Augmented IL-15Rα expression by CD40 activation is critical in synergistic CD8 T cell-mediated antitumor activity of anti-CD40 antibody with IL-15 in TRAMP-C2 tumors in mice

Abstract

IL-15 has potential as an immunotherapeutic agent for cancer treatment because it is a critical factor for the proliferation and activation of NK and CD8(+) T cells. However, monotherapy of patients with malignancy with IL-15 that has been initiated may not be optimal, because of the limited expression of the private receptor, IL-15Rα. We demonstrated greater CD8 T cell-mediated therapeutic efficacy using a combination regimen of murine IL-15 administered with an agonistic anti-CD40 Ab (FGK4.5) that led to increased IL-15Rα expression on dendritic cells (DCs), as well as other cell types, in a syngeneic established TRAMP-C2 tumor model. Seventy to one hundred percent of TRAMP-C2 tumor-bearing wild-type C57BL/6 mice in the combination group manifested sustained remissions, whereas only 0-30% in the anti-CD40-alone group and none in the murine IL-15-alone group became tumor free (p < 0.001). However, the combination regimen showed less efficacy in TRAMP-C2 tumor-bearing IL-15Rα(-/-) mice than in wild-type mice. The combination regimen significantly increased the numbers of TRAMP-C2 tumor-specific SPAS-1/SNC9-H(8) tetramer(+)CD8(+) T cells, which were associated with the protection from tumor development on rechallenge with TRAMP-C2 tumor cells. Using an in vitro cytolytic assay that involved NK cells primed by wild-type or IL-15Rα(-/-) bone marrow-derived DCs, we demonstrated that the expression of IL-15Rα by DCs appeared to be required for optimal IL-15-induced NK priming and killing. These findings support the view that anti-CD40-mediated augmented IL-15Rα expression was critical in IL-15-associated sustained remissions observed in TRAMP-C2 tumor-bearing mice receiving combination therapy.

Figure 1. The combination therapy that involved mIL-15 and an agonistic anti-CD40 antibody led to the regression of established TRAMP-C2 tumors in wild type C57BL/6 mice

The therapy was started when the tumors were well established with an average volume of 80mm³. (A) Changes of tumor volumes during the therapeutic course and (B) Kaplan-Meier survival plot of the mice in one of the two therapeutic studies (n=9–10). Treatment with mIL-15 alone (○) at a dose of 2.5µg/mouse, 5 days a week for 2 weeks inhibited the tumor growth slightly and prolonged survival of the TRAMP-C2 tumor-bearing mice when compared with the mice in the PBS control group (♦) (p<0.05) while treatment with the anti-CD40 antibody (▲) at a dose of 200 µg/mouse on day 0, then 100 µg/mouse on days 3, 7 and 10 provided greater inhibition of the tumor growth and prolonged survival of the TRAMP-C2 tumor-bearing mice when compared with the mice in the PBS control or mIL-15 alone group (p<0.001). Furthermore, combination therapy (△) with both mIL-15 and the anti-CD40 antibody provided a greater therapeutic efficacy as demonstrated by the fact that all of the mice in the combination group were alive at day 60 with 80% becoming tumor free while only 20% of the mice in the anti-CD40 antibody alone group and none of the mice in the PBS control and mIL-15 alone groups were alive at that time. (C). Bioluminescence imaging of the TRAMP-C2/luc-GFP tumor-bearing mice confirmed efficacy of combination treatment. Groups of 6 mice bearing TRAMP-C2/luc-GFP tumors were treated using the same therapeutic protocol. The bioluminescence images were taken at different time points. Treatment with anti-CD40 antibody significantly delayed the tumor growth. However, at day 40 after initiation of the therapy, only one out of 6 mice in the anti-CD40 group was tumor free. In contrast, the combination treatment led to the regression of the tumors with all of the 6 mice in the group becoming and remaining tumor free.

Figure 2. TRAMP-C2 tumor-bearing IL-15Rα−/− mice showed reduced efficacy with the combination regime when compared to wild type mice

(A) Changes of tumor volumes during the therapeutic course and (B) Kaplan-Meier survival plot of the mice in one of the two therapeutic studies (n=10). The protocol for this study was the same as that in Figure1. Treatment with mIL-15 alone (○) showed very little therapeutic efficacy in the TRAMP-C2 tumor-bearing IL-15Rα −/− mice. Treatment with the anti-CD40 antibody (▲) significantly delayed the tumor growth and prolonged the survival of the TRAMP-C2 tumor-bearing mice when compared with the mice in either PBS control (♦) or mIL-15 alone group (p<0.001). Furthermore, combination therapy (△) with both mIL-15 and the anti-CD40 antibody provided a greater therapeutic efficacy as seen by the prolonged survival with 20% of the mice becoming tumor free whereas none of the mice in other groups was tumor free (p<0.001). The results were from one of two experiments.

Figure 3. CD8⁺ T-cells played a major role in the combination regimen mediated antitumor activity in the TRAMP-C2 model

TRAMP-C2 tumor-bearing mice were treated with anti-asialo-GM1 (50 µL) or purified rat anti-mouse CD8 antibody (200 µg/injection), together with the combination regimen of mIL-15 and the anti-CD40 antibody. (A) Representative FACS analysis of PBMC from the mice at day 19 after initiation of the therapy. The results showed that the addition of an anti-CD8 antibody was associated with the depletion of more than 90% of CD8⁺ cells. Administration of anti-asialo-GM1 reduced the number of CD8⁺ cells, especially CD44^highCD8⁺ and TRAMP-C2 tumor specific SPAS-1/SNC9-H₈ tetramer⁺CD8⁺ cells. (B) Kaplan-Meier survival plot of the mice. Compared with the group (○) receiving the combination therapy alone, depletion of CD8⁺ T-cells (▲) with the anti-CD8 antibody in association with combination treatment nearly abrogated the antitumor efficacy (p<0.001) and administration of anti-asialo-GM1 (△) reduced the antitumor activity (p<0.001). The experiment was repeated and the results of the two experiments were pooled together.

