Journal: Frontiers in Immunology

Article Title: A Unique Cellular and Molecular Microenvironment Is Present in Tertiary Lymphoid Organs of Patients with Spontaneous Prostate Cancer Regression

doi: 10.3389/fimmu.2017.00563

Figure Lengend Snippet: IL17 production, B cell activation, and CD8 T cell accumulation are evident in prostate tumor-associated tertiary lymphoid organs (TLO) . The 5-μm thick paraffin sections were stained with antibodies against proliferating cell nuclear antigen (PCNA, red), CD8 (green), and CD20 (white) to identify B cells and CD8 T cells in tumor-associated TLO. Serial sections were stained with antibodies against CD21 (red), IL17 (green), and CD3 (white). Representative 200× magnification pictures from the same TLO areas showed in H&E stains were taken with a Zeiss Axioplan Microscope and recorded with a Hamamatsu Camera. (A,E) Well-populated B cells follicles with small numbers of small proliferating B cells, small follicular dendritic cell (FDC) networks, and intrafollicular CD8 T cells are appreciated in prostatic intraepithelial neoplasia (PIN) prostatectomy specimens. (B,F) Large PCNA + CD20 + B blasts, and CD8 T cells interspersed inside B cell follicles are detected in prostate samples from patients at intermediate stages of cancer. (C,G) A germinal center with a concentric FDC network, containing multiple large proliferating B blasts and surrounded by CD8 T cells was found inside some TLO of prostatectomy specimens from patients afflicted by advanced cancer. (D,H) Although proliferating B cells are notably reduced, B cell and CD8 T cells are still organized in TLO from evanescent prostate cancer patients. Yellow arrows point to proliferating B cells, while white arrow is depicting a proliferating CD8 T cell. Scale bars represent 100 μm.

Article Snippet: The primary antibodies were as follows: goat anti-human CD105 (AF1097, R&D Systems), rabbit anti cyclooxygenase 2 (GTX15191, GeneTex), mouse anti-human CD68 (clone PG-M1, GeneTex), goat anti-CD3 epsilon (clone M-20, Santa Cruz Biotechnology), rabbit anti-T bet (H-210, Santa Cruz biotechnology), rat anti-human Foxp3 (PCH101, eBioscience), goat-anti proliferating cell nuclear antigen (PCNA) (clone C-20, Santa Cruz Biotechnology), mouse anti-human Ki-67 (clone MIB-1, Dakocytomation), rabbit anti-human CD8 (clone SP16, Thermo Fisher Scientific), mouse anti-human CD20 (clone L-26, Abcam), rabbit anti-human PD-L1 (Invitrogen, PA5-28115), rabbit anti-IL17 (H-132, Santa Cruz Biotechnology), mouse anti-human CD21 (clone 2G9, Thermo Fisher Scientific), rat anti-peripheral node addressin (clone MECA-79, BD Pharmigen), rabbit anti-human CD138 (RB-9422-P1, Thermo Fisher Scientific), mouse anti-podoplanin (clone D2-40, GTX31231, GeneTex), rabbit anti-granzyme B (clone EPR8260, Abcam), rat anti-human DC-LAMP (clone 1010E1.01, Novus Biologicals), rabbit anti-human prostate stem cell antigen (PSCA) (GTX15168, GeneTex), rabbit anti-human CXCL10 (GTX31176, GeneTex), biotin-mouse anti-smooth muscle actin (clone 1A4, Thermo Fisher Scientific), and mouse anti-human plasma cell (clone LIV3G11, Thermo Fisher Scientific).

