rabbit anti mouse ifnβ detection antibody  (R&D Systems)

Journal: Clinical & Translational Immunology

Article Title: Intratumoral administration of the Toll‐like receptor 7/8 agonist 3M‐052 enhances interferon‐driven tumor immunogenicity and suppresses metastatic spread in preclinical triple‐negative breast cancer

doi: 10.1002/cti2.1177

Figure Lengend Snippet: Combination 3M‐052/PD‐1 blockade increases primary tumor immunogenicity and adaptive immune responses. BALB/c mice were injected with 1 × 10 5 4T1.2 cells into the 4th mammary fat pad, and palpable tumors were i.t. injected with vehicle or 3M‐052 on day 7 post‐inoculation. Mice were treated with four doses of isotype or anti‐PD1 mAb in a neoadjuvant setting before primary tumor resection on day 14. Primary tumors were assessed for (a) number of CD206 + F4/80 + cells; (b) number of TCRβ + CD8 + cells; (c) ratio of TCRβ + CD8 + : FoxP3 + CD25 + TCRβ + CD4 + cells; (d) MHC‐I (H2‐K d ) and (e) MHC‐II expression of F4/80 + cells; MHC‐I expression of (f) CD11b + DCs and (g) isolated tumor cells (mCherry + ); number of (h) CD69 + TCRβ + CD4 + cells, (i) CD69 + TCRβ + CD8 + cells and (j) IFN‐γ + TCRβ + CD8 + cells; and (k) ratio of tumor: TCRβ + CD8 + cells. (l) TCRβ + CD8 + cells from day 18 peripheral blood (PB) was assessed for specificity against 4T1.2 cells denoted by IFN‐γ production. Experiments were independently repeated twice, and representative data are shown. MFI: mean fluorescence intensity. All experiments n = 7 mice/group except (c, k) n = 8 or 9 mice/group, (l) n = 10 mice/group. Statistical analysis was performed by one‐way ANOVA with post hoc Tukey's multiple comparison test. Error bars are SEM. * P < 0.05; **P < 0.01 ; *** P < 0.001; and **** P < 0.0001.

Article Snippet: IFN‐α and IFN‐β enzyme‐linked immunosorbent assay was performed using standard molecular biology techniques as described previously ; IFN‐α capture antibody (clone RMMA‐1, 1/500, 0.16 μg mL −1 ; PBL Assay Science, Piscataway, NJ, USA), IFN‐α detection antibody (1/500 rabbit polyclonal mouse IFN‐α: 32100‐1; 4 μg mL −1 ; PBL Assay Science), IFN‐β capture antibody (clone RMMB‐1, 1/500, PBL Assay Science), IFN‐β detection antibody (1/500 rabbit polyclonal mouse: 32400‐1; PBL Assay Science).

Techniques: Immunopeptidomics, Injection, Expressing, Isolation, Fluorescence, Comparison

Journal: Clinical & Translational Immunology

Article Title: Intratumoral administration of the Toll‐like receptor 7/8 agonist 3M‐052 enhances interferon‐driven tumor immunogenicity and suppresses metastatic spread in preclinical triple‐negative breast cancer

doi: 10.1002/cti2.1177

Figure Lengend Snippet: CD8 + T‐cell depletion does not impact 3M‐052 antiprimary tumor effects but attenuates enhanced long‐term survival. (a) Experimental intervention timeline. BALB/c mice were injected with 1 × 10 5 4T1.2 cells into the 4th mammary fat pad, and palpable tumors were i.t. injected with vehicle or 3M‐052 on day 7 post‐inoculation. Mice were treated with five doses of isotype or anti‐CD8 mAb, and primary tumors were resected on day 13. (b) Day 5 flow cytometry analysis of peripheral blood (PB) TCRβ + CD8 + cell proportions. Primary tumors were assessed for (c) weight (mg); (d) IFN‐γ + TCRβ + CD8 + T cells; (e) CD206 + F4/80 + macrophages; and (f) CD11b + and (g) CD103 + DC MHC‐I and (h) F4/80 + macrophage MHC‐I (H2‐K d ) expression. (i) Kaplan–Meier survival curve comparing metastasis‐free survival. (j) A proportion of lungs were taken at endpoint for H&E staining to assess metastatic burden; tumor regions are denoted by dotted line and ‘T’ (4× and 20× magnification, scale bars = 200 µm, representative images shown). (k) Lungs were taken at endpoint and assessed for tumor‐specific CD103 + CD8 + T cells in an ICS for IFN‐γ by flow cytometry. MΦ: macrophage. MFI: mean fluorescence intensity. Data are representative of one experiment. (b, c, i) n = 8 mice/group; (d–h, k) n = 6 mice/group; and (j) n = 2 mice/group. Statistical analysis was performed by (b) the Student's two‐tailed t ‐test; (c–h, k) one‐way ANOVA with post hoc Tukey's multiple comparison test; and (i) the Mantel–Cox log‐rank test. Error bars are SEM. * P < 0.05; **P < 0.01 ; *** P < 0.001; and **** P < 0.0001.