Figure 4. The combination of mIL-15 with the anti-CD40 antibody led to a significant increase in the absolute numbers of TRAMP-C2 tumor specific SPAS-1/SNC9-H₈ tetramer⁺CD8⁺ cells in the spleens of TRAMP-C2 tumor-bearing wild type mice

TRAMP-C2 tumor-bearing mice were treated using the same therapeutic protocol as used in Figure 1. At day 12 after initiation of the therapy, all of the mice (4 mice/group) were sacrificed and spleens were taken. Splenocytes were separated and counted. The percentages of CD8⁺, CD44^highCD8⁺, SPAS-1/SNC9-H₈ tetramer⁺CD8⁺ were analyzed by flow cytometry. (A) Total cell numbers per spleen. (B) Absolute numbers of CD8⁺ cells per spleen. (C) Absolute numbers of CD44^highCD8⁺ cells per spleen. (D) Absolute numbers of SPAS-1/SNC9-H₈ tetramer⁺CD8⁺ cells per spleen. Treatment with mIL-15 or anti-CD40 antibody or their combination increased the total cell numbers and the absolute numbers of CD8⁺ cells and CD44^highCD8⁺ cells in the spleen when compared with the control group (p<0.05). Critically, only combination regimen significantly increased the numbers of SPAS-1/SNC9-H₈ tetramer⁺CD8⁺ cells when compared with all other groups (p<0.05).

Figure 5. Enhanced TRAMP-C2 tumor specific SPAS-1/SNC9-H₈ tetramer⁺CD8⁺ T-cells were associated with protection from a rechallenge with TRAMP-C2 tumor cells

(A) Kaplan-Meier survival plot of mice that received rechallenge of TRAMP-C2 cells at day 40 after initiation of the original therapy. When compared with control mice (♦) which did not receive TRAMP-C2 cells and any treatment previously, mice surviving the first challenge with TRAMP-C2 cells (○) demonstrated resistance to TRAMP-C2 tumor development (p<0.001). Depletion of CD8 cells (▲) nearly abrogated protection mediated by combination therapy (p<0.01). (B) Kaplan-Meier survival plot of mice that received rechallenge of MC38 cells at day 40 after original therapy. Mice surviving the challenge with TRAMP-C2 cells did not show resistance to an irrelevant tumor, MC38, development (p>0.5). (C) Representative FACS analysis of PBMC from mice just before rechallenge. Mice that initially received the TRAMP-C2 tumor that became and remained tumor free after receiving the combination regimen manifested an increase in the percentages of CD8⁺, CD44^highCD8⁺ T cells, especially the SPAS-1/SNC9-H₈ tetramer⁺CD8⁺ T-cells when compared with mice in the control group that had not received either the TRAMP-C2 tumor cells or any treatment previously. (D) Bioluminescence images. Surviving tumor free mice in the combination group from Fig. 1C were rechallenged with TRAMP-C2/luc-GFP cells at day 40 after initiation of the therapy. The mice surviving the first challenge with TRAMP-C2/luc-GFP cells demonstrated resistance to the tumor development when compared with the mice in the control group.

Figure 6. CD40 activation induced IL-15Rα expression and increased levels of IL-15Rα on the BMDCs appear critical for NK cell lytic activity

(A) Histograms of flow cytometry analysis of IL-15Rα expression on cell surfaces. C57BL/6 mice were treated with PBS or with the anti-CD40 antibody at a dose of 200µg on day 0, followed by 100µg on days 3. At day 5, the splenocytes were separated from the mice for flow cytometry analysis. Treatment with the anti-CD40 antibody increased the expression of IL-15Rα (solid line) on all cell types tested (CD19+, CD11b+ and CD3+CD8+ cells) when compared with that observed in the mice treated with PBS (grey shade). (B) Cytolytic activities of NK cells primed by wild type or IL-15Rα−/− BMDCs stimulated by LPS, anti-CD40 or medium alone. Compared with BMDCs cultured with medium, wild type BMDCs stimulated with LPS or anti-CD40 antibody enhanced cytolytic activity of NK cells while IL-15Rα−/− comparably stimulated BMDCs did not. (C) Cytolytic activities of NK cells cocultured with wild type BMDCs in presence or absence of anti-CD40 antibody or mIL-15 or their combination. With anti-CD40 antibody or mIL-15, NK cells showed enhanced cytolytic activity toward target cells when compared with NK cells cocultured with BMDCs in medium alone. Furthermore, with both anti-CD40 antibody and mIL-15, NK cells showed greatest cytolytic activity toward target cells when compared with NK cells in all other cocultures. (D) Cytolytic activities of NK cells cocultured with IL-15Rα−/− BMDCs in presence or absence of anti-CD40 antibody or mIL-15 or their combination. In contrast to wild type BMDCs, NK cells cocultured with IL-15Rα−/− BMDCs in presence of anti-CD40 antibody did not show increased lysis activity toward target cells when compared with NK cells cocultured with IL-15Rα−/− BMDCs in medium alone. With both anti-CD40 antibody and mIL-15, NK cells only showed a comparably increased lysis activity toward target cells as NK cells cocultured with IL-15Rα−/− BMDCs in presence of mIL-15 alone, suggesting that expression of IL-15Rα by BMDCs is critical for the synergetic effect of the combination regimen on activation of NK cells.