Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: The effect of FGL2 expression on survival and the correlation between FGL2 and immune checkpoints. ( A ) The expression of FGL2 in a human liver tissue microarray was detected by mIHC. Representative images were shown. Scale bar, 20 μm. Kaplan-Meier analysis of overall survival ( B ) and progression-free survival ( C ) of HCC patients based on FGL2 expression ( n = 85). The Gene Expression Profiling Interactive Analysis server was used to analyze the correlation between Fgl2 and Pdcd1 (encoding PD1, ( D ), Cd274 (encoding PD-L1, ( E ), Pdcd1lg2 (encoding PD-L2, ( F ), Ctla4 ( G ), Tigit ( H ), Entpd1 (encoding CD39, ( I ), Havcr2 (encoding TIM3, ( J ), Btla ( K ) and Lag3 ( L ) at the mRNA expression level, respectively. R=Pearson’s correlation coefficient

Article Snippet: After washing and blocking, the cells were incubated with the following primary antibodies: FGL2 (1:200) (H00010875-M01, Abnova), PD-L1 (1:200) (17952-1-AP, Proteintech), LAMP1 (1:200) (ab25630, Abcam), TFEB (1:200) (13372-1-AP, Proteintech) and mTOR (1:100) (66888-1-Ig, Proteintech).

Techniques: Expressing, Microarray, Gene Expression

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 knockout inhibited tumor growth and downregulated the expression of PD-L1. ( A ) Hepa1-6 cells were inoculated orthotopically into the left liver lobe of WT and Fgl2 -/- mice. The concentration of FGL2 in tumor tissue homogenate was detected by ELISA ( n = 5). ( B ) The livers from C57BL/6 mice bearing orthotopic tumors were shown ( n = 8). ( C ) Representative immunohistochemical images for PD-L1 staining in orthotopic tumors ( n = 4). ( D ) The expression level of PD-L1 on CD45- tumor cells was measured by flow cytometry ( n = 4). Data were represented as median fluorescence intensity (MFI). ( E ) Hepa1-6 cells were inoculated subcutaneously (s.c.) into the right inguinal fold region of WT and Fgl2 -/-mice. The concentration of FGL2 in tumor tissue homogenate was shown ( n = 5). ( F ) Gross images of s.c. tumors of WT or Fgl2 -/- mice ( n = 8). Tumor volume ( G ) and tumor weight ( H ) of the indicated group of C57BL/6 mice ( n = 8). ( I ) The expression level of PD-L1 on tumor cells in the s.c. transplanted hepatomas was detected by flow cytometry ( n = 5). ( J ) WT and Fgl2 -/- mice bearing s.c. tumors were treated with anti-CD3 neutralizing antibodies (200 µg/mice) or IgG1 isotype control (200 µg/mice). Gross images of s.c. tumors in mice treated as indicated ( n = 6). Tumor volume ( K ) and tumor weight ( L ) of the indicated group ( n = 6). Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After washing and blocking, the cells were incubated with the following primary antibodies: FGL2 (1:200) (H00010875-M01, Abnova), PD-L1 (1:200) (17952-1-AP, Proteintech), LAMP1 (1:200) (ab25630, Abcam), TFEB (1:200) (13372-1-AP, Proteintech) and mTOR (1:100) (66888-1-Ig, Proteintech).

Techniques: Knock-Out, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Flow Cytometry, Fluorescence, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 upregulated PD-L1 expression in HCC cells and attenuated the cytotoxicity of T cells. Protein levels of FGL2 (FLAG) and PD-L1 were analyzed by Western blot (WB) in Huh7 ( A ) and MHCC97H cells ( B ) stably expressing FGL2-FLAG. Lentivirus expressing shRNA against FGL2 (sh-FGL2) was used for FGL2 knockdown, and nontargeting shRNA(sh-control) was added as control. FGL2 and PD-L1 expression in MHCC97H cells were detected by WB ( C ), immunofluorescence (IF) ( D ) and flow cytometry (FCM) ( E ). Scale bar, 25 μm. FGL2 knockdown or nontargeting shRNA-transfected MHCC97H cells were treated with IFN-γ (50 ng/mL) for 24 h. The expression of PD-L1 was detected by WB ( F ) and FCM ( G ). ( H ) T-cell cytotoxicity assay against MHCC97H cells. Apoptotic MHCC97H cells were detected by FCM. Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001

Article Snippet: After washing and blocking, the cells were incubated with the following primary antibodies: FGL2 (1:200) (H00010875-M01, Abnova), PD-L1 (1:200) (17952-1-AP, Proteintech), LAMP1 (1:200) (ab25630, Abcam), TFEB (1:200) (13372-1-AP, Proteintech) and mTOR (1:100) (66888-1-Ig, Proteintech).

