Journal: Cancer Immunology Research

Article Title: Rescue of Notch-1 Signaling in Antigen-Specific CD8+ T Cells Overcomes Tumor-Induced T-cell Suppression and Enhances Immunotherapy in Cancer

doi: 10.1158/2326-6066.cir-14-0021

Figure Lengend Snippet: Figure 1. Induction of Notch-1 and Notch-2 regulates CD8þ T-cell functions and is inhibited in tumor-infiltrating T cells. A, CD3þ T cells were activated with plate-bound anti-CD3/CD28 (0.5 mg each) in the presence of increasing concentrations of GSI, Z-Ile-Leu-CHO. Proliferation was determined after 72 hours by [3H]-thymidine uptake. Activated T cells cultured with DMSO and nonstimulated T cells (NS) were used as controls. Results represent mean SD from three similar independent experiments. , P < 0.001. B, CFSE-labeled CD4þ or CD8þ T cells were activated as shown in A with 30 mmol/L GSI, and proliferation was determined 72 hours later by flow cytometry. Histograms are a representative result from three experiments. C, Notch isoform mRNAs were measured in T cells activated for 48 hours. Results represent mean SD from two experiments. , P < 0.001. D, CD3þ, CD4þ, or CD8þ T cells were activated with anti-CD3/CD28, and whole-cell extracts were harvested after 48 and 72 hours. Western blot analyses are representative results of four repeats. E andF, CFSE- labeled CD3þ T cells from floxed and conditional-null Notch-1 and Notch-2 mice were activated as shown in A and monitored for cell proliferation by CFSE. Supernatants were harvested and IFNg levels were measured by ELISA. Results represent mean SD from three independent experiments. , P < 0.001. G, T lymphocytes were isolated from tumors and spleen of mice bearing s.c. 3LL tumors for 17 days or spleens from mice without tumors. T cells were activated with anti-CD3/CD28 for 24 hours and tested for Notch-1 and Notch-2 mRNA by real-time PCR. Results represent mean SD from four different animals, tested in triplicates. , P < 0.001. TBM, tumor-bearing mice.

Article Snippet: Lewis lung carcinoma (3LL) and EL-4 thymoma cells were obtained from the American Type Culture Collection and maintained in RPMI-1640 (Lonza-BioWhittaker) supplemented with 10% fetal calf serum (Hyclone), 25 mmol/L HEPES (Invitrogen, Life Technologies), 4 mmol/L L-glutamine (Invitrogen, Life Technologies), and 100 U/mL of penicillin, streptomycin (Invitrogen, Life Technologies).

Techniques: Cell Culture, Labeling, Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Isolation, Real-time Polymerase Chain Reaction

Journal: Cancer Immunology Research

Article Title: Rescue of Notch-1 Signaling in Antigen-Specific CD8+ T Cells Overcomes Tumor-Induced T-cell Suppression and Enhances Immunotherapy in Cancer

doi: 10.1158/2326-6066.cir-14-0021

Figure Lengend Snippet: Figure 5. Transgenic N1IC in activated antigen-specific CD8þ T cells block tumor growth. A, 106 3LL or 3LL-OVA cells were s.c. injected in N1IC or N1ICf/f mice. Tumor volumes were measured using calipers, as described in Materials and Methods. Results represent mean SD from two independent experiments (N1ICf/f n ¼ 7; N1ICf/f n ¼ 7). ns, nonstatistical significance, P < 0.001; , P < 0.001. B, CD45.1þ mice were injected s.c. with 3LL-OVA for 7 days, after which they were adoptively transferredwith naïve CD8þ T cells from N1IC or N1ICf/f mice (CD45.2þ), and immunizedwith siinfekl. Results represent mean SD from three independent experiments. N1ICf/f n ¼ 22; N1ICf/f n ¼ 22. , P < 0.001; , P < 0.01. C, lymph nodes collected 10 days after immunization from B were challenged with siinfekl and the production of IFNg was measured using ELISpot. Results represent mean SD from three independent experiments. , P < 0.01.

Article Snippet: Lewis lung carcinoma (3LL) and EL-4 thymoma cells were obtained from the American Type Culture Collection and maintained in RPMI-1640 (Lonza-BioWhittaker) supplemented with 10% fetal calf serum (Hyclone), 25 mmol/L HEPES (Invitrogen, Life Technologies), 4 mmol/L L-glutamine (Invitrogen, Life Technologies), and 100 U/mL of penicillin, streptomycin (Invitrogen, Life Technologies).

