baf466  (R&D Systems)

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 2. Upregulation of CXCL10 and MHC class II in human neutrophils in urine during BCG infusion therapy. (a,b) Comprehensive analysis of mRNA expression in urine-derived neutrophils compared to peripheral blood neutrophils. Blood and urine were collected from three patients after one week from the 6th BCG infusion. Comprehensive analysis of mRNA in neutrophils was performed using a DNA tip microarray. (a) Cluster analysis after adjustment and standardiza- tion. The mRNA expression in neutrophils obtained from urine samples (vertical axis) or periph- eral blood (horizontal axis) was analyzed. White lines indicate the thresholds for genes that are upregulated or downregulated > 2-fold between urine- and blood-derived neutrophils. A relatively higher expression in urine-derived neutrophils is indicated using arrows, including expression for CXCR3 ligands (CXCL9 and CXCL10) and MHC class II (HLA-DRB1, HLA-DPA1, and HLA-DQA1). (b) Volcano plot depicting the differentially expressed genes between peripheral blood-derived and urine-derived neutrophils after the 6th BCG infusion. The horizontal axis denotes the fold change in mRNA expression in neutrophils from the urine and blood, while the vertical axis represents the –log10 (p-value) for a t-test of differences in neutrophils from the blood and urine. These data represent the top 6000 genes of the –log10 (p-value). The gene expressions of CXCR3 ligands (CXCL9, CXCL10, and CXCL11) and MHC class II (HLA-DQA2, HLA-DPA1, and HLA-DQA1) were also detected as characteristic features of urine-derived neutrophils (arrows). (c,d) Representative data of intracellular- stained neutrophilic cells obtained via flow cytometric analysis. The CD33+CD15+ neutrophilic cells in the blood (c) or urine (d) samples were obtained from the same patient who was treated with 4th BCG infusions and are presented as CXCL10 MFI (upper panels) and HLA-DR MFI (lower panels). Gray-closed histograms indicate each background staining, and light blue line histograms denote the staining of CXCL10 or HLA-DR. (e–g) Comparison of intracellular expression of (e,f) CXCL10 and (g) HLA-DR in neutrophilic cells from the blood (open circle) and urine (closed circle) samples. These samples were collected after one week from the 2nd to the 6th BCG infusions (after each infusion). (e) ∆CXCL10 MFI was calculated as follows: ∆CXCL10 MFI = (MFI of PE-conjugated anti-CXCL10 mAb staining) −(MFI of PE-conjugated control IgG staining). (f) The neutrophilic cells

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Expressing, Derivative Assay, Microarray, Staining, Comparison, Control

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 3. Effect of BCG on CXCL10 and MHC-II expression in human or mice neutrophilic cells in vitro. Human (a,d) or mouse (b,e) peripheral blood was diluted ten-fold in 10% FCS RPMI1640, or mouse bone marrow cells (4 × 106/mL; c,f) were incubated with or without 4 µg/mL of BCG for 20 h. Following incubation, the expression levels of CXCL10 (a–c) and MHC class II (d–f) in human (CD33+CD15+) or mouse neutrophils (CD45+Ly6G+) were analyzed, as described in Figure S2. Statistical significance was calculated with the paired t-test, * p < 0.05 (n = 3). Abbreviations: BCG, Bacillus Calmette–Guérin; CXCL10, chemokine (C-X-C motif) ligand 10; HLA-DR, human major histocompatibility complex class II cell surface receptor; MFI, mean fluorescence intensity; and I-A/I-E, mouse major histocompatibility complex class II cell surface receptor.

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Expressing, In Vitro, Incubation, Immunopeptidomics, Cell Surface Receptor Assay

