BiTE teclistamab (2 ug/mL). Paracrine killing was quantified by RFP counts of the BCMA-negative HeLa targets in the IncuCyte. Data from E:T ratio of 1:16 is shown. (C) Data from paracrine killing of TAK1 KO HeLa targets at an E:T ratio of 1:256 is shown. (D) Experiment in (B) was independently performed three times. Bar graphs represent mean RFP counts in the wells at endpoint from three biological replicates. Data from E:T ratios of 1:16 and 1:256 are shown. (E) HeLa cells were transiently transfected with expression plasmids encoding FLAG-YOPJ-T2A-ZSG or hCD19-T2A-ZSG as negative control. The next day, cells were stimulated with 10 ng/mL TNF and IFNγ for 24 hours followed by flow cytometry analysis as described in (A). Dot plot shown is representative of four experiments that were performed. Bar chart shows the percentage loss in ZSG signal in cytokine-treated samples relative to media for each transfected DNA from four experiments. Values are mean ± S.D. p values from Mann-Whitney tests are shown for data in (A, D and E).

Article Snippet: Antagonist antibodies used were from following sources: murine-TNF (clone XT3.11, Bio X cell BE0058) murine IFNγ (clone XMG1.2, Bio X Cell BE0055), human-TNF (Bio X Cell SIM0001) and human IFNγ (Bio X Cell BE0235).

Techniques: Transfection, Expressing, Negative Control, Flow Cytometry, Fluorescence, Labeling, Cell Culture, MANN-WHITNEY