Th9 cells promote antitumor immune responses in vivo

Abstract

Th9 cells are a subset of CD4+ Th cells that produce the pleiotropic cytokine IL-9. IL-9/Th9 can function as both positive and negative regulators of immune response, but the role of IL-9/Th9 in tumor immunity is unknown. We examined the role of IL-9/Th9 in a model of pulmonary melanoma in mice. Lack of IL-9 enhanced tumor growth, while tumor-specific Th9 cell treatment promoted stronger antitumor responses in both prophylactic and therapeutic models. Th9 cells also elicited strong host antitumor CD8+ CTL responses by promoting Ccl20/Ccr6-dependent recruitment of DCs to the tumor tissues. Subsequent tumor antigen delivery to the draining LN resulted in CD8+ T cell priming. In agreement with this model, Ccr6 deficiency abrogated the Th9 cell-mediated antitumor response. Our data suggest a distinct role for tumor-specific Th9 cells in provoking CD8+ CTL-mediated antitumor immunity and indicate that Th9 cell-based cancer immunotherapy may be a promising therapeutic approach.

Figure 1. IL-9–neutralized mice are more susceptible to developing lung melanoma.

C57BL/6 mice (n = 4–5/group) receiving control IgG or α–IL-9 every other day beginning 1 day before i.v. challenge of 1 × 10⁵ B16 melanoma cells were analyzed on day 18 after challenge. The P values in the graphs show comparisons between IgG and α–IL-9 groups. (A) Images and weights of lungs show increased tumor development in the lungs of mice treated with α–IL-9. NT, untreated. (B) Number of total leukocytes, CD8⁺ T cells, and CD4⁺ T cells in the lung leukocyte fraction analyzed with FACS. (C) Cell numbers of myeloid population subsets in the lung leukocyte fractions analyzed by FACS. (D) Expression of CD44 on CD8⁺ T cells from the lung. Numbers above scale bars indicate percentage of CD44^hi cells. (E) RT-PCR analysis of mRNA expression of chemokines and their receptors in the lung tumor tissues. Data shown were normalized to the β-actin gene. Representative results from 1 of 2 performed experiments are shown.

Figure 2. Prophylactic effect of tumor-specific Th9 cells.

(A) OVA-specific Th9 cells used in each experiment typically contained about 25% IL-9–expressing CD4⁺ OT-II T cells. (B–F) C57BL/6 mice (n = 4–5/group) received PBS or 3 × 10⁶ Τh9 cells on the same day when mice were i.v. challenged with 1 × 10⁵ B16-OVA cells, and were analyzed on day 19 after challenge. (B) Comparison of tumor foci numbers in the lung between PBS- and Th9 cell–treated mice. (C) Number of total leukocytes, CD8⁺ T cells, and CD4⁺ T cells in the lung leukocyte fraction analyzed by FACS. (D) Cell numbers of myeloid population subsets in the lung leukocyte fraction analyzed by FACS. (E) Expression of CD44 on CD4⁺ and CD8⁺ T cells from the lung. (F) RT-PCR analysis of mRNA expression of chemokines and their receptors in lung tumor tissues. Data shown were normalized to the β-actin gene. (G) Th1 or Th9 cells (2 × 10⁶) were s.c. injected on the same day and at the same location where 5 × 10⁵ B16-OVA tumor cells had been implanted s.c. Tumor growth curves are shown. P = 0.0033 for Th1 versus Th9 on day 21. (H) Th1 or Th9 cells (3 × 10⁶) were i.v. transferred on the same day that 2 × 10⁵ B16-OVA tumor cells were s.c. injected. Tumor growth curves are shown. P = 0.012 for Th1 versus Th9 on day 21. Representative results from 1 of 3 repeated experiments are shown. The P values in the graphs show comparisons with PBS.

Figure 3. Therapeutic effect of tumor-specific Th9 cells.

PBS or 3 × 10⁶ Th1 or Th9 cells were transferred i.v. to C57BL/6 mice bearing 5-day established pulmonary B16-OVA melanoma. Mice (n = 4–5/group) were analyzed on day 19 after challenge. (A) Comparison of tumor foci numbers in the lung between treated mice. P = 0.042, Th1 versus Th9. (B) Number of total leukocytes, CD8⁺ T cells, and CD4⁺ T cells in the lung leukocyte fraction analyzed by FACS. P = 0.0014, Th1 versus Th9 for CD8⁺ T cells. (C) Expression of CD44 on CD4⁺ and CD8⁺ T cells from the LLN and lung leukocyte fraction. For Th1 versus Th9, P = 0.011 (LLN CD4⁺ T cells), P = 0.022 (LLN CD8⁺ T cells), P = 0.020 (lung CD4⁺ T cells), and P = 0.0035 (lung CD8⁺ T cells). (D) Cell numbers of myeloid population subsets in the lung leukocyte fraction analyzed by FACS. P = 0.00031, Th1 versus Th9 for CD8α⁺ DCs. (E) RT-PCR analysis of mRNA expression of chemokines and their receptors in the lung tumor tissues. Data shown were normalized to β-actin gene. For Th1 versus Th9, P = 0.014 (Ccl20), P = 0.025 (Ccr6). Representative results from 1 of 3 repeated experiments are shown. P values in the graphs show comparisons with PBS.

