Journal: Molecular Therapy Oncology

Article Title: Engineering Proteus mirabilis improves antitumor efficacy via enhancing cytotoxic T cell responses

doi: 10.1016/j.omton.2024.200770

Figure Lengend Snippet: P. mirabilis FlaB increases expression of PD-L1 on tumor-infiltrating immune cells in the murine tumor model (A) Mouse tumors were stained with the PD-L1 antibody and analyzed by flow cytometry 24 h after intraperitoneal injection of with l -arabinose (n = 3). (B) Assessment of expression of PD-L1 in immune cells (ICs) and tumor cells (TCs) from five high-power fields (n = 3). ∗p < 0.05, ∗∗p < 0.01. (C) Representative IHC results of samples stained with PD-L1; scale bar, 50 μm.

Article Snippet: The following antibodies were used: CD3- fluorescein isothiocyanate (FITC; F2100301, MultiSciences, Hangzhou, China), CD8α-PE (F2100802, MultiSciences), CD4-APC 1/20 (F2100403, MultiSciences), and PD-L1-APC (F2127403, MultiSciences).

Techniques: Expressing, Staining, Flow Cytometry, Injection

Journal: Molecular Therapy Oncology

Article Title: Engineering Proteus mirabilis improves antitumor efficacy via enhancing cytotoxic T cell responses

doi: 10.1016/j.omton.2024.200770

Figure Lengend Snippet: Co-treatment with P. mirabilis FlaB and PD-L1 blockade confers synergistic antitumor activity by increasing levels of tumor-infiltrating CD8 + T cells (A) Study protocol. Mice were intravenously injected with P. mirabilis FlaB or PBS when the tumor had grown to ∼150 mm 3 in the murine CT26 tumor model. The PD-L1 antibody (aPD-L1) or IgG isotype control antibody was intraperitoneally administered on days 0, 4, 8, and 12 after bacterial injection. Three days after bacterial injection, the mice in the P. mirabilis FlaB group received daily intraperitoneal injection of l -arabinose for 8 consecutive days. (B) Images of tumors from representative mice from each group (n = 6). (C) Tumor volumes in each group were monitored every other day. (D) Representative plot of IHC staining of CD8 and PD-L1 in tumor tissue 24 h after PD-L1 blockade treatment. Numbers of CD8 + T cells and PD-L1 + ICs were statistically assessed from five high-power fields. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: The following antibodies were used: CD3- fluorescein isothiocyanate (FITC; F2100301, MultiSciences, Hangzhou, China), CD8α-PE (F2100802, MultiSciences), CD4-APC 1/20 (F2100403, MultiSciences), and PD-L1-APC (F2127403, MultiSciences).

Techniques: Activity Assay, Injection, Control, Immunohistochemistry

Journal: Journal of Clinical Investigation

Article Title: CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1–positive regulator for tumor immune evasion

doi: 10.1172/jci167728

Figure Lengend Snippet: Figure 3. ATXN3 selectively functions as a HIF-2α deubiquitinase to promote tumoral PD-L1 transcription. (A and B) A549 cells were cultured under normoxia and hypoxia (hyp) (1% pO2) for 48 hours, and surface PD-L1 levels were analyzed by flow cytometry and quantification. (C) ATXN3 specif- ically interacts with HIF-2α. HA–HIF-2α expression plasmid was cotransfected with or without FLAG-ATXN3 into HEK293T cells. Their interactions were examined by co-IP with anti-FLAG antibodies and by Western blotting with anti-HA antibodies. (D) The interaction between ATXN3 and HIF-1α was tested in transfected HEK293T cells. (E) Endogenous interaction between ATXN3 and HIF-2α in A549 cells. (F) HA-ubiquitin, FLAG–HIF-2α, and Myc-ATXN3 plasmids were cotransfected into HEK293T cells. HIF-2α ubiquitination was determined by immunoprecipitation of HIF-2α with anti-FLAG antibodies and immunoblotting with anti-HA antibody. (G and H) HIF-2α was cotransfected with or without ATXN3 plasmids into HEK293T cells. The transfected cells were treated with CHX for different times. The protein levels of HIF-2α (top panel) and ATXN3 (middle panel) were analyzed by West- ern blotting. β-Actin was used as a loading control (bottom panel). (I and J) Immunoblot analysis of HIF-2α protein stability in WT and ATXN3-KO A549 cells. (K) ATXN3 enhances hypoxia-induced PD-L1 expression through protecting HIF-2α from ubiquitination-induced protein degradation. B: Ordinary 1-way ANOVA; H and J: 2-tailed unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Primary antibodies used for immunohistochemistry were antibodies against PD-L1 (1:200; Cusabio, catalog CSBMA878942A1m), ATXN3 (1:500; Proteintech, catalog 13505-1-AP), IRF1 (1:100; Cell Signaling Technology, catalog 847S), and HIF-2α (1:100; Thermo Fisher Scientific, catalog PA1-16510).