Techniques: Activation Assay, Staining, Microscopy

Journal: Frontiers in Immunology

Article Title: A Unique Cellular and Molecular Microenvironment Is Present in Tertiary Lymphoid Organs of Patients with Spontaneous Prostate Cancer Regression

doi: 10.3389/fimmu.2017.00563

Figure Lengend Snippet: Enumeration of CD8 T cells in tertiary lymphoid organs (TLO) and tumor areas of prostate cancer . CD8 T cells were counted in all TLO contained in individual prostatectomy specimens or in five to eight randomly selected tumor areas lacking TLO (200× magnification). (A) Average number of CD8 T cells inside TLO and (B) average number of CD8 T cells in tumor regions are shown. n = 10–19 measurements/patient cohort. Bars represent mean ± SEM. Statistically significant differences (*** p ≤ 0.0005, **** p < 0.0001) were calculated by using two-tailed Student’s t -test with GraphPad Prism.

Article Snippet: The primary antibodies were as follows: goat anti-human CD105 (AF1097, R&D Systems), rabbit anti cyclooxygenase 2 (GTX15191, GeneTex), mouse anti-human CD68 (clone PG-M1, GeneTex), goat anti-CD3 epsilon (clone M-20, Santa Cruz Biotechnology), rabbit anti-T bet (H-210, Santa Cruz biotechnology), rat anti-human Foxp3 (PCH101, eBioscience), goat-anti proliferating cell nuclear antigen (PCNA) (clone C-20, Santa Cruz Biotechnology), mouse anti-human Ki-67 (clone MIB-1, Dakocytomation), rabbit anti-human CD8 (clone SP16, Thermo Fisher Scientific), mouse anti-human CD20 (clone L-26, Abcam), rabbit anti-human PD-L1 (Invitrogen, PA5-28115), rabbit anti-IL17 (H-132, Santa Cruz Biotechnology), mouse anti-human CD21 (clone 2G9, Thermo Fisher Scientific), rat anti-peripheral node addressin (clone MECA-79, BD Pharmigen), rabbit anti-human CD138 (RB-9422-P1, Thermo Fisher Scientific), mouse anti-podoplanin (clone D2-40, GTX31231, GeneTex), rabbit anti-granzyme B (clone EPR8260, Abcam), rat anti-human DC-LAMP (clone 1010E1.01, Novus Biologicals), rabbit anti-human prostate stem cell antigen (PSCA) (GTX15168, GeneTex), rabbit anti-human CXCL10 (GTX31176, GeneTex), biotin-mouse anti-smooth muscle actin (clone 1A4, Thermo Fisher Scientific), and mouse anti-human plasma cell (clone LIV3G11, Thermo Fisher Scientific).

Techniques: Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Costimulatory pathways in multiple sclerosis: distinctive expression of PD-1 and PD-L1 in patients with different patterns of disease.

doi: 10.4049/jimmunol.0901038

Figure Lengend Snippet: FIGURE 1. MBP-stimulated CD4 and CD8 expressing PD-1. Representative results of MBP-stimulated PBMC of patients with either AMS or SMS are shown. Top panels, PD-1-expressing MBP-stimulated CD4 T lymphocytes. Bottom panels, PD-1-expressing MBP-stimulated CD8 T lymphocytes. In the upper right corners, the percentage of CD4/PD-1 and CD8/PD-1 T cells relative to the total number of lymphocytes are indicated. Summary results obtained in AMS and SMS patients are presented in A and B. The boxes stretch from the 25th to the 75th percentile; the lines across the boxes indicate the median values; the lines stretching from the boxes indicate extreme values. Statistical significance is shown.

Article Snippet: The following mAbs were used in this study: anti-human CD4 (clone 13B8.2; mouse IgG2a isotype), anti-human CD14 (clone 116; mouse IgG1 isotype), anti-human CD19 (clone J4.119; mouse IgG1 isotype) coupled to R-PE-Cyanine 5 Tandem (PE-Cy5; Caltag Laboratories); anti-human CD8 (clone SFCI21thy2D3; mouse IgG1 isotype) PE-Cyanin-7 (BeckmanCoulter); anti-human CD86 (clone BU63; mouse IgG1 isotype), antihuman CD80 (clone 3H5; mouse IgG1 isotype; Serotec) coupled to FITC; anti-human PD-L1 (gift from Dr. L. Chen) and anti-mouse IgG (H L chain) coupled to FITC (eBioscience); anti-human-B7H3 (gift from Dr. L. Chen) coupled FITC anti-human PD-1 (clone MIH4) coupled to PE (mouse-IgG1 isotype; eBioscience); and anti-human annexin V coupled to FITC (mouse-IgG1 isotype; Beckman Coulter).

