Journal: Advanced Science

Article Title: Chemotherapy‐Mediated Induction of PD‐L1 via SEI1 Facilitates Myeloma Immune Evasion

doi: 10.1002/advs.202411082

Figure Lengend Snippet: Chemotherapy inhibits cytotoxic T lymphocyte activity. A) Gene set enrichment analysis (GSEA) of public RNA‐seq datasets ( GSE161801 ( n = 30) and GSE223060 ( n = 41)) of the immune‐response‐gene‐related signatures between newly diagnosed patients, undergoing treatment and those in relapse. NES, normalized enrichment score. B) Representative two patient's UMAP plot of the expression of CD3D , CD8 , CD69 , and IFNG in a public scRNA‐seq data ( GSE161801 ) ( n = 30). UMAP: uniform manifold approximation and projection. C,D) Pathway enrichment analysis of public RNA‐seq datasets of the myeloma cells treated with or without melphalan ( GSE178292 ) or bortezomib ( GSE171837 ). E) Immunofluorescent staining of healthy and myeloma patients bone marrow samples before or after chemotherapy with 4',6‐diamidino‐2‐phenylindole dihydrochloride (DAPI) and antibodies against CD138 (myeloma cell marker) or granzyme B (GZB, activity of T cell) ( n = 5). Scale bar, 10 µm. F) Box plots showing the relative mean fluorescence intensity (MFI) of CD138 (left panel) or granzyme B (right panel) in the biopsy segment ( n = 5). G) The relative expression of TIM3 , TOX , TIGIT , PD‐1 , or LAG3 in CD8 + T cells isolated from the bone marrow aspirates of another 20 myeloma patients before or after chemotherapy.

Article Snippet: CD138 antibody was purchased from R&D Systems (#AF3190).

Techniques: Activity Assay, RNA Sequencing, Expressing, Staining, Marker, Fluorescence, Isolation

Journal: Advanced Science

Article Title: Chemotherapy‐Mediated Induction of PD‐L1 via SEI1 Facilitates Myeloma Immune Evasion

doi: 10.1002/advs.202411082

Figure Lengend Snippet: Melphalan or bortezomib promotes the expression of PD‐L1 in myeloma cells and inhibit cytotoxic T lymphocyte activity. A) A published GEO dataset ( GSE124510 ) analysis shows the expression profile of upregulated genes in the MM.1S cells treated with or without melphalan or bortezomib. All gene names were listed in Table (Supporting Information). B) PD‐L1 messenger RNA (mRNA) levels of the MM.1S cells treated with or without melphalan or bortezomib ( GSE124510 ). C,D) The expression of PD‐L1 in primary myeloma cells ( n = 6) or myeloma cell lines treated with or without melphalan or bortezomib. E) Relative mRNA and F) protein expression of PD‐L1 in ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. G) Membrane PD‐L1 expression by flow cytometric analysis after melphalan (20 µ m ) or bortezomib (10 n m ) treatment for 24 h. IgG, immunoglobulin G. H) Immunofluorescent staining of myeloma cell lines (ARH‐77 or IM‐9) with DAPI and antibodies against PD‐L1. Scale bar, 3 µm. I) Immunofluorescent staining of myeloma patients bone marrow samples before or after treatment with DAPI and antibodies against CD138 or PD‐L1 ( n = 5). Scale bar, 5 µm. J) T‐cell‐mediated cancer cell killing assay. Pretreatment of myeloma cell lines with melphalan (20 µ m ) or bortezomib (10 n m ) for 12 h, ARH‐77 or IM‐9 cells co‐cultured with activated T cell for 48 h were subjected to flow cytometric analysis. D–H,J) Representative of three independent experiments. Data are averages ± standard deviation (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. B,C) p ‐values were determined by Student's t ‐test. D,E,J) p ‐values were determined using one way ANOVA. ns, not significant.

Article Snippet: CD138 antibody was purchased from R&D Systems (#AF3190).

