A Novel p27^kip1-Smad3 Regulated Pathway Is Involved in Induction of T Cell Tolerance.

Induction and maintenance of peripheral tolerance is essential for homeostasis of the immune system. In vivo studies demonstrate the significance of tolerance induction in preventing autoimmunity, graft rejection and GVHD. Upregulation of the cyclin-dependent kinase inhhibitor, p27, correlates with induction of T cell tolerance in vitro and in vivo. p27 interacts with cdk2, cdc2, grb2, and Rho family GTPases. Extensive studies support an essential role of cdks, particularly cdk2, in cell cycle re-entry. Cdk2 promotes cell cycle progression in part by phosphorylating Rb and related pocket proteins thereby reversing their ability to sequester E2F transcription factors. Recent work indicates that cdk2 phosphorylates Smad2 and Smad3. Smad3 inhibits progression from G1 to S phase, and impaired phosphorylation on the cdk-specific sites renders it more effective in executing this function. In contrast, cdk-mediated phosphorylation of Smad3 reduces Smad3 transcriptional activity and antiproliferative function. In spite the strong correlation between p27 expression level and T cell tolerance, it remains unclear whether p27 has a causative role in induction of tolerance. Here, we examined the role of p27 during induction of tolerance of naïve T cells in vivo, using RAG2 deficient, DO11.10 TCR-transgenic T cells that lack the cyclin-cdk-binding domain of p27 (p27Δ) thereby disrupting only the interactions of p27 with cyclin-cdk complexes. We adoptively transferred CD4⁺ T cells from RAG2^−/−DO11.10 TCR-transgenic mice (DO11.10) or RAG2^−/−DO11.10 TCR-transgenic p27Δ mice (DO11.10/p27Δ) into syngeneic wild-type recipients and compared the development of immune responses to immunogenic or tolerizing stimulus in vivo. Following exposure to immunogenic or tolerizing stimulus, DO11.10 and DO11.10/p27Δ CD4⁺ T cells underwent equal numbers of divisions in vivo, and both cell types exhibited reduced number of divisions in response to tolerizing stimulus. Strikingly, only wild-type DO11.10 TCR-transgenic T cells were tolerized as determined by impaired cyclin E activation, proliferation, and IL-2 production upon antigen-specific rechallenge. Compared to primed wild-type DO11.10 cells, tolerized wild-type DO11.10 cells exhibited impaired cdk2 and cdc2 activity, reduced levels of Smad3 phosphorylation on cdk-specific sites, and increased Smad3-transactivation leading to upregulation of the cdk4/6-specific cdk inhibitor p15. In contrast, after either priming or tolerizing stimulus, DO11.10/p27Δ cells exhibited comparable cdk2 and cdc2 activity, cdk-mediated phosphorylation of Smad3, low-level Smad3 transactivation, and no upregulation of p15. Furthermore, knockdown of Smad3 by expression of Smad3 shRNA in wild-type DO11.10 T cells recapitulated the functional and molecular findings observed in DO11.10/p27Δ cells, preventing induction of tolerance and upregulation of p15, and resulting in production of IL-2 and cell cycle progression. In contrast, expression of Smad3 mutant resistant to cdk-mediated phosphorylation in DO11.10/p27Δ cells recapitulated the molecular and functional effects of tolerance and resulted in inhibition of IL-2 production, upregulation of p15 and blockade of cell cycle progression. These results show that p27 plays a causative role in the induction of tolerance of naïve T cells and Smad3 is a critical component of a pathway downstream of p27 regulating the induction of tolerance in vivo.