Journal: International Journal of Molecular Sciences

Article Title: Quantifying PD1 Saturation by PDL1 in Tumor Tissue Using a Novel RNA Aptamer-Based Assay

doi: 10.3390/ijms27125469

Figure Lengend Snippet: Correlation between PD1 LIRECAP assay (C:P aptamer ratio) and PD1 signaling in the Jurkat-Raji co-culture system ( A ) Schematic illustration of the PD1 LIRECAP assay. Equimolar concentrations of P and C aptamers were added to biospecimens, bound aptamers extracted and aptamers amplified together using a single set of primers and quantified using unique TaqMan colorimetric probes. The ratio of bound aptamers (C:P ratio) was determined. ( B ) Raji-PDL1 and Raji-Null cells were combined in varying ratios (0–100% PDL1 positivity) and co-cultured with Jurkat-PD1 cells at a 1:1 Raji:Jurkat ratio. Jurkat-PD1 T cell inhibition mediated through the PD1 signaling was quantified as the percentage decrease in luciferase activity relative to the 0% Raji-PDL1 control in co-culture conditioned medium. After supernatant was collected, cell pellets were formalin-fixed and analyzed using the PD1 LIRECAP assay to assess the C:P ratio. ( C ) Raji-PDL1 cells correlate with inhibition of Jurkat-PD1 T cell activation (mean ± SEM from n = 4 independent sets). ( D ) Increasing the percentage of Raji-PDL1 increases the C:P ratio as determined by the PD1 LIRECAP assay (mean ± SEM from n = 4 independent sets). ( E ) C:P ratio as determined by the PD1 LIRECAP assay correlates with PD1 signaling-mediated T cell inhibition in the Raji-Jurkat co-culture system (mean ± SEM from n = 4 independent sets).

Article Snippet: The ratio of bound C18 to P17 (C:P ratio), as determined by the PD1 LIRECAP assay (schema in A), was evaluated using the Jurkat-Lucia TCR-hPD1 cell and Raji cell co-culture system (Invivogen, San Diego, CA, USA), which also enables evaluation of PD1 signaling.

Techniques: Co-culture Assay, Amplification, Cell Culture, Inhibition, Luciferase, Activity Assay, Control, Activation Assay, ATP Bioluminescent Assay, TaqMan Probe Assay

Journal: International Journal of Molecular Sciences

Article Title: Quantifying PD1 Saturation by PDL1 in Tumor Tissue Using a Novel RNA Aptamer-Based Assay

doi: 10.3390/ijms27125469

Figure Lengend Snippet: Correlation between PD1 LIRECAP assay (C:P aptamer ratio) and PD1–PDL1 proximity in FFPE biospecimens from Jurkat-Raji co-culture system ( A ) Schematic illustration of the preparation and analysis of FFPE sections from the Jurkat-Raji co-culture system used to assess the correlation between the PD1 LIRECAP assay and PD1–PDL1 proximity. ( B ) C:P ratio as measured by PD1 LIRECAP assay correlates with PD1 saturation by PDL1 (mean ± SEM from n = 2 independent sets) ( C ) Representative images from PD1–PDL1 PLA of FFPE from cell pellets (Blue—DAPI; Red—PLA positivity) ( n = 2). ( D ) Quantification of PLA positivity per cell, expressed as percent positive cells (mean ± SEM from n = 2 independent sets). ( E ) PD1–PDL1 PLA correlates with C:P aptamer ratio, as measured by PD1 LIRECAP assay (mean ± SEM from n = 2 independent sets).

Article Snippet: The ratio of bound C18 to P17 (C:P ratio), as determined by the PD1 LIRECAP assay (schema in A), was evaluated using the Jurkat-Lucia TCR-hPD1 cell and Raji cell co-culture system (Invivogen, San Diego, CA, USA), which also enables evaluation of PD1 signaling.

Techniques: Co-culture Assay, Analysis, Formalin-fixed Paraffin-Embedded, Proximity Ligation Assay

Journal: Journal of Clinical Medicine

Article Title: A New Histology-Based Prognostic Index for Aggressive T-Cell lymphoma: Preliminary Results of the “TCL Urayasu Classification”

doi: 10.3390/jcm13133870

Figure Lengend Snippet: Summary of the tumor immunohistochemical findings in the patients with TCL ( n = 16).