Techniques: Expressing, Activity Assay, Membrane, Staining, Cell Culture, Standard Deviation

Journal: Advanced Science

Article Title: Chemotherapy‐Mediated Induction of PD‐L1 via SEI1 Facilitates Myeloma Immune Evasion

doi: 10.1002/advs.202411082

Figure Lengend Snippet: Melphalan or bortezomib induces upregulation of myeloma cell PD‐L1 expression through activation of DNA damage and cGAS–STING pathway. A) GSEA analysis of public RNA‐seq datasets ( GSE178292 , GSE171837 , and GSE124510 ) of the sister chromatid segregation genes between myeloma cells treated with or without melphalan or bortezomib. B) Immunofluorescent staining of myeloma cell lines (ARH‐77 or IM‐9) with DAPI and antibodies against phosphorylated histone H2A.X. Scale bar, 3 µm. C) Western blot analysis of PD‐L1 in ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. D) Immunofluorescent staining of myeloma patients’ bone marrow samples before or after treatment with DAPI and antibodies against CD138 or phosphorylated histone H2A.X. Scale bar, 3 µm. E) The relative expression of BRCA1 in primary myeloma cells treated with or without melphalan or bortezomib ( n = 6). F,G) The relative expression of cGAS or STING in ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. H) Western blot analysis of cGAS–STING pathway‐associated proteins and PD‐L1 expression in ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. I) Correlation coefficient of the mRNA levels of PD‐L1 and the expression of cGAS or STING ( n = 30) in a public RNA‐seq dataset ( GSE179929 ). The correlations were evaluated using Pearson coefficient. r , correlation coefficient. J) The relative expression of cGAS in primary myeloma cells treated with or without melphalan or bortezomib ( n = 6). K) Immunofluorescent staining of myeloma patients bone marrow samples before or after treatment with DAPI and antibodies against CD138, STING, or phosphorylated histone H2A.X ( n = 5). Scale bar, 10 µm. L) Expression of STING or IRF7 in nonspecific, STING or IRF7 shRNA‐expressing myeloma cells. M,N) The relative expression of STING or PD‐L1 in STING knockdown ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. O) The relative expression of PD‐L1 in IRF7 knockdown ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. P) Immunofluorescent analysis of ARH‐77 or IM‐9 myeloma cell lines with STING or IRF7 knockdown, treated with or without melphalan or bortezomib, and stained with DAPI and PD‐L1 antibody. Scale bar, 5 µm. Q) Western blot analysis of PD‐L1 in ARH‐77 or IM‐9 myeloma cell lines with STING or IRF7 knockdown, treated with or without melphalan or bortezomib. R) The relative expression of PD‐L1 in ARH‐77 or IM‐9 myeloma cell lines, treated with or without melphalan, bortezomib, or H‐151. S) Schematic of the melphalan or bortezomib induces DNA damage/cGAS–STING/IRF7/PD‐L1 signaling axis. B,C,F–H,L–R) Representative of three independent experiments. Data are averages ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. E,J) p ‐values were determined by Student's t ‐test. F,G,M–O,R) p ‐values were determined using one way ANOVA. ns, not significant.

Article Snippet: CD138 antibody was purchased from R&D Systems (#AF3190).

Techniques: Expressing, Activation Assay, RNA Sequencing, Staining, Western Blot, shRNA, Knockdown

Journal: Advanced Science

Article Title: Chemotherapy‐Mediated Induction of PD‐L1 via SEI1 Facilitates Myeloma Immune Evasion

