Simultaneous blockade of multiple immune system inhibitory checkpoints enhances antitumor activity mediated by interleukin-15 in a murine metastatic colon carcinoma model

Abstract

Purpose: Interleukin 15 (IL-15) is a promising cytokine for immunotherapy of cancer due to its ability to stimulate the immunity of natural killer, B, and T cells. Its effectiveness, however, may be limited by inhibitory checkpoints and pathways that can attenuate immune responses. Finding strategies to abrogate these negative regulators and enhance the efficacy of IL-15 is a critical challenge.

Experimental design: In a preclinical study, we evaluated IL-15 combined with antibodies to block the negative immune regulators cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death ligand 1 (PD-L1) in a metastatic murine CT26 colon carcinoma model.

Results: IL-15 treatment resulted in a significant prolongation of survival in mice with metastatic tumor. Administration of IL-15, however, also increased expression of PD-1 on the surface of CD8(+) T cells including CD8(+)CD44(high) memory phenotype T cells. Moreover, IL-15 also increased the secretion of the immunosuppressive cytokine, IL-10. Combining IL-15 with anti-PD-L1 and anti-CTLA-4 (multiple immune checkpoint blockade) exhibited greater CTL killing and IFNγ secretion. Moreover, this combination resulted in a significant reduction in surface expression of PD-1 on CD8(+) T cells, a decrease in IL-10 secretion, and led to significantly longer survival of tumor-bearing animals compared with mice treated with IL-15 alone or combined singularly with anti-PD-L1 or anti-CTLA-4.

Conclusions: Combining the immune stimulatory properties of IL-15 with the simultaneous removal of 2 critical immune system inhibitory checkpoints, we showed enhancement of immune responses leading to increased antitumor activity.

Conflict of interest statement

Disclosure of potential conflicts of Interest

The authors disclosed no potential conflicts of Interest.

©2010 AACR.

Figures

Figure 1. CD8+ T-cells are involved in the anti-tumor response to CT26 mediated by IL-15

Expression of MHC class I on CT26 cells was analyzed by flow cytometry using FITC-anti-mouse-H-2Kd (open area). An antibody of the same isotype was used as a negative control (filled area). MHC class I mean fluorescence intensity (MFI) of CT26 cells either A; in vitro or B; obtained ex vivo from lung nodules of tumor-bearing animals treated with PBS is shown. C; Survival of CT26 tumor-bearing mice treated with mIL-15 with and without CD8+ T-cells depletion is shown. One day after tumor inoculation, mice received 5 µg/day of mIL-15 injected five times a week for three weeks (mIL-15, diamonds). One group of mice were depleted of CD8+ T-cells (mIL-15 + anti-CD8, squares) using anti-mouse-CD8 antibody administered on days 0 and 1, and then three time a week for three weeks. PBS treated animals served as controls (PBS, dots). The data represent three independent experiments.

Figure 2. Negative immune checkpoint blockade combined with mIL-15 down-regulated surface PD-1 expression on CD8+ T-cells and CD8+CD44high T-cells that had been mediated by mIL-15

Flow cytometry was used to analyze the PD-1 expression on splenic CD8+ T-cells as well as on the CD8+CD44high population from the CT26-bearing mice following treatment with PBS, mIL-15, mIL-15 and anti-PD-L1, or mIL-15, anti-PD-L1 and anti-CTLA-4 on day 10 after CT26 tumor inoculation. Surface expression of PD-1 on CD8+ T-cells was detected using APC-anti-CD8 and PE-anti-PD-1 (open area, Fig. 2A). The CD8+CD44high population was stained with APC-anti-CD8 and PE-Cy5.5-anti-CD44 antibodies (open area, Fig. 2B). An isotype matched IgG was used as a negative control (filled area). All the samples were analyzed on gated CD8+ T-cells or CD8+CD44high cells as indicated. MFI is shown. The figure is representative of two independent experiments wherein each group contained three to five animals. Statistical analysis was performed based on PD-1 expression on CD8+ T cells (Fig. 2C) and CD8+ CD44high population (Fig. 2D). * P<0.05 and ** P<0.01.

Figure 3. The combination of anti-PD-L1 and anti-CTLA-4 antibodies that block negative checkpoints enhanced mIL-15 induced IFNγ secretion as well as down-regulated IL-10 secretion by CD8+ T-cells

A and B; Splenic CD8+ T-cells obtained ex vivo from CT26 tumor-bearing mice treated with PBS, mIL-15, mIL-15 and anti-PD-L1, or mIL-15 combined with anti-PD-L1 and anti-CTLA-4 were stimulated with 10 µg/ml plate-bound anti-CD3 plus 1 µg/mL of a soluble anti-CD28 antibody and the supernatants were collected after 72 hours. IFNγ and IL-10 secretion was quantitated by ELISA. Cells were co-cultured with the same concentration of isotype-matched antibodies as a background control; *P<0.05; C, Naïve splenic CD8+ T-cells were stimulated with anti-CD3 (10 µg/mL, black dots; 4 µg/mL, grey diamonds) plus 1 µg/mL of a soluble anti-CD28 antibody in the presence of different amounts of mIL-15 as indicated. IL-10 secretion was quantitated by ELISA. Cells cultured with the same concentration of an isotype control antibody were set up as controls. D, anti-PD-L1 and anti-CTLA-4 (10 ng/mL) were added with 20 ng/mL mIL-15 present in wells at the same stimulation conditions as above (black bars). IL-10 secretion in the supernatants was quantitated by ELISA. Media alone with no mIL-15 was set up as a control, *P<0.05. ND: non-detectable (>16pg/ml). The data is representative of three independent experiments.

Figure 4. mIL-15 induced tumor-specific IFNγ secretion and lytic activity

On day 21, CT26-bearing mouse splenocytes were harvested and incubated with irradiated CT26 cells at a ratio of 50:1. Cultures were incubated for 4 days. A, 1 × 106 effector cells were cultured with CT26-tumor cells at a ratio of 20:1 for 6 h. Brefeldin A (10 µg/mL) was added to the cultures for the last 5 h. Cells from each group were first stained with APC conjugated anti-CD8, then intracellularly stained with PE-anti-IFNγ. Five hundred thousand events were collected for each sample by a flow cytometer. B; tumor-specific CD8+ T-cell target cell killing activities were detected by a non-radioactive cytotoxicity assay. PBS (open triangles), mIL-15 (open squares), mIL-15 + anti-PD-L1 (filled triangles) and mIL-15 + anti-PD-L1 + anti-CTLA4 (filled diamonds) groups are shown in panel B. The figures are representative of three independent experiments.

Figure 5. IL-15 treatment reduced the number of tumor nodules in the lungs

A; On day 21, pulmonary metastases were counted; each group included 3 to 5 mice, *P<0.05. The data represents three independent experiments. B; Representative lung samples showing pulmonary metastases at day 21.

Figure 6. IL-15 treatment with blockade of multiple inhibitory checkpoints prolonged the survival of CT26 tumor-bearing animals

Kaplan-Meier survival by treatment illustrating tumor bearing animal survival after the different treatments. PBS control (open diamonds), anti-CTLA-4 (open squares), mIL-15 and anti-CTLA-4 (grey squares), mIL-15 (black triangles), anti-PD-L1 (black diamond), mIL-15 and anti-PD-L1 (grey diamonds) and mIL-15 combined with anti-PD-L1 and anti-CTLA-4 (grey circles). The data represent three independent experiments.