Adoptive transfer of Tc1 or Tc17 cells elicits antitumor immunity against established melanoma through distinct mechanisms

Abstract

Adoptive cell transfer (ACT) of ex vivo-activated autologous tumor-reactive T cells is currently one of the most promising approaches for cancer immunotherapy. Recent studies provided some evidence that IL-17-producing CD8(+) (Tc17) cells may exhibit potent antitumor activity, but the specific mechanisms have not been completely defined. In this study, we used a murine melanoma lung-metastasis model and tested the therapeutic effects of gp100-specific polarized type I CD8(+) cytotoxic T (Tc1) or Tc17 cells combined with autologous bone marrow transplantation after total body irradiation. Bone marrow transplantation combined with ACT of antitumor (gp100-specific) Tc17 cells significantly suppressed the growth of established melanoma, whereas Tc1 cells induced long-term tumor regression. After ACT, Tc1 cells maintained their phenotype to produce IFN-γ, but not IL-17. However, although Tc17 cells largely preserved their ability to produce IL-17, a subset secreted IFN-γ or both IFN-γ and IL-17, indicating the plasticity of Tc17 cells in vivo. Furthermore, after ACT, the Tc17 cells had a long-lived effector T cell phenotype (CD127(hi)/KLRG-1(low)) as compared with Tc1 cells. Mechanistically, Tc1 cells mediated antitumor immunity primarily through the direct effect of IFN-γ on tumor cells. In contrast, despite the fact that some Tc17 cells also secreted IFN-γ, Tc17-mediated antitumor immunity was independent of the direct effects of IFN-γ on the tumor. Nevertheless, IFN-γ played a critical role by creating a microenvironment that promoted Tc17-mediated antitumor activity. Taken together, these studies demonstrate that both Tc1 and Tc17 cells can mediate effective antitumor immunity through distinct effector mechanisms, but Tc1 cells are superior to Tc17 cells in mediating tumor regression.

Figure 1

Strong anti-tumor effect was mediated by ACT with Tc1 or Tc17 cells in the combination of TBI and BMT. (A) B6 mice were inoculated i.v. with luciferase-transduced B16-F10 melanoma cells. Six days later, tumor-bearing mice received 1200 cGy (split doses). One day after, Tc1 and Tc17 cells generated from Pmel-1/Thy1.1 mice were transferred into tumor-bearing mice together with TCD-BM cells from normal B6 mice. In the non-myeloablative regiment, Tc1 and Tc17 cells were transferred into tumor bearing mice with sublethally irradiation and no BMT. Tumor growth was monitored by in vivo bioluminescent imaging 14 days after ACT. (B) Recipient mice were injected i.v. with 5× 10⁵ B16F10 tumor cells. Seven days later, 2×10⁶ gp100 Ag-specific Tc1 and Tc17 cells (Thy 1.1) in combination with BM (Ly5.1) were adoptively transferred into mice bearing established metastases. Fourteen days later, spleen and lungs from individual animals were harvested, and single cell suspensions were made as described in “Materials and Methods”. Cells were stained with anti-Thy 1.1, anti-CD8, anti-ly5.1. Gates were set on the ly5.1^-Thy 1.1⁺CD8⁺ T cell populations. Data shown are from a representative experiment of 2 repeating experiments showing the percentages of lung and spleen-derived Thy 1.1⁺CD8⁺ T cells.

Figure 2

Generation and Tc1 and Tc17 Responses of antigen-specific to tumor antigen stimulation ex vivo. (A) Tc1 and Tc17 cells were generated as described in “Materials and Methods”, and their phenotype in terms of IFNγ and IL-17 production was shown on gated Pmel-1 (Thy1.1⁺) cells. (B) In a separate experiment, pmel-1 Tc1 and Tc17 cells were labeled with CSFE and transferred into B16 tumor-bearing B6 mice (n = 4). Five days after cell transfer, mice were euthanized and their splenocytes were stained for CD8 and Thy1.1. The CSFE profile is shown on gated live CD8⁺Thy1.1⁺ cells for one of four representative mice. (C) A set of B6 mice (n = 3) were inoculated i.v. with luciferase transduced B16 F10 cells and received Tc1 or Tc17 cells combined with BMT. Mice were euthanized on day 14 after treatment. Splenocytes were isolated and processed for cytokine-release ELISPOT assays. Gp100-specific IFNγ- and IL-17-producing cells were detected after co-culture with gp100-pulsed and unpulsed EL4 for 20 hours. Experiments were repeated 2 times with similar results. (D) The graph shows the average number of IFNγ- and IL-17-producing spots from triplicate wells with 1SD as error bars.