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 4. Upregulation of CXCL10 and MHC class II in monocytes and neutrophils in peritoneal effusion cells after BCG injections. Mice were injected with B16F10 cells (5 × 104 cells/100 µL/head), and the PECs were collected after two weeks. The PECs induced after one injection of BCG (40 µg/head) after 16 h and the PECs induced after five repeated injections of BCG (40 µg/head) after 16 h from the final injection are presented as “1-shot” and “5-shots”, respectively. These PECs were intracellularly stained with each antibody, and the relative expression (MFI) of CXCL10 and I-A/I-E was analyzed in CD45+Ly6C+ cells and CD45+Ly6G+ cells, respectively. (a–i) Representative flow cytometric analysis of mouse monocytes (Ly6C+ cells) and neutrophils (Ly6G+ cells) via flow cytometry. The (a–c) panels present flow cytometric analysis of the PECs induced 2 weeks after B16F10 cell injection (presented as “Tumor”). The (d–f) panels show representative flow cytometric analysis of the PECs induced 16 h after the administration of BCG (presented as “1-shot). The (g–i) panels indicate representative flow cytometric analyses of the PECs induced via five repeated BCG injections at one-week intervals. The PECs were collected 16 h after the final BCG admin- istration (presented as “5-shots”). The left panels (a,d,g) show CD45+ leukocytes presented with the gates of Ly6C+ cells (monocytic cells) and Ly6G+ cells (neutrophilic cells). (j–l) The number and proportion of myeloid cells (Ly6C+ and Ly6G+ cells) of the PECs. The peritoneal effusion cells obtained after injection of B16F10 cells are presented as “tumor” (open circles). The cells induced 16 h after a single administration of BCG are presented in the group “1-shot” (closed circles). The cells induced via five repeated injections of BCG are presented in the group “5-shots” (closed triangles). The (j) number of the cells in peritoneal fluid were counted using a hemocytometer, and the proportions of (k) Ly6C+ cells and (l) Ly6G+ cells in CD45+ leukocytes were analyzed via flow cytometry. (m–p) The intracellular expression levels of CXCL10 and MHC-II (I-A/I-E) in mouse monocytes (Ly6C+ cells) and neutrophils (Ly6G+ cells) after BCG injection. These PECs were intracellularly stained with each anti- body, and the relative expression (MFI) of (m,n) CXCL10 and (o,p) I-A/I-E was analyzed in (m,o) CD45+Ly6C+ cells and (n,p) CD45+Ly6G+ cells, respectively. Statistical analyses were per- formed using the Kruskal–Wallis test with the Dunn’s post-hoc test. Each bar is presented as the mean of data. * p < 0.05; ** p < 0.01; and ns, not significant. Abbreviations: PECs, peritoneal exudate cells; CXCL10, C-X-C motif chemokine ligand 10; BCG, Bacillus Calmette–Guérin; and MFI, mean fluorescence intensity.

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Injection, Staining, Expressing, Cytometry

Journal: Biomedicines

Article Title: Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin .

doi: 10.3390/biomedicines11113062

Figure Lengend Snippet: Figure 7. CXCL10 and MHC class II expression in neutrophils induced via BCG was inhibited via partial neutrophil depletion using anti-Ly6G mAbs. BCG (40 µg/100 µL/head) was injected into the peritoneal cavity, following which the antibodies (100 µg/50 µL/head; control mAb, open circle; or anti-Ly6G mAb, closed circle) were injected into the

Article Snippet: The antibodies used in this study were as follows: fluorescein isothiocyanate (FITC)anti-human CD14 mAb (MφP9), phycoerythrin (PE)-anti-human CD16 mAb (3G8), and allophycocyanin (APC)-anti-human human leukocyte antigen DR isotype (HLA-DR) mAb (G46-6) from BD Biosciences; FITC-anti-human CD15 mAb (HI98), APC- or FITC-antihuman CD16 mAb (3G8), brilliant violet 421-anti-human CD33 mAb (WM53), PE-antihuman CD163 mAb (GHI/61), PE-anti-human CD197 mAb (G043H7), PE-anti-human C-X-C motif chemokine ligand 10 (CXCL10) (J034D6), APC-anti-mouse I-A/I-E mAb (M5/114.15.2), FITC-anti-mouse CD45 mAb (30-F11), PerCP-Cy5.5-anti-mouse Ly6C mAb (HK1.4), brilliant violet 421-anti-mouse Ly6G mAb (1A8), brilliant violet 421-anti-mouse Gr-1 mAb (RB6-8C5), and PE-streptavidin from BioLegend; APC-anti latency-associated peptide-1 (LAP; the N-terminal region of transforming growth factor-β1 precursor) mAb (#27232) and biotin-anti-mouse CXCL10 goat Ab (#BAF466) from R&D systems (Minneapolis, MN, USA); and PE-anti-human GPI-80 mAb (3H9) from MBL (Nagoya, Japan).

Techniques: Expressing, Injection, Control