Article Figures & Data
Figures
- Figure 1.
Induction of Notch-1 and Notch-2 regulates CD8+ T-cell functions and is inhibited in tumor-infiltrating T cells. A, CD3+ T cells were activated with plate-bound anti-CD3/CD28 (0.5 μg 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 CD4+ or CD8+ T cells were activated as shown in A with 30 μmol/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, CD3+, CD4+, or CD8+ 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 and F, CFSE-labeled CD3+ 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 IFNγ 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.
- Figure 2.
Transgenic N1IC in CD8+ T cells does not regulate activation and proliferation, but induces markers linked to central memory. A, spleens from N1ICf/f or N1IC mice were cultured in the presence or absence of siinfekl (2 μg/mL) for 72 hours, after which eGFP expression was determined in gated CD8+ T cells. Flow cytometry histograms are a representative experiment of five independent repeats. B, cells from N1ICf/f or N1IC mice were activated as A and extracts were collected after 48 and 72 hours and tested for Notch-1 and Notch-2 by Western blotting. A representative experiment of four repeats is shown. C, expression of CD25 and CD69 was determined in siinfekl-activated N1ICf/f or N1IC cells (24 hours). A representative flow cytometry histogram from five experiments is shown. D, eFluor 670-labeled N1ICf/f or N1IC cells were cultured in the presence or absence of siinfekl for 72 hours, after which cell proliferation was established in CD8+ T cells by flow cytometry. Results represent mean ± SD of the percentage of cells proliferating from three independent experiments. ns, nonstatistical significance, P > 0.01. E, CD45.1+ mice previously injected with 5 × 106 CD8+ T cells from CD45.2+ N1IC or N1ICf/f mice were immunized with 0.5 μg siinfekl in IFA. Four days later, mice were injected i.p. with BrdU, and the percentage of CD45.2+ CD8+ BrdU+ cells was established by flow cytometry. Results represent mean ± SD from two similar independent experiments (N1ICf/f n = 16; N1ICf/f n = 14). Ns, nonstatistical significance, P > 0.01. F, representative flow cytometry dot plot experiment showing the baseline expression of CD44, CD62L, CD122, and CD127 in CD8+ T cells from N1ICf/f or N1IC mice. Experiment was repeated with 6 mice, obtaining similar results. G, hematoxylin and eosin (H&E) staining to evaluate spleen morphology in 9-week old N1ICf/f (n = 5, top left) and N1IC (n = 5, bottom left) mice, wild-type mice transferred with 5 × 106 preactivated N1IC (n = 5, 6 weeks after transfer, top right), and N1IC-CD2 Cre recombinase (n = 5, bottom right, 6 weeks after birth). Grnz B, granzyme B.
- Figure 3.
Conditional expression of N1IC in activated antigen-specific CD8+ T cells directly promotes cytotoxic T-cell responses. A, splenocytes from N1ICf/f or N1IC mice were activated with siinfekl (2 μg/mL) for 72 hours, after which CD8+ T cells were negatively sorted and cocultured at different ratios with 51chromium-labeled EL4 tumor cells loaded with siinfekl. Supernatants were collected 8 hours later and counts per minute calculated. Results represent mean ± SD from three similar independent experiments. ***, P < 0.001. B, 5 × 106 N1IC or N1ICf/f CD8+ T cells activated with siinfekl for 72 hours were adoptively transferred into mice. The same mice then received 3 × 106 cells of a 1:1 ratio of siinfekl-loaded splenocytes labeled with high CFSE (1 μmol/L) and control splenocytes labeled with low CFSE (0.1 μmol/L). The presence of CFSE-labeled cells was determined 24 hours later by flow cytometry in CD8Neg cells. Flow cytometry histograms are a representative experiment of three replicates. C, N1ICf/f or N1IC cells were cultured in the presence or absence of siinfekl for 72 hours, after which supernatants were collected and tested for IFNγ by ELISA. Results represent mean ± SD of three independent experiments. ***, P < 0.001. D, 5 × 106 CD8+ T cells from CD45.2+ N1IC or N1ICf/f mice were adoptively transferred into CD45.1+ mice, followed by vaccination with 0.5 μg siinfekl in IFA. Spleens were harvested 4 days later and challenged with siinfekl for 24 hours, after which the percentage of CD45.2+ CD8+ IFNγ+ cells was monitored by flow cytometry. Results represent mean ± SD from two similar independent experiments (N1ICf/f n = 10; N1ICf/f n = 9). ***, P < 0.001. E, expression of CD107a was determined in cells activated as described in C. Results represent mean ± SD from three independent experiments. ***, P < 0.001. F and G, CD8+ T cells from N1IC or N1ICf/f mice were activated with siinfekl for 24 to 72 hours, after which whole-cell extracts were collected and used for immunoblot. Representative illustrations from three independent experiments are shown. H, ChIP assays to detect the endogenous binding of Notch-1 to granzyme B promoter were assessed in N1IC or N1ICf/f cells cultured with and without siinfekl for 48 hours, as described in Materials and Methods. Results represent mean ± SD from three independent experiments. ***, P < 0.001. Grnz B, granzyme B.