doi: 10.1002/advs.202411082

Figure Lengend Snippet: The cGAS–STING–IRF7 signaling axis promotes SEI1 expression, which upregulates PD‐L1 expression. A) A published GEO dataset ( GSE124510 ) analysis shows SEI1 mRNA levels of the MM.1S cells treated with or without melphalan or bortezomib. B) SEI1 mRNA levels in primary myeloma cells treated with or without melphalan or bortezomib ( n = 6). C) The relative expression of SEI1 in ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. D,E) Western blot and immunofluorescent analyses of SEI1 in ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. Scale bar, 5 µm. F) Gene tracks showing representative ATAC‐seq profiles at SEI1 gene loci in primary CD138 + plasma cells from two relapsed myeloma patients following treatment with bortezomib. G) ChIP PCR assay showing phosphor‐IRF7 enrichment on SEI1 promoter of ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. H,I) Schematic of the SEI1 promoter luciferase reporter. Red crosses: mutations of nucleotides. The luciferase activity of Luc‐ SEI1 constructs was set at 1. SP‐IRF7, simulation of phosphorylation of IRF7. J,K) The relative expression of SEI1 in STING or IRF7 knockdown ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. L,M) The relative expression of SEI1 or PD‐L1 in SEI1 knockdown ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. N–P) Western blot and immunofluorescent analysis of PD‐L1 in ARH‐77 or IM‐9 myeloma cell lines with SEI1 knockdown, treated with or without melphalan or bortezomib. Scale bar, 3 µm. Q) Immunofluorescent staining of myeloma patients bone marrow samples before or after treatment with DAPI and antibodies against CD138, SEI1, or PD‐L1 ( n = 5). Scale bar, 5 µm. R) Schematic of the melphalan or bortezomib induces DNA damage/cGAS–STING/IRF7/SEI1/PD‐L1 signaling axis. C–E,G,I–P) Representative of three independent experiments. Data are averages ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. A,B) p ‐values were determined by Student's t ‐test. C,G,J,K) p ‐values were determined using one way ANOVA. ns, not significant.

Article Snippet: CD138 antibody was purchased from R&D Systems (#AF3190).

Techniques: Expressing, Western Blot, Clinical Proteomics, Luciferase, Activity Assay, Construct, Phospho-proteomics, Knockdown, Staining

Journal: Advanced Science

Article Title: Chemotherapy‐Mediated Induction of PD‐L1 via SEI1 Facilitates Myeloma Immune Evasion

doi: 10.1002/advs.202411082

Figure Lengend Snippet: SEI1 bridges the interaction between CBP/p300 and PAF1 complex to regulate PD‐L1 transcription. A) HA‐SEI1 overexpressed IM‐9 cells were lysed, and the total protein lysate was immunoprecipitated with an agarose‐immobilized HA antibody and analysis with mass spectrometry. The proteins identified are indicated. B,C) Co‐immunoprecipitation of SEI1, PAF1, CDC73, or p300 in ARH‐77 or IM‐9 cells treated with melphalan or bortezomib. D) Co‐immunoprecipitation of SEI1 along with PAF1, CDC73, or p300 in HEK293T cells co‐transfected with SEI1 and either PAF1 , CDC73 , or p300 plasmid. E) Schematic of the truncations including ΔCABD, ΔSD, ΔPBID, and ΔCT fragments. F) Western blotting showing different truncations of SEI1 (full length, ΔCABD, ΔSD, ΔPBID, and ΔCT) in HEK293T cells. G–K) Pulldown of PAF1, CDC73, or p300 with different truncations of SEI1 (full length, ΔCABD, ΔSD, ΔPBID, and ΔCT) in HEK293T cells. L) Pulldown of MYC‐p300 with CDC73 or PAF1 in HEK293T cells. M) Gene tracks showing representative ATAC‐seq profiles at PD‐L1 gene loci in primary CD138 + plasma cells from two relapsed myeloma patients following treatment with bortezomib. N,O) ChIP PCR assay showing SEI1, p300, or H3K27ac enrichment on PD‐L1 promoter of ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. P) ChIP‐seq profiles show H3K27ac signal tracks at the PD‐L1 gene loci for ARH‐77 cells treated with or without melphalan or bortezomib. Q) ChIP PCR assay showing H3K27ac enrichment on PD‐L1 promoter of ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. R) ChIP PCR assay showing enrichment of H3K27ac on PD‐L1 promoter in sh Ctrl or sh SEI1 ARH‐77 or IM‐9 cells. S) Expression of PAF1 in nonspecific, PAF1 shRNA‐expressing myeloma cells. T,U) The relative expression of PD‐L1 in PAF1 knockdown ARH‐77 and IM‐9 cells treated with or without melphalan or bortezomib. V,W) ChIP PCR assay showing enrichment of V) SEI1 or W) H3K27ac on PD‐L1 promoter in sh Ctrl or sh PAF1 ARH‐77 and IM‐9 cells. X) Schematic of the SEI1 bridges the interaction between CBP/p300 and PAF1 complex to regulate PD‐L1 transcription. B–D,F–L,N,O,Q–W) Representative of three independent experiments. Data are averages ± SD. *** p < 0.001 and **** p < 0.0001. All p ‐values were determined using one way ANOVA.