Techniques: Cell Culture, Flow Cytometry, Expressing, Plasmid Preparation, Co-Immunoprecipitation Assay, Western Blot, Transfection, Ubiquitin Proteomics, Immunoprecipitation, Control

Journal: Oncology Letters

Article Title: Clinicopathological analysis of epithelioid inflammatory myofibroblastic sarcoma

doi: 10.3892/ol.2018.8530

Figure Lengend Snippet: Primary antibodies used for immunohistochemistry.

Article Snippet: Sections were observed under a light microscope with the magnifications of 40, 100, 200 and ×400. table ft1 table-wrap mode="anchored" t5 Table I. caption a7 Target Supplier Catalog number Dilution Staining ALK Origene Technologies, Inc., Beijing, China TA801287 Ready to use + PD-L1 Origene Technologies, Inc. TA507087 Ready to use + PD-1 Origene Technologies, Inc. TA314461 Ready to use Lymphocyte + Ki-67 Origene Technologies, Inc. UM870033 1:100 Index 40% P53 Gene Tech Co., Ltd., Shanghai, China GM700101 Ready to use + DES Origene Technologies, Inc. TA502328 1:60 Focally + CK Origene Technologies, Inc. ZM-0069 1:80 Focal + EMA Gene Tech Co., Ltd. {"type":"entrez-nucleotide","attrs":{"text":"GM061329","term_id":"240150502","term_text":"GM061329"}} GM061329 1:200 − CAM5.2 Origene Technologies, Inc. ZM-0316 Ready to use Focal + Myoglobin Origene Technologies, Inc. ZA-0192 Ready to use − CALDES Gene Tech Co., Ltd. {"type":"entrez-nucleotide","attrs":{"text":"GM355701","term_id":"221808897","term_text":"GM355701"}} GM355701 Ready to use − Vimentin Gene Tech Co., Ltd. {"type":"entrez-nucleotide","attrs":{"text":"GM072529","term_id":"221566844","term_text":"GM072529"}} GM072529 1:120 + CD30 Gene Tech Co., Ltd. {"type":"entrez-nucleotide","attrs":{"text":"GM075129","term_id":"222040140","term_text":"GM075129"}} GM075129 1:35 − CD3 Origene Technologies, Inc. ZM-0417 1:120 Lymphocyte + CD20 Gene Tech Co., Ltd. {"type":"entrez-nucleotide","attrs":{"text":"GM075529","term_id":"221304020","term_text":"GM075529"}} GM075529 1:200 Little lymphocyte + CD4 Origene Technologies, Inc. ZM-0418 Ready to use Lymphocyte + CD8 Gene Tech Co., Ltd. {"type":"entrez-nucleotide","attrs":{"text":"GT211202","term_id":"261529303","term_text":"GT211202"}} GT211202 Ready to use Lymphocyte + MC Gene Tech Co., Ltd. ZM-0386 Ready to use − Calretinin Origene Technologies, Inc. TA353630 1:60 Focal + WT-1 Gene Tech Co., Ltd. {"type":"entrez-nucleotide","attrs":{"text":"GM356102","term_id":"221291992","term_text":"GM356102"}} GM356102 Ready to use + D2-40 Gene Tech Co., Ltd. {"type":"entrez-nucleotide","attrs":{"text":"GM361929","term_id":"221292291","term_text":"GM361929"}} GM361929 1:60 − HMB45 Origene Technologies, Inc. ZM-0187 Ready to use − CD117 Origene Technologies, Inc. ZA-0523 Ready to use − DOG1 Origene Technologies, Inc. ZM-0371 Ready to use − SMA Origene Technologies, Inc. ZM-0003 Ready to use − Open in a separate window CALDES, caldesmon; CK, cytokeratins; DES, desmin; EMA, epithelial membrane antigen; MYF4, myogenin; PD, programmed death; PD-L1, programmed death-ligand 1; CD, cluster of differentiation; MC, mesothelial cells; WT-1, Wilms' tumor 1; DOG1, anoctamin-1; SMA, smooth muscle actin.