Techniques: Expressing, Western Blot, Stable Transfection, shRNA, Knockdown, Control, Immunofluorescence, Flow Cytometry, Transfection, Cytotoxicity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 disruption induced lysosome-dependent degradation of PD-L1. HCC cells with FGL2 overexpression ( A ) or knockdown ( B ) were treated with CHX (25 mmol/L) for the indicated time points and then subjected to WB for quantitation of PD-L1 level. The expression of PD-L1 in MHCC97H cells after treatment with proteasome inhibitor (MG132) ( C ) or lysosomal inhibitors, NH4Cl ( D ) and chloroquine ( E ) was detected by WB. ( F ) Images and quantification of LysoTracker red staining in MHCC97H cells were displayed. Scale bar, 100 μm. ( G ) The protein levels of LAMP1 and CTSD were determined by WB. ( H ) The colocalization between PD-L1 and LAMP1 in MHCC97H cells was detected by immunofluorescence assay. The intensity profiles of PD-L1 and LAMP1 along the white line were plotted on the middle panel. The statistical result of the colocalization factor (Pearson’s R value) was shown on the right panel. Scale bar, 25 μm. Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After washing and blocking, the cells were incubated with the following primary antibodies: FGL2 (1:200) (H00010875-M01, Abnova), PD-L1 (1:200) (17952-1-AP, Proteintech), LAMP1 (1:200) (ab25630, Abcam), TFEB (1:200) (13372-1-AP, Proteintech) and mTOR (1:100) (66888-1-Ig, Proteintech).

Techniques: Disruption, Over Expression, Knockdown, Quantitation Assay, Expressing, Staining, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 disruption promoted the expression of nuclear TFEB. The cytosolic and nuclear distribution of TFEB ( A ) and MITF ( B ) in MHCC97H cells was detected by WB following FGL2 knockdown. Quantification of nuclear TFEB or MITF to Histone-H3 was shown. ( C ) Images and quantification of nuclear TFEB in MHCC97H cells were displayed. Scale bar, 50 μm. The expression levels of TFEB ( D ) and MITF ( E ) were determined by WB in mouse liver cancer tissues ( n = 6). The transcriptional levels of Lamp1, Atp6v0d2 , Ctns and Ctsb were detected by qPCR in mouse liver cancer tissues ( F ) and MHCC97H cells ( G ). TFEB siRNA or control siRNA was transfected to MHCC97H cells pretreated with FGL2 shRNA or control shRNA. The protein levels of LAMP1, CTSD ( H ), and PD-L1 ( I ) were determined by WB. The relative quantification of LAMP1, CTSD or PD-L1 to GAPDH was shown. ( J ) The expression of TFEB in an HCC tissue microarray was detected by mIHC. Representative images were shown. Scale bar, 20 μm. Kaplan-Meier analysis of the progression-free survival ( K ) and overall survival ( L ) of HCC patients based on nuclear TFEB expression ( n = 85). Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: After washing and blocking, the cells were incubated with the following primary antibodies: FGL2 (1:200) (H00010875-M01, Abnova), PD-L1 (1:200) (17952-1-AP, Proteintech), LAMP1 (1:200) (ab25630, Abcam), TFEB (1:200) (13372-1-AP, Proteintech) and mTOR (1:100) (66888-1-Ig, Proteintech).

Techniques: Disruption, Expressing, Knockdown, Control, Transfection, shRNA, Quantitative Proteomics, Microarray