Article Snippet: The primary antibodies against the major proteins involved in anticancer drug metabolism included (1) GRP94: Proteintech (Rosemont, IL 60018, USA), clone 1H10B7 (this monoclonal antibody was generated against the N-terminal region of full-length HSP90b1); (2) CYP3A4: Sigma-Aldrich (St. Louis, MO 63103, USA), SAB1400064 (this polyclonal antibody was generated against CYP3A4); (3) AKR1C3: Proteintech, 11194-1-AP (this polyclonal antibody was generated against AKRC3); (4) MDR1 (P-glycoprotein): Proteintech, 22336-1-AP (this polyclonal antibody was generated against MDR1); (5) MRP1 (CD9): Proteintech, 60232-1-IG (this monoclonal antibody was generated against the N-terminal region of full-length MRP1); (6) TGF beta1: Proteintech, 21898-1-AP (this polyclonal antibody was generated against TGF-beta); (7) GRP78: Proteintech, 66574-1-IG (this monoclonal antibody was generated against the N-terminal region of full-length GRP78); (8) glutathione S-transferase kappa1 (GST): Proteintech, 14535-1-AP (this polyclonal antibody was generated against GST1); (9) thymidine phosphorylase: Abcam (Cambridge, UK), ab226917 (this polyclonal antibody was generated against thymidine phosphorylase); (10) MRP4 (ABCC4): SANTA CRUZ BIOTECHNOLOGY (Dallas, TX 75220, USA), SC-376262 (this monoclonal antibody was generated against the N-terminal region of full-length MRP4 (amino acid 1-280)); (11) CYP2B6: LifeSpan BioSciences, Inc. (Seattle, WA 98121, USA), LS-C352084 (this polyclonal antibody was generated against CYP2B6); (12) TNF1 alpha: Sigma-Aldrich, SAB4502982 (this polyclonal antibody was generated against TNF1 alpha); (13) PD-1; (14) PD-L1: Proteintech (Rosemont, IL, USA), 66248-1-IG, mouse IgG1 monoclonal antibody, clone 2B11D11; (15) PD-L2: Proteintech (Rosemont, IL, USA), 18251-1-AP 16, rabbit IgG polyclonal antibody; (16) P53: Cell Signaling Technology, Inc. (3 Trask Lane Danvers, MA 01923, USA), DO-7 mouse monoclonal antibody #48818; (17) c-MYC: Abcam (Kendall Sq Cambridge, MA 02139, USA), Y69 clone ab32072; (18) ENT-1 (equilibrative nucleoside transporter 1): Proteintech (Rosemont, IL, USA), 1337-1-AP rabbit IgG polyclonal antibody; (19) AKR1B1: Sigma-Aldrich (3050 Spruce Street Saint Louis, MO 63103, USA), rabbit polyclonal antibody HPA052751; (20) AKR1B10: Sigma-Aldrich (3050 Spruce Street Saint Louis, MO 63103, USA), rabbit monoclonal antibody HPA020280.

Techniques: Immunohistochemical staining, Silver Staining

Journal: Frontiers in Immunology

Article Title: CMTM6-Deficient Monocytes in ANCA-Associated Vasculitis Fail to Present the Immune Checkpoint PD-L1

doi: 10.3389/fimmu.2021.673912

Figure Lengend Snippet: Less PD-L1+ monocytes in patients with AAV. (A) Percentages of PD-L1+ monocytes in HC (n=20) and AAV patients (n=26). (B) Surface expression of PD-L1 (MFI) on classical (CD14+CD16-), intermediate (CD14+CD16+), and non-classical monocytes (CD14dim CD16++) in healthy control donors (n=20) and AAV patients (n=26). (C–E) Percentages of PD-L2+, CD80+, and CD86+ monocytes in HC and AAV patients (n=9-10 each cohort). Mann-Whitney (A, B, D) test or unpaired t-test (C, E) were applied. **P<0.01; ***P<0.001. Bar graph shows mean ± SEM. AAV, ANCA-associated vasculitis; HC, healthy control donors; MFI, mean fluorescence intensity; PD-L1, Programmed death-ligand 1; ns, statistically not significant.

Article Snippet: The following antibodies or stainings were used: CMTM6 (biorbyt), PD-L1, PD-L2, CD80, CD86, CD4, CD25 (all BioLegend), and CFSE (Molecular Probes).