Techniques: Immunohistochemistry, Staining

Journal: Science advances

Article Title: PD-L1 methylation restricts PD-L1/PD-1 interactions to control cancer immune surveillance.

doi: 10.1126/sciadv.ade4186

Figure Lengend Snippet: Fig. 1. PD-L1 Lys 162 mono-methylation attenuates PD-1/PD-L1 interaction. (A) Forest plot of ORR in patients treated with anti–PD-1 or anti–PD-L1 monotherapy versus control in NSCLC. (B) Human embryonic kidney (HEK) 293T cell transfected with hemagglutinin (HA)–PD-L1, and then treated with or without 3-deazaneplanocin A (Dznep), immunoprecipitation (IP) and immunoblot (IB) analysis measuring PD-1/PD-L1 interaction. (C) Presenting potential modification sites from mass spectrometry analysis of PD-L1. (D) HEK293T cell transfected with various HA–PD-L variants and IP and IB analysis measuring PD-1/PD-L1 interaction. (E) HEK293T cells transfected with HA–PD-L wild-type (WT) or HA–PD-L1 K162R variant, followed by IP and IB analysis. (F) Flow cytometry (left) measuring PD-L1 or PD-1 binding on the membrane of RKOPD-

Article Snippet: The primary antibodies for PD-L1 (29122 and 13684; Cell Signaling Technology and CSB-MA878942A0m; CUSABIO), pan phosphoserine/threonine rabbit mAb (AP0893; ABclonal), pan phospho-tyrosine rabbit mAb (AP1162; ABclonal), ABclonal), pan monomethyl lysine (A18293; ABclonal), SETD7 (A9225; ABclonal), LSD2(A19820; ABclonal), glyceraldehyde-3-phosphate dehydrogenase (sc-47724; Santa Cruz Biotechnology), H3K4me1 (A2355; ABclonal), flag tag (AE063; ABclonal and 14793S; Cell Signaling Technology), pan acethyl lysine (ab190479, abcam), HA tag (AE036; ABclonal and 3724; Cell Signaling Technology), αtubulin (AC007; ABclonal), PCNA (A12427; ABclonal), biotin (5597; Cell Signaling Technology, 20684; ABclonal), caveolin-1 (A19006; ABclonal), and CD8 (ab237709; Abcam) were purchased from commercial company.

Techniques: Methylation, Control, Transfection, Immunoprecipitation, Western Blot, Modification, Mass Spectrometry, Variant Assay, Flow Cytometry, Binding Assay, Membrane

Journal: Science advances

Article Title: PD-L1 methylation restricts PD-L1/PD-1 interactions to control cancer immune surveillance.

doi: 10.1126/sciadv.ade4186

Figure Lengend Snippet: Fig. 2. PD-L1 Lys 162 methylation restricts its immunosuppressive function. (A and B) T cell–mediated tumor cell killing assay in RKOPD-L1 WT or RKOPD-L1 K162R cells. The representative images (A) and the quantitative ratio (B) of dead cells were shown; n = 3, P = 0.0015. (C and D) Activated T cells cocultured with pretreated RKO cells. The representative images (C) and statistical analysis (D) were shown; n = 3, P = 0.0002. Ig, immunoglobulin. (E and F) Measuring granzyme B (GZMB) of T cells [from experiment (A)] by flow cytometry. The representative images (E) and statistical analysis (F) were shown; n = 3, P = 0.0057. FSC-A, forward scatter area. (G to I) In vitro xenograft tumor assays in immunocompetent humanized peripheral blood mononuclear cell (huPBMC)-NCG mice. The treatment protocol was summarized (G); the tumor growth rate (H) and weight (I) were shown; n = 3, P = 0.0044 (H), P = 0.0013 (I). (J and K) Representative images for number of intratumor CD8+ T cells by im- munofluorescence (IF) (J). Statistical analysis was shown (K); n = 3. (L and M) In vitro xenograft tumor assays in immunodeficient NCG mice. The tumor growth rate (L) and weight (M) were shown. (N and O) In vitro xenograft tumor assays using LewishWT or LewishK162R cells in C57BL/6hPD1 mice; the tumor growth rate (N) and weight (O) were shown; n = 5, P = 0.0079 (N), P = 0.0079 (O). (P and Q) Flow cytometry measuring PD-1+ TIM3+ and GZMB expression of tumor-infiltrating CD8+ T cells; statistical analysis was shown; n = 5, P = 0.025 (P), P = 0.0089 (Q). (R to T) In vitro xenograft tumor assays using LewishWT or LewishK162R cells; anti-CD8 antibody to block CD8+ T cell. The treatment protocol was summarized (R); the tumor growth rate (S) and weight (T) were shown; n = 6, P = 0.0053 (O) or ns, P = 0.0067 or ns (P). Error bars are means ± SD. Statistical significance was assessed using Student’s two-tailed t test. ns, not significant.

