IFI16/IFI202 blockade suppresses tumor growth through CD8+ T-cell-mediated immunity

Abstract

Background: Tumor-associated macrophages (TAMs) are key drivers of immunosuppressive inflammation in the breast cancer microenvironment, promoting tumor progression and resistance to immune checkpoint blockade. Our previous work identified extracellular IFI16/IFI202 as tumor-derived damage-associated molecular patterns which activate pro-inflammatory signaling in TAMs via Toll-like receptor 2, thereby facilitating immune evasion. In this study, we investigated the therapeutic potential of a monoclonal antibody (mAb) targeting extracellular IFI202 to suppress tumor-promoting inflammation and to restore antitumor immunity.

Methods: We developed a mAb against IFI202 and evaluated its functions in bone marrow-derived macrophages (BMDMs) using ELISA and western blotting. In vivo efficacy was assessed in the mouse mammary tumor virus-polyoma virus middle T-antigen breast cancer model by treating with IFI202 mAb, or anti–programmed death-1 (PD-1) antibody, as monotherapies or in combination. Tumor volume, metastasis, cytokine levels, and immune cell infiltration were analyzed. Statistical significance was assessed using Mann–Whitney U test or ANOVA, with P < 0.05 considered significant.

Results: Conditioned medium obtained from 4T1 breast cancer cells pre-incubated with IFI202 mAb suppressed secretion of IL-6 and TNF-α, and inhibited activation of ERK and NF-κB in BMDMs. Intraperitoneal injection of IFI202 mAb in mouse mammary tumor virus-polyoma middle T-antigen mice significantly reduced tumor growth and lung metastasis. In addition, IL-1β expression, CD8⁺ T cell infiltration, and levels of granzyme B and interferon-γ, were enhanced in the tumors of IFI202 mAb-treated mice, indicating that IFI202 mAb restored cytotoxic function of CD8⁺ T-cells. Combination of IFI202 mAb with PD-1 mAb significantly improved antitumor efficacy compared to monotherapy.

Conclusions: Neutralization of extracellular IFI202 suppresses TAM-mediated inflammation and supports a tumor microenvironment favorable for T-cell–mediated immunity. In combination with anti–PD-1 therapy, IFI202 mAb further enhances antitumor responses, suggesting a promising and tumor-selective strategy that may help overcome resistance to immune checkpoint blockade in breast cancer.

Supplementary Information: The online version contains supplementary material available at 10.1186/s13058-025-02213-4.

Keywords: Breast cancer; CD8+ t cells; IFI202; Neutralizing antibody; Tumor-associated macrophages.

Fig. 1

Function of IFI202 mAb in suppression of IL-6 production in BMDMs. A The inhibitory effect of IFI202 mAb on IL-6 induction was examined by a neutralization assay. rIFI202 protein (200 ng/mL) was pre-treated with increasing concentrations of IFI202 mAb for 1 h and then applied to culture of BMDMs that had been differentiated for 7 days. After 24 h of incubation, cell supernatants were collected and IL-6 levels were quantified by ELISA. Data are presented as mean ± SD (n = 3). The neutralization curve was fitted using a four-parameter logistic model. B CM was collected from 4T1 breast cancer cells cultured for 48 h. The concentration of IFI202 in the 4T1-CM was 27.5 ± 1.9 pg/mL, as determined by ELISA. The CM was then pre-treated with either control IgG (5 µg/mL) or IFI202 mAb (5–10 µg/mL) for 1 h, and subsequently applied to BMDMs that had been differentiated for 7 days. After 24 h of incubation, BMDM-CM was collected and analyzed for cytokine levels by ELISA. Data are presented as mean ± SD (n = 3). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. **P < 0.01, *P < 0.05 vs. 4T1-CM treated with IgG; ^###P < 0.001 vs. 4T1-CM treated with 5 µg/mL IFI202 mAb. C Schematic representation of the experiment (top). 4T1-CM (middle) or rIFI202 protein (200 ng/mL) (bottom) was incubated with either control IgG (10 µg/mL) or IFI202 mAb (10 µg/mL) for 1 h, and then applied to BMDMs for the indicated time period. Expression levels of pERK/ERK and pp65/p65 were assessed by western blotting. Quantification of band intensities from three independent experiments is shown. Data are presented as mean ± SD (n = 3). Statistical significance was determined by two-way ANOVA with Bonferroni post hoc tests. ***P < 0.001, **P < 0.01, *P < 0.05 vs. 4T1-CM or rIFI202 treated with IgG of each time point; ^###P < 0.001 vs. 4T1-CM or rIFI202 treated with IgG

