Article Figures & Data
Figures
- Figure 1.
hetIL15 promotes tumor infiltration and persistence of adoptively transferred Pmel-1 and endogenous CD8+ T cells in the absence of lymphodepletion. A, ACT therapy in B16 melanoma-bearing mice. Pmel-1 cells (5 × 106) were adoptively transferred comparing three treatment protocols: (i) cell transfer without lymphodepletion (ACT, gray symbols), (ii) cell transfer in irradiated host (ACT+XRT, red symbols), and (iii) cell transfer plus IP hetIL15 administration (ACT+hetIL15, blue symbols). Mice were sacrificed at day 5, 7, and 12 for tumor and spleen analysis. B, The frequency of tumor-infiltrating Pmel-1 cells was determined by flow cytometry at the indicated time points after ACT for each treatment group. The number of Pmel-1 cells in each tumor was normalized per million of cells present in the tumor suspension. Bars represent mean ± SEM. Data of two independent experiments were combined. *, P < 0.05; **, P < 0.01. C, The proportion of Pmel-1 cells present in the tumors over time was calculated as percentage of the mean value at day 5 after ACT for each treatment group. Mean values ± SEM are shown. For each treatment group, r2 and significant deviation from zero were calculated by linear regression. Comparisons of the different treatment groups were performed using two-way ANOVA. **, P < 0.01; ns, nonsignificant. D, The frequency of tumor-infiltrating endogenous CD8+ T cells was determined by flow cytometry at the indicated time points after ACT for each treatment group. The number of endogenous CD8+ T cells in each tumor was normalized per million of cells present in the tumor suspension. Individual animal values and mean ± SEM are shown from data of two independent combined experiments. **, P < 0.01. E, Tumor sections from mice in the ACT (left), ACT+XRT (middle), and ACT+hetIL15 (right) treatment groups. TILs were identified by immunohistochemical staining using antibodies specific for CD3+ (white), CD4+ (orange), CD8+ (magenta), and CD90.1+ (green, staining Pmel-1 cells). A representative image (20× magnification) from one mouse/group is shown. F, Higher magnification of the selected region in ACT+hetIL15 group (white square) shows cells identified as T cells (CD3+, labeled ∧), CD4+ T cells (CD3+ CD4+, labeled +), endogenous CD8+ T cells (CD3+CD8+, labeled #), and Pmel-1 T cells (CD3+CD8+ CD90.1+, labeled *). G, The mean values of the Pmel-1 cell (left) and endogenous CD8+ T-cell (right) counts from 9–15 tumor images are shown. Five to six tumors in each treatment group were analyzed. InForm software was used to enumerate each cell type. *, P < 0.05; **, P < 0.01.
- Figure 2.
Tumor-resident Pmel-1 cells are preferentially targeted by hetIL15. A, Fold difference in Pmel-1 and endogenous CD8+ T-cell counts in tumors and spleens for mice in the ACT+hetIL15 (left, blue) and in the ACT+XRT (right, red) groups normalized to ACT alone. Bars represent mean fold change (±SEM) compared with the mean level of the animals in the ACT group (set as 1). Data were combined from three independent experiments (day 12 after ACT). **, P < 0.01. B, The percentage of Pmel-1 cells (defined by the expression of CD90.1) within the CD8+ T-cell population was assessed by flow cytometry in tumors (left), spleens (middle), and lungs (right) at days 5 and 12. Representative mice from the ACT+hetIL15 group are shown. C, The ratio of Pmel-1 cells to endogenous CD8+ T cells in tumor, spleen, and lung of mice receiving ACT+hetIL15 treatment was determined. Values from individual animals (combining data from day 5 and day 12 after ACT) and mean ± SEM are shown. Data were combined from two independent experiments. *, P < 0.05; **, P < 0.01. D, Mice implanted with B16 melanoma cells and MC38 colon carcinoma cells on opposite flanks underwent ACT+hetIL15 treatment. Fold increase in Pmel-1/CD8+ T-cell ratio was calculated for B16 tumor and MC38 tumor in comparison with spleen (set as 1) for each mouse. Analysis was performed at day 9 after ACT. **, P < 0.01.
- Figure 3.
hetIL15 increases cytotoxic potential and IFNγ production of adoptively transferred Pmel-1 cells in the tumor. A, Tumor-resident Pmel-1 cells were analyzed for the expression of CD62L and KLRG1 markers. A representative animal for each treatment group is shown. B, The frequency of GzmB+ Pmel-1 cells in the tumor (% of total Pmel-1 cells) was determined by intracellular staining followed by flow cytometry. A representative animal for each treatment group is shown. C, The frequency of GzmB+ Pmel-1 cell in tumors is expressed as the percentage of total Pmel-1 cells (left) and number of GzmB+ Pmel-1 cells normalized per million of cells present in the tumor suspension (right); mean values ±SEM are shown for the three groups. Data collected from day 7 and day 12 after ACT were combined. *, P < 0.05; **, P < 0.01. D, The frequency of IFNγ−producing Pmel-1 cells (left) and endogenous CD8+ T cells (middle) in tumor and of Pmel-1 cells in inguinal lymph nodes (right) was determined upon 6 hours (tumor) or 12 hours (lymph node) ex vivo cultures in medium only or in the presence of the hgp10025–33 peptide. Analysis was performed at day 7 after ACT. ACT: n = 3; ACT+XRT: n = 5; and ACT+hetIL15: n = 5. *, P < 0.05; **, P < 0.01.