Article Snippet: The primary antibodies for PD-L1 (29122 and 13684; Cell Signaling Technology and CSB-MA878942A0m; CUSABIO), pan phosphoserine/threonine rabbit mAb (AP0893; ABclonal), pan phospho-tyrosine rabbit mAb (AP1162; ABclonal), ABclonal), pan monomethyl lysine (A18293; ABclonal), SETD7 (A9225; ABclonal), LSD2(A19820; ABclonal), glyceraldehyde-3-phosphate dehydrogenase (sc-47724; Santa Cruz Biotechnology), H3K4me1 (A2355; ABclonal), flag tag (AE063; ABclonal and 14793S; Cell Signaling Technology), pan acethyl lysine (ab190479, abcam), HA tag (AE036; ABclonal and 3724; Cell Signaling Technology), αtubulin (AC007; ABclonal), PCNA (A12427; ABclonal), biotin (5597; Cell Signaling Technology, 20684; ABclonal), caveolin-1 (A19006; ABclonal), and CD8 (ab237709; Abcam) were purchased from commercial company.

Techniques: Methylation, Flow Cytometry, In Vitro, Expressing, Blocking Assay, Two Tailed Test

Journal: Science advances

Article Title: PD-L1 methylation restricts PD-L1/PD-1 interactions to control cancer immune surveillance.

doi: 10.1126/sciadv.ade4186

Figure Lengend Snippet: Fig. 3. PD-L1 is methylated at Lys 162 by SETD7. (A) Whole cell lysis (WCL) were collected for IP with PD-L1 or SETD7 antibody, followed by IB analysis. (B) Coincubating His–PD-L1 and SETD7 proteins, followed by IB analysis. (C) Fixed RKO cells stained with PD-L1 and SETD7 antibodies, followed by immunofluorescence (IF) assays. (D) HEK293T cells transfected with HA–PD-L1, and then cotransfected with Flag-SETD7, Flag-EZH2, Flag-NSD2, or Flag-SETDB1, followed by IP and IB analysis. (E) Transfecting Flag-SETD7 or Flag-SETD7 H297A into H1975 cells, WCE were collected for IB analysis. (F) Transfecting Flag-SETD7 into RKOPD-L1 WT or RKOPD-L1 K162R cells, WCE were collected for IB analysis. (G) Silencing cellular SETD7 expression, WCE were collected for IP with PD-L1 antibody, followed by IB analysis. (H) Immunoprecipitated SETD7 WT or SETD7 H297A protein from HEK293 cells was incubated with S-adenosyl-L-methionine along with His–PD-L1 protein for in vitro methylation assay. PD- L1 methylation was analyzed by IB analysis using with PD-L1 K162 mono-methylation–specific antibody. (I) RKO cells treated by IL-6 (10 ng/ml) in a time-dependent manner, followed by IP and IB analysis. All IBs are performed three times, independently, with similar results.

Article Snippet: The primary antibodies for PD-L1 (29122 and 13684; Cell Signaling Technology and CSB-MA878942A0m; CUSABIO), pan phosphoserine/threonine rabbit mAb (AP0893; ABclonal), pan phospho-tyrosine rabbit mAb (AP1162; ABclonal), ABclonal), pan monomethyl lysine (A18293; ABclonal), SETD7 (A9225; ABclonal), LSD2(A19820; ABclonal), glyceraldehyde-3-phosphate dehydrogenase (sc-47724; Santa Cruz Biotechnology), H3K4me1 (A2355; ABclonal), flag tag (AE063; ABclonal and 14793S; Cell Signaling Technology), pan acethyl lysine (ab190479, abcam), HA tag (AE036; ABclonal and 3724; Cell Signaling Technology), αtubulin (AC007; ABclonal), PCNA (A12427; ABclonal), biotin (5597; Cell Signaling Technology, 20684; ABclonal), caveolin-1 (A19006; ABclonal), and CD8 (ab237709; Abcam) were purchased from commercial company.