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 interacted with mTOR kinase and affected the phosphorylation of mTORC1 pathway. ( A ) Immunoblot analysis of (p-)mTOR, (p-)P70S6K, (p-)4EBP1 and (p-)AKT in MHCC97H cells following FGL2 knockdown. ( B ) Total P70S6K, 4EBP1 and AKT proteins as well as their phosphorylated forms were detected in liver cancer tissues from WT and Fgl2 -/- mouse(n = 6). ( C ) The phosphorylation level of TFEB Ser211 was detected by WB. The expression levels of (p-)P70S6K, (p-)4EBP1 ( D ) and PD-L1 ( E ) were detected in MHCC97H cells treated with FGL2 shRNA or control shRNA in the absence or presence of mTOR signaling activator (MHY1485). ( F ) Co-immunoprecipitation analysis of the interaction between FGL2 and mTOR in MHCC97H cells stably expressing FGL2-FLAG. ( G ) The colocalization of FGL2 and mTOR in MHCC97H cells was detected by immunofluorescence. Scale bar, 25 μm.( H ) 293T cells were co-transfected with HA-mTOR, FLAG-FGL2 or vector plasmids. Cell lysates were subjected to co-immunoprecipitation using anti-HA or anti-FLAG antibodies. ( I ) 293T cells were co-transfected with HA-mTOR truncations and FLAG-FGL2 plasmids. Cell lysates were subjected to co-immunoprecipitation using anti-FLAG antibodies. ( J ) MHCC97H cell lysates were subjected to immunoprecipitation​ with anti-mTOR antibodies, followed by immunoblot analysis. Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: After washing and blocking, the cells were incubated with the following primary antibodies: FGL2 (1:200) (H00010875-M01, Abnova), PD-L1 (1:200) (17952-1-AP, Proteintech), LAMP1 (1:200) (ab25630, Abcam), TFEB (1:200) (13372-1-AP, Proteintech) and mTOR (1:100) (66888-1-Ig, Proteintech).

Techniques: Phospho-proteomics, Western Blot, Knockdown, Expressing, shRNA, Control, Immunoprecipitation, Stable Transfection, Immunofluorescence, Transfection, Plasmid Preparation

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 inhibition enhanced antitumor immune responses of anti-PD1 therapy in HCC. ( A ) WT and Fgl2 -/- mice bearing s.c. tumors were treated with anti-PD1 monoclonal antibodies (αPD1, 100 µg/mice) or IgG isotype control (100 µg/mice). Gross images of tumors in mice treated as indicated ( n = 6). Tumor volume ( B ) and tumor weight ( C ) of the indicated group. ( D ) Representative immunohistochemical images of subcutaneous tumors for PD-L1 staining ( n = 3). Representative flow cytometric plots and quantification of the proportion of IFN-γ+ CD8+ cells ( E ), granzyme B+ CD8+ cells ( F ) and CD107a+ CD8+ cells ( G ) in tumor tissues in response to different treatments. ( H ) Representative flow cytometric plots and quantification of the proportion of Tregs (CD4+ CD25+ Foxp3+) in tumor tissues from the indicated groups. The proportions of Th1 (CD4+ IFN-γ+ T cells) ( I ) and Th2 (CD4+ IL-4+ T cells) cells ( J ) in tumor tissues were shown. Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After washing and blocking, the cells were incubated with the following primary antibodies: FGL2 (1:200) (H00010875-M01, Abnova), PD-L1 (1:200) (17952-1-AP, Proteintech), LAMP1 (1:200) (ab25630, Abcam), TFEB (1:200) (13372-1-AP, Proteintech) and mTOR (1:100) (66888-1-Ig, Proteintech).

Techniques: Inhibition, Bioprocessing, Control, Immunohistochemical staining, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: Schematic of FGL2-mediated PD-L1 regulation and therapy potential of combination interference of FGL2 and PD1. In HCC cells, FGL2 stabilizes the mTOR-Raptor interaction and activates mTORC1 signaling. Activated mTORC1 phosphorylates TFEB, thereby suppressing its nuclear translocation and subsequent lysosomal biogenesis. This lysosomal dysfunction​ attenuates PD-L1 degradation, leading to elevated PD-L1 abundance and the inhibition of T cell–mediated cytotoxicity. In HCC mouse models, FGL2 inhibition exerts synergistic antitumor effects with anti-PD1 therapy by downregulating tumorous expression of PD-L1, promoting the activity of tumor-infiltrating lymphocytes, and suppressing the accumulation of Tregs

Article Snippet: After washing and blocking, the cells were incubated with the following primary antibodies: FGL2 (1:200) (H00010875-M01, Abnova), PD-L1 (1:200) (17952-1-AP, Proteintech), LAMP1 (1:200) (ab25630, Abcam), TFEB (1:200) (13372-1-AP, Proteintech) and mTOR (1:100) (66888-1-Ig, Proteintech).

