Noncanonical K27-linked polyubiquitination of TIEG1 regulates Foxp3 expression and tumor growth

Abstract

Earlier, we demonstrated the essential role of Kruppel-like transcription factor, TIEG1, in TGF-β-induced regulatory T cell (Treg) development. In this article, we demonstrate that IL-6, which promotes Th17 development, abrogated TIEG1 nuclear translocation and inhibited TGF-β-induced Treg development. Tyrosine kinase Tyk2-mediated phosphorylation of TIEG1 at Tyr179 promoted noncanonical K-27-linked polyubiquitination, which inhibited TIEG1 nuclear translocation. To test the role of TIEG1-regulated Treg/Th17 development in antitumor immunity, we analyzed TRAMP-C2 tumor growth in TIEG1(-/-) mice. The defective Treg development and elevated Th17 response resulted in enhanced immune reactivity in the tumor and inhibition of TRAMP-C2 tumor growth in TIEG1(-/-) mice. Thus, our results uncovered a novel regulatory mechanism that modulates Tregs and may regulate tumor progression.

FIGURE 1

IL-6–Tyk2 pathway-mediated phosphorylation regulates TIEG1 nuclear localization and Treg/Th17 differentiation. A, Naive CD4⁺CD25^– T cells were stimulated with anti-CD3 and anti-CD28 in the presence of TGF-β (5 ng/ml) alone or TGF-β (5 ng/ml) plus IL-6 (20 ng/ml). The nuclear and cytoplasmic fractions were immunoblotted with anti-TIEG1 Ab. The membranes were reprobed with anti-histone H3 and anti-actin Ab. C, cytoplasmic; N, nuclear. B, CD4⁺CD25^– T cells were treated as in A. Endogenous TIEG1 from total-cell lysates was immunoprecipitated with anti-TIEG1 Ab and immunoblotted with anti–phos-tyrosine Ab. Blots were reprobed using anti-TIEG1 Ab, p-TIEG1, or tyrosine-phosphorylated TIEG1. C, 293T cells were transfected with plasmids encoding His-TIEG1, Flag-Tyk2, or Flag-Tyk2/K930I, alone or in combination, for 24 h. TIEG1 was immunoprecipitated with anti-His Ab and immunoblotted with anti–phos-tyrosine Ab. Blot was stripped and reprobed with anti-TIEG1. The amount of Tyk2 in total-cell lysates was determined by immunoblotting. Actin was used as an equal loading control. D, Naive CD4⁺CD25– T cells isolated from Tyk2^+/+ and Tyk2^–/– mice were stimulated with anti-CD3 and anti-CD28 in the presence of TGF-β (5 ng/ml) alone or TGF-β (5 ng/ml) plus IL-6 (20 ng/ml). TIEG1 was immunoprecipitated using anti-TIEG1 Ab and immunoblotted using anti–phos-tyrosine Ab. E, TIEG1 interacts with Tyk2 in primary CD4 T cells. CD4⁺CD25^– T cells were stimulated with TGF-β or TGF-β plus IL-6, as described above. Endogenous TIEG1 or Tyk2 was immunoprecipitated with corresponding Abs and analyzed by immunoblotting with anti-Tyk2. The same blot was stripped and reprobed with anti-TIEG1. The amount of TIEG1 and Tyk2 in total-cell extracts was determined by Western blotting. Actin was used as an equal loading control.

FIGURE 2

Tyk2 phosphorylates TIEG1 at Tyr179. A, Truncated mutants of TIEG1. Each tyrosine residue in individual truncated mutants is indicated. 6×His and Xpress represent His₆ tag and Xpress tag, respectively. B, 293T cells were cotransfected with plasmids encoding Flag-Tyk2 and truncated TIEG1 for 24 h. TIEG1 was precipitated by Ni-NTA affinity purification and immunoblotted with anti–phos-tyrosine Ab (upper panel). The same blot was stripped and reprobed with anti-Xpress (middle panel). C, 293T cells were cotransfected with plasmids encoding Flag-Tyk2 and TIEG1 or TIEG1/Y179F mutant for 24 h. TIEG1 was immunoprecipitated with anti-His Ab and analyzed by immunoblotting with anti–phos-tyrosine Ab. The results shown are from one representative experiment that was conducted three times.

FIGURE 3

Tyk2-mediated phosphorylation regulates TIEG1 nuclear localization and Treg/Th17 differentiation. A, Naive TIEG1^–/– CD4⁺ CD25^– cells were reconstituted with WT or TIEG1-Y179F mutant by retroviral transduction. Nuclear/cytoplasmic localization of TIEG1 was analyzed in FACS-sorted GFP⁺ cells following treatment with TGF-β or TGF-β + IL-6. B, GFP⁺ cells were stimulated with TGF-β (Treg-inducing) and TGF-β + IL-6 (Th17-inducing) conditions for 7 d. The effect of Y179F mutation on Foxp3 versus IL-17 expression was analyzed by real-time PCR. The results shown are from one representative experiment that was conducted three times.