Techniques: Methylation, Lysis, Staining, Immunofluorescence, Transfection, Expressing, Immunoprecipitation, Incubation, In Vitro

Journal: Science advances

Article Title: PD-L1 methylation restricts PD-L1/PD-1 interactions to control cancer immune surveillance.

doi: 10.1126/sciadv.ade4186

Figure Lengend Snippet: Fig. 4. SETD7 regulates PD-L1 and PD-1 interaction and antitumor immunity. (A) IP and IB analysis measuring PD-1/PD-L1 interaction in short hairpin RNA–negative control (shNC) or short hairpin RNA–SETD7 (shSETD7) cells. (B to D) Representative images (B) showing bound PD-1/Fc fusion proteins on the membrane of shNC or shSETD7 cells. Statistical analysis was shown (C and D); n = 9, P < 0.0001 (C), P < 0.0001 (D). (E) T cell–mediated tumor cell killing assay in shNC or shSETD7 cells. The quantitative ratio of dead cells was showed; n = 3, P < 0.0001. (F) Activated T cells cocultured with shNC or shSETD7 cells; measuring GZMB of T cell by flow cytometry. Statistical analysis was shown; n = 3, P < 0.001. (G to I) In vitro xenograft tumor assays using shNC or shSETD7 cells in immunocompetent huPBMC-NCG mice. The treatment protocol was summarized (G); the tumor weight (H) and growth rate (I) were shown; n = 5, P < 0.0005 (H), P < 0.01 (I). (J) Immunohistochemistry analysis for number of intratumor CD8+ T cells. statistical analysis was shown; n = 15, P < 0.0001. (K to M) In vitro xenograft tumor assays using shNC or shSETD7 LewishWT

Article Snippet: The primary antibodies for PD-L1 (29122 and 13684; Cell Signaling Technology and CSB-MA878942A0m; CUSABIO), pan phosphoserine/threonine rabbit mAb (AP0893; ABclonal), pan phospho-tyrosine rabbit mAb (AP1162; ABclonal), ABclonal), pan monomethyl lysine (A18293; ABclonal), SETD7 (A9225; ABclonal), LSD2(A19820; ABclonal), glyceraldehyde-3-phosphate dehydrogenase (sc-47724; Santa Cruz Biotechnology), H3K4me1 (A2355; ABclonal), flag tag (AE063; ABclonal and 14793S; Cell Signaling Technology), pan acethyl lysine (ab190479, abcam), HA tag (AE036; ABclonal and 3724; Cell Signaling Technology), αtubulin (AC007; ABclonal), PCNA (A12427; ABclonal), biotin (5597; Cell Signaling Technology, 20684; ABclonal), caveolin-1 (A19006; ABclonal), and CD8 (ab237709; Abcam) were purchased from commercial company.

Techniques: shRNA, Negative Control, Membrane, Flow Cytometry, In Vitro, Immunohistochemistry

Journal: Science advances

Article Title: PD-L1 methylation restricts PD-L1/PD-1 interactions to control cancer immune surveillance.

doi: 10.1126/sciadv.ade4186

Figure Lengend Snippet: Fig. 5. Methylation of PD-L1 K162 is required for SETD7-mediated antitumor immunity. (A) Transfecting vector or Flag-SETD7 plasmids into RKOPD-L1 WT or RKOPD-L1

Article Snippet: The primary antibodies for PD-L1 (29122 and 13684; Cell Signaling Technology and CSB-MA878942A0m; CUSABIO), pan phosphoserine/threonine rabbit mAb (AP0893; ABclonal), pan phospho-tyrosine rabbit mAb (AP1162; ABclonal), ABclonal), pan monomethyl lysine (A18293; ABclonal), SETD7 (A9225; ABclonal), LSD2(A19820; ABclonal), glyceraldehyde-3-phosphate dehydrogenase (sc-47724; Santa Cruz Biotechnology), H3K4me1 (A2355; ABclonal), flag tag (AE063; ABclonal and 14793S; Cell Signaling Technology), pan acethyl lysine (ab190479, abcam), HA tag (AE036; ABclonal and 3724; Cell Signaling Technology), αtubulin (AC007; ABclonal), PCNA (A12427; ABclonal), biotin (5597; Cell Signaling Technology, 20684; ABclonal), caveolin-1 (A19006; ABclonal), and CD8 (ab237709; Abcam) were purchased from commercial company.

