Tbet and IL-36γ cooperate in therapeutic DC-mediated promotion of ectopic lymphoid organogenesis in the tumor microenvironment

Abstract

We have previously reported that direct injection of dendritic cells (DC) engineered to express the Type-1 transactivator Tbet (i.e., DC.Tbet) into murine tumors results in antitumor efficacy in association with the development of structures resembling tertiary lymphoid organs (TLO) in the tumor microenvironment (TME). These TLO contained robust infiltrates of B cells, DC, NK cells, and T cells in proximity to PNAd⁺ blood vessels; however, they were considered incomplete, since the recruited B cells failed to organize into classic germinal center-like structures. We now report that antitumor efficacy and TLO-inducing capacity of DC.Tbet-based i.t. therapy is operational in peripheral lymph node-deficient LTA^-/- mice, and that it is highly dependent upon a direct Tbet target gene product, IL-36γ/IL-1F9. Intratumoral DC.Tbet fails to provide protection to tumor-bearing IL-36R^-/- hosts, or to tumor-bearing wild-type recipient mice co-administered rmIL-1F5/IL-36RN, a natural IL-36R antagonist. Remarkably, the injection of tumors with DC engineered to secrete a bioactive form of mIL-36γ (DC.IL36γ) also initiated therapeutic TLO and slowed tumor progression in vivo. Furthermore, DC.IL36γ cells strongly upregulated their expression of Tbet, suggesting that Tbet and IL-36γ cooperate to reinforce each other's expression in DC, rendering them competent to promote TLO formation in an "immunologically normalized," therapeutic TME.

Keywords: Dendritic cells; immunotherapy; interleukin (IL)-36γ; tbet; tertiary lymphoid organ; tumor.

Figure 1.

Characterization of therapeutic DC.Tbet. Bone marrow-derived DC were generated in GM-CSF + IL-4 cultures for 5–6 d, before being infected for 48 h with rAd.mTbet or control rAd.EGFP or empty rAd.ψ5, or they were left untransduced, as indicated. In (A), DC were treated, as indicated, with the addition of LPS + IFNγ for the second 24 h of infection. Affymetrix gene array analyses were performed on DC.Tbet (A), with transcripts increased >5-fold compared with control DC reported. DC.Tbet generated from Tbet-ZsG (H-2^b) reporter mice were then analyzed by IFM for intracellular expression of the Tbet reporter (green) and IL-36γ (red), with DAPI staining of nuclei (B). DC.Tbet or control DC were cultured at 4 × 10⁵ cells/mL. After 48 h of infection, supernatant was harvested and mIL-12p70 (C) or mIL-36γ (D) production was analyzed by ELISA. **p < 0.05 for DC.Tbet vs. control DC (t-test). In E, DC.Tbet, control DC.ψ5, or PBS were then injected intratumorally into mice bearing d7 established s.c. MCA205 sarcomas on days 7 and 14 post-tumor inoculation. Tumor size was measured over time. **p < 0.05 for DC.Tbet vs. PBS or control DC.ψ5 treatment on days ≥ 11 (ANOVA). Data are representative of those obtained in two independent experiments performed in each case.

Figure 2.

DC.Tbet injected i.t. promote the rapid infiltration of lymphocytes and the development of PNAd⁺ blood vessels in association with enhanced locoregional induction of IL-36γ. MCA205 tumor-bearing wild-type C57BL/6 mice or Tbet-ZsG reporter mice were treated by i.t. delivery of 1 × 10⁶ DC.Tbet or DC.ψ5 7 d post-tumor inoculation. In (A), tumors were harvested from Tbet-ZsG mice at 4h, 10h or 24h after treatment with DC.Tbet and tissue sections analyzed by IFM for CD4⁺ T cells, CD8⁺ T cells, CD19⁺ B cells, and PNAd⁺ HEV. In (B) and (C), expression and localization of peripheral node addressin (PNAd), CD11c⁺ DC, CD3⁺ T cells, and B220⁺ B cells were analyzed in tumor sections from Tbet reporter mice on days 5 (B) and 12 (C) post-treatment with DC.Tbet. In (D), day 5 (post-DC.Tbet treatment) tumor sections from Tbet-ZsG mice were analyzed by IFM for co-expression of the Tbet reporter (green) and IL-36γ (red, using a specific polyclonal antibody). In (E), IL-36γ protein levels were assayed by quantification of fluorescence from IFM, or transcript levels were assayed by real-time PCR in total tumor RNA isolated from wild-type C57BL6/J hosts, at the indicated time points following DC.Tbet or DC.ψ5 treatment. Data are representative of those obtained in two to three independent experiments performed in each case.