Techniques: Expressing, Control, MANN-WHITNEY, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Identification of genetic modifiers enhancing B7-H3-targeting CAR T cell therapy against glioblastoma through large-scale CRISPRi screening

doi: 10.1186/s13046-024-03027-6

Figure Lengend Snippet: TNFSF15 is an immunostimulatory factor that enhances CAR T cell cytotoxicity. A Cell surface staining of CD69 and CD25 in CAR T cells stimulated by plate-bound recombinant B7-H3-Fc protein with or without recombinant trimeric TNFSF15 protein. ( n = 3 for 0, 100 ng/mL and = 2 for 400 ng/mL). Error bars denote SEM. B Tumor killing of U87 MG cells by B7-H3-targeting CAR T cells or control T cells at an E: T ratio of 1:2 after two-day co-culture with or without exogenous addition of recombinant TNFSF15 protein ( n = 4). Error bars denote SEM. C Gene expression correlation between TNFSF15 and the T cell activation signature ( GZMB 、 GZMK 、 GZMA 、 IFNG 、 TNF 、 IL2 、 IL2R 、 CD69 and CD137 ) in human GBM samples. Data were obtained from TCGA. D Schematic of a recent study that performed single-cell RNA sequencing on mouse skin-draining lymph nodes for probing cellular response to various cytokines, including TNFSF15 (TL1A). E Violin plots showing the expression levels of genes involved in T cell activation and NF-κB pathway in CD8 + T cells following PBS or TNFSF15 treatment in vivo. F-I Gene expression correlation between TNFSF15 and NFKB2 ( F ), NFKB1 ( G ), RELB ( H ) and ICAM1 ( I ) in human GBM samples. Data were obtained from TCGA. J Schematic of our proposed model. Inhibiting ARPC4 or NDUFV1 in GBM cells upregulates TNFSF15. TNFSF15 acts as an immunostimulatory factor that activates the NF-κB pathway in CAR T cells, leading to increased production and release of proinflammatory and cytotoxic factors, thus enhancing the anti-tumor activity of CAR T cells

Article Snippet: Non-tissue culture-treated 24-well plates were coated with 0.5 μg/mL recombinant human B7-H3-Fc proteins with or without recombinant trimeric TL1A/TNFSF15 protein (MCE, Cat. no. HY-P78447) in 100 ng/mL or 400 ng/mL at 4 °C for 24 h. Plates were washed with DPBS and T cell medium, and 5 × 10 5 B7-H3.CAR T cells were added onto the plate for stimulation.

Techniques: Staining, Recombinant, Control, Co-Culture Assay, Gene Expression, Activation Assay, RNA Sequencing, Expressing, In Vivo, Activity Assay

Journal: Genes, chromosomes & cancer

Article Title: Genomic alterations of the JAK2 and PDL loci occur in a broad spectrum of lymphoid malignancies.

doi: 10.1002/gcc.22345

Figure Lengend Snippet: Figure 4. QRT-PCR analysis of JAK2, PDL1 and PDL2 performed in two T-NHL patients with 9p24.1 amplification. (A) Both JAK2 and PDL1 are significantly upregulated in a case 2.6 (PTCL NOS), as compared to the average expression in five non-malignant lymph nodes. (B) Only PDL1 is significantly upregulated in a case 2.9 (ALK1 ALCL), as compared to the average expression in five nonmalignant lymph nodes. JAK2 and PDL2 are significantly downregulated in this patient.

Article Snippet: Immunohistochemistry was performed on paraffin-embedded tissue sections with antibodies against PDL1 and PDL2 (ProSci Incorporated, Poway, CA).

Techniques: Quantitative RT-PCR, Expressing

Journal: Scientific Reports

Article Title: Selective HDAC6 inhibitors improve anti-PD-1 immune checkpoint blockade therapy by decreasing the anti-inflammatory phenotype of macrophages and down-regulation of immunosuppressive proteins in tumor cells

doi: 10.1038/s41598-019-42237-3

Figure Lengend Snippet: The up-regulation of PD-L1 in anti-PD-1 treated mice is mediated by IFNγ. ( A ) C57BL/6 mice were subcutaneously injected with 1 × 10 6 SM1 murine melanoma tumor cells. Mice were treated with 15 mg/kg anti-PD-1 or a vehicle control for 21 days. Tumor nodules were isolated to evaluate the expression of IFNγ by qRT-PCR ( B ), and PD-L1, PD-L2, B7-H3, B7-H4, OX40L, and GAPDH by immunoblot ( C ). SM1 melanoma cells were treated in vitro with NextA or vehicle and then co-cultured with CD3/CD28 activated splenocytes in the presence or absence of IFNγ blocking antibody at 1:1000 and 1:100 dilutions. Then, the expression of PD-L1 was analyzed by qRT-PCR ( D ), and the expression of IFNγ by ELISA ( E ).

Article Snippet: The antibodies used for immunoblotting included: PD-L1 (ProSci, 4059), PD-L2 (ProSci, 4063), CD70 (Abcam, ab175389), B7-H3 (ThermoFisher Scientific, PA551098), B7-H4 (Abbiotec, 250473), Galectin-9 (Abcam, ab9630), ICOS-L (Abcam, ab138354), alpha-Tubulin (Cell Signaling, 3873) and acetyl-alpha Tubulin (Cell Signaling, 3971).

