The Cyclin-Dependent Kinase Inhibitor p27^KIP1 Is Mandatory for Tolerance Induction In Vivo.

Elucidating the signals that influence the choice between T cell activation and tolerance in vivo will facilitate the design of novel therapies for the induction of transplantation tolerance. The cyclin-dependent kinase (cdk) inhibitor p27 is an important negative regulator of cell cycle progression in T lymphocytes. We have previously observed that in vitro generated anergic T cells express high levels of p27 and that forced expression of p27 renders T cells incapable of progressing through the cell cycle and transcribing the IL-2 gene. Therefore, we sought to determine the role of p27 on the decision of naive T cells between activation and tolerance in vivo. In order to eliminate the ability of p27 to regulate the cell cycle without disrupting other interactions of the protein, we used mice in which only the cdk-binding domain of p27 has been deleted resulting in a truncated form of p27 (p27tr). To generate a T cell population, which uniformly expresses a specific TCR that allows antigen-specific stimulation in vivo, we bred the DO11.10 TCR transgene onto the p27tr background. DO11.10 mice express a MHC class II-restricted transgenenic (Tg) TCR specific for OVA_323–339 peptide that can be detected by a clonotypic antibody. To generate animals with T cells exclusively expressing the DO11.10 TCR, we crossed the DO11.10 TCR-Tg mice onto RAG2−/− background which eliminates endogenous TCRs. Naive T cells from DO11.10+RAG2−/−p27tr mice (termed p27tr/Tg) expressed markers identical to those of naive T cells from wild type DO11.10+RAG2−/− (termed WT-Tg) mice, allowing the accurate comparison of naive cells that differ only in their ability to express the p27 cdk-binding domain. Tg T cells from WT-Tg or p27tr/Tg mice were adoptively transferred into syngeneic recipients, which were injected with OVA_323–339 either subcutaneously (primed) or intravenously (tolerized) according to established protocol. Draining lymph nodes were harvested and Tg cells were stimulated with OVA_323–339 to determine their ability to respond on antigen-specific rechallenge. WT-Tg and p27tr/Tg cells from the primed treatment group had robust proliferation and production of IL-2. Strikingly, in the tolerant treatment group, although WT-Tg cells proliferated poorly and had significantly reduced IL-2 production, p27tr/Tg cells had proliferation and IL-2 production that were indistinguishable from the p27tr/Tg primed treatment group. Next we used blockade of costimulation to induce tolerance in vivo. Tg T cells from WT-Tg or p27tr/Tg mice were adoptively transferred into syngeneic recipients, which were treated with either PBS (primed) or with anti-CD40L mAb plus CTLA4-Ig (tolerized) and were immunized with OVA_323–339 subcutaneously. Subsequent rechallenge with OVA_323–339 revealed that, after in vivo treatment of anti-CD40L mAb and CTLA4-Ig, only WT-Tg cells became tolerant and were incapable of proliferating and producing IL-2, whereas responses of p27tr/Tg cells were not suppressed. Detailed analysis revealed that the failure of p27tr/Tg cells to undergo tolerance induction in the syngeneic recipients was not due to defective suppression by Treg, but due to the intrinsic inability of p27tr/Tg T cells to undergo arrest at the G1 phase of the cell cycle during tolerogenic treatment. These results demonstrate a key role for p27 in determining the outcome of naive T cell encounter with a tolerogenic stimulus in vivo and have significant implications for developing of immune based therapies geared towards tolerance induction.