Article Snippet: CD138 antibody was purchased from R&D Systems (#AF3190).

Techniques: Immunoprecipitation, Mass Spectrometry, Transfection, Plasmid Preparation, Western Blot, Clinical Proteomics, ChIP-sequencing, Expressing, shRNA, Knockdown

Journal: Advanced Science

Article Title: Chemotherapy‐Mediated Induction of PD‐L1 via SEI1 Facilitates Myeloma Immune Evasion

doi: 10.1002/advs.202411082

Figure Lengend Snippet: SEI1 facilitates tumor immune evasion through chemotherapy‐induced PD‐L1 induction, both in vivo and in clinical relevance. A,B) ARH‐77 or IM‐9 cell lines were subcutaneously injected into the NSG mice, and intraperitoneal administration of bortezomib (0.5 mg kg −1 bodyweight) or melphalan (1 mg kg −1 bodyweight) three times per week for a duration of 2 weeks, beginning 2 weeks after cell injection. Shown are the relative expressions of TP53BP1 , PARP1 , BRCA1 , cGAS , STING , SEI1 , and PD‐L1 in tumor tissue of NSG mice ( n = 3 mice per group). C,D) Immunofluorescent staining of a mentioned subcutaneous tumor with DAPI and antibodies against phosphorylated histone H2A.X. Scale bar, 10 µm. E) Immunofluorescent staining of a mentioned subcutaneous tumor with DAPI and antibodies against STING, SEI1, or PD‐L1. Scale bar, 10 µm. 6 week old male C57BL/6J mice were intravenously injected vk12598 cells ( n = 3 mice per group), followed by intraperitoneal administration of bortezomib (0.5 mg kg −1 bodyweight), melphalan (1 mg kg −1 bodyweight), or H‐151 (10 mg kg −1 bodyweight) three times per week for a duration of 2 weeks, beginning 2 weeks after cell injection. F) The experimental schematic. G) ELISA analysis shown the concentrations of M‐protein in mouse sera. H,I) The relative expressions of SEI1 or PD‐L1 in spleen tumor cells of vk12598 mice ( n = 3 mice per group). J) Immunofluorescent staining of F mentioned spleen tumor cells of vk12598 mice with DAPI and antibodies against CD138, SEI1, or PD‐L1. Scale bar, 10 µm. K) Immunofluorescent staining of F mentioned spleen tumor cells of vk12598 mice with DAPI and antibodies against CD138 or granzyme B. Scale bar, 10 µm. L–O) The relative expression of BRCA1 , cGAS , SEI1 , or PD‐L1 in primary myeloma cells isolated from the bone marrow aspirates of 20 myeloma patients before or after chemotherapy. P–S) Correlation coefficient of the mRNA levels of cGAS and mRNA levels of BRCA1 , SEI1 , or PD‐L1 or mRNA levels of SEI1 and mRNA levels of PD‐L1 in myeloma patients ( n = 20). The correlations were evaluated using Pearson coefficient. r , correlation coefficient. p ‐value was determined by Pearson coefficient. T,U) Kaplan–Meier analysis of T) overall and U) progression‐free survivals in myeloma patients with high ( n = 50) or low ( n = 50) SEI1 or PD‐L1 expression. Data are averages ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. M–P) p ‐values were determined by Student's t ‐test. A,B,G–I) p ‐values were determined using one way ANOVA. T,U) p‐ values were determined using a two‐sided log‐rank test.

Article Snippet: CD138 antibody was purchased from R&D Systems (#AF3190).