Techniques: Methylation, Plasmid Preparation

Journal: Science advances

Article Title: PD-L1 methylation restricts PD-L1/PD-1 interactions to control cancer immune surveillance.

doi: 10.1126/sciadv.ade4186

Figure Lengend Snippet: Fig. 6. LSD2 demethylates PD-L1 K162 methylation. (A) WCE were collected for IP with PD-L1 or LSD2 antibody, followed by IB analysis with (top) or without (bottom) PNGase F reaction. (B) Fixed RKO cells stained with PD-L1 and LSD2 antibodies, followed by IF assays. (C) Transfecting HA–PD-L1 WT or HA–PD-L1 K162R plasmids into HEK293T cells, WCE were collected for IP assays with HA antibody, followed by IB analysis. (D) IP and IB analysis measuring LSD-2/PD-L1 interaction in shNC or shSETD7 cells. (E) IP and IB analysis measuring PD-1K162me level in vector or LSD2 cells. (F) WCE were collected from shNC or shLSD2 cells, followed by IB analysis. (G) Flow cytometry measuring PD-L1/PD-1 binding on the membrane of shNC or shLSD2 cells. (H to J) Representative images (H) for confocal image showing bound PD-1/Fc fusion proteins on the membrane of shNC or shLSD2 cells. Statistical analysis was shown (I and J); n = 9, P < 0.0001 (I), P < 0.0001 (J). (K) T cell–mediated tumor cell killing assay in indicated RKO cells; the quantitative ratio of dead cells was shown; P = 0.0004; n = 3, P = 0.0004. (L) Activated T cells cocultured with indicated RKO cells; measuring GZMB of T cell by flow cytometry. Statistical analysis was shown; n = 3, P < 0.01. (M to O) In vitro xenograft tumor assays using vector or LSD2 LewishWT cells in C57BL/6hPD1 mice. Anti–PD-L1 antibody was used at indicated time. The treatment protocol was summarized (M). The representative bioluminescence images (N) and tumor growth rate (O) were shown; n = 5, P = 0.0011 or = 0.0001. All IBs are performed three times, independently, with similar results. Error bars are means ± SD. Statistical significance was assessed using Student’s two-tailed t test.

Article Snippet: The primary antibodies for PD-L1 (29122 and 13684; Cell Signaling Technology and CSB-MA878942A0m; CUSABIO), pan phosphoserine/threonine rabbit mAb (AP0893; ABclonal), pan phospho-tyrosine rabbit mAb (AP1162; ABclonal), ABclonal), pan monomethyl lysine (A18293; ABclonal), SETD7 (A9225; ABclonal), LSD2(A19820; ABclonal), glyceraldehyde-3-phosphate dehydrogenase (sc-47724; Santa Cruz Biotechnology), H3K4me1 (A2355; ABclonal), flag tag (AE063; ABclonal and 14793S; Cell Signaling Technology), pan acethyl lysine (ab190479, abcam), HA tag (AE036; ABclonal and 3724; Cell Signaling Technology), αtubulin (AC007; ABclonal), PCNA (A12427; ABclonal), biotin (5597; Cell Signaling Technology, 20684; ABclonal), caveolin-1 (A19006; ABclonal), and CD8 (ab237709; Abcam) were purchased from commercial company.

Techniques: Methylation, Staining, Plasmid Preparation, Flow Cytometry, Binding Assay, Membrane, In Vitro, Two Tailed Test

Journal: Science advances

Article Title: PD-L1 methylation restricts PD-L1/PD-1 interactions to control cancer immune surveillance.

