Fig 1: Cell proliferation and binding assays for cytotopically modified IL-15. (A,B) Representative cell proliferation assay carried out by incubation of different concentrations of the protein of interest with murine CTLL-2 cells for 72 h. Proliferation was quantified by measuring optical densities at 490 nm. The tables show the mean EC50s (±1 SD) for each protein calculated from individual curves from 3 to 4 independent experiments (**p < 0.01 unpaired t-test). (C) Murine RBCs were incubated with 1 µg of the protein of interest followed by incubation with an anti-IL-15 antibody conjugated with PE. Fluorescence intensity was measured using a FACSCalibur flow cytometer. Mod IL-15: modified IL-15, mod IL-15 Gen: modified IL-15 purified from inclusion bodies; Cyto-IL15, cytotopically modified IL-15; cyto-IL15 Gen, cytotopically modified IL-15 purified from inclusion bodies; IL-15, commercially available IL-15 from Peprotech.
Fig 2: Effects of cytotopically modified in house–produced IL-15 on growth of TRAMP-C2 subcutaneous prostate tumors. (A) Tumor volumes up to day 17 post-treatment. Data are means + 1 SEM for all the tumors per group (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 two-way ANOVA with Dunnett’s multiple comparisons post-test). (B) Survival curves of mice post-treatment. The table shows the median survival of each group and comparisons of equality of individual survival curves with the survival curve of vehicle or IL-15 Gen [**p < 0.01, ***p < 0.001, Log-rank (Mantel-Cox) test]. Note: IL-15 Gen is same as mod IL-15 Gen.
Fig 3: Resveratrol-ßcd induced macrophages to secrete IL-18 and promote CD8T bystander activation. (A) CD8T activation in co-cultured Raw264.7-derived M1 or M2 macrophages with the indicated resveratrol-ßcd and CD8T cells derived from the spleen. Error bar = mean ± S.D. (B) Activation of tumor-derived CD8T cells and cell proliferation (C) in co-culture with M1 macrophages or M1 macrophages + resveratrol-ßcd. Error bar = mean ± S.D. (D) M1 or M2 macrophages were activated and incubated with the indicated resveratrol-ßcd concentrations for 48 h. The levels of IL-12, IL-15, and IL-18 in the supernatant were analyzed by ELISA. (E) Subcutaneous tumors were harvested 4 days after the first treatment. The intratumoral cytokine levels were measured by ELISA. n = 5. Error bar = mean ± S.D. (F) IL-18 in the supernatant of M1 macrophages was neutralized by the indicated anti-IL-18 antibody (aIL-18) and the effect of neutralization was analyzed. Error bar = mean ± S.D. (G) After neutralization, M1 macrophages treated with resveratrol-ßcd were co-cultured with spleen-derived CD8Ts. CD8T activation was analyzed 24 h after co-culture. Error bar = mean ± S.D., **p < 0.01, ***p < 0.001.
Fig 4: Evaluation of purity and cytotopic modification of IL-15. An SDS-PAGE gel (left) was run to evaluate the purity of the recombinant modified protein. Also, immunoblotting (right) using antibodies against IL-15 (anti-IL-15) and the peptide chain of the cytotopic molecule PTL3146 (anti-PTL3146) was performed to evaluate the efficacy of the cytotopic modification. Two batches of recombinant protein were used: protein obtained using the optimized protocol described in this paper (mod IL-15, 1) and protein obtained from inclusion bodies (mod IL-15 Gen, 3). Samples (2) and (4) correspond to the reduced forms of (1) and (3), respectively. Reduction was achieved by adding 100 mM DTT to the samples 2 and 4 before loading into the SDS-PAGE.
Fig 5: Schematic representation of modified human IL-15, the cytotopic peptide PTL3146, and the final cytotopically modified IL-15 (cyto-IL-15). In this work, a modified human IL-15 was expressed in E. coli. The recombinant protein consists of human IL-15 (dark grey), a linker (orange), a histidine tag to help with protein purification (blue), and a cysteine residue (yellow). The cytotopic peptide contains a cysteine residue as well (yellow), a positively charged peptide (blue) to interact with the cell membrane and two myristoyl groups (light grey).
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