Article Snippet: IFN‐α and IFN‐β enzyme‐linked immunosorbent assay was performed using standard molecular biology techniques as described previously ; IFN‐α capture antibody (clone RMMA‐1, 1/500, 0.16 μg mL −1 ; PBL Assay Science, Piscataway, NJ, USA), IFN‐α detection antibody (1/500 rabbit polyclonal mouse IFN‐α: 32100‐1; 4 μg mL −1 ; PBL Assay Science), IFN‐β capture antibody (clone RMMB‐1, 1/500, PBL Assay Science), IFN‐β detection antibody (1/500 rabbit polyclonal mouse: 32400‐1; PBL Assay Science).

Techniques: Injection, Flow Cytometry, Expressing, Staining, Fluorescence, Two Tailed Test, Comparison

Journal: Clinical & Translational Immunology

Article Title: Intratumoral administration of the Toll‐like receptor 7/8 agonist 3M‐052 enhances interferon‐driven tumor immunogenicity and suppresses metastatic spread in preclinical triple‐negative breast cancer

doi: 10.1002/cti2.1177

Figure Lengend Snippet: 3M‐052 acts indirectly on tumor cells through cytokine production from DCs. (a) 4T1.2 cells were treated with various IFN agonists for 48 h and assessed for MHC‐I (H2‐K d ) expression. Poly I:C‐transfected cells were compared with Lipofectamine‐treated control cells; (b) BMDCs and (c) BM macrophages were stimulated for 24 h with various IFN inducers and assessed for MHC‐I (H2‐K d ) expression by flow cytometry and production of (d) IFN‐β by enzyme‐linked immunosorbent assay and (e) IL‐6, (f) IL‐1β and (g) TNF‐α by cytometric bead array (CBA). Conditioned medium (s/n) from 3M‐052‐treated (h) 4T1.2, (i) BMDCs and (j) BM macrophages was transferred onto fresh 4T1.2 cells ± IFNAR1 blocking mAb, MAR1‐5A3. Following 48 h, recipient 4T1.2 MHC‐I (H2‐K d ) expression was assessed by flow cytometry. BM MΦ: bone marrow‐derived macrophages. MFI: mean fluorescence intensity. n = 3–6 biological replicates/group. Experiments were independently repeated twice, and representative data are shown. All data n = 3 replicates/group except (d) n = 6 replicates/group. Statistical analysis was performed by one‐way ANOVA with post hoc Tukey's multiple comparison test. Error bars are SEM. * P < 0.05; **P < 0.01 ; *** P < 0.001; and **** P < 0.0001.

Article Snippet: IFN‐α and IFN‐β enzyme‐linked immunosorbent assay was performed using standard molecular biology techniques as described previously ; IFN‐α capture antibody (clone RMMA‐1, 1/500, 0.16 μg mL −1 ; PBL Assay Science, Piscataway, NJ, USA), IFN‐α detection antibody (1/500 rabbit polyclonal mouse IFN‐α: 32100‐1; 4 μg mL −1 ; PBL Assay Science), IFN‐β capture antibody (clone RMMB‐1, 1/500, PBL Assay Science), IFN‐β detection antibody (1/500 rabbit polyclonal mouse: 32400‐1; PBL Assay Science).

