Naive CD4(+) T cells give rise to T-helper-cell subsets with functions that are tailored to their respective roles in host defence. The specification of T-helper-cell subsets is controlled by networks of lineage-specifying transcription factors, which bind to regulatory elements in genes that encode cytokines and other transcription factors. The nuclear context in which these transcription factors act is affected by epigenetic processes, which allow programmes of gene expression to be inherited by progeny cells that at the same time retain the potential for change in response to altered environmental signals. In this Review, we describe these epigenetic processes and discuss how they collaborate to govern the fate and function of T helper cells.

Regulatory T cells (T reg ) expressAcquired immune tolerance depends upon a subset of T cells that suppress the differentiation and function of pathogenic effector T cells. These regulatory T cells (T reg ) specifically express Foxp3, a member of the forkhead family of DNA-binding proteins that appears to be necessary and sufficient for T reg lineage choice (1-3). Humans with mutations in the foxp3 gene suffer from an X-linked complex of immune dysregulation, polyendocrinopathy, and enteropathy, a syndrome that results in part from the lack of T reg and leads to eventual death of these patients in childhood (4). Similarly, T reg are crucial for the inhibition of immunopathology in experimental models of organ transplantation and early onset autoimmune disorders and have been implicated in the control of childhood (type 1) autoimmune diabetes mellitus (5, 6).Recent studies have shown that Foxp3 overexpressed in transformed cell lines can repress transcription from artificial forkhead, nuclear factor of activated T cell (NFAT), 3 or NFB consensus binding elements in transiently transfected plasmids (7-9). These data suggest that Foxp3, like Foxp1 and Foxp2 (10), can function as a transcriptional repressor. However, expression of Foxp3 also leads to the induction of multiple genes, but the molecular basis for this activity is not known. It is also clear that Foxp3 expression alone is not sufficient for regulatory activity, as Foxp3ϩ T reg must receive antigenic signaling to exert suppressor function (11), but the basis for this activation requirement is not clear.We have used chromatin immunoprecipitation (ChIP) to analyze binding and remodeling of the chromatin at several Foxp3-responsive genes. Our studies provide important insight into how Foxp3 may regulate anergy in T reg and suggest a potential explanation for why T reg require T cell receptor (TCR) activation for their suppressive functions. EXPERIMENTAL PROCEDURESCells-A Jurkat human T cell line expressing the ecotropic retroviral receptor (mCAT-1) (12) was used for these studies. CD4ϩCD25Ϫ and CD4ϩCD25ϩ were purified from C57BL/6 mice using a T reg isolation kit (Miltenyi). T reg were also induced in vitro by culturing CD4ϩCD25Ϫ for 3-4 days on platebound anti-CD3 in the presence of soluble anti-CD28 (0.5 g/ml each) and TGF␤ (5 ng/ml).Retroviral Transduction-Murine Foxp3 cDNA was amplified from C57BL/6 thymus and cloned into the murine stem cell virus (MSCV)-based MIGR1 and MINR1 retroviral vectors (13), and versions of each vector were constructed containing an in-frame, N-terminal FLAG epitope. For generation of retrovirus, constructs were cotransfected with the pCLeco (Invitrogen) helper plasmid into the 293T-based Phoenix ecotropic packaging cell line (provided by G. Nolan, Stanford University). CD4ϩCD25Ϫ T cells were activated with phorbol 12-myristate 13-acetate (3 ng/ml), ionomycin (1 M), and IL-2 (10 units/ml) for 24 h, washed, and transduced by spinfection

Cell division drives T cell clonal expansion and differentiation, and is the result of concerted signaling from Ag, costimulatory, and growth factor receptors. How these mitogenic signals are coupled to the cell cycle machinery in primary T cells is not clear. We have focused on the role of p27kip1, a major cyclin-dependent kinase binding protein expressed by CD4+ T cells. Our studies using p27kip1 gene dosage demonstrate that early after activation, p27kip1 acts to promote, rather than inhibit, G1 to S phase progression within the first division cycle. However, throughout subsequent cell divisions p27kip1 behaves as a negative regulator, directly establishing the threshold amount of growth factor signaling required to support continued cell division. During this phase, signals from CD28 and IL-2R cooperate with the TCR to “tune” this threshold by inducing the degradation of p27kip1 protein, and we show that agents that block these pathways require elevated p27kip1 levels for their full antiproliferative activity. Finally, we show that p27kip1 opposes the development of CD4+ T cell effector function, and is required for the full development of anergy in response to a tolerizing stimulus. Our results suggest that p27kip1 plays a complex and important role in the regulation of cell division and effector function in primary CD4+ T cells.

The cyclin-dependent kinase (CDK) inhibitor p27kip1 is an important negative regulator of the cell cycle that sets a threshold for mitogenic signals in T lymphocytes, and is required for T cell anergy in vitro. To determine whether p27kip1 is required for tolerance in vivo, we performed cardiac allograft transplantation under conditions of combined CD28/CD40L costimulatory blockade. Although this treatment induced long-term allograft survival in wild-type recipients, costimulatory blockade was no longer sufficient to induce tolerance in mice lacking p27kip1. Rejected allografts from p27kip1−/− mice contained more CD4+ T lymphocytes and exhibited more tissue damage than allografts from tolerant, wild-type mice. Infiltrating p27kip1-deficient T cells, but not wild-type T cells, exhibited nuclear expression of cyclins E and A, indicating uncontrolled T cell cycle progression in the graft. The failure of tolerance in p27kip1−/− mice was also accompanied by markedly increased numbers of allospecific, IFN-γ-producing cells in the periphery, and occurred despite apparently normal regulatory T cell activity. These data demonstrate that the CDK inhibitor p27kip1 enforces the costimulatory requirement for the expansion and differentiation of alloimmune effector T lymphocytes in vivo, and point to CDKs as novel targets for immunosuppressive or tolerance-inducing therapies.