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PI3-kinase and the control of T cell growth and proliferation by FoxOs

Bulletin du Cancer. Volume 93, Number 5, 10036-8, Mai 2006, Electronic Journal of Oncology

Summary

Author(s) : Stéphanie Fabre, Valérie Lang, Georges Bismuth , Institut Cochin, Départment de biologie cellulaire, Inserm U567, CNRS UMR8104. Université René-Descartes, équipe labellisée par la Ligne nationale contre le cancer, 22 rue Méchain, 75014 Paris.

Summary : Numerous cancers are caused by an uncontrolled uncontrolled activity of the PI3-kinase pathway. The proto-oncogene Akt, one of its main effectors, commands several molecular switches involved in cell survival and proliferation. One of these switches is represented by a group of related molecules belonging to the Forkhead family of transcription factors, called FoxOs. FoxOs negatively control cell cycle entry and this process emerges now as a mainstream mechanism used by various cell types to escape cell quiescence. In the light of recent works, FoxOs seem also to have a key role in the proliferative response of immune cells, especially in the clonal expansion of T lymphocytes induced by antigen. Experimental evidence supporting a relationship in T cells between PI3-kinase metabolism and these growth suppressive genes will be described in this mini-review.

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ARTICLE

Auteur(s) : Stéphanie Fabre, Valérie Lang, Georges Bismuth

Institut Cochin, Départment de biologie cellulaire, Inserm U567, CNRS UMR8104. Université René-Descartes, équipe labellisée par la Ligne nationale contre le cancer, 22 rue Méchain, 75014 Paris

Antigen recognition leads to a sustained activation of 3’-phosphoinositide metabolism in T cells

Activation of naive T lymphocytes is triggered by T cell receptor (TCR) recognition of MHC-peptide complexes present at the surface of antigen presenting cells (APC). The interaction usually starts with discrete contacts between the two cell types, and the engagement of a small number of TCR molecules, rapidly followed by the formation of a large and more or less organized interacting zone, called the immunological synapse (IS) [1]. Thereafter this interaction remains stable during several hours, as revealed by numerous in vitro studies and also confirmed more recently in vivo by dual photon microscopy analysis of secondary lymphoid organs [2]. In this sequence, the first signaling events (i.e. calcium rise or tyrosine kinase activation) are detected very early, before the formation of the prolonged IS. However, we also know that this prolonged interaction step is a critical parameter to trigger in the T lymphocyte a wide range of metabolic events that ultimately result in proliferation. An increasing body of evidence now suggests that activation of the phosphoinositide (PI)-3-kinase (PI3K) metabolic pathway is decisive to support this T cell clonal expansion triggered by IS formation.

PI3Ks catalyze the formation of 3’-PI, a well-known class of phospholipids almost undetectable in naïve T cells. 3’-PI recruits at the plasma membrane PH domain-containing proteins, such as the serine threonine kinase Akt, a key downstream effector of this metabolic pathway. The PH domain of Akt fused to GFP has been used to provide evidence for an extensive production of 3’-PI in T cells during conjugate formation [3, 4]. Remarkably, 3’-PI metabolism in T cells escapes the traditional paradigm so far established in neutrophils or fibroblasts. In these cell types, an uneven stimulus usually gives a transient and restricted accumulation of 3′-PI at their leading edge, whereas T cells show an accumulation of 3′-PI in the whole plasma membrane, far beyond the IS, which lasts several hours. These discrepancies can be explained by the fact that in T cells PI3K class IA regulatory subunits (such as p85α) are stably recruited for hours within the IS, where a sustained accumulation of phosphotyrosines is observed, and also by a rapid and permanent diffusion of 3’-PI lipids from their site of production in the IS, to the whole plasma membrane [5].

PI3Ks affect T cell proliferation

PI3Ks, especially those of class IA, appear to be major players in lymphocyte homeostasis as

revealed by genetic studies in mice. Thus, invalidation of PIK3r1, the gene encoding p85α and its splice variants p50α and p55α, results in a reduction of the number of mature B cells and immunoglobulin production [6]. Intriguingly, T cells are much less affected. It was speculated that PI3Ks might not be essential for T cell functions, but mice expressing catalytically inactive p110δ subunits show a defect both in their B cell and T cell responses to antigen stimulation [7]. Moreover, studies using pharmacological inhibitors also demonstrate the role of PI3Ks in the stimulatory effects of some cytokines such as IL-2 or IL-12 on T cell survival and proliferation [8, 9]. Interestingly, inhibition of PI3Ks does not prevent the formation of the IS and many biological events induced by antigen, with the exception of T cell blastogenesis [3, 4]. This is consistent with the fact that invalidation of the 3′-PI phosphatase PTEN in T cells induces a lethal lymphoproliferative syndrome [10] and ultimately suggests that PI3K metabolism has a major impact on the biological processes conditioning T cell proliferation. Many downstream PI3K biological effects in T cells are controlled by Akt activated after its interaction with 3’-PI at the plasma membrane. Akt phosphorylates numerous substrates influencing protein synthesis, cell survival and proliferation. Cooperatively, many of them may contribute to the control of cell growth and proliferation by PI3Ks. However, there is currently a growing interest for transcriptional factors of the FoxO (Forkhead box subgroup O) family.