Techniques: In Vivo, Injection, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Isolation

Journal: Advanced Science

Article Title: Chemotherapy‐Mediated Induction of PD‐L1 via SEI1 Facilitates Myeloma Immune Evasion

doi: 10.1002/advs.202411082

Figure Lengend Snippet: Sequential treatment with chemotherapy followed by PD‐L1 antibodies on myeloma cells augments activated T cells cytotoxicity. A,B) T‐cell‐mediated cancer cell killing assay. Myeloma cells (ARH‐77 or IM‐9) were pretreated with melphalan (10 µ m ) and bortezomib (5 n m ) or PD‐L1 antibodies (1 µg mL −1 ) in various sequences, followed by co‐culture with activated T cells for 24 h were subjected to flow cytometric analysis ( n = 3 biological replicates). C) 6 week old male C57BL/6J mice were intravenously injected vk12598 cells ( n = 3 mice per group), followed by intraperitoneal administration of bortezomib (0.5 mg kg −1 bodyweight) and melphalan (0.5 mg kg −1 bodyweight) or PD‐L1 antibodies (10 mg kg −1 bodyweight) in different sequences three times for 1 week each. D) ELISA analysis shown the concentrations of M‐protein in mouse sera. E) Kaplan–Meier analysis of overall mouse survival ( n = 5 mice per group). F) Immunofluorescent staining of mouse spleen samples using DAPI, CD138 antibodies, or granzyme B (GZB) antibodies. Scale bar, 5 µm. G) Depiction of signaling pathways involved in SEI1 facilitates tumor immune evasion through chemotherapy‐induced PD‐L1 induction. Data are averages ± SD. * p < 0.05, ** p < 0.01, and **** p < 0.0001. B) p ‐value was determined by the Student's t ‐test. D) p ‐value was determined using two‐way ANOVA. E) p‐ value was determined using a two‐sided log‐rank test.

Article Snippet: CD138 antibody was purchased from R&D Systems (#AF3190).

Techniques: Co-Culture Assay, Injection, Enzyme-linked Immunosorbent Assay, Staining, Protein-Protein interactions

Journal: Advanced Science

Article Title: Engineered Biomimetic Fibrillar Fibronectin Matrices Regulate Cell Adhesion Initiation, Migration, and Proliferation via α 5 β 1 Integrin and Syndecan‐4 Crosstalk

doi: 10.1002/advs.202300812

Figure Lengend Snippet: Syndecan‐4 crosstalks with α 5 β 1 integrin to sense the fibrillarity and mechanical stiffness of FN. a,b) Adhesion forces of pKO‐ β 1 fibroblasts to heparin‐treated 2D globular FN substrates, 2.5D fibrillar FN matrices, and 3D fibrillar FN matrices a) without and b) with crosslinking. Reference adhesion forces of pKO‐ β 1 fibroblasts to non‐treated FN substrates or matrices are given in gray. Bottom p values compare given and reference data. Middle p values compare 2.5D fibrillar or 3D fibrillar FN matrices with 2D globular FN substrates. Top p values compare 2.5D fibrillar and 3D fibrillar FN matrices. AS indicates the slope of a linear regression and s.e. of adhesion strengthening rate (pN s −1 ). c) Adhesion forces of pKO‐ β 1 fibroblasts depleted from syndecan‐1 (SDC1 KO), syndecan‐2 (SDC2 KO), syndecan‐3 (SDC3 KO), or syndecan‐4 (SDC4 KO) to 2D globular, 3D fibrillar, and crosslinked 3D fibrillar (3DX) FN substrates. Adhesion forces of pKO‐ β 1 fibroblasts to respective FN substrates are given in gray as reference. p values compare given and reference data. d) Adhesion forces of pKO‐ β 1 SDC4 KO fibroblasts rescued with syndecan‐4 to 2D globular, 3D fibrillar, and crosslinked 3D fibrillar (3DX) FN substrates. Adhesion forces of pKO‐ β 1 fibroblasts to respective FN substrates are given in gray as reference. Bottom p values compare given and reference data. Middle p values compare 2.5D fibrillar or 3D fibrillar FN matrices with 2D globular FN substrates. Top p values compare 2.5D fibrillar and 3D fibrillar FN matrices. Dots represent adhesion forces of individual fibroblasts and red bars the median. n indicates the number of fibroblasts tested. p values were calculated using two‐tailed Mann–Whitney test.