Techniques: Translocation Assay, Inhibition, Expressing, Activity Assay

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: PD-L1 expression in GBM and PD-1 recruitment to the CARIS with GBM. A, Constitutive (UPN01) and inducible (UPN06) surface expression of PD-L1 in primary GBM cells after 24–48 hours of IFN-γ exposure. Representative results from two samples shown. UPN, unique patient number. B, PD-L1 median fluorescent intensity (MFI) on primary GBM ( n = 14) before and at 48 hours of IFN-γ (10 ng/mL) exposure; ***, P < 0.001, Wilcoxon signed-rank test. C, Fold-change in PD-L1 MFI in GBM cells from baseline, at 24, 48, and 72 hours. Each color represents a single patient donor, with some measured at multiple time points. Data are shown as individual values with the mean ± SD. D, PD-L1 expression in WT LN229-GBM cells and LN229 with PD-L1 deletion (LN229-PD-L1 KO) at baseline and at 24 hours of exposure to IFN-γ (10 ng/mL) or CAR28ζ T cells. E, MFI of PD-1 in the immune synapse between CARζ cells and WT LN229-GBM cells at 15, 30, and 60 minutes (****, P < 0.0001; ns, P > 0.5, Kruskal–Wallis and Wilcox pairwise); ≥20,000 events were captured, and 300–1,000 CAR + conjugates were examined for PD-1 recruitment to CARIS. The white box represents the IQR with horizontal lines at 25%, 50%, and 75%. F, Representative image capture showing the CARIS with tumor cell (WT LN229-GBM) and other parameters evaluated. Gating strategy is shown in the Supplementary material. G, Spearman correlation between PD-1 intensity and CAR intensity in the immune synapse at 15, 30, and 60 minutes. H, Spearman correlation between PD-1 intensity and actin intensity in the CARIS at 15, 30, and 60 minutes. ns, not significant.

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Design and functional screening of PD-1 TR and CPR28 molecules. A, Schematic representation of the bicistronic vectors encoding for the HER2-CAR with truncated PD-1 (PD-1 TR ) or CPR28. The CPR28 dimer included a membrane proximal extracellular cysteine residue (C141) required for CD28 homodimerization, as indicated. B, Flow cytometry analysis showing coexpression of HER2-CAR and PD-1 CPR on T cells. CAR28ζ and NT T cells from the same donor were used as controls for assessment of CPR expression determined by surface PD-1 detection. C, Long-term cytolytic function of CAR28ζ/PD-1 TR , compared with CAR28ζ cells, against U373-GBM cells at effector to target ratios of 1:5 and 1:10 assessed using a cell-impedance based assay (xCELLigence). D, Comparison of the cytolytic ability of patient-derived CAR28ζ cells ( n = 3) coexpressing CPR28 dimer or CPR28 monomer against autologous HER2 + GBM cells at an effector to target ratio of 1:10 by assessment of tumor cell viability in an xCELLigence assay. In C and D , error bars represent the mean ± SD at each time point. ****, P < 0.0001, two-way ANOVA with the Tukey multiple comparisons test. E, IL-2 and ( F ) IFN-γ release by CAR28ζ, CAR28ζ/PD-1 TR , and CAR28ζ/CPR28 cells (100,000 T cells/well) upon stimulation with Fc-conjugated HER2 (0–2 μg/mL) and PD-L1 (0–5 μg/mL) proteins. Median values from a representative donor shown. G, CAR28ζ/CPR28 cell–induced lysis of Raji cells modified to express HER2 or HER2 and PD-L1 across different T-cell to tumor cell ratios but not the WT Raji cells (HER2 − /PD-L1 − ) or those expressing PD-L1 alone in a 51 Cr-release assay. T cells with CPR28 alone had no cytotoxic effect against HER2 + or PD-L1 + Raji cells, like NT T cells from the same donor. A, Created in BioRender. Navai, S. (2024) BioRender.com/l86h941 .

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Functional Assay, Membrane, Residue, Flow Cytometry, Expressing, Comparison, Derivative Assay, Lysis, Modification, Release Assay

