IFN-γ-dependent tumor-antigen cross-presentation by lymphatic endothelial cells promotes their killing by T cells and inhibits metastasis

Abstract

Tumor-associated lymphatic vessels promote metastasis and regulate antitumor immune responses. Here, we assessed the impact of cytotoxic T cells on the local lymphatic vasculature and concomitant tumor dissemination during an antitumor response. Interferon-γ (IFN-γ) released by effector T cells enhanced the expression of immunosuppressive markers by tumor-associated lymphatic endothelial cells (LECs). However, at higher effector T cell densities within the tumor, T cell-based immunotherapies induced LEC apoptosis and decreased tumor lymphatic vessel density. As a consequence, lymphatic flow was impaired, and lymph node metastasis was reduced. Mechanistically, T cell-mediated tumor cell death induced the release of tumor antigens and cross-presentation by tumor LECs, resulting in antigen-specific LEC killing by T cells. When LECs lacked the IFN-γ receptor expression, LEC killing was abrogated, indicating that IFN-γ is indispensable for reducing tumor-associated lymphatic vessel density and drainage. This study provides insight into how cytotoxic T cells modulate tumor lymphatic vessels and may help to improve immunotherapeutic protocols.

Fig. 1.. Boosting tumor-specific T cell responses promotes tumor LEC apoptosis.

(A to E) C57BL/6 mice were injected or not with in vitro activated CD8⁺ OT-1 CD45.1⁺ cells (1 × 10⁶ cells) 9 days after B16F10-OVA⁺VC⁺ inoculation. (A) Tumor growth. (B) LVD (number of LECs per grams of tumor, normalized to control group) and (C) frequency and median of cleaved casp-3⁺ (casp-3) LECs (CD45^neg CD31⁺ GP38⁺) in tumors at day 14 by flow cytometry. Graphs represent a pool of two experiments with 10 to 18 mice per group. (D and E) Representative immunofluorescent labeled tumor sections stained against OT-1 (CD45.1; green), LECs (Lyve-1; red), cleaved casp-3 (c-casp-3) (white), and merge without (−) or upon OT-1 transfer at day 4 after transfer. (D) LVD measured as Lyve-1⁺ cell counts per square millimeter of tumor and relative tumor LEC distribution in individual tumors (T1 to T4). (E) LEC cleaved casp-3 activity [mean fluorescence intensity (MFI)] per tumor and per individual tumor LECs. (F to H) C57BL/6 mice were vaccinated or not with CpG-B + OVA at day 5 after B16F10-OVA⁺VC⁺ inoculation. (F) Tumor growth. (G) LVD and (H) frequency and median of cleaved casp-3⁺ LECs in tumors at day 12 by flow cytometry. Graphs represent a pool of two experiments with 10 to 11 mice per group. (A and F) Two-way analysis of variance (ANOVA) test; (B to E, G, and H) Mann-Whitney test. Error bars show means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2.. Positive correlation between intratumoral CTL densities and tumor LEC killing.

(A to C) C57BL/6 mice were injected or not with different doses of in vitro activated OT-1 cells (0.05 × 10⁶ or 0.5 × 10⁶ cells) 9 days after B16-OVA-VC inoculation and euthanized 3 or 5 days later. (A) Tumor size, (B) frequency of cleaved casp-3⁺ tumor LECs, and (C) LEC density 3 and 5 days after injection. Data represent a pool of three experiments (A and B) with N = 16 to 17 mice per group and one experiment (C) with 5 to 6 mice per group. (D to F) Correlations between (D) OT-1, (E) OT-1–producing IFN-γ, and (F) OT-1–producing granzyme-B densities and the frequency of cleaved casp-3⁺ tumor LECs 5 days after transfer of 0.5 × 10⁶ activated OT-1 cells. Data represent a pool of six experiments, N = 59 mice. (A to C) One-way ANOVA test; (D to F) linear regression. Error bars show means ± SEM. N.S., not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.. Tumor LV drainage functions are impaired upon adoptive T cell transfer.

(A and B) C57BL/6 mice were injected or not with different doses of in vitro activated OT-1 cells (0.05 × 10⁶ or 0.5 × 10⁶ cells) 9 days after B16F10-OVA⁺VC⁺ inoculation and euthanized 5 days later. FITC-labeled beads (0.5 mm) were injected intratumorally 24 hours before euthanasia. Frequencies of (A) FITC beads⁺ DCs (CD11c⁺MHCII⁺) and (B) total DCs in TdLNs (pool of inguinal and axillary LN) by flow cytometry. Data represent two pooled experiments, N = 10 to 11 mice per group. (C) C57BL/6 mice were inoculated with B16F10-OVA⁺VC⁺ and were injected or not with 0.5 × 10⁶ in vitro activated OT-1 cells 9 days later. CTV-labeled B16F10-VC⁺ tumor cells were injected peritumorally at day 13, and mice were euthanized at day 14. Frequencies of GP38⁺ CTV⁺ cells in the TdLNs (inguinal and axillary). Data represent a pool of three experiments, N = 14 to 15 mice per group. (A and B) One-way ANOVA; (C) Mann-Whitney statistical test. Error bars show means ± SEM. *P < 0.05, **P < 0.01.

Fig. 4.. IFN-γR signaling in LECs is indispensable to ATT-mediated tumor LV destruction.