Article Snippet: The detached fibroblasts were resuspended in SCFS media with FBS (1% w/v) and recovered from detachment process at 37 °C for at least 30 min. After recovery, cells were pelleted and 10 6 fibroblasts were resuspended in flow cytometry buffer (100 μL, EDTA (2 m m ) and BSA (2% w/v) in PBS) containing antibodies as follows; phycoerythrin (PE)‐labeled integrin subunit β 1 (1:6, BioLegend, 102 208), SDC1 (1:10, R&D systems, FAB2966P) with rat IgG control (1:10, R&D systems, IC005P), or SDC4 (1:50, Abcam, ab279590) with rabbit IgG control (1:50, Abcam, ab209478) antibodies/Alexa Fluor 647‐conjugated SDC2 (1:20, R&D systems, FAB6585R) with sheep IgG control (1:20, R&D systems, IC016R) antibodies at 4 °C for 1 h. For SDC3, 10 6 fibroblasts were incubated with SDC 3 (1:400, ThermoFisher, PA5‐47377) with Goat IgG control (1:400, ThermoFisher, 02–6202) antibodies in flow cytometry buffer at 4 °C for 1 h, washed by flow cytometry media twice, and incubated with Alexa Fluor 488 rabbit anti‐goat IgG (Invitrogen, A11078) at 4 °C for 1 h. After incubation, fibroblasts were washed by flow cytometry buffer twice, followed by measuring their fluorescence intensity employing LSRFortessa (BD AG).

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Breast cancer research : BCR

Article Title: Tumor necroptosis-mediated shedding of cell surface proteins promotes metastasis of breast cancer by suppressing anti-tumor immunity.

doi: 10.1186/s13058-023-01604-9

Figure Lengend Snippet: Fig. 4 Necroptosis promotes tumor metastasis by activating cell surface proteases. a, b ELISA of soluble JAM-A, CD138 and E-cad in TIF(a) and serum (b) from WT and Mlkl KO MMTV-PyMT mice at 15 weeks (n = 6, each). c, d ELISA of soluble JAM-A, CD138 and E-cad in TIF (c) and serum (d) from MMTV-PyMT mice treated with vehicle or GW280264X (n = 5, each). e qPCR analysis of IFN-γ mRNA expression in tumor-infiltrating CD8+ T cells of MMTV-PyMT mice treated with vehicle or GW280264X (n = 5, each; left panel). Flow cytometry analysis of tumor-infiltrating GZMB+ CD8+ T cells in MMTV-PyMT mice treated with vehicle or GW280264X (n = 5, each; right panel). f qPCR analysis of IFN-γ and T-bet mRNA expression in tumor-infiltrating CD4+ T cells of MMTV-PyMT mice treated with vehicle or GW280264X (n = 5, each). g Flow cytometry analysis of peripheral blood GZMB+ CD8+ T cells and IFN-γ+ CD4+ T cells MMTV-PyMT mice treated with vehicle or GW280264X (n = 5, each). h Left panel shows the representative images of H&E-stained lung sections from MMTV-PyMT mice treated with vehicle or GW280264X.Scale bar, 2 mm. Right panel shows the quantification of metastatic foci in lungs from MMTV-PyMT mice treated with vehicle or GW280264X (n = 5, each). Data presented as the mean ± sem and p value was determined by two-tailed t test (a–h)

Article Snippet: Samples were then analyzed for mouse JAM-A (ab277080, Abcam), mouse CD138 (NBP2-76610, Novus) and mouse E-cadherin (ab197751, Abcam) according to manufacturer’s instruction.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Staining, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Suppression of Pathological Allergen-Specific B Cells by Protein-Engineered Molecules in a Mouse Model of Chronic House Dust Mite Allergy