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Costimulatory pathways in multiple sclerosis: distinctive expression of PD-1 and PD-L1 in patients with different patterns of disease.

doi: 10.4049/jimmunol.0901038

Figure Lengend Snippet: FIGURE 4. Annexin V- and PD-1-expressing MBP-stimulated CD4 and CD8 T lymphocytes. Representative results of MBP-stimulated PBMC of patients with either AMS or SMS are shown. Top panels, MBP-stimulated CD4 T lymphocytes expressing annexin V and PD-1. Bottom panels, MBP-stimulated CD8 T lymphocytes expressing annexin V and PD-1. Summary results obtained in AMS and SMS patients are presented in A and B. The boxes stretch from the 25th to the 75th percentile; the lines across the boxes indicate the median values; the lines stretching from the boxes indicate extreme values. Statistical significance is shown.

Article Snippet: The following mAbs were used in this study: anti-human CD4 (clone 13B8.2; mouse IgG2a isotype), anti-human CD14 (clone 116; mouse IgG1 isotype), anti-human CD19 (clone J4.119; mouse IgG1 isotype) coupled to R-PE-Cyanine 5 Tandem (PE-Cy5; Caltag Laboratories); anti-human CD8 (clone SFCI21thy2D3; mouse IgG1 isotype) PE-Cyanin-7 (BeckmanCoulter); anti-human CD86 (clone BU63; mouse IgG1 isotype), antihuman CD80 (clone 3H5; mouse IgG1 isotype; Serotec) coupled to FITC; anti-human PD-L1 (gift from Dr. L. Chen) and anti-mouse IgG (H L chain) coupled to FITC (eBioscience); anti-human-B7H3 (gift from Dr. L. Chen) coupled FITC anti-human PD-1 (clone MIH4) coupled to PE (mouse-IgG1 isotype; eBioscience); and anti-human annexin V coupled to FITC (mouse-IgG1 isotype; Beckman Coulter).

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Costimulatory pathways in multiple sclerosis: distinctive expression of PD-1 and PD-L1 in patients with different patterns of disease.

doi: 10.4049/jimmunol.0901038

Figure Lengend Snippet: FIGURE 5. pAkt-expressing MBP-stimulated CD4 and CD8 T lymphocytes. Representative results of MBP-stimulated PBMC of patients with either AMS or stable SMS are shown. Top panels, MBP-stimulated CD4 T lymphocytes expressing pAkt. Bottom panels, MBP-stimulated CD8 T lymphocytes expressing pAkt. Summary results obtained in AMS and SMS patients are presented in A and B. The boxes stretch from the 25th to the 75th percentile; the lines across the boxes indicate the median values; the lines stretching from the boxes indicate extreme values. Statistical significance is shown.

Article Snippet: The following mAbs were used in this study: anti-human CD4 (clone 13B8.2; mouse IgG2a isotype), anti-human CD14 (clone 116; mouse IgG1 isotype), anti-human CD19 (clone J4.119; mouse IgG1 isotype) coupled to R-PE-Cyanine 5 Tandem (PE-Cy5; Caltag Laboratories); anti-human CD8 (clone SFCI21thy2D3; mouse IgG1 isotype) PE-Cyanin-7 (BeckmanCoulter); anti-human CD86 (clone BU63; mouse IgG1 isotype), antihuman CD80 (clone 3H5; mouse IgG1 isotype; Serotec) coupled to FITC; anti-human PD-L1 (gift from Dr. L. Chen) and anti-mouse IgG (H L chain) coupled to FITC (eBioscience); anti-human-B7H3 (gift from Dr. L. Chen) coupled FITC anti-human PD-1 (clone MIH4) coupled to PE (mouse-IgG1 isotype; eBioscience); and anti-human annexin V coupled to FITC (mouse-IgG1 isotype; Beckman Coulter).