Figure 3

Tc1 cells mediated stronger anti-tumor immunity responses than Tc17 cells. (A) Schema for treatment regimen of B16 melanoma. B6 mice were inoculated i.v. with luciferase-transduced B16-F10 melanoma cells. Six days later, tumor-bearing mice received 1200 cGy (split doses). One day after, Tc1 and Tc17 cells generated from Pmel-1/Thy1.1 mice were transferred into tumor-bearing mice together with TCD-BM cells from normal B6 mice. (B) Percentage survival of tumor bearing mice was presented as pooled data from 2 replicate experiments combined. (C) Tumor growth was monitored by in vivo bioluminescent imaging (n = 4); one representative experiment from a total of 5 experiments is shown.

Figure 4

Requirement of IFNγR on tumor cells for Tc1- or Tc17-mediated anti-tumor responses. (A) B6 mice were inoculated i.v. with IFNγR^DN B16 tumor cells. gp100 specific Tc1 and Tc17 cells were administered 7 days after tumor implantation. Overall survival rate was observed. (B) Ag-specific IL-17 cytokine-release was analyzed by an ELISPOT assay using splenocytes from IFNγR^DN B16 tumor bearing mice. ELISPOT was performed using several stimulator cells: IFNγ-treated (100 U/mL, 20 hours), and non-treated and IFNγ-treated (100 U/mL, 20 hours) B16, and non-treated B16 IFNγR^DN B16 tumor. (C) Ag-specific IFNγ and cytokine-release was analyzed by ELISPOT assays using splenocytes from IFNγR^DN B16 tumor bearing mice. ELISPOT was performed using several stimulator cells: IFNγ-treated (100 U/mL, 20 hours), and non-treated B16 and non-treated B16 IFNγR^DN B16 tumor.

Figure 5

Role of IFN-γ and IL-17 in Tc17-mediated anti-tumor effect. WT C57/B6 mice (n = 4 per group) were given B16 (A) and IFNγR^DN B16 tumor (B) i.v. by tail vein. Seven days later, tumor-bearing mice were adoptively transferred with Tc1 and Tc17 cells in the combination of BM. Anti-IFNγ mAb and anti-IL-17 mAb was administered intraperitoneally on days 0 and continuously twice a week for 4 weeks total. Overall survival was monitored. Corresponding groups of untreated tumor-bearing mice served as controls.

Figure 6

Activation and expansion of antigen-specific Tc1 and Tc17 cells in vivo. Normal B6 mice with established WT or IFNγR^DN B16 tumor received Tc1 or Tc17 cells in combination with BMT. Four mice were euthanized on day 14 and 28 after ACT. (A) Leukocytes isolated from spleen or lung on day 14 were stained with anti-Thy 1.1, anti-CD8, anti-IFNγ, anti-IL-17 and TNFα. Intracellular cytokine expression was shown on gated Thy 1.1⁺ cells. Flow plots display 1 of 3-4 mice in each group. (B) Percentages of IFNγ⁺, /IL-17⁺, IFNγ⁺/IL-17⁺and TNFα⁺ on gated Thy 1.1⁺ cells are shown from 3-4 mice per group on day 14 in spleen and lung. (C) Absolute numbers of adoptive transferred Tc1 or Tc17 cells are shown from 3-4 mice per group in spleen and lung on day 14 and 28. The data represent 1 of 3 experiments using similar settings. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 7

Phenotypes of transferred Tc1 and Tc17 cells in vivo. The experiment was set as in figure 6. Leukocytes isolated from recipient lung on day 14 were stained for surface expression with Thy 1.1, CD8, CXCR3, CCR6 and CD107a. Percentages of CXCR3⁺ (A), CCR6⁺ (B), and CD107a⁺ cells (C) on gated Thy 1.1⁺ population are shown from 3-4 mice per group. The data represent 1 of 3 experiments using similar settings. * p < 0.05, ** p < 0.01, and *** p < 0.001.