Techniques: Expressing, Transfection, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Blocking Assay, Derivative Assay, Fluorescence, Comparison

Journal: Clinical & Translational Immunology

Article Title: Intratumoral administration of the Toll‐like receptor 7/8 agonist 3M‐052 enhances interferon‐driven tumor immunogenicity and suppresses metastatic spread in preclinical triple‐negative breast cancer

doi: 10.1002/cti2.1177

Figure Lengend Snippet: Type I IFN signalling critical for 3M‐052 primary tumor and long‐term survival effects. (a) Experimental intervention timeline. BALB/c mice were injected with 1 × 10 5 4T1.2 cells into the 4th mammary fat pad, and palpable tumors were i.t. injected with vehicle or 3M‐052 on day 7 post‐inoculation. Mice were treated with four doses of isotype or MAR1‐5A3 mAb and primary tumors resected on day 13. Resected primary tumors were assessed for (b) weight (mg); (c) Ki67 and c‐caspase‐3 staining of formalin‐fixed primary tumors (20× magnification, scale bars = 100 µm, representative images shown); (d) proportion of CD69 + TCRβ − NKp46 + cells; (e) proportion of CD11b + TCRβ − NKp46 + cells; (f) number of CD206 + F4/80 + macrophages; and (g) tumor cell, (h) F4/80 + macrophage, (i) CD11b + CD11c + and (j) CD103 + CD11c + DC MHC‐I (H2‐K d ) expression; and (k) number of IFN‐γ + TCRβ + CD8 + T cells. (l) Day 20 bioluminescence imaging of the thoracic cavity and (m) Kaplan–Meier survival curve comparing metastasis‐free survival. MΦ: macrophage. MFI: mean fluorescence intensity. Data are representative of one experiment. (b, l, m) n = 7 mice/group; (c) n = 2 mice/group; and (d–k) n = 5 mice/group. Statistical analysis was performed by one‐way ANOVA with post hoc Tukey's multiple comparison test and (m) the Mantel–Cox log‐rank test. Error bars are SEM. * P < 0.05; **P < 0.01 ; *** P < 0.001; and **** P < 0.0001.

Article Snippet: IFN‐α and IFN‐β enzyme‐linked immunosorbent assay was performed using standard molecular biology techniques as described previously ; IFN‐α capture antibody (clone RMMA‐1, 1/500, 0.16 μg mL −1 ; PBL Assay Science, Piscataway, NJ, USA), IFN‐α detection antibody (1/500 rabbit polyclonal mouse IFN‐α: 32100‐1; 4 μg mL −1 ; PBL Assay Science), IFN‐β capture antibody (clone RMMB‐1, 1/500, PBL Assay Science), IFN‐β detection antibody (1/500 rabbit polyclonal mouse: 32400‐1; PBL Assay Science).

Techniques: Injection, Staining, Expressing, Imaging, Fluorescence, Comparison

Journal: Clinical & Translational Immunology

Article Title: Intratumoral administration of the Toll‐like receptor 7/8 agonist 3M‐052 enhances interferon‐driven tumor immunogenicity and suppresses metastatic spread in preclinical triple‐negative breast cancer

doi: 10.1002/cti2.1177

Figure Lengend Snippet: Human TNBC cell lines enhance immunogenicity in response to type I IFN or 3M‐052. MCF7, CAL‐120, MDA‐MB‐231, MDA‐MB‐453 and MDA‐MB‐468 cells were stimulated with either DMSO, 3M‐052 or IFN‐α for 48 h, and expression of (a) HLA‐ABC and (b) PD‐L1 was assessed by flow cytometry. (c) TLR7 and (d) TLR8 expression of various human TNBC cell lines and THP1. MFI: mean fluorescence intensity. Experiments were independently repeated twice, and representative data are shown. (a, b) n = 4 biological replicates/group and (c, d) n = 3 biological replicates/group. Data are one representative of two independent experiments. Statistical analysis was performed by one‐way ANOVA with post hoc Tukey's multiple comparison test. Error bars are SEM. * P < 0.05; **P < 0.01 ; *** P < 0.001; and **** P < 0.0001.

Article Snippet: IFN‐α and IFN‐β enzyme‐linked immunosorbent assay was performed using standard molecular biology techniques as described previously ; IFN‐α capture antibody (clone RMMA‐1, 1/500, 0.16 μg mL −1 ; PBL Assay Science, Piscataway, NJ, USA), IFN‐α detection antibody (1/500 rabbit polyclonal mouse IFN‐α: 32100‐1; 4 μg mL −1 ; PBL Assay Science), IFN‐β capture antibody (clone RMMB‐1, 1/500, PBL Assay Science), IFN‐β detection antibody (1/500 rabbit polyclonal mouse: 32400‐1; PBL Assay Science).

Techniques: Immunopeptidomics, Expressing, Flow Cytometry, Fluorescence, Comparison