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Costimulatory pathways in multiple sclerosis: distinctive expression of PD-1 and PD-L1 in patients with different patterns of disease.

doi: 10.4049/jimmunol.0901038

Figure Lengend Snippet: FIGURE 6. Blockade of the PD-1-PD-L1 pathway. pAKT-expressing, MBP-stimulated CD4 and CD8 T lymphocytes.

Article Snippet: The following mAbs were used in this study: anti-human CD4 (clone 13B8.2; mouse IgG2a isotype), anti-human CD14 (clone 116; mouse IgG1 isotype), anti-human CD19 (clone J4.119; mouse IgG1 isotype) coupled to R-PE-Cyanine 5 Tandem (PE-Cy5; Caltag Laboratories); anti-human CD8 (clone SFCI21thy2D3; mouse IgG1 isotype) PE-Cyanin-7 (BeckmanCoulter); anti-human CD86 (clone BU63; mouse IgG1 isotype), antihuman CD80 (clone 3H5; mouse IgG1 isotype; Serotec) coupled to FITC; anti-human PD-L1 (gift from Dr. L. Chen) and anti-mouse IgG (H L chain) coupled to FITC (eBioscience); anti-human-B7H3 (gift from Dr. L. Chen) coupled FITC anti-human PD-1 (clone MIH4) coupled to PE (mouse-IgG1 isotype; eBioscience); and anti-human annexin V coupled to FITC (mouse-IgG1 isotype; Beckman Coulter).

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Armored Inducible Expression of IL-12 Enhances Antitumor Activity of Glypican-3-Targeted Chimeric Antigen Receptor-Engineered T Cells in Hepatocellular Carcinoma.

doi: 10.4049/jimmunol.1800033

Figure Lengend Snippet: FIGURE 6. GPC3-m28Z-mNFAT-mIL-12 T cells improved antitumor immunity against established murine HCC tumor. (A) Left panel, Experimental scheme of in vivo immunocompetent model. Murine T cells at different doses were transferred i.v. into female C57BL/6 mice 8 d after tumor cell in- oculation (n = 6). Right panel, Tumor growth curve for Hepa1-6-GPC3 xenografts treated with indicated murine T cells. Arrow indicates murine T cells infusion. (B) The tumor weight was measured at the study end point. (C) Body weight of each group was measured every 3–4 d (baseline = 100%). (D) The amounts of mIFN-g and mIL-12 (p70) in the sera of treated mice 8 d after therapy were assessed by ELISA. Data shown are the mean 6 SEM. (E) CAR copy numbers in genomic DNA of residual tumors 8 d after therapy were measured by real-time PCR. (F) The sections of formalin-fixed, paraffin- embedded tumor tissue were immunostained with anti-mouse CD8a Ab or anti-mouse Foxp3 Ab. The images were obtained under original magnification 3400. Data shown are from three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: To detect murine CD8+ T cells and regulatory T cells (Tregs), the sections of formalin-fixed, paraffin-embedded tumor tissue were immunostained with anti-mouse CD8a Ab (Cell Signaling Technology) or anti-mouse Foxp3 mAb (eBioscience), followed by goat anti-rat IgG-HRP (Santa Cruz Biotechnology) or HRP-conjugated rabbit anti-mouse IgG (H&L) (KangChen Biotech).