Techniques: Injection, Control, Isolation, Expressing, Quantitative RT-PCR, Western Blot, In Vitro, Cell Culture, Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Selective HDAC6 inhibitors improve anti-PD-1 immune checkpoint blockade therapy by decreasing the anti-inflammatory phenotype of macrophages and down-regulation of immunosuppressive proteins in tumor cells

doi: 10.1038/s41598-019-42237-3

Figure Lengend Snippet: NextA improves the anti-tumor activity of anti-PD-1 immune checkpoint blockade. ( A ) C57BL/6 mice were subcutaneously injected with 1 × 10 6 SM1 murine melanoma tumor cells. Mice were treated with a vehicle control, 3 mg/kg anti-PD-1, 15 mg/kg NextA, or a combination of both agents for 25 days. ( B ) Kaplan-Meier survival plot of the previous study. ( C ) Individual group plots representation for the previous study. ( D – H ) Tumors were collected at the end point from mice treated with a vehicle control, anti-PD-1, NextA, or combination of both agents and the presence of the costimulatory markers PD-L1, PD-L2, OX40L, MHC class I, and MHC class II was evaluated by flow cytometry. ( I ) The expression of PD-L1, PD-L2 and GAPDH was evaluated by immunoblot. ( J ) Tumors were collected at the end point from mice treated with vehicle control, anti-PD-1, NextA, or a combination of both agents and the presence of M1 and M2 surface markers were evaluated by flow cytometry. M1/M2 ratios were calculated from three independent populations from M1 and M2. ( K – O ) Total RNA was isolated from the same aforementioned sample conditions and qRT-PCR was done to evaluate the expression of IFNγ, IL-2, IL-12, IL-10 and TGFβ.

Article Snippet: The antibodies used for immunoblotting included: PD-L1 (ProSci, 4059), PD-L2 (ProSci, 4063), CD70 (Abcam, ab175389), B7-H3 (ThermoFisher Scientific, PA551098), B7-H4 (Abbiotec, 250473), Galectin-9 (Abcam, ab9630), ICOS-L (Abcam, ab138354), alpha-Tubulin (Cell Signaling, 3873) and acetyl-alpha Tubulin (Cell Signaling, 3971).

Techniques: Activity Assay, Injection, Control, Flow Cytometry, Expressing, Western Blot, Isolation, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Selective HDAC6 inhibitors improve anti-PD-1 immune checkpoint blockade therapy by decreasing the anti-inflammatory phenotype of macrophages and down-regulation of immunosuppressive proteins in tumor cells

doi: 10.1038/s41598-019-42237-3

Figure Lengend Snippet: Schematic representation of the role of HDAC6 in the TME. Combination treatment of anti-PD-1 blocking antibody and selective HDAC6i. ( A ) Treatment with anti-PD-1 antibody enhances T cell function and the release of pro-inflammatory cytokines. These mediators induce the production of several immunosuppressive proteins in tumor cells, including PD-L1 and PD-L2. Additionally, anti-PD-1 treatment reduces the pro-inflammatory phenotype of macrophages. ( B ) The addition of selective HDAC6i neutralizes the negative feedback induced by IFNγ and de-activates immune inhibitory check-point signals.

Article Snippet: The antibodies used for immunoblotting included: PD-L1 (ProSci, 4059), PD-L2 (ProSci, 4063), CD70 (Abcam, ab175389), B7-H3 (ThermoFisher Scientific, PA551098), B7-H4 (Abbiotec, 250473), Galectin-9 (Abcam, ab9630), ICOS-L (Abcam, ab138354), alpha-Tubulin (Cell Signaling, 3873) and acetyl-alpha Tubulin (Cell Signaling, 3971).

Techniques: Blocking Assay, Cell Function Assay

Journal: Scientific Reports

Article Title: Selective HDAC6 inhibitors improve anti-PD-1 immune checkpoint blockade therapy by decreasing the anti-inflammatory phenotype of macrophages and down-regulation of immunosuppressive proteins in tumor cells

doi: 10.1038/s41598-019-42237-3

Figure Lengend Snippet: Primers used for qRT-PCR.

Article Snippet: The antibodies used for immunoblotting included: PD-L1 (ProSci, 4059), PD-L2 (ProSci, 4063), CD70 (Abcam, ab175389), B7-H3 (ThermoFisher Scientific, PA551098), B7-H4 (Abbiotec, 250473), Galectin-9 (Abcam, ab9630), ICOS-L (Abcam, ab138354), alpha-Tubulin (Cell Signaling, 3873) and acetyl-alpha Tubulin (Cell Signaling, 3971).

Techniques: Sequencing