Fig. 2

IFI202 mAb inhibits tumor growth and metastasis in MMTV-PyMT mice. A Experimental scheme for antibody treatment. Nine-week-old MMTV-PyMT female mice were received intraperitoneal injection of either control IgG (100 µg/mL) or IFI202 mAb (100 µg/mL) three times per week for 4 weeks (left). Tumor volumes were measured by caliper (middle). Data are presented as mean ± SEM (n = 5). Statistical significance was determined by two-way ANOVA with Bonferroni post hoc tests. ***P < 0.001, *P < 0.05 vs. IgG-treated mice of each time point; ^###P < 0.001 vs. IgG-treated mice. At the endpoint, total tumor weight was measured (right). Data are presented as mean ± SEM (n = 5). Statistical significance was determined by Mann–Whitney U test. **P < 0.01 vs. IgG-treated mice. B Expression levels of pERK/ERK and pp65/p65 in tumors were assessed by western blotting (left). Quantification of the band intensity is shown (right). Data are presented as mean ± SEM (n = 5). Statistical significance was determined by Mann–Whitney U test. *P < 0.05 vs. IgG-treated mice. C Representative hematoxylin and eosin-stained images of lung sections showing metastatic lesions (left). Quantification of metastatic area and number of metastatic nodules was performed using ImageJ software (right). Data are presented as mean ± SEM (n = 5). Statistical significance was determined by Mann–Whitney U test. **P < 0.01 vs. IgG-treated mice. Scale bar, 2 mm. D Experimental scheme for antibody treatment. Five-week-old female BALB/c mice were inoculated with 4T1 cells, and once the tumors became palpable, they received intraperitoneal injections of either control IgG (100 µg/mL) or IFI202 mAb (100 µg/mL) three times per week for 12 days (left). Tumor volumes were measured by caliper (middle). Data are presented as mean ± SEM (n = 7–8). Statistical significance was determined by two-way ANOVA with Bonferroni post hoc tests. ***P < 0.001 vs. IgG-treated mice of each time point; ^###P < 0.001 vs. IgG-treated mice. At the endpoint, total tumor weight was measured (right). Data are presented as mean ± SEM (n = 7–8). Statistical significance was determined by Mann–Whitney U test. ***P < 0.01 vs. IgG-treated mice

Fig. 3

IFI202 mAb treatment promotes CD8⁺ T cell activation in vivo. Immunohistochemistry was performed to assess expression levels of IL-1β, CD8, granzyme B, and IFN-γ in mammary tumor tissues as shown in Fig. 2. Representative immunohistochemistry images of tumor sections are shown (top). Staining intensity was quantified from three images per tumor section using ImageJ software (bottom). Data are presented as mean ± SEM (n = 5). Statistical significance was determined by Mann–Whitney U test. **P < 0.01 and *P < 0.05 vs. IgG-treated mice. Scale bar, 50 μm

Fig. 4

IFI202 mAb enhances the PD-1 mAb-induced tumor suppression. A Experimental scheme for antibody treatment. Nine-week-old MMTV-PyMT female mice received intraperitoneal injection of either IgG (100 µg/mL), IFI202 mAb (50 µg/mL), PD-1 mAb (50 µg/mL), or a combination of IFI202 mAb and PD-1 mAb (each at 50 µg/mL), three times per week for 5 weeks (left). Tumor volumes were measured by caliper (middle). Data are presented as mean ± SEM (n = 4–6). Statistical significance was determined by two-way ANOVA. ***P < 0.001 vs. IgG-treated mice; ^###P < 0.001 and ^#P < 0.05 vs. IFI202 mAb-treated mice; ⁺⁺⁺P < 0.001 vs. PD-1 mAb-treated mice. At the endpoint, total tumor weight was measured (right). Data are presented as mean ± SEM (n = 4–6). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. ***P < 0.001 vs. IgG-treated mice; ^###P < 0.001, ^##P < 0.01 vs. IFI202 mAb-treated mice; ⁺⁺⁺P < 0.001 vs. PD-1 mAb-treated mice. B Expression levels of IL-1β, IL-6, IFN-γ, and granzyme B in tumors were assessed by western blotting (top). Quantification of the band intensity is shown (bottom). Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. ***P < 0.001, **P < 0.01, *P < 0.05 vs. IgG-treated mice; ^###P < 0.001, ^##P < 0.01, ^#P < 0.05 vs. IFI202 mAb-treated mice; ⁺P < 0.05, ⁺⁺⁺P < 0.001 vs. PD-1 mAb-treated mice