doi: 10.1126/sciadv.ade4186

Figure Lengend Snippet: Fig. 7. PD-L1 K162 hypermethylation is a predictive biomarker of PD-1/PD-L1 blockade resistance therapy. (A to D) Ten NSCLC samples and its WCE were collected; measuring PD-L1 and PD-L1 k162 methylation level by IB analysis; investigating cytotoxicity of tumor-infiltrating CD8+ T cells in tumor specimens by flow cytometry analysis. Pearson’s correlation between PD-L1 K162me/PD-L1 ratio and the number/cytotoxicity of tumor-infiltrating CD8+ T cells was shown; n = 10. (E and F) Collecting 70 clinic samples from patients with NSCLC receiving anti–PD-1 treatment, IF assays measuring PD-L1, PD-L1 K162me, SETD7, and LSD2 expression level; Pearson’s correlation between PD-L1 K162me/PD-L1 ratio and SETD7 (E) or LSD2 (F) was shown; n = 70, P = 0.021 (E), P = 0.01 (F). (G) Kaplan-Meier survival analysis was shown. Patients were grouped by PD-L1 expression level; n = 33 (high), n = 33 (low), P = 0.9345. (H) Shown is the PD-L1 relative expression level of patients with anti–PD-1 treatment resistance or sensitivity; n = 45 (sensitivity), n = 22 (resistance), P = 0.0155. (I) Shown is the PD-L1 K162me/PD-L1 relative ratio of patients with anti–PD-1 treatment resistance or sensitivity; n = 45 (sensitivity), n = 22 (resistance), P < 0.0001. (J) Receiver operating characteristic curve analysis for the indicated parameters in patients receiving anti–PD-1 treatment. (K) Shown is the PD-L1 and PD-L1 K162me expression level of patients with NSCLC receiving anti–PD-1 treatment; red (sensitivity), black (resistance); the axes were separated by the Youden index. (L) Shown is the PD-L1 and PD-L1 K162me/PD-L1 expression level of patients with NSCLC receiving anti–PD-1 treatment; red (sensitivity), black (resistance); the axes were separated by the Youden index. (M) Kaplan-Meier survival analysis was shown; patients were grouped by the PD-L1 K162me/PD-L1 ratio; n = 33 (high), n = 33 (low), P = 0.0261. (N) Working model of SETD7-catalyzed LSD2-antagonized PD-L1 K162 methylation cross-talk with host antitumor immunity and anti-PD-(L)1 treatment prognosis. Error bars are means ± SD. Statistical significance was assessed using Student’s two-tailed t test.

Article Snippet: The primary antibodies for PD-L1 (29122 and 13684; Cell Signaling Technology and CSB-MA878942A0m; CUSABIO), pan phosphoserine/threonine rabbit mAb (AP0893; ABclonal), pan phospho-tyrosine rabbit mAb (AP1162; ABclonal), ABclonal), pan monomethyl lysine (A18293; ABclonal), SETD7 (A9225; ABclonal), LSD2(A19820; ABclonal), glyceraldehyde-3-phosphate dehydrogenase (sc-47724; Santa Cruz Biotechnology), H3K4me1 (A2355; ABclonal), flag tag (AE063; ABclonal and 14793S; Cell Signaling Technology), pan acethyl lysine (ab190479, abcam), HA tag (AE036; ABclonal and 3724; Cell Signaling Technology), αtubulin (AC007; ABclonal), PCNA (A12427; ABclonal), biotin (5597; Cell Signaling Technology, 20684; ABclonal), caveolin-1 (A19006; ABclonal), and CD8 (ab237709; Abcam) were purchased from commercial company.

Techniques: Biomarker Discovery, Methylation, Flow Cytometry, Expressing, Two Tailed Test

Journal: Journal of vascular and interventional radiology : JVIR

Article Title: Impact of Chemoembolic Regimen on Immune Cell Recruitment and Immune Checkpoint Marker Expression following Transcatheter Arterial Chemoembolization in a VX2 Rabbit Liver Tumor Model

doi: 10.1016/j.jvir.2022.03.026

Figure Lengend Snippet: Primary Antibodies Used for Immunohistochemistry

Article Snippet: PD-L1 , UMAB229 , Mouse , 1:25 , TB , Origene , UM800121.

Techniques:

Journal: Frontiers in Immunology

Article Title: Targeting PD-L1 in solid cancer with myeloid cells expressing a CAR-like immune receptor

doi: 10.3389/fimmu.2024.1380065

Figure Lengend Snippet: CARIR expression in THP-1 macrophages enabled significant cytotoxicity against PD-L1 + target cells. Non-modified- (Ctrl), CARIR-Δz, or CARIR-z-engineered THP-1 effector cells were co-cultured in the presence of 5ng/ml PMA for 3 days with RM-1 hPD-L1 or WT-RM-1 target cells at effector to target ratio of 5:1. Following the co-culture, the cells were stained with APC anti-human CD11b, Brilliant Violet 605 anti-human PD-L1, and Zombie NIR viability dye. The number of the remaining live CD11b - target cells following the co-culture was quantified by flow cytometry with the use of absolute counting beads. (A) Representative contour plots of triplicate experiments show the percentage change for PD-L1 + , PD-L1 int , and PD-L1 - RM-1 hPD-L1 tumor cells following the co-culture. (B) BAR graphs summarize the percentage data shown on (A) . (C) BAR graphs summarize the absolute cell count data shown on (A) . (D) Representative contour plots of triplicate experiments show the percentage change of RM-1 tumor cells (PD-L1 - ) following the co-culture. (E) BAR graph summarizes the percentage data shown on (D) . (F) BAR graph summarizes the absolute cell count data shown on (D) . Tumor cells were gated on non-beads, live, singlets, and CD11b - . Data were presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by one-way ANOVA.