Figure 3.

I.t.-delivered DC.Tbet mediates antitumor efficacy in secondary lymph node-deficient LTA^−/− mice that is both CD4⁺ and CD8⁺ T cell-dependent. In (A), lymphotoxin-α (LTA)^−/− mice bearing established day 7 MCA205 sarcomas were left untreated, or they were treated (D) on days 7 and 14 by i.t. injection of 10⁶ DC.Tbet or control DC.ψ5 cells alone, with tumor size (mean +/− SD from five mice/group) then monitored over time. In (B), this same model was left untreated, or treated on days 7 and 14 with DC.ψ5 or DC.Tbet cells (+/− systemic i.p. administration of depleting anti-CD4⁺ or anti-CD8⁺ mAbs) and time-to-euthanasia (as an index of overall survival) reported over time. **p < 0.05 for DC.Tbet vs. all other cohorts (ANOVA). Data are representative of those obtained in two independent experiments performed.

Figure 4.

The antitumor efficacy and TLO promoting activity associated with i.t. DC.Tbet-based therapy is ablated by the IL-36R antagonist IL-1F5 in vivo. WT C57BL6/J mice bearing established day 7 MCA205 sarcomas were left untreated, or they were treated on days 7 and 14 by i.t. injection of 10⁶ DC.Tbet or DC.ψ5 alone or with DC.Tbet plus co-injection (i.t.) of rmIL-1F5 (0.1 μg or 1 μg in 50 μL PBS) followed by (i.t.) injections of the respective doses of IL-1F5 alone in 50 μL PBS on days 8, 9, 15, and 16, with tumor growth (A) monitored over time (n = 5 mice/group). **p < 0.05 for DC.Tbet vs. all other cohorts (ANOVA) on days ≥ 17. In (B)and (C), tumors were harvested from the indicated treatment cohorts on day 25 post-tumor inoculation (i.e., 9 d following the final injection of rmIL-1F5) and analyzed by IFM for the presence of PNAd⁺ vessels (B) and CD4⁺ T cells and CD8⁺ T cells (C). Data are representative of three independent assays performed in each case.

Figure 5.

The antitumor efficacy and TLO promoting activity associated with i.t. DC.Tbet-based therapy is absent in the IL-36R^−/− recipient mice. 10⁵ MC38 colon carcinoma cells were injected into the flanks of syngenic wild-type C57BL/6 (A) mice and allowed to establish. Tumor-bearing mice were randomized into groups of five animals/cohort on day 7 post-implantation, with all cohorts exhibiting comparable mean tumor size. These animals were then left untreated, or were treated on days 7 and 14 by i.t. injection of 10⁶ control DC.EGFP or DC.Tbet/EGFP, with tumor growth subsequently monitored over time (n = 5 mice/group). **p < 0.05 for DC.Tbet vs. all other cohorts on days ≥ 13 for C57BL/6 recipients. In (B), the experiment from (A)was repeated, with the addition of co-treatment cohorts including LTβR-Ig (100 μg) or an isotype control antibody (100 μg), injected i.t. 3 h before each injection of DC.Tbet. *p < 0.05 for DC.Tbet + Iso-Ig vs. DC.EGFP on days ≥ 11; **p < 0.05 for DC.Tbet + LTβR-Ig vs. DC.EGFP on days 11–18 (NS on day 20); ***p < 0.05 for DC.Tbet + Iso-Ig vs. DC.Tbet + LTβR-Ig on day 20. In (C), tumors harvested 5 d following i.t. injections of DC.Tbet/EGFP or control DC.EGFP were cryosectioned and analyzed by IFM for the presence of PNAd⁺ HEV, CD3⁺ T cells, and CD11c⁺ DC (C). In (D), 10⁵ MC38 colon carcinoma cells were injected into the flanks of syngenic IL-36R^−/− and allowed to establish and were treated as described in (A). p = NS for DC.Tbet vs. control cohorts in IL-36R^−/− mice at all time points (ANOVA). In (E), day 18–21 tumors harvested from the indicated treatment cohorts were cryosectioned and then H&E stained as described in Materials and Methods. Robust TIL populations (C) and (E) were observed in cortical regions of MC38 tumors, only in DC.Tbet-treated C57BL/6 hosts. In (F)and (G), DC.Tbet/EGFP-treated tumors were harvested from wild-type C57BL6/J hosts at day 20 and analyzed by IFM for the presence of PNAd⁺ HEV, CD3⁺ T cells, and CD11c⁺ DC (F) or PNAd⁺ HEV, CD3⁺ T cells, and DC.Tbet/EGFP (G). Data are representative of those obtained inthree independent experiments performed in each case.

