STAT3 Inhibits CD103+ cDC1 Vaccine Efficacy in Murine Breast Cancer

Abstract

Conventional dendritic cells (cDCs) are a critical immune population, composed of multiple subsets, and responsible for controlling adaptive immunity and tolerance. Although migratory type 1 cDCs (CD103⁺ cDC1s in mice) are necessary to mount CD8⁺ T cell-mediated anti-tumor immunity, whether and how tumors modulate CD103⁺ cDC1 function remain understudied. Signal Transducer and Activator of Transcription 3 (STAT3) mediates the intracellular signaling of tumor-associated immunosuppressive cytokines, such as interleukin (IL)-10; thus, we hypothesized that STAT3 restrained anti-tumor immune responses elicited by CD103⁺ cDC1s. Herein, we show that in vitro-derived STAT3-deficient (Stat3^∆/∆) CD103⁺ cDC1s are refractory to the inhibitory effects of IL-10 on Toll-like receptor 3 (TLR3) agonist-induced maturation responses. In a tumor vaccination approach, we found Stat3^∆/∆ CD103⁺ cDC1s restrained mammary gland tumor growth and increased mouse survival more effectively than STAT3-sufficient CD103⁺ cDC1s. In addition, vaccination with Stat3^∆/∆ CD103⁺ cDC1s elicited increased amounts of tumor antigen-specific CD8⁺ T cells and IFN-γ⁺ CD4⁺ T cells in tumors and tumor-draining lymph nodes versus phosphate-buffered saline (PBS)-treated animals. Furthermore, IL-10 receptor-deficient CD103⁺ cDC1s controlled tumor growth to a similar degree as Stat3^∆/∆ CD103⁺ cDC1s. Taken together, our data reveal an inhibitory role for STAT3 in CD103⁺ cDC1 maturation and regulation of anti-tumor immunity. Our results also suggest IL-10 is a key factor eliciting immunosuppressive STAT3 signaling in CD103⁺ cDC1s in breast cancer. Thus, inhibition of STAT3 in cDC1s may provide an important strategy to improve their efficacy in tumor vaccination approaches and cDC1-mediated control of anti-tumor immunity.

Keywords: CD103+ dendritic cells; STAT3; breast cancer; cDC1; immunosuppression; immunotherapy; tumor vaccine.

Figure 1

Evaluation of Signal Transducer and Activator of Transcription 3 (STAT3) function in CD103⁺ type 1 conventional dendritic cell (cDC1) maturation. In vitro-generated, fluorescence activated cell sorting (FACS)-purified Stat3-sufficient (Stat3^fl/fl) or -deficient (Stat3^∆/∆) CD103⁺ cDC1s were stimulated with or without 10 ng/mL murine interleukin (IL)-10, 20 μg/mL polyinosinic:polycytidylic acid (poly I:C), or 10 ng/mL IL-10 and 20 μg/mL poly I:C for 16 hours (h), as indicated. (A) Representative flow cytometry plots (left) and cumulative data (right) of cell surface expression of co-stimulatory markers on CD103⁺ cDC1s following the indicated treatment. n = 8 (Stat3^fl/fl), n = 9 (Stat3^∆/∆) for all conditions, combined from 4 independent experiments. (B) Mean cytokine and chemokine abundance in cell culture supernatants from (A), determined by multiplex analysis; cytokines and chemokines that were not detected within observable limits are not shown. n = 4 (phosphate buffered saline, PBS), n = 5 (IL-10), n = 6 (poly I:C), n = 6 (poly I:C + IL-10) for both genotypes, combined from 4 independent experiments. Data shown in panel (A) (right) represent mean ± S.E.M. (A) results analyzed by two-way ANOVA and Bonferroni’s multiple comparison test, comparing all treatments within each genotype, and comparing the two genotypes within a given treatment. Results were considered significant when p < 0.05. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 2

Efficacy of Stat3-deficient CD103⁺ cDC1 vaccine in murine breast cancer. Mice bearing bilateral Polyomavirus middle T-antigen (PyMT) tumors, engineered to overexpress ovalbumin (OVA, PyMT-OVA) received intratumoral (i.t.) injection of PBS, the Stat3^fl/fl CD103⁺ cDC1 vaccine, or the Stat3^∆/∆ CD103⁺ cDC1 vaccine in right side tumors only, 7 days (d) after tumor establishment. (A) Representative mean tumor size (mm²) of treated (T) and non-treated (NT) tumors, from one of two independent experiments. (B) Individual tumor sizes (mm²) from (A). (A,B) n = 7 (PBS), n = 7 (Stat3^fl/fl), n = 8 (Stat3^∆/∆). (C) Cumulative mouse survival from two independent experiments. n = 14 (PBS), n = 14 (Stat3^fl/fl), n = 16 (Stat3^∆/∆). (D) Tumor mass (mg) on the indicated day following i.t. PBS or CD103⁺ cDC1 vaccine injection; cumulative results from three independent experiments shown. Left panels, day 4; n = 15 (PBS T, NT; Stat3^fl/fl T, NT; Stat3^∆/∆ NT), n = 16 (Stat3^∆/∆ T). Right panels, day 10; n = 14 (PBS T, NT), n = 15 (Stat3^fl/fl T, NT; Stat3^∆/∆ T, NT). (A,D) Data shown are the mean ± S.E.M. Results analyzed by two-way ANOVA (A) or one-way ANOVA and Tukey’s multiple comparisons test (D). (C) Data analyzed by log-rank (Mantel–Cox) test. Results were considered significant when p < 0.05. * p < 0.05; *** p < 0.001; **** p < 0.0001.

