IL-33 synergizes with TCR and IL-12 signaling to promote the effector function of CD8+ T cells

Abstract

The effector functions of CD8(+) T cells are influenced by tissue inflammatory microenvironments. IL-33, a member of the IL-1 family, acts as a danger signal after its release during cell necrosis. The IL-33/ST2 axis has been implicated in various Th2 responses. Its role in CD8(+) T-cell-mediated immune response is, however, not known. Here we find that type 1 cytotoxic T (Tc1) cells cultured in vitro unexpectedly express high levels of the IL-33 receptor ST2. Interestingly, the expression of ST2 in Tc1 cells is dependent on T-bet, a master Th1/Tc1 transcription factor. In addition, IL-33 enhances TCR-triggered IFN-γ production. IL-33 together with IL-12 can stimulate IFN-γ production in Tc1 cells. Moreover, IL-33 synergizes with IL-12 to promote CD8(+) T-cell effector function. The synergistic effect of IL-33 and IL-12 is partly mediated by Gadd45b. Together, these in vitro data establish a novel role of IL-33 in promoting effector type 1 adaptive immune responses.

Figure 1. Expression of ST2 in effector CD8⁺ T cells

(A and B) Naïve CD8⁺T cells were cultured in Tc0, Tc1, Tc2, and Tc17 conditions for 4 days. (A) Total RNA was made and subject to real time RT-PCR analysis of ST2 mRNA levels. RQ refers to relative quantity of ST2 mRNA, calculated by software provided by Applied Biosystem. RQ for Tc2 was set as 1. (B) Surface expression of ST2 was determined by flow cytometry. Isotype control is presented as the shaded area. (C) CD8⁺ T cells were cultured in Tc1 condition for 4 days (4d). These cells were either stimulated with anti-CD3 for 4h (4d + 4h), or cultured with the addition of fresh IL-2 (10 units/ml) for 3 more days (7d). At the end of 7 day culture, cells were extensive washed and cultured in the Tc1 polarizing condition for an additional 4 days (2° 4d). Total RNA was made from cells in each group and subjected to real time RT-PCR analysis of ST2 mRNA. RQ for Tc1 day 4 was set as 1. (D and E) Naïve CD8⁺ T cells were cultured in Tc1 or Tc2 polarizing conditions for 7 days. The cells were then subject to a second round of Tc1 or Tc2 polarization for 4 days. (D) Total RNA was made and analyzed by real time RT-PCR for levels of ST2 mRNA. RQ for Tc2 was set as 1. (E) Surface expression of ST2 was determined by flow cytometry. Isotype control is presented as the shaded area. Data in A, C and D are presented as the mean ±SEM of triplicate samples and are representative of three independent experiments. **P < 0.001, two-tailed unpaired Student’s t-test.

Figure 2. Role of Tbet and Eomes in ST2 expression in Tc1 cells

(A, B) Naive CD8⁺ T cells from C57BL/6WT (WT), T-bet ^−/− (TKO), Eomes ^−/−(EKO), and T-bet/Eomes doubly deficient (DKO) mice were cultured in Tc1 or Tc2 for 4 days. (A) Total RNA was made and subjected to real time RT-PCR analysis for ST2. (B) Surface ST2 expression was analyzed by flow cytometry. (C) CD8⁺ T cells cultured in Tc1 condition with or without anti-IFN-γ mAb (10μg/ml) for 4 days. Total RNA was made and subjected to real time RT-PCR analysis for ST2. Data in (A) and (C) are presented as the mean±SEM of triplicate samples. Results are representative of three independent experiments.

Figure 3. IL-33 synergizes with TCR signaling or IL-12 to promote IFN-γ production

Naive CD8⁺ T cells were cultured in Tc1 conditions for 4 days and were then stimulated with IL-33 and anti-CD3 for 16h alone or in combination. IFN-γ production was detected by (A) flow cytometry and (B) ELISA. Naive CD8⁺ T cells were cultured in Tc1 condition for 4 days and were subsequently stimulated with IL-33 or IL-12 alone or in combination for 24h. IFNγ production was measured by (C) flow cytometry or (D) ELISA. (E) Naive CD8⁺ T cells cultured in Tc1 condition for 4 days and were subsequently stimulated with IL-33 or IL-12 alone or in combination for 48h. Cell viability was determined by Trypan blue exclusion. (F) Naive CD8⁺ T cells from Pmel-1 WT and Pmel-1 T-bet^−/− (TKO) mice cultured with APCs and the gp100 peptide in Tc1 polarizing condition for 4 days. The activated cells were then stimulated with IL-33 or IL-12 alone or in combination for 24h. WT Tc1 cells stimulated by anti-CD3 for 16h were used as a positive control. IFN-γ production was measured by ELISA. ND, non-detectable. Data are presented as the mean ±SEM of triplicate samples and representative of three independent experiments. **, P < 0.01, two-tailed unpaired Student’s t-test.

Figure 4

IL-33 and IL-12 synergistically drive the effector fate of Tc1 cells. Naive CD8⁺ T cells cultured in Th1 condition for 4 days were then stimulated with IL-33 or IL-12 alone or combined together for (A-D) 4h or (E-G) 24h. mRNAs for (A) IFNγ, (B) T-bet, (C) Blimp1, (D) Eomes, (E) Lef1, (F) TCF-1, and (G) IL7R were quantified by Real-time PCR. Data are presented as mean ±SEM of triplicate samples and are representative of three independent experiments. *P<0.05, **P<0.01, ***P< 0.001, two-tailed unpaired Student’s t-test.

Figure 5. Role of Gadd45b in IL-33/IL-12-stimulated IFN-γ production in Tc1 cells

(A) Naive CD8⁺ T cells were purified from WT mice and cultured in Tc1 condition for 4 days. Then these cells were stimulated with IL-33, or IL-12, IL-12 plus IL-33 for 4h. Gadd45b mRNA levels were measured by Real-time PCR. (B and C) Naive CD8⁺ T cells were purified from WT and Gadd45b deficient mice and cultured in Tc1 condition for 4 days. Cells were then stimulated with IL-33 plus IL-12 for 15 minutes, 30 minutes and 60 minutes. Phosphorylated (phospho)-P38 (pP38) level was measured by (B) Western blot and (C) the mean relative intensity of pP38 was calculated. (D and E) TC1 cells generated as in (B) were stimulated with or without IL-33 or IL-12 for 24h. (D) IFN-γ mRNA was detected by Real-time PCR, and (E) IFN-γ protein was measured by ELISA. (F) Naive CD8⁺ T cells were cultured in Tc1 conditions for 4 days. These cells were then cultured with IL-12 and IL-33 in the presence or absence of P38 inhibitor (50 μM, 10 μM, 1 μM) for 24 h; IFN-γ production was detected by ELISA. ND, undetectable. KO, Gadd45b ^−/−. Data are presented as mean ±SEM of triplicate samples and are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired Student’s t-test.