FoxOs regulate T cell growth induced by antigen

Various transcription factors are regulated by an increased 3’-PI metabolism, among which a subgroup of proteins called FoxO, belonging to the forkhead family of transcription factors, can be distinguished. Four FoxO molecules have been described in mammals, FoxO1, FoxO3, FoxO4 and FoxO6, which can be phosphorylated by Akt on three consensus serine/threonine residues. In cells where the PI3K/Akt pathway is not activated, FoxOs are unphosphorylated and essentially nuclear (( figure 1 )). In this localization, they control the transcription of multiple genes, coding in particular for proteins blocking cell cycle entry, p27kip, p130/Rb and cyclin G2 [11]. FoxOs are therefore considered as critical players in the control of cell quiescence. However, Akt-mediated phosphorylation of FoxOs inhibits these activities by inducing their nuclear exclusion and cytoplasmic retention through a Ran GTPase-dependent nuclear export and 14-3-3 protein chaperoning. Different studies have now revealed that this process represents a key event to trigger the proliferative effect of the PI3K pathway. Thus, the permanent growth of PTEN-deficient tumor cell lines caused by a persistent accumulation of 3’-PI is blocked by constitutively active FoxO molecules that cannot be excluded from the nucleus after their mutation on the three Akt phosphorylation sites. Also PTEN deficiency leads to aberrant localization of FoxOs to the cytoplasm, and restoration of PTEN expression brings FoxOs back to the nucleus and restores transcriptional activation [12]. More generally, FoxOs are now considered as true tumour suppressor factors, as also suggested by their direct implication in certain cancers. For instance, the t(2;13)(q35;q14) translocation, giving a chimeric transcript between FoxO1 and PAX3, is frequently observed in alveolar rhabdomyosarcoma [13]. Other translocations, in particular those involving FoxO3 and the MLL gene, are also observed in acute myeloid leukaemias [14] as well as translocations of FoxO4 on the X chromosome in childhood lymphomas [15].

Until recently, our knowledge of the role of FoxOs in the homeostasis of the immune system was limited, but things are rapidly evolving. Thus, a recent study has shown that FoxO3 deficiency in mice leads to a spontaneous inflammatory syndrome and lymphoproliferation [16]. The specific role of FoxOs in the context of T cells responding to antigen after synapse formation was also challenged using live video imaging microscopy analysis of FoxO1 localization in primary human T cells interacting with APCs [5]. This study reported that antigenic stimulation induces an extensive nuclear exclusion of FoxO1, starting a few minutes after the beginning of the contact between the two cell types and lasting for hours. This kinetics was fully consistent with the rapid and prolonged PI3K activation seen in T cells stimulated by antigen. As expected, in this cell system also, mutation of the residues phosphorylated by Akt completely prevents FoxO1 relocalisation to the cytoplasm. Mainly, this constitutively active mutant strongly impairs T cell growth induced by antigen.

Conclusion

Downstream of PI3Ks, FoxO transcription factors appear to be at the crossroads of numerous biological processes controlling survival, growth and cellular longevity in various organisms and cellular systems [17]. They role in lymphocyte physiology is far from being perfectly understood, but recent studies clearly indicate that sustained PI3K activation at the IS in naive T cells is essential to maintain 3’-PI cellular levels at sufficiently high concentrations in order to safely sequester FoxOs outside the nucleus, thereby allowing clonal expansion in response to APCs. A similar phenomenon has also been reported in murine B cells expressing active forms of FoxO1 and FoxO3 [18], suggesting that the control of FoxOs shuttling may represent a general strategy used by lymphocytes to switch from quiescence to cell cycle progression after stimulation by antigen. This has not been yet demonstrated in vivo, but in lymphoid organs, activated T cells undergo numerous mitoses and FoxO inactivation is likely to represent an important outcome to favor this process under physiological conditions. But, many other questions remain unresolved. For instance, do the various FoxOs regulate T cell growth through redundant mechanisms? Do they exert similar control over the different T cell populations (i.e. naive versus memory T cells)? Are there some specific targets of FoxOs in T cells influencing parameters other than quiescence and growth in the development of an adaptive immunity? Clarifying these issues could probably lead us not only to uncover new aspects of the mechanisms controlling the homeostasis of the immune system, but would also help us to better understand the function of these PI3K-dependent transcriptional factors in the tumoral process.

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