Article Snippet: The following monoclonal antibodies (mAbs) and their isotype controls were used for flow cytometry: APC human PD-1, APC human PD-L1, Brilliant Violet human PD-L1, APC human CD86, APC human CD11b, PE human CD247 (CD3z), APC mouse CD11b; PE human EGFR from R&D Systems; APC mouse PD-L1 from Tonbo Biosciences.

Techniques: Expressing, Modification, Cell Culture, Co-Culture Assay, Staining, Flow Cytometry, Cell Counting

Journal: Frontiers in Immunology

Article Title: Targeting PD-L1 in solid cancer with myeloid cells expressing a CAR-like immune receptor

doi: 10.3389/fimmu.2024.1380065

Figure Lengend Snippet: CARIR expression in primary human macrophages enhanced phagocytosis against human PD-L1 + target cells. (A) Schematic illustrations show different CARIR constructs varying in the intracellular signaling domain(s). (B) Flow dot plots show the efficiency of CARIR transduction in human HSCs based on staining for PD-1. (C) Representative flow plots showing the % phagocytosis of CARIR expressing primary human macrophage against RM1 or RM1 hPD-L1 target cells. For generating CARIR modified human macrophages to serve as the effector cells, human CD34 + hematopoietic stem cells (HSCs) were engineered to express CARIR through lentiviral transduction, and then differentiated into macrophages. Fluorescent dye labeled effector cells and target cells were co-cultured for 3 hours before flow analysis for the % phagocytosis. The cells were gated on FSC/SSC, singlets, live, and PD-1 + Violet + macrophages. The CellTrace Violet and CFSE double positive population represent macrophages that have phagocytosed target cells. Data shown are representatives of triplicate experiments (B, C) . (D) The bar graph summarizes the flow data shown in (C) ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by unpaired student t test with two tailed distributions.

Article Snippet: The following monoclonal antibodies (mAbs) and their isotype controls were used for flow cytometry: APC human PD-1, APC human PD-L1, Brilliant Violet human PD-L1, APC human CD86, APC human CD11b, PE human CD247 (CD3z), APC mouse CD11b; PE human EGFR from R&D Systems; APC mouse PD-L1 from Tonbo Biosciences.

Techniques: Expressing, Construct, Transduction, Staining, Modification, Labeling, Cell Culture, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Targeting PD-L1 in solid cancer with myeloid cells expressing a CAR-like immune receptor

doi: 10.3389/fimmu.2024.1380065

Figure Lengend Snippet: CARIR expression in human THP-1 macrophages increased phagocytosis against PD-L1 + human tumor cells. (A, C) Histogram shows the detection of cell surface PD-L1 expression by flow cytometry in cultured human tumor cell lines. Data shown are representatives of 3 independent experiments. (B, D) Phagocytosis activity against PD-L1 + tumor cell lines MDA-MB-231 and NCI-H358 (B) , or PD-L1 - tumor cell lines Hs578T and SK-MEL-28 (D) . Human monocytic THP-1 cells were either nonmodified (Ctrl THP-1) or engineered to express either CARIR-Δz or CARIR-z through lentiviral transduction, and then differentiated toward macrophages by culturing in the presence of 1 ng/ml PMA for 24 hours. CellTrace Violet labeled THP-1 effectors were co-cultured with CellTrace Yellow labeled indicated tumor cells for 4 hours, followed by flow cytometry analysis of the % phagocytosis. Cells were gated on live, singlets, and violet + cells. The events that were double positive for CellTrace Violet and CellTrace Yellow were considered as phagocytic events. The phagocytosis activity was normalized to the non-modified THP-1 condition. The data was presented as mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.00 by one-way ANOVA.

Article Snippet: The following monoclonal antibodies (mAbs) and their isotype controls were used for flow cytometry: APC human PD-1, APC human PD-L1, Brilliant Violet human PD-L1, APC human CD86, APC human CD11b, PE human CD247 (CD3z), APC mouse CD11b; PE human EGFR from R&D Systems; APC mouse PD-L1 from Tonbo Biosciences.

Techniques: Expressing, Flow Cytometry, Cell Culture, Activity Assay, Transduction, Labeling, Modification