Figure 4. Th9 cells maintain cytokine expression profile in tumor-bearing mice.

(A) PBS or 3 × 10⁶ Th1 or Th9 cells were i.v. transferred to mice (wild-type and Ifng^–/– mice) with 5-day established pulmonary B16-OVA melanoma. On day 4 after transfer, total LLN and lung cells were harvested and restimulated with OVA peptides for 36 hours. Cytokine levels in culture supernatants were tested using ELISA. (B) OVA-specific CFSE-labeled Th1 or Th9 cells (3 × 10⁶) were i.v. transferred into mice with 5-day pulmonary B16-OVA melanoma. On day 4 after transfer, CFSE⁺ Th1 or Th9 cells and CFSE^– endogenous cells were sorted from LLNs with flow cytometer. CFSE⁺ Th1 or Th9 cells were maintained with 10 U/ml IL-2, and CFSE^– cells were restimulated with 5 μg/ml OVA_323–339 and OVA_257–264 peptides for 36 hours. Cytokine levels in the supernatant were tested using ELISA. (C) mAbs neutralizing IL-9, IL-10, or control IgG were i.p. injected to C57BL/6 wild-type mice bearing 5-day established pulmonary B16-OVA melanoma; some groups of mice were also i.v. transferred with 3 × 10⁶ Th9 cells. The graph shows the comparison of the numbers of tumor foci in the lungs of mice (n = 4/group) receiving the indicated treatments. Representative results from 1 of 2 performed experiments are shown.

Figure 5. Th9 cells promote tumor-specific CD8⁺ CTL response in tumor-bearing mice.

(A–C) FACS analysis of the frequencies of CD8⁺ T cells staining positive for tumor infiltrating OVA tetramer (K^b-SIINFEKL), relative to total CD8⁺ T cells, (A) in therapeutic lung models, (B) in the total leukocytes in prophylactic s.c. tumor models when T cells were s.c. injected, or (C) in the total leukocytes in prophylactic s.c. tumor models when T cells were i.v. transferred. Graphs show the total number of tumor-infiltrating OVA tetramer–positive CD8⁺ T cells and/or granzyme B–producing CD8⁺ T cells in the leukocyte fraction. n = 4 mice/group. (D) PBS or 3 × 10⁶ Th1 or Th9 cells were i.v. transferred into mice bearing 5-day established pulmonary B16-OVA melanoma. A group of mice transferred with Th9 cells also received depleting mAbs against CD8 every 3 days starting from 1 day before T cell transfer. Shown are the lung foci numbers observed on day 19 after challenge (n = 4 mice/group). Representative results from 1 of 2 performed experiments are shown. In D, P values indicate comparisons with PBS.

Figure 6. Th9 cell treatment enhances tumor-specific CD8⁺ CTL differentiation.

(A and C) CFSE-labeled OT-I CD8⁺ T cells (3 × 10⁶) were i.v. transferred into mice bearing 5-day pulmonary B16-OVA melanoma, which were also i.v. transferred at the same day with 3 × 10⁶ Th1 or Th9 cells. (B and D) CFSE-labeled OT-I CD8⁺ T cells (3 × 10⁶) were i.v. transferred into mice s.c. injected with 5 × 10⁵ B16-OVA cells, together with s.c. injection of Th1 or Th9 cells at the same site of tumor cell inoculation on the same day. All mice (n = 3/group) were sacrificed 3 days later, and OT-I cells from TDLNs were analyzed by FACS after restimulation. (A and B) Upper panels show the frequency (%) of IFN-γ–producing CFSE^lo (proliferated) OT-I cells; lower panels show total CFSE⁺ OT-I cells and total CFSE^lo IFN-γ–producing OT-I cells recovered from TDLNs. (C and D) Histograms show granzyme B production by CFSE⁺ OT-I cells; graphs show mean fluorescence intensity of granzyme B expression by CFSE⁺ OT-I cells recovered from TDLNs. P values are shown as indicated.

Figure 7. Th9 cells regulate DC function in a Ccr6-dependent manner.

(A) C57BL/6 mice (n = 3/group) were i.v. challenged with 1 × 10⁵ B16-OVA-GFP tumor cells and, on the same day, were i.v. transferred with 3 × 10⁶ Th1 or Th9 cells. LLNs were tested using FACS 3 days later for DC populations. Histograms show the percentage of GFP⁺ DCs gated on CD11c⁺CD8α⁺ DCs and CD11c⁺CD11b⁺ DCs. (B) Number of total and GFP⁺ cells for each DC population was calculated from A. Representative results from one of two performed experiments are shown. (C) PBS or 3 × 10⁶ Th9 cells were transferred to mice (wild-type and Ccr6^–/– mice; n =4/group) bearing 5-day established pulmonary B16-OVA melanoma. Shown are the lung weights on day 19 after challenge. (D) Representative images of the lungs of mice receiving different treatments. (E) LLN cells from C were analyzed for DC populations using FACS. The numbers of total CD8α⁺ DCs and CD11b⁺ DCs from mice (n = 4/group) are shown. Unless otherwise indicated, P values are shown as indicated.