Journal: Oncoimmunology

Article Title: Reduction of CD11b + myeloid suppressive cells augments anti-neuroblastoma immune response induced by the anti-GD 2 antibody ch14.18/CHO

doi: 10.1080/2162402X.2020.1836768

Figure Lengend Snippet: Evaluation of tumor infiltrating CD11b + cells. (a-d) Representative immunohistochemical images of tumor infiltrating leukocytes (a) and CD11b + cells (c) and respective negative controls (b and (d). Primary tumors obtained from A/J mice inoculated subcutaneously with NXS2-HGW NB cells were stained with either anti-CD45- (A) or anti-CD11b Ab (C) for detection of leukocytes and CD11 + cells, respectively. Magnification of 100 ×. (e) Gating strategy of flow cytometric analysis of CD11b + cell subsets (GD 2 − /CD45 + /CD11b + ). A dot plot of GD 2 and CD45 expression showing a living cell fraction (left) was used to define a GD 2 − /CD45 + cell population that was next characterized in terms of CD11b expression (open black curve) using a histogram (right). Isotype Ab served as a negative control (filled gray curve). (f) Quantitative analysis of leukocytes and CD11b + cells infiltrating primary tumors in untreated mice (0.9% NaCl; white columns). Leukocytes were calculated as a percent of the viable GD 2 -negative CD45-positive cells relative to all viable cells detected in primary tumor tissue. Two subsets of CD11b + (CD11b + cells (CD11b + ) and cells showing high expression of CD11b (CD11b high )) were calculated as a percent of viable GD 2 − /CD45 + /CD11b + cells relative to all viable leukocytes detected in tumor tissue

Article Snippet: Mice were untreated (0.9% NaCl, i.p.) or treated with ch14.18/CHO alone (provided by Polymun Scientific, Klosterneuburg, Austria), 15 mg/kg, i.p., days 4, 5, 6, 7, and 8 after tumor cell injection), anti-CD11b Ab alone (Bio X Cell, West Lebanon, NH, USA, clone M1/70, 25 mg/kg, i.p., days 4, 7, 11, 14, and 18), 5-FU alone (Merck, Darmstadt, Germany, 50 mg/kg, i.p., days 4, 11, and 18), or ch14.18/CHO and 5-FU.

Techniques: Immunohistochemical staining, Staining, Expressing, Negative Control

Journal: Oncoimmunology

Article Title: Reduction of CD11b + myeloid suppressive cells augments anti-neuroblastoma immune response induced by the anti-GD 2 antibody ch14.18/CHO

doi: 10.1080/2162402X.2020.1836768

Figure Lengend Snippet: Antitumor effects of the ch14.18/CHO treatment in combination with reduction of suppressive myeloid cells in vivo . (a) Schematic overview of the treatment protocol. Four days after tumor cell injection, mice received either ch14.18/CHO or anti-CD11b Ab or 5-FU or combination of ch14.18/CHO and 5-FU. (b) Analysis of tumor growth in mice treated with either anti-CD11b Ab (open squares) or 5-FU (open circles). Controls received 0.9% NaCl (closed squares). Data are shown as mean values ± SEM. Statistical analysis of differences between anti-CD11b and control, 5-FU and control as well anti-CD11b and 5-FU groups was performed with one-tailed t-test: * P < .05 for 0.9% NaCl vs. anti-CD11b; § P < .05, §§ P < .01, §§§ P < .001 for 0.9% NaCl vs. 5-FU; # P < .05, ## P < .01 for anti-CD11b vs. 5-FU. (c) Analysis of tumor infiltrating effector cells of mice treated with anti-CD11b Ab (anti-CD11b, black columns) or 5-FU (5-FU, gray columns) in comparison with the untreated controls (0.9% NaCl, white columns). Leukocytes shown in % of all viable CD45 + /GD2 − cells and CD11b + cells shown in % of all viable CD45 + /CD11b + /GD2 − cells were assessed in tumor tissue using flow cytometry analysis. To show impact of anti-CD11b and 5-FU treatments on effector cell count, t-test was used. *** P < .001 vs. 0.9% NaCl. (d) Analysis of tumor growth in mice treated with either ch14.18/CHO alone (gray circles) or ch14.18/CHO in combination with 5-FU (closed circles). Controls received 0.9% NaCl (closed squares). Data are shown as mean values ± SEM. Statistical analysis of differences between ch14.18/CHO and control, combined treatment (ch14.18/CHO + 5-FU) and control as well as combined treatment (ch14.18/CHO + 5-FU) and 5-FU groups was performed with one-tailed t -test: * P < .05, * P < .01, * P < .001 for 0.9% NaCl vs. ch14.18/CHO group; # P < .05 for ch14.18/CHO vs. combined treatment group (ch14.18/CHO + 5-FU); § P < .05, §§ P < .01, §§§ P < .001 for 0.9% NaCl vs. combined treatment group (ch14.18/CHO + 5-FU)

Article Snippet: Mice were untreated (0.9% NaCl, i.p.) or treated with ch14.18/CHO alone (provided by Polymun Scientific, Klosterneuburg, Austria), 15 mg/kg, i.p., days 4, 5, 6, 7, and 8 after tumor cell injection), anti-CD11b Ab alone (Bio X Cell, West Lebanon, NH, USA, clone M1/70, 25 mg/kg, i.p., days 4, 7, 11, 14, and 18), 5-FU alone (Merck, Darmstadt, Germany, 50 mg/kg, i.p., days 4, 11, and 18), or ch14.18/CHO and 5-FU.