(A) Lyve-1 staining of ndLN (non dLN) sections from IFN-γR^WT and IFN-γR^Δprox-1 mice 4 weeks after tamoxifen treatment. (B to F) Tamoxifen-treated IFN-γR^WT and IFN-γR^Δprox-1 mice were injected or not with 0.5 × 10⁶ in vitro activated OT-1 cells 8 days after B16F10-OVA⁺VC⁺ inoculation. (B) Tumor growth. Data are representative of three experiments. (C) Tumor LEC density and (D) frequency of cleaved casp-3⁺ tumor LEC 5 days after T cell injection. Data represent two pooled experiments, N = 6 to 10 mice per group. (E) Black color intensity analysis of inguinal, axillary, and brachial TdLN from indicated mice 6 days after OT-1 transfer. Pictures show representative images, and histograms show the quantification of the intensity for individual LNs. Data are pooled from three experiments, N = 16 mice per group. (F) Gp100 staining of inguinal, axillary, and brachial TdLNs from indicated mice 9 days after OT-1 transfer. Pictures show representative images, and histograms show the quantification of Gp100⁺ cells in LNs. Data are from six mice per group. (A, C, and D) Mann-Whitney statistical test; (B) two-way ANOVA test; (E) one-way ANOVA test. Error bars show means ± SEM.*P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 5.. CTLs induce LEC apoptosis through antigen-specific interactions.

(A) LEC/FRC cultures were loaded or not with OVA peptide (OVA) and incubated with either different ratios of in vitro activated OT-1 cells (1:10, 1:3, and 3:1 OT1:LEC/FRC) or soluble IFN-γ. Frequencies of cleaved casp-3⁺ LECs after 14 hours. Data are representative of two independent experiments. Each point represents a technical replicate. (B) WT (LC3-GFP⁺) or β₂m^KO (GFP^neg) LEC/FRC cultures were loaded or not with OVA and incubated or not with anti–IFN-γ antibodies and granzyme-B inhibitor. In vitro activated OT-1 cells were added in the cultures and frequencies of cleaved casp-3⁺ WT and β₂m^KO LECs (gated on CD45^negCD31⁺gp38⁺ cells) 14 hours later. Data are from one experiment. (C) Sorted WT (LC3-GFP⁺) and β₂m^KO (GFP^neg) LEC cocultures (ratio of 1:1) were loaded with OVA and in vitro activated OT-1 cells were added (OT-1:LEC ratio of 10:1), and time-lapse imaging was performed (see movie S1). Representative images at indicated time points of bright field, all LECs, WT LC3-GFP⁺ LECs, and cleaved casp-3 activity. The arrows indicate one example of β₂m^KO (GFP^neg) LECs. Graphs represent the relative MFI of cleaved casp-3 substrate (left y axis) and the relative frequency of OT-1 touching LECs (right y axis) in WT and β₂m^KO LECs. Data are representative pooled from three experiments. (A) Mann-Whitney statistical test. Error bars show means ± SEM. (C) Linear regression. *P < 0.05, ****P < 0.0001.

Fig. 6.. CTLs induce the apoptosis of tumor-antigen cross-presenting tumor LECs in an IFN-γ–dependent manner.

(A) Tamoxifen-treated IFN-γR^WT and IFN-γR^Δprox-1 mice were injected or not with 0.5 × 10⁶ OT-1 cells 9 days after B16F10-OVA⁺VC⁺ inoculation. mRNA levels of indicated genes in tumor LECs 4 days later. Data are from one experiment, N = 3 to 4 mice per group. (B and C) C57BL/6 mice were injected or not with 0.05 × 10⁶ or 0.5 × 10⁶ OT-1 cells 9 days after B16F10-OVA⁺VC⁺ inoculation. (B) MHCI-OVA complex levels in tumor LECs at days 3 and 5 after OT-1 transfer. (C) Frequencies of cleaved casp-3 among MHCI-OVA^neg (Neg) and MHCI-OVA^pos (Pos) LECs 5 days after transfer. (D) Tamoxifen-treated IFN-γR^WT and IFN-γR^Δprox-1 mice were injected with 0.5 × 10⁶ OT-1 cells 9 days after B16F10-OVA⁺VC⁺ inoculation. MHCI-OVA complex levels in tumor LECs at days 5, 8, and 16 after OT-1 transfer. Data are from one experiment, N = 4 to 10 mice per group. (A, C, and D) Mann-Whitney statistical test; (B) one-way ANOVA test. Error bars show means ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 7.. T cell–mediated LEC apoptosis requires MHCI expression on tumor LECs.

(A and B) C57BL/6 and β₂m^KO were injected or not with 0.5 × 10⁶ OT-1 cells 8 days after B16F10-OVA⁺VC⁺ inoculation. (A) Tumor growth and (B) frequencies of cleaved casp-3⁺ tumor LECs 4 days after OT-1 transfer. Data are from one experiment, N = 6 to 7 mice per group. (C to F) Tamoxifen-treated β₂m^WT and β₂m^Δprox-1 mice were injected with 0.5 × 10⁶ OT-1 cells 9 days after B16F10-OVA⁺VC⁺ inoculation. (C) Tumor growth and (D) frequencies of cleaved casp-3⁺ tumor LECs 5 days after OT-1 transfer. Data are from one experiment, N = 6 to 7 mice per group. (E) The percentage of metastasis-positive and metastasis-negative (free of metastasis) LNs according to black color. Pictures show representative images. Scale bars, 0.5 cm. (F) Immunofluorescent Gp100 staining of inguinal, axillary, and brachial TdLNs (iLN, aLN, and bLN, respectively) 5 days after OT-1 transfer. Histograms represent the quantification of Gp100⁺ cells in LNs. (A and C) Two-way ANOVA test; (B, D, and E) Mann-Whitney statistical test. Error bars show means ± SEM. *P < 0.05, **P < 0.01.