Natural Killer T-cell Immunotherapy in Combination with Chemotherapy-Induced Immunogenic Cell Death Targets Metastatic Breast Cancer

Abstract

Natural killer T (NKT) cells are glycolipid-reactive lymphocytes that promote cancer control. In previous studies, NKT-cell activation improved survival and antitumor immunity in a postsurgical mouse model of metastatic breast cancer. Herein, we investigated whether NKT-cell activation could be combined with chemotherapeutic agents to augment therapeutic outcomes. Gemcitabine and cyclophosphamide analogues enhanced the potential immunogenicity of 4T1 mammary carcinoma cells by increasing the expression of antigen-presenting molecules (MHC-I, MHC-II, and CD1d) and promoting exposure or release of immunogenic cell death markers (calreticulin, HMGB1, and ATP). In 4T1 primary tumor and postsurgical metastasis models, BALB/c mice were treated with cyclophosphamide or gemcitabine. NKT cells were then activated by transfer of dendritic cells loaded with the glycolipid antigen α-galactosylceramide (α-GalCer). Chemotherapeutic treatments did not impact NKT-cell activation but enhanced recruitment into primary tumors. Cyclophosphamide, gemcitabine, or α-GalCer-loaded dendritic cell monotherapies decreased tumor growth in the primary tumor model and reduced metastatic burden and prolonged survival in the metastasis model. Combining chemotherapeutics with NKT-cell activation therapy significantly enhanced survival, with surviving mice exhibiting attenuated tumor growth following a second tumor challenge. The frequency of myeloid-derived suppressor cells was reduced by gemcitabine, cyclophosphamide, or α-GalCer-loaded dendritic cell treatments; cyclophosphamide also reduced the frequency of regulatory T cells. Individual treatments increased immune cell activation, cytokine polarization, and cytotoxic responses, although these readouts were not enhanced further by combining therapies. These findings demonstrate that NKT-cell activation therapy can be combined with gemcitabine or cyclophosphamide to target tumor burden and enhance protection against tumor recurrence. .

The novel agonistic iNKT-cell antibody NKT14m induces a therapeutic antitumor response against B-cell lymphoma

“…These observations are in line with previous studies, which have demonstrated that cyclophosphamide does not induce any significant changes in the total numbers of these different cell subsets. 34 Although it was reported that cyclophosphamide has immunostimulatory activities 39,40 and can induce the activation of CD4 + and CD8 + T cells, including iNKT cells, 34 its administration alone in our B-cell lymphoma model did not have any antitumor effect (Figure 3(c)), in line with previous data. 34 The combination treatment with NKT14m antibody and cyclophosphamide resulted in a greater antitumor effect compared to either treatment alone (Figure 3(c)).…”

“…34 Although it was reported that cyclophosphamide has immunostimulatory activities 39,40 and can induce the activation of CD4 + and CD8 + T cells, including iNKT cells, 34 its administration alone in our B-cell lymphoma model did not have any antitumor effect (Figure 3(c)), in line with previous data. 34 The combination treatment with NKT14m antibody and cyclophosphamide resulted in a greater antitumor effect compared to either treatment alone (Figure 3(c)). In contrast to previous studies using α-GalCer, 34 iNKT-cell activation with NKT14m antibody seems to be more potent and its combination with a chemotherapeutic agent, such as cyclophosphamide, has a synergistic activity increasing IFN-γproducing iNKT and T cells (Figure 3(d)-(f)), and establishing a long-lasting memory (Figure 4(b)), suggesting that this combination could foster a coordinated antitumor response which translates into a better outcome.…”

“…34 The combination treatment with NKT14m antibody and cyclophosphamide resulted in a greater antitumor effect compared to either treatment alone (Figure 3(c)). In contrast to previous studies using α-GalCer, 34 iNKT-cell activation with NKT14m antibody seems to be more potent and its combination with a chemotherapeutic agent, such as cyclophosphamide, has a synergistic activity increasing IFN-γproducing iNKT and T cells (Figure 3(d)-(f)), and establishing a long-lasting memory (Figure 4(b)), suggesting that this combination could foster a coordinated antitumor response which translates into a better outcome.…”

“…In this setting, we did not observe any reduction in the proportion and total numbers of iNKT, CD4 + and CD8 + T cells in spleen of treated mice 24 hours after antibody administration (day 12), suggesting that Cy does not have any detrimental effect on these cell subsets (Figure 3(b) and supplemental figure S3), as other studies have shown. 34 In addition, toxicity was not observed in mice receiving cyclophosphamide (i.e., no changes in body weight, motility, behaviour and organ morphology). NKT14m combined with Cy significantly enhanced the antitumor effect observed with Cy alone (87% vs. 37% survival, respectively; Figure 2.…”

Immunooncology in Breast Cancer: Active and Passive Vaccination Strategies

“…However, in the last few years, several reports has been published about stem cell-derived NK cells that may have a better cytotoxic effect against tumor cells [36,37]. Furthermore, the combination with chemotherapy might lead to higher responses in metastatic breast cancer patients [38].…”