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Effect of decoupling signal 2 from the CAR on T-cell function. A, Illustration depicting the bipartite T-cell activation through signal 1 delivery from CAR engaging the HER2 antigen and signal 2 from binding of CPR with PD-L1 (or PD-L2). B, Percent increase in IFN-γ production by CAR28ζ/CPR28 and CARζ/CPR28 compared with CAR28ζ cells (50,000 T cells/well) at 24 hours of coculture with autologous GBM cells ( n = 5 patients). Effector (100,000 T cells) to target ratio of 1:1. **, P < 0.01; ****, P < 0.0001, one-way ANOVA with the Tukey multiple comparisons test. C, Cytolytic function of CAR28ζ/CPR28 and CARζ/CPR28 cells assessed using 4-hour 51 Cr-release assay at baseline and at 7 days of persistent T-cell stimulation through the CAR. NT T cells had poor viability after prolonged stimulation without added homeostatic cytokines and were not evaluable. ****, P < 0.0001, two-way ANOVA with the Tukey multiple comparisons test. D, Percent of total T cells expressing HER2-CAR on cell surface, and percent of CAR + T cells detected with PD-1 (surrogate marker for CPR), respectively, amongst different CPR/CART groups ( n = 4 patients) using flow cytometry. E, Median fluorescent intensity (MFI) of HER2-CAR detected in transduced T cells shown in D . In D and E , only statistically significant differences are shown, *, P < 0.05; **, P < 0.01; ****, P < 0.0001, one-way ANOVA with the Tukey multiple comparisons test. F, Multiplex analysis for proinflammatory cytokines (IL-2, MIP-1α, TNF-α, and GM-CSF) in autologous T-cell and GBM coculture ( n = 4 patients) supernatants at 24 hours. UPN, unique patient number. *, P < 0.05; **, P < 0.01; ****, P < 0.0001, two-way ANOVA with the Tukey multiple comparisons test. G, Assessment of long-term cytolytic function of CPR/CART against autologous GBM cells ( n = 3 patients) using cell impedance–based xCELLigence assay. Statistical differences (denoted by the color key) shown are in comparison to control treatment CAR28ζ cells overtime. ****, P < 0.0001, two-way ANOVA with the Tukey multiple comparisons test. In G, black arrow indicates addition of T cells. A, Created in BioRender. Navai, S. (2019) BioRender.com/p40z816 .

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Cell Function Assay, Activation Assay, Binding Assay, Release Assay, Cell Stimulation, Expressing, Marker, Flow Cytometry, Multiplex Assay, Comparison, Control

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Phenotypic and functional profile of CART receiving bipartite activation signals through CPR41BB costimulation. A, In vivo functional screening of CARζ and CAR28ζ cells coexpressing CPR28 or CPR41BB against orthotopic xenografts of HER2 + PD-L1 + U373-GBM in SCID mice ( n = 5 per group). B, Fold change in tumor burden after treatment relative to the tumor volume before intratumoral injection of T cells (day 0), quantified by serial BLI. *, P < 0.05; **, P < 0.01, two-way ANOVA with the Tukey multiple comparisons test. Data are shown as the mean ± SD. C, CD8 + : CD4 + ratio in CAR-expressing T cells among CPR/CART from patients with GBM ( n = 4) as assessed by flow cytometry. Box plots show minimum to maximum with individual values. ns, P > 0.5, one-way ANOVA with the Tukey multiple comparisons test. D, Pie graph demonstrating immunophenotype distribution of CARζ/CPR41BB cells ( n = 4 patients) at baseline. Percentages shown represent the mean value. Immunophenotype is defined as follows: naïve, CCR7 + /CD45RA + ; central memory, CCR7 + /CD45RA − ; effector memory, CCR7 − /CD45RA − ; and terminal effector, CCR7 − /CD45RA + . E, The percentage CAR + CD8 + central memory (CCR7 + /CD45RA − ) T cells did not significantly differ amongst different CPR/CART groups ( n = 4 patients). The mean ± SEM is shown. ns, P > 0.5, one-way ANOVA with the Dunnett multiple comparisons test. F, Pie graph demonstrating immunophenotype distribution of CARζ/CPR41BB cells ( n = 4 patients) after 7 days of continued stimulation by repeat cocultures with autologous GBM cells. Percentages shown represent the mean value. On day 7 of repeated stimulation, CARζ/CPR41BB cells ( n = 4 patients) demonstrated ( G ) a significantly higher proportion of CAR + /CD8 + cells with the central memory phenotype compared with other CPR/CART groups and CAR28ζ, and ( H ) a significantly lower proportion of CAR + /CD8 + cells with the effector memory phenotype compared with CAR28ζ/CPR28 cells. The mean ± SEM is shown. *, P < 0.05; **, P < 0.01, one-way ANOVA with the Dunnett multiple comparisons test. I, Log-change in the median fluorescent intensity (MFI) of T-cell surface PD-1, LAG3, and TIM3 at 7 days of repeat coculture with autologous GBM cells ( n = 4 patients) from baseline. Box plots show minimum to maximum with individual values. *, P < 0.05; **, P < 0.01, two-way ANOVA with the Tukey multiple comparisons test. ns, not significant.