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Inhibition of PCSK9 enhances the antitumor effect of PD-1 inhibitor in colorectal cancer by promoting the infiltration of CD8 + T cells and the exclusion of Treg cells

doi: 10.3389/fimmu.2022.947756

Figure Lengend Snippet: In vivo antitumor effect of anti-PD-1 antibody. (A) Tumor volume and tumor weight in the MC38 tumor model. Anti-PD-1 mAb administration was 5 mg/kg, n = 4 mice/group. (B) Tumor volume and tumor weight of CT26 tumor model. Anti-PD-1 mAb administration was 5 mg/kg, n = 3 mice/group. (C, D) Flow cytometry analysis of tumor-infiltrating T cells for mice treated with PBS or anti-PD-1 antibody in MC38 tumor model (C) and in CT26 tumor model (D) . (E, F) IHC staining of CD8a and Foxp3 in CT26 tumors. "*" means p-value < 0.05 and "**" means p-value < 0.01.

Article Snippet: The following were the antibodies used: rabbit anti-mouse CD8a antibody (Servicebio, GB11068), rabbit anti-mouse Foxp3 antibody (Servicebio, GB112325), rabbit anti-mouse CSF1R antibody (Servicebio, GB11581), rabbit anti-mouse NK1.1 antibody (abcam, ab289542), rabbit anti-mouse CD11b antibody (Servicebio, GB11581), and rabbit anti-mouse CD4 antibody (Servicebio, GB13064-2).

Techniques: In Vivo, Flow Cytometry, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Inhibition of PCSK9 enhances the antitumor effect of PD-1 inhibitor in colorectal cancer by promoting the infiltration of CD8 + T cells and the exclusion of Treg cells

doi: 10.3389/fimmu.2022.947756

Figure Lengend Snippet: (A) IHC staining of CD8a in tumor of the MC38 tumor model treated with anti-PD-1 or anti-PCSK9 antibody and quantitative analysis of positive particles and (B) for the CT26 tumor model. (C) Flow cytometry analysis of CD45 + , CD3 + , and CD8 + T-cell infiltration in MC38 tumors. "*" means p-value < 0.05 and "**" means p-value < 0.01.

Article Snippet: The following were the antibodies used: rabbit anti-mouse CD8a antibody (Servicebio, GB11068), rabbit anti-mouse Foxp3 antibody (Servicebio, GB112325), rabbit anti-mouse CSF1R antibody (Servicebio, GB11581), rabbit anti-mouse NK1.1 antibody (abcam, ab289542), rabbit anti-mouse CD11b antibody (Servicebio, GB11581), and rabbit anti-mouse CD4 antibody (Servicebio, GB13064-2).

Techniques: Immunohistochemistry, Flow Cytometry

Journal: Oncology Letters

Article Title: Survival analysis with regard to PD-L1 and CD155 expression in human small cell lung cancer and a comparison with associated receptors

doi: 10.3892/ol.2019.9910

Figure Lengend Snippet: Immunofluorescence double staining of PD-1/TIGIT and CD8 in small cell lung cancer. Nuclear staining with DAPI (blue); CD8 staining with TRITC-goat anti-rabbit second antibody (red) or FITC-donkey anti-rabbit second antibody (green); PD-1 staining with FITC-goat anti-mouse second antibody (green); TIGIT staining with TRITC-donkey anti-goat second antibody (red). Sections were photographed at magnification, ×400. CD, cluster of differentiation; PD, programmed death; TIGIT, T cell immunoreceptor with immunoglobulin and ITIM domains; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine; TILs, tumor-infiltrating lymphocytes.

Article Snippet: Sections were incubated with primary anti-TIGIT antibody and anti-CD8 antibody (1:75 dilution; cat. no. 17335-1-AP; ProteinTech Group, Inc.), or with anti-PD-1 antibody and anti-CD8 antibody (1:75 dilution; cat. no. 17335-1-AP; ProteinTech Group, Inc.) at 4°C.

Techniques: Immunofluorescence, Double Staining, Staining