T-bet-dependent expression of osteopontin contributes to T cell polarization

Abstract

The osteopontin (Opn) glycoprotein has been implicated in diverse physiological processes, including vascularization, bone formation, and inflammatory responses. Studies of its role in immune responses has suggested that Opn can set the early stage of type-1 immune (cell-mediated) responses through differential regulation of IL-12 and IL-10 cytokine gene expression in macrophages. Although Opn has been suggested to play a role in the development of type-1 immunity, little is known about control of Opn gene expression. Here, we report that Opn gene expression in activated T cells, but not macrophages, is regulated by T-bet, a transcription factor that controls CD4+ T helper (Th1) cell lineage commitment. We also find that T-bet-dependent expression of Opn in T cells is essential for efficient skewing of CD4+ T and CD8+ T cells toward the Th1 and type 1 CD8+ T cells (Tc1) pathway, respectively. Taken together, these findings begin to delineate the genetic basis of Opn expression in T cells and further clarify the role of Opn in Th and Tc1 development.

Fig. 1.

T-bet up-regulates Opn production in activated T cells. (A) T cells were incubated (2.5 × 10⁵ cells per ml) with (○ and •) or without (▵ and ▴) plate-coated 5 μg/ml anti-CD3 and 1 μg/ml anti-CD28 Abs, and daily levels of Opn protein and mRNA were determined by real-time PCR of cDNA samples from triplicates from CD4⁺ T cells (○ and ▵) and CD8⁺ T cells (• and▴). (B) Opn mRNA levels expressed by activated (•) and resting (▴) T-bet^+/+ (WT) T cells and activated (○) and resting (▵) T-bet^-/- T cells. Plate-coated CD3/28 Abs were used for T cell activation as described in A. (C) Levels of secreted Opn expressed by T-bet^+/+ (▪) and T-bet^-/- (□) T cells determined by ELISA 4 d after incubation as above with anti-CD3/28 Abs. (D) Effect of adding indicated concentrations of IFN-γ to Opn response of T-bet^+/+ (filled bars) and T-bet^-/- (○) CD4⁺ T or CD8⁺ T cells on day 6. T cells were activated with plate-coated anti-CD3/28 Abs. (E) Purified CD4⁺ T cells (10⁶ cells per ml) from T-bet^+/+ (filled bars) and T-bet^-/- (empty bars) mice with 3 μg/ml anti-CD3 Ab and 1 μg/ml anti-CD28 Ab. Culture supernatants and cells were harvested on day 4. Figures here are representative of at least three repeats.

Fig. 2.

T-bet induces Opn at the mRNA level. (A) Opn production was assessed after reconstituting T-bet expression in stimulated T-bet-deficient T cells. GFP-RV-T-bet (RV-T-bet) and negative control GFP-RV were used to transduce primary T-bet^-/- T cells before activation with plate-bound 1 μg/ml anti-CD3 and 1 μg/ml anti-CD28 Abs. (B) CD4⁺ T cells positively purified from T-bet^-/- mice were stimulated and transduced with RV, RV-T-bet, or RV-T-bet Y525F. (C) Opn promoter (nucleotides -777 to +79) activity was assessed by luciferase assay with or without T-bet ectopic expression by using EL-4 cells. Values are averages of three independent experiments with error bars of mean ± SEM. Data are representative of multiple repeats.

Fig. 3.

T-bet does not regulate Opn in macrophages. (A) BALB/c splenic DCs from T-bet^+/+ and T-bet^-/- mice were incubated (10⁶ cells per ml) for 12 h, and Opn levels in supernatants were examined by ELISA. (B and C) Peritoneal macrophages isolated from naive BALB/c mice as described in Materials and Methods, plated (10⁶ cells per ml) with 30 ng/ml of LPS. Levels of Opn mRNA (B) and secreted Opn protein in culture supernatants (C) are shown with T-bet^+/+ (•) and T-bet^-/- (○) macrophages. (D) Levels of Opn mRNA and secreted protein were compared between T cells and macrophages (thioglycollate-elicited) on day 6. Live cells were selected by Nycodenz spin and plated on day 5 as the same concentration (10⁶ per ml) for both cell types, as described in Materials and Methods. (E) The 2D2 TCR transgenic CD4⁺ T cells were i.v. injected into B6 Rag2^-/- mice (2 × 10⁶ cells per mouse). Two weeks later, the mice were received 100 μgofMOG peptide by s.c. injection in complete Freund's adjuvant, and peripheral blood was collected at the indicated times for serum Opn measurements; three mice per group. Groups of recipients with (•) and without (○) 2D2 CD4⁺ T cell transfer. Bars indicate mean ± SEM.

Fig. 4.

Opn^-/- T cells fail to elicit Th1 response. (A) Levels of IFN-γ and IL-10 were measured for day 4 cell culture supernatants. Cells of indicated types were plated as 2.5 × 10⁵ cells per ml in precoated wells with 2.5 μg/ml of anti-CD3 Ab. Values show ratios of IFN-γ/IL-10 of ELISA. (B) EAE was induced in B6(N16)2D2⁺ mice (Opn-WT/HZ, n = 19, •; Opn deficient, n = 14, ○) as described in Materials and Methods. EAE scores are shown as mean ± SEM. (C) MOG-specific cytokine production by LN cells from EAE-induced Opn-deficient (□) and Opn WT/HZ (▪) 2D2 TCR transgenic mice. (D) 2D2 CD4⁺ T cell proliferation from the same set of Opn-WT (•) and Opn-deficient (○) cells in C.

Fig. 5.

Opn deficiency enhances IL-10 production and attenuates CTL cytotoxicity. (A and B) CD8⁺ T cells (2.5 × 10⁵ cells per ml) from Opn-WT (•) and Opn-deficient (○) mice were stimulated with plate-bound anti-CD3 Ab at the indicated concentrations and 1 μg/ml anti-CD28 Ab before culture supernatants were harvested on day 6. Levels of IFN-γ (A) and IL-10 (B) were assessed by ELISA. (C) Comparison of IL-10 production by Opn-WT (▪) vs. Opn-deficient (□) T cells upon antigen-specific activation by DC-pulsed with OT-1 peptide. OT-1 CD8⁺ T cells were purified from 16-generation mice backcrossed to B6. DCs were purified from Opn^-/- mice and activated overnight with 10 μg/ml anti-CD40 Ab, followed by pulsing with OT-1 peptide (20 μg/ml). Peptides were washed off, and DCs were irradiated (3,000 rads) before setting up coculture with OT-1 cells. Levels of IL-10 were assessed from day-4 culture supernatants. (D) Comparison of Opn^+/+ (○) and Opn^-/- (•) CTL cytotoxicity against male HY antigen. Results are representatives of triplicate experiments.