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Functional Assay, Activation Assay, In Vivo, Injection, Expressing, Flow Cytometry

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Dynamics of CARζ/CPR41BB T-cell activation and CARIS formation in comparison with CAR41BBζ cells. A, CARζ/CPR41BB cells ( n = 3 donors) demonstrated significantly lower IL-2 and IFN-γ release compared with CAR41BBζ cells at 24 hours of coculture with LN229-GBM cells, with CAR28ζ cells consistently showing the higher Th1 cytokine production. Data are shown as the mean ± SD. **, P < 0.000; ****, P < 0.0001; two-way ANOVA with the Tukey multiple comparisons test. B, Western blot analysis for CAR-phosphoCD3ζ (pCD3) in T cells in a resting state (maintained in culture with IL-7/IL-15). The pCD3 to CD3 ratio is normalized to CARζ in each donor. *, P < 0.05; one-way ANOVA with the Tukey multiple comparisons test. C, Representative image capture showing the CARIS with tumor cell (LN229-GBM) and different CARζ/CPR41BB immune synapse parameters evaluated. Gating strategy is shown in the Supplementary Material. Spearman correlation ( D ) between the intensity of CAR and CPR at the CARIS and ( E ) between the intensity of actin and CPR at the CARIS, both assessed by imaging flow cytometry at 15, 30, and 60 minutes. F, CARζ/CPR41BB and CAR41BBζ cells show significantly higher percent (%) of F-actin at the CARIS compared with CARζ cells. ns, P > 0.5; **, P < 0.01; ***, P < 0.0001, two-way ANOVA with the Tukey post hoc test. Data are shown as the mean with 95% confidence interval (CI). G, CARζ/CPR41BB cells show significantly higher CPR intensity in the CARIS with WT LN229 GBM cells at 15, 30, and 60 minutes compared with conjugates with LN229-PD-L1 KO (Kruskal–Wallis and Wilcox pairwise). CPR intensity increased over time in both conditions. ns, not significant.

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Activation Assay, Comparison, Western Blot, Imaging, Flow Cytometry

Journal: Cancer Research Communications

Article Title: A Checkpoint Reversal Receptor Mediates Bipartite Activation and Enhances CAR T-cell Function

doi: 10.1158/2767-9764.CRC-24-0125

Figure Lengend Snippet: Metabolomic parameters of CARζ/CPR41BB cells in comparison with CAR41BBζ cells. A, OCR measurements of resting (cultured in media containing in IL-7 and IL-15) CARζ/CPR41BB and CAR41BBζ cells ( n = 3 donors; 200,000 T cells per well) under basal metabolic conditions and after the addition of mitochondrial inhibitors. B, Comparison of maximal respiration between CARζ/CPR41BB cells and CAR41BBζ cells at baseline (day 0). P = 0.059, Student two-tailed t test. C, Basal OCR, maximal respiration, and SRC between CAR41BBζ and CARζ/CPR41BB cells after 7 days of stimulation with Fc-conjugated HER2 and PD-L1 proteins. ns, P > 0.05, Student two-tailed t test. D, The ECAR in resting CARζ/CPR41BB and CAR41BBζ cells. E, The ECAR of CARζ/CPR41BB and CAR41BBζ at baseline and at 7 days of continued stimulation with plate-bound HER2 and PD-L1 proteins. ns, P > 0.05; **, P < 0.01, Student two-tailed t test. F, Basal OCR to ECAR ratio in CARζ/CPR41BB compared with CAR41BBζ cells at rest and at 7 days of continued stimulation with HER2 and PD-L1 proteins. ns, P > 0.05; *, P < 0.05, Student two-tailed t test. G, The OCR in CD8 + CARζ/CPR41BB and CAR41BBζ cells ( n = 3 donors; 150,000 T cells per well) following 48-hour coculture with LN229-GBM cells (effector to tumor ratio 1:2). H, The SRC of CD8 + CARζ/CPR41BB and CAR41BBζ cells after 48 hours of coculture with LN229 GBM cells. ns, P > 0.05, Student two-tailed t test. The OCR and ECAR measurements are shown as the mean ± SEM. Data shown denote the IQR in the violin plot and the mean ± SEM in the bar graphs. ns, not significant.

Article Snippet: PD-L1 knockout (KO) LN229 cells were generated using the CRISPR-mediated PD-L1 (CD274) Human Gene Knockout Kit (KN213071, OriGene) according to the manufacturer’s instructions.

Techniques: Comparison, Cell Culture, Two Tailed Test