Figure 3

Maturation and migration of Stat3-deficient CD103⁺ cDC1s after intratumoral injection. Bilateral PyMT-OVA tumor-bearing CD45.1⁺ mice received i.t. injection of PBS, the CD45.2⁺ Stat3^fl/fl CD103⁺ cDC1 vaccine, or the CD45.2⁺ Stat3^∆/∆ CD103⁺ cDC1 vaccine in right side tumors only, 7 d after tumor establishment. The vaccine-derived CD45.2⁺ CD103⁺ cDC1s were identified in tumors and tumor-draining lymph nodes (TdLNs) by analysis of CD45.2⁺ cells 40 h after i.t. injection. (A) Cumulative and (B) representative cell surface expression of the indicated co-stimulatory molecules on vaccine-derived CD45.2⁺ CD103⁺ cDC1s. (C) Number of vaccine-derived CD45.2⁺ CD103⁺ cDC1s in tumors and TdLNs, as indicated. (D) Representative flow cytometry plots showing vaccine-derived CD103⁺ cDC1s as a percentage of live cells. (A,C) Data shown are the mean ± S.E.M. from two independent experiments. n = 14 (Stat3^fl/fl tumor, TdLN; Stat3^∆/∆ tumor), n = 13 (Stat3^∆/∆ TdLN). Data analyzed by unpaired, two-tailed t test. Results were considered significant when p < 0.05. * p < 0.05.

Figure 4

T cell responses after vaccination with Stat3-deficient CD103⁺ cDC1s. T cell subsets were analyzed in tumors and TdLNs 4 d and 10 d following i.t. vaccination of mice bearing bilateral PyMT-OVA tumors with Stat3^fl/fl CD103⁺ cDC1s, Stat3^∆/∆ CD103⁺ cDC1s or PBS. (A) Cumulative amounts and (B) representative flow cytometry plots of OVA-specific CD8⁺ T cells (identified by SIINFEKL/H2-Kb pentamer staining) (CD45⁺ CD3⁺ CD4⁻ CD8⁺ Pentamer⁺) within tumors and TdLNs on 4 d and 10 d following vaccination, as indicated. Data shown are from two independent experiments (A). Tumors, d 4, n = 11 for all groups; TdLNs, d 4, n = 11 (Stat3^fl/fI T, NT; Stat3^∆/∆ T; PBS NT), n = 10 (PBS T, Stat3^∆/∆ NT). Tumors, d 10, n = 10 (PBS T, NT; Stat3^fl/fI NT), n = 11 (Stat3^fl/fl T), n = 7 (Stat3^∆/∆ T), n = 8 (Stat3^∆/∆ NT). TdLNs, d 10, n= 10 (PBS T, NT), n = 11 (Stat3^fl/fl T, NT; Stat3^∆/∆ T, NT). (C) Cumulative amounts and (D) representative flow cytometry plots of IFN-γ⁺ CD4⁺ T cells (CD45⁺ CD3⁺ CD8⁻ CD4⁺ IFN-γ⁺) within tumors and TdLNs on 4 d and 10 d following vaccination, as indicated. Data shown are from three independent experiments (C). Tumors, d 4, n = 15 (PBS T, NT; Stat3^fl/fl NT; Stat3^∆/∆ T, NT), n = 16 (Stat3^fl/fl T). TdLNs, d 4, n = 15 for all groups. Tumors, d 10, n = 14 (PBS T, NT), n = 15 (Stat3^fl/fl T), n = 11 (Stat3^∆/∆ T), n = 13 (Stat3^fl/fl NT), n = 12 (Stat3^∆/∆ NT). TdLNs, d 10, n = 13 (PBS T), n = 14 (PBS NT), n = 15 (Stat3^fl/fl T, NT; Stat3^∆/∆ T, NT) (A,C) Data shown are the mean ± S.E.M. Data analyzed by one-way ANOVA and Tukey’s multiple comparisons test. Results were considered significant when p < 0.05. * p < 0.05; ** p < 0.01.

Figure 5

Efficacy of IL-10 receptor β subunit-deficient (Il10rb^−/−) CD103⁺ cDC1 vaccine in murine breast cancer. (A) Cytokine amounts in PyMT-OVA culture supernatant determined by multiplex analyses. Data are from three independent experiments, n = 3. (B,C) Bilateral PyMT-OVA tumor-bearing mice received i.t. injection of PBS, the Stat3^fl/fl CD103⁺ cDC1 vaccine, the Stat3^∆/∆ CD103⁺ cDC1 vaccine, or the Il10rb^−/− CD103⁺ cDC1 vaccine in right side tumors only, 7 d after tumor establishment. (B) Representative mean tumor size (mm²) from one of two independent experiments. (C) Individual tumor sizes from (B), n = 7 (PBS, Il10rb^−/−), n = 9 (Stat3^fl/fl, Stat3^∆/∆). (A,B) Data shown are the mean ± S.E.M. (B) Data analyzed by two-way ANOVA and Tukey’s multiple comparisons test. Results were considered significant when p < 0.05. **** p < 0.0001.