Figure 6.

DC.IL36γ produce/secrete bioactive IL-36γ and upregulate intrinsic transcription of Tbet. Real-time PCR (A) and Western blot analysis (B) for IL-36γ were performed on lysates of DC.IL36γ/EGFP vs. control DC.null or DC.EGFP to confirm transduction efficacy. In (C), Affymetrix gene array analyses were performed as outlined in Materials and Methods, with transcripts increased >5-fold compared in DC.IL36γ vs. control DC.null reported. In (D) and (E), real-time PCR (D) and Western blot analysis (E) for Tbet were performed on lysates of DC.IL36γ/EGFP and/or DC.Tbet/EGFP and/or control DC.EGFP and/or control DC.null. In (A) and (D), mean ± SD data are reported; **p < 0.05 (t-test). In (F), DC were differentiated for 5 d in vitro, with CD11c⁺ cells then isolated and either transduced with rAd to express EGFP, mTbet, and/or mIL-36γ, or untransfected control DC were treated for 24 h with the indicated TLR agonists or agonist anti-CD40 (FGK45) antibody. Cell-free supernatants were then analyzed by mIL-36γ ELISA. *p < 0.05 for DC.Tbet and DC.IL36γ vs. all other treatment groups (ANOVA). In (G), cell-free supernatants were recovered from engineered DC and analyzed for bioactivity by addition to cultures of bulk splenocytes isolated from Tbet (ZsG) reporter mice; i.e., 10⁶ splenocytes were cultured in 200 μL of basal media (negative control), media containing LPS + IFNγ (positive control) or cell-free media harvested from DC.null or DC.IL36γ cells 48 h after rAd infection. After overnight culture, splenocytes were analyzed by flow cytometry for upregulation of intracellular Tbet reporter expression. In (H), DC.IL36γ cells were analyzed by IFM as described in Fig. 1(B) to detect coordinate expression of Tbet and IL-36γ protein. All data are representative of those obtained in two to three independent experiments performed in each case.

Figure 7.

I.t. delivery of DC.IL36γ is therapeutic and promotes TLO in wild type, but not in IL-36R^−/−hosts. 10⁵ MC38 colon carcinoma cells were established s.c. in the flanks of syngenic wild-type C57BL/6 (A) mice. After randomization of the tumor-bearing animals on day 7 to cohorts (five mice/group) exhibiting comparable mean tumor sizes, mice were treated by i.t. injection with 10⁶ control DC.EGFP or DC.IL36γ/EGFP on days 7 and 14, and tumor growth monitored over time. **p < 0.05 for DC.IL36γ/EGFP vs. DC.EGFP on days ≥ 11 (ANOVA). After euthanasia on day 20 post-tumor inoculation, tumors were cryosectioned and analyzed by IFM for the presence of TLO based on the presence of PNAd⁺ HEV surrounded by CD3⁺ T cells, CD11c⁺ DC, and DC.IL36γ/EGFP in the tumor cortex (B). In C, the experiment performed in (A)was repeated in IL-36R^−/− host animals. NS for DC.IL36γ/EGFP vs. DC.EGFP at all time points (ANOVA). In (D), MC38 tumor sections harvested on d18 (IL-36R^−/− hosts) or d20 (WT C56BL/6J hosts) post-treatment with DC.IL36γ/EGFP or control DC.EGFP were then H&E stained to identify TIL in the cortical regions of the tumors. In (E), BMDC generated from wild-type or Tbet^−/− mice were infected with control rAd.EGFP or rAd.mIL36γ/EGFP and injected i.t. into established MC38 tumors in wild-type C57BL/six mice on days 7 and 14 and tumor growth (mean +/− SEM in mm²) was monitored over time. **p < 0.05 for WT or Tbet^−/− DC.IL36γ/EGFP vs. DC.EGFP on days ≥11; NS for WT vs. Tbet^−/− DC.IL36γ/EGFP at all time points (ANOVA). Data are representative of those obtained in two to three independent experiments performed in each case.