Techniques: Transgenic Assay, Blocking Assay, Injection, Enzyme-linked Immunospot

Journal: Cancer Immunology Research

Article Title: Rescue of Notch-1 Signaling in Antigen-Specific CD8+ T Cells Overcomes Tumor-Induced T-cell Suppression and Enhances Immunotherapy in Cancer

doi: 10.1158/2326-6066.cir-14-0021

Figure Lengend Snippet: Figure 6. Expression of N1IC in antigen-specific T cells enhances the efficacy of T cell–based immunotherapy. A, 5 106 CD8þ T cells N1IC or N1ICf/f preactivated in vitro for 48 hours were adoptively transferred into mice bearing 3LL-OVA tumors for 7 days. Tumor volume was monitored, as described in Materials and Methods. Results represent mean SD from two similar experiments. N1ICf/f n ¼ 8; N1ICf/f n ¼ 8. , P < 0.001. B and C, single-cell suspensions from tumors represented in A were collected and monitored for the percentage of CD45.2þ CD8þ T cells (B) and CD44 and CD62L in CD45.2þ cells by flow cytometry. C, results represent mean SD from three independent experiments. N1ICf/f n ¼ 6; N1ICf/f n ¼ 6. , P < 0.001. D and E, spleens (D) and lymph nodes (E) were harvested 10 days after T-cell transfer and challenged with siinfekl for 24 hours, after which they were tested for CD107a (D) and production of IFNg (E) by flow cytometry and ELISpot, respectively. Results represent mean SD from two similar independent experiments. , P < 0.001.

Article Snippet: Lewis lung carcinoma (3LL) and EL-4 thymoma cells were obtained from the American Type Culture Collection and maintained in RPMI-1640 (Lonza-BioWhittaker) supplemented with 10% fetal calf serum (Hyclone), 25 mmol/L HEPES (Invitrogen, Life Technologies), 4 mmol/L L-glutamine (Invitrogen, Life Technologies), and 100 U/mL of penicillin, streptomycin (Invitrogen, Life Technologies).

Techniques: Expressing, In Vitro, Cytometry, Enzyme-linked Immunospot

Journal: Cancer Immunology Research

Article Title: Rescue of Notch-1 Signaling in Antigen-Specific CD8+ T Cells Overcomes Tumor-Induced T-cell Suppression and Enhances Immunotherapy in Cancer

doi: 10.1158/2326-6066.cir-14-0021

Figure Lengend Snippet: Figure 7. N1IC in T cells overcomes the tolerogenic effect induced by MDSC. A, MDSC were isolated from tumors and spleens of mice bearing s.c. 3LL cells for 17 days using anti-Gr1 kits. Then, total RNA was isolated and tested for Notch ligands by quantitative PCR. Results represent mean SD from four independent animals and tested in triplicate. , P < 0.001. B, activated CD3þ T cells were cocultured at different ratios with tumor-infiltrating MDSC for 48 hours. Then, T cells were negatively isolated using anti-CD11b beads and whole-protein extracts were harvested and used for detection of Notch-1 and Notch-2 isoforms by Western blotting. A representative experiment of three repeats is shown. C, activated T cells cocultured with MDSC at a 1:1/2 ratio were treated with L-NMMA (500 mmol/L), D-NMMA (500 mmol/L), and NN (200 mmol/L) for 48 hours. Then, extracts were isolated and used as shown in B. Representative results are from three similar experiments. D, CD8þ T cells from CD45.2þ N1IC or N1ICf/f mice were adoptively transferred into CD45.1þ

Article Snippet: Lewis lung carcinoma (3LL) and EL-4 thymoma cells were obtained from the American Type Culture Collection and maintained in RPMI-1640 (Lonza-BioWhittaker) supplemented with 10% fetal calf serum (Hyclone), 25 mmol/L HEPES (Invitrogen, Life Technologies), 4 mmol/L L-glutamine (Invitrogen, Life Technologies), and 100 U/mL of penicillin, streptomycin (Invitrogen, Life Technologies).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Western Blot