- Figure 4.
hetIL15 treatment sustains tumor-resident PD-1lowGzmB+Ki-67+ Pmel-1 cells and increases tumor Pmel-1/Treg ratio. A, Percentage of Pmel-1 cells in tumor expressing the proliferation marker Ki-67 for the mice in each of the three treatment groups at day 12 after ACT. Bars represent mean ± SEM. Data from two independent experiments were combined. **, P < 0.01. B, Pmel-1 cells infiltrating the tumor were analyzed for the expression of PD-1, Ki-67, and GzmB by flow cytometry. The GzmB+ Pmel-1 cells (red dots) were overlaid on the total Pmel-1 cell population (black contour). Representative animals from the ACT (left), ACT+XRT (middle), and ACT+hetIL15 (right) treatment groups at day 12 after ACT are shown. C, The percentage of proliferating and cytotoxic Pmel-1 cells characterized by low expression of PD-1 (PD-1lowGzmB+Ki-67+) was determined in tumors at day 12 after ACT (left). The percentage of Pmel-1 cells with a phenotype consistent with exhaustion (PD-1highGzmB−Ki-67−) was also determined in the tumor at day 12 after ACT (right). The values from individual animal and mean ± SEM are shown. **, P < 0.01. D, The frequency of tumor-infiltrating Treg cells was determined by flow cytometry at day 12 after ACT for each treatment group. The number of Treg cells in each tumor was normalized per million of cells present in the tumor suspension. Bars represent mean ± SEM (left). The Pmel-1/Treg ratio was determined in tumor at day 12 after ACT for each treatment group. Bars represent mean ± SEM. **, P < 0.01 (right).
- Figure 5.
hetIL15 and ACT promote tumor control in the absence of lymphodepletion. Mice were implanted with 5 × 105 B16 cells SC at day −5. Mice were randomized in different treatment groups: PBS administration (black, n = 10), ACT alone (gray, n = 7), hetIL15 alone (green, n = 7), and ACT+hetIL15 (blue, n = 8) for A, and ACT alone (gray, n = 5), XRT alone (black, n = 7), ACT+XRT (red, n = 9), and ACT+hetIL15 (blue, n = 10) for B. Splenic-derived Pmel-1 cells (1 × 106/mouse) were administered at day 0. Injections of hetIL15 were performed 3 times per week for a total of 8 doses (3 μg/dose/mouse). Tumor measurements were performed every 2 to 3 days. Mean ± SEM for each time point are shown. One of three similar experiments is shown. Statistical significance was calculated using repeated measures one-way ANOVA. The P values were corrected for multiple comparisons using Holm–Sidak test (*, P < 0.05; **, P < 0.01).
- Figure 6.
IL2 coadministration with ACT results in tumor accumulation and proliferation of Pmel-1 cells similar to hetIL15, but significantly increases the frequency of tumor-associated Tregs (A). Pmel-1 cells (5 × 106) were adoptively transferred comparing 3 treatment protocols: cell transfer without lymphodepletion (ACT, gray symbols), cell transfer plus IP hetIL15 administration (ACT+hetIL15, blue symbols), and cell transfer plus IP IL2 administration (9 μg/dose, green symbols). Mice were sacrificed at day 10 for tumor analysis. The frequency of tumor-infiltrating Pmel-1 cells was determined by flow cytometry for each treatment group. The number of Pmel-1 cells in each tumor was normalized per million of cells present in the tumor suspension. Bars represent mean ± SEM. B, Percentage of Pmel-1 cells in tumor expressing the proliferation marker Ki-67 for the mice in each of the three treatment groups at day 10 after ACT. Bars represent mean ± SEM.C, The frequency of tumor-infiltrating Tregs was determined by flow cytometry at day 10 after ACT for each treatment group. The number of Tregs in each tumor was normalized per million of cells present in the tumor suspension. Bars represent mean ± SEM (left). D, The Pmel-1/Treg ratio was determined in tumor for each treatment group at day 10 after ACT. Bars represent mean ± SEM. E, Mice were implanted with 5 × 105 B16 cells subcutaneously at day −5. Three treatment groups were compared: no treatment (gray, n = 10), ACT+hetIL15 (blue, n = 10), and ACT+IL2 (green, n = 10). Splenic-derived Pmel-1 cells (1 × 106/mouse) were administered at day 0. Intraperitoneal injections of hetIL15 and IL2 were performed 3 times per week for a total of 8 doses (3 μg/dose/mouse). Tumor measurements were performed every 2 to 3 days. Mean ± SEM for each time point is shown. *, P < 0.05; **, P < 0.01.
Additional Files
Supplementary Data
- Supplementary Figures 1-5, Supplementary Table 1 - Supplementary Figure 1. IL-15 availability promotes proliferation and maintenance of transferred CD8+ T in the spleen of recipient mice; Supplementary Figure 2. Gating strategy for the identification of adoptively transferred Pmel-1 cells and endogenous CD8+ T cells infiltrating the tumor; Supplementary Figure 3. Absolute counts of splenic Pmel-1 and CD8+ T cells are profoundly affected by hetIL-15 treatment; Supplementary Figure 4. Evaluation of PD-1, Ki67 and GzmB expression by tumorinfiltrating Pmel-1 cells; Supplementary Figure 5. Evaluation of PD-1, Ki67 and GzmB expression by tumorinfiltrating CD8+ T cells; Supplementary Table 1. Hematological parameters during ACT treatments.
Volume 23, Issue 11