doi: 10.3390/ijms252413661

Figure Lengend Snippet: Phenotyping of immune cells in the lungs by FACS analyses. ( A ) Characterization of myeloid cell types—alveolar macrophages ( I ), SiglecF high CD11c low ( III ), SiglecF med CD11c− ( IV ) and total eosinophils ( II ), and neutrophils ( V )—presented as the percentage of the parent population. ( B ) Analysis of B and antibody-secreting cells—percentages of CD19 cells ( I ), CD19 CD80 cells ( II ), CD19 IgE cells ( III ), CD138 cells ( IV ), plasma cells ( V ), plasmablasts ( VI ), and CD32 mean fluorescent intensity (MFI) of CD19 ( VII ) and CD19 IgE cells ( VIII ). ( C ) Phenotyping of T cells—CD3 cells ( I ), CD3 CD4 cells ( II ), CD3 CD8 cells ( III ), CD4 CD69 − CD25 + cells ( IV ), CD4 CD69 + CD25 + cells ( V ), CD4 CD69 + CD25 − cells ( VI ), CD8 CD69 − CD25 + cells ( VII ), CD8 CD69 + CD25 − cells ( VIII ), and CD4 CD69 + CD25 + cells ( IX ). Data are shown as mean ± SD of 8–9 mice per group. The differences between the groups were evaluated using a one-way ANOVA followed by Tukey’s multiple comparisons, or a Kruskal–Wallis test followed by Dunn’s multiple comparisons test, depending on the normality of the data; p values are indicated on the graphs: * p < 0.05; ** p < 0.01; *** p < 0.001. Data are representative of at least 4 independent experiments.

Article Snippet: The following were used for the fluorescence-activated cell sorting (FACS) experiments: Fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD16/32 antibody (Cat# 35-0161-U100, clone 2.4G2, Tonbo Bioscience, San Diego, CA, USA), anti-rat IgG-FITC antibody (Cat# 405404, BioLegend, San Diego, CA, USA), anti-mouse FITC-conjugated CD11c (Cat# 117306, clone N418, BioLegend, San Diego, CA, USA), CD25 (Cat# 102006, clone PC61, BioLegend, San Diego, CA, USA), and CD45R (B220) (Cat# 103206, clone RA3-6B2, BioLegend, San Diego, CA, USA); Phycoerythrin (PE)-conjugated CD19 (Cat# 115508, clone 6D5, BioLegend, San Diego, CA, USA), SiglecF (Cat# 12-1702-80, clone 1RNM44N, eBioscience, Frankfurt, Germany), CD69 (Cat# 104508, clone H1.2F3, BioLegend, San Diego, CA, USA), and CD138 (Cat# FAB2966P, clone 300506, R&D Systems, Minneapolis, MN, USA); PE-Cyanine 7 (PE-Cy7)–conjugated CD3 (Cat# 100320, clone 145-2C11, BioLegend, San Diego, CA, USA) and CD8 (Cat# 100722, clone 53-6.7, BioLegend, San Diego, CA, USA); Pacific Blue-conjugated Ly6G (Cat# 127612, clone 1A8, BioLegend, San Diego, CA, USA), CD3 (Cat# 100334, clone 145-2C11, BioLegend, San Diego, CA, USA), and CD80 (Cat# 104724, clone 16-10A1, BioLegend, San Diego, CA, USA); PE-Cyanine 5 (PE-Cy5)–conjugated CD4 (Cat# 100410, clone GK1.5, BioLegend, San Diego, CA, USA) and Streptavidin (Cat# 15-4317-82, eBioscience, San Diego, CA, USA); and biotin-conjugated IgE (Cat# 13-5992-85, clone 23G3, eBioscience, San Diego, CA, USA) mAbs.

Techniques: Clinical Proteomics

Journal: Cancer Management and Research

Article Title: Distribution of CD8 T Cells and NK Cells in the Stroma in Relation to Recurrence or Metastasis of Nasopharyngeal Carcinoma

doi: 10.2147/CMAR.S365230

Figure Lengend Snippet: CD8 T cells, NK cell, macrophages, and plasma cell were stained in tumor core and stromal in NPC by immunohistochemistry. ( A ). CD8 200× ( B ). CD8 400× ( C ). CD56 200× ( D ). CD56 400× ( E ). CD68 200× ( F ). CD68 400× ( G ). CD138 200× ( H ). CD138 400×.

Article Snippet: These TIICs were stained with different antibodies as follows: an anti-human CD8 mouse monoclonal antibody (TA802079S, dilution 1:150, Origene), an anti-human CD56 mouse monoclonal antibody (TA805376S, dilution 1:150, Origene), an anti-human CD68 mouse monoclonal antibody (TA802949S, dilution 1:500, Origene), and an anti-human CD138 mouse monoclonal antibody (TA813799S, dilution 1:1000, Origene).

Techniques: Staining, Immunohistochemistry