Techniques: In Vivo, Injection, Control, One-tailed Test, Comparison, Flow Cytometry, Cell Counting

Journal: Oncoimmunology

Article Title: Reduction of CD11b + myeloid suppressive cells augments anti-neuroblastoma immune response induced by the anti-GD 2 antibody ch14.18/CHO

doi: 10.1080/2162402X.2020.1836768

Figure Lengend Snippet: Impact of the ch14.18/CHO treatment and reduction of suppressive myeloid cells on survival and gene expression. The GD 2 expressing murine NB cells NXS2-HGW were injected subcutaneously into A/J mice followed by the treatment with the chimeric anti-GD 2 Ab ch14.18/CHO, anti-CD11b Ab, 5-FU, or combination of ch14.18/CHO and 5-FU. (a) Comparison of OS probabilities of mice treated with either ch14.18/CHO alone (black dashed line) or 5-FU alone (gray dashed line) or ch14.18/CHO in combination with 5-FU (black solid line) with control mice (0.9% NaCl, gray solid line). Death ahead of schedule and a tumor volume of 750 mm 3 were defined as event. LogRank test. * P < .05 vs. ch14.18/CHO, *** P < .001 vs. 5-FU, $$$ P < .001 vs. 0.9% NaCl, # P < .05 vs. 0.9% NaCl. (b) Comparison of mRNA expression levels of suppressive myeloid cell-associated and modulating genes between primary tumor tissue and NB cells NXS2-HGW used for tumor establishment. Tumor samples were collected from tumor bearing mice treated with ch14.18/CHO, anti-CD11b, 5-FU, or ch14.18/CHO in combination with 5-FU as well as control mice. Expression levels of Arg1, IL-1β, IL-10, CCL2, NOS2, IFN-γ, M-CSFr, IL-6 r, IL-8, IDO, GM-CSF, IL-4, M-CSF, IL-6, TGF-β1, and VEGF-A were determined using RT-PCR analysis relative to the internal control GAPDH and are represented as colors. The color spectrum between light- and dark gray was used to represent the different levels of expression with light gray for low- and dark gray for high expression of the respective gene. Statistical analysis of expression level differences between anti-CD11b and control, 5-FU and control, anti-CD11b and 5-FU as well as ch14.18/CHO and ch14.18/CHO in combination with 5-FU groups was performed with one-tailed t -test: * P < .05 vs. 0.9% NaCl, # P < .05 vs. anti-CD11b

Article Snippet: Mice were untreated (0.9% NaCl, i.p.) or treated with ch14.18/CHO alone (provided by Polymun Scientific, Klosterneuburg, Austria), 15 mg/kg, i.p., days 4, 5, 6, 7, and 8 after tumor cell injection), anti-CD11b Ab alone (Bio X Cell, West Lebanon, NH, USA, clone M1/70, 25 mg/kg, i.p., days 4, 7, 11, 14, and 18), 5-FU alone (Merck, Darmstadt, Germany, 50 mg/kg, i.p., days 4, 11, and 18), or ch14.18/CHO and 5-FU.

Techniques: Gene Expression, Expressing, Injection, Comparison, Control, Reverse Transcription Polymerase Chain Reaction, One-tailed Test

Journal: Oncoimmunology

Article Title: Reduction of CD11b + myeloid suppressive cells augments anti-neuroblastoma immune response induced by the anti-GD 2 antibody ch14.18/CHO

doi: 10.1080/2162402X.2020.1836768

Figure Lengend Snippet: PCR conditions and primer sequences of CD11b+ myeloid cell-associated and modulating genes. To investigate mRNA expression of CD11b+ myeloid cell-associated and modulating genes in tumor tissue and the neuroblastoma cells NXS2-HGW, RT-PCR analysis was used

Article Snippet: Mice were untreated (0.9% NaCl, i.p.) or treated with ch14.18/CHO alone (provided by Polymun Scientific, Klosterneuburg, Austria), 15 mg/kg, i.p., days 4, 5, 6, 7, and 8 after tumor cell injection), anti-CD11b Ab alone (Bio X Cell, West Lebanon, NH, USA, clone M1/70, 25 mg/kg, i.p., days 4, 7, 11, 14, and 18), 5-FU alone (Merck, Darmstadt, Germany, 50 mg/kg, i.p., days 4, 11, and 18), or ch14.18/CHO and 5-FU.

Techniques: Expressing