- Figure 4.
Transgenic N1IC regulates granzyme B expression by amplification of canonical and noncanonical pathways. A, ChIP assays to monitor endogenous binding of RBP-J and NF-κB to granzyme B promoter were assessed in N1IC and N1ICf/f CD8+ T cells cultured with or without siinfekl for 48 hours. Results represent mean ± SD from three independent experiments. ***, P < 0.001. B, immunoprecipitations were achieved using 200 μg of protein extracts from activated N1IC and N1ICf/f CD8+ T cells and 2 μg anti-Notch-1 or control IgG antibodies. After overnight incubation, protein G plus–captured complexes were analyzed for Notch-1 (cleaved and transgenic), RBP-J, and NF-κB p65 by Western blotting. As input controls, we used 10 μg of extracts from each experimental group before immunoprecipitation. Representative illustrations are from two experiments. C, kinetics for RBP-J and NF-κB p65 in activated N1IC or N1ICf/f cells. Representative blotting from three independent repeats. D, N1IC and N1ICf/f cells were activated with siinfekl in the presence or absence of PTDC (150 nmol/L). Granzyme B levels were tested 72 hours later. Representative illustrations from three independent experiments are shown. Grnz B, granzyme B.
- Figure 5.
Transgenic N1IC in activated antigen-specific CD8+ 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 = 7; N1ICf/f n = 7). ns, nonstatistical significance, P < 0.001; ***, P < 0.001. B, CD45.1+ mice were injected s.c. with 3LL-OVA for 7 days, after which they were adoptively transferred with naïve CD8+ T cells from N1IC or N1ICf/f mice (CD45.2+), and immunized with siinfekl. Results represent mean ± SD from three independent experiments. N1ICf/f n = 22; N1ICf/f n = 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 IFNγ was measured using ELISpot. Results represent mean ± SD from three independent experiments. **, P < 0.01.
- Figure 6.
Expression of N1IC in antigen-specific T cells enhances the efficacy of T cell–based immunotherapy. A, 5 × 106 CD8+ 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 = 8; N1ICf/f n = 8. ***, P < 0.001. B and C, single-cell suspensions from tumors represented in A were collected and monitored for the percentage of CD45.2+ CD8+ T cells (B) and CD44 and CD62L in CD45.2+ cells by flow cytometry. C, results represent mean ± SD from three independent experiments. N1ICf/f n = 6; N1ICf/f n = 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 IFNγ (E) by flow cytometry and ELISpot, respectively. Results represent mean ± SD from two similar independent experiments. ***, P < 0.001.
- 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 CD3+ 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 μmol/L), D-NMMA (500 μmol/L), and NN (200 μmol/L) for 48 hours. Then, extracts were isolated and used as shown in B. Representative results are from three similar experiments. D, CD8+ T cells from CD45.2+ N1IC or N1ICf/f mice were adoptively transferred into CD45.1+ congeneic recipients. Following transfer, mice were vaccinated with a mix of DCs and/or MDSC pulsed with siinfekl, as described in Materials and Methods, and the draining lymph nodes were harvested, activated with siinfekl, and tested for the production of IFNγ using ELISpot. Results represent mean ± SD from two similar independent experiments. ns, nonstatistical significance, P < 0.001; ***, P < 0.001.
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Supplementary Data
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Volume 2, Issue 8