FIGURE 4

Tyk2-mediated phosphorylation-dependent polyubiquitination of TIEG1 is K27 linked. A, 293T cells were transfected with the indicated combination of plasmids encoding Flag-Tyk2, His-TIEG1, Myc-Itch, and HA-Ub for 48 h. Ubiquitinated TIEG1 was purified by Ni-NTA precipitation and analyzed by immunoblotting with anti-HA Ab. The expression of Tyk2 and Itch from total-cell lysates is shown (bottom two panels). B, 293T cells were transfected with plasmids encoding His-TIEG1, Myc-Itch, HA-Ub, and increased amounts of Flag-Tyk2. The ubiquitinated TIEG1 was purified by Ni-NTA precipitation and analyzed by immunoblotting with anti-HA Ab. C, 293T cells were transfected with plasmids encoding His-TIEG1, Myc-Itch, Flag-Tyk2, and HA-Ub or Ub-K48 or Ub-K63 (Ub-K48, only lysine K48 present, all other lysine mutated into arginine; Ub-K63, only lysine K63 present, all others mutated into arginine] for 40 h. Ubiquitinated TIEG1 was precipitated by Ni-NTA beads and analyzed by Western blotting with anti-HA Ab. The same blot was stripped and reprobed with anti-TIEG1. D, 293T cells were transfected with plasmids encoding His-TIEG1, Myc-Itch, Flag-Tyk2, and various lysine mutants of HA-ubiquitin (K0, all lysines are mutated into arginine; K6, K11, K27, K29, K33, K48, and K63, a single lysine present, all other lysines mutated into arginine). Ubiquitinated TIEG1 was precipitated and blotted with anti-HA Ab. E, 293T cells were transfected with Flag-Tyk2, Myc-Itch, and His-TIEG1 and HA-Ub WT, K0, K27, or K27R (K27R, only lysine K27 mutated into arginine, all other lysines present). The polyubiquitination of TIEG1 was determined by immunoblotting with anti-HA. F, 293T cells were transfected with Flag-Tyk2, Myc-Itch, HA-UbK27, His-TIEG1 WT, or His-TIEG1/Y179F mutant in 293T cells. The polyubiquitination of TIEG1 was determined by immunoblotting with anti-HA. The results shown are from one representative experiment that was conducted three times.

FIGURE 5

Defective TGF-β induced Treg development in TIEG1^–/– naive CD4 T cells. A, Experimental procedures. TRAMP-C2 cells were cultured for 3 d without change of the medium. CM was collected and incubated with naive CD4⁺CD25^– T cells isolated from spleen and lymph nodes of TIEG1^+/+ and TIEG1^–/– mice in the presence of anti-CD3 and anti-CD28. After 3–5 d of culture with CM, T cells were harvested for Foxp3 analysis. B, Real-time PCR analysis of Foxp3 expression. CD4⁺CD25^– T cells were stimulated with or without CM as in A, and Foxp3 expression level was analyzed by real-time PCR. The increased folds of Foxp3 mRNA were normalized to that of actin mRNA. C, In a similar experiment to B, anti-TGF-β (15 μg/ml) or control isotype Ab was added to the cultures. Foxp3 mRNA was analyzed by real-time PCR. D, In a similar experiment to A, IL-6 was added to the T cell cultures. IL-17 mRNA was analyzed by real-time PCR.

FIGURE 6

Elevated antitumor immune response in TIEG1^–/– mice. A, Kaplan–Meier curves of tumor-bearing TIEG1^+/+ mice (n = 16) and TIEG1^–/– mice (n = 16). Male mice were injected s.c. with TRAMP-C2 cells. Tumors were monitored three times per week. Tumor incidence in each group was calculated from days 17–60. B, Representative image of tumor-bearing TIEG1^+/+ and TIEG1^–/– mice. The photograph was taken at day 60 after initial inoculation. C, Foxp3⁺ Tregs in the spleen (upper panels) and lymph nodes (lower panels) from tumor-bearing (+TRAMP-C2) or control mice (–TRAMP-C2) were analyzed using FACS following intracellular staining using anti-Foxp3 Ab. D, CD4⁺Foxp3+ cell numbers in the spleen and lymph nodes of TIEG1^+/+ and TIEG1^–/– mice. Each symbol represents an individual mouse. E, Tumor tissues were collected from TIEG1^+/+ mice or TIEG1^–/– mice, and Foxp3, IFN-γ, and IL-17 expression was analyzed by real-time PCR. The results shown are from one representative experiment that was conducted three times.

FIGURE 7

Adoptive transfer of TIEG1-deficient T cells inhibits TRAMP-C2 tumor growth. Rag1^–/– mice were injected s.c. with TRAMP-C2 cells on day 0. On day 3, naive CD4⁺CD25^– (3 × 10⁶) and CD8⁺ T cells (3 × 10⁶) from TIEG1^+/+ and TIEG1^–/– mice were adoptively transferred (i.v.) into these Rag^–/– mice (n = 5). A, The kinetics of tumor growth. B, Representative image of tumor-bearing Rag1^–/– mice. C, Percentage of CD4⁺Foxp3⁺ cells in tumor-infiltrated cells. D, Relative mRNA levels of IFN-γ and IL-17 in tumor-infiltrated lymphocytes.