PTEN regulates natural killer cell trafficking in vivo

Phosphatase and tensin homolog (PTEN) is a critical negative regulator of the phosphoinositide-3 kinase pathway, members of which play integral roles in natural killer (NK) cell development and function. However, the functions of PTEN in NK cell biology remain unknown. Here, we used an NK cell-specific PTEN-deletion mouse model to define the ramifications of intrinsic NK cell PTEN loss in vivo. In these mice, there was a significant defect in NK cell numbers in the bone marrow and peripheral organs despite increased proliferation and intact peripheral NK cell maturation. Unexpectedly, we observed a significant expansion of peripheral blood NK cells and the premature egress of NK cells from the bone marrow. The altered trafficking of NK cells from peripheral organs into the blood was due to selective hyperresponsiveness to the blood localizing chemokine S1P. To address the importance of this trafficking defect to NK cell immune responses, we investigated the ability of PTEN-deficient NK cells to traffic to a site of tumor challenge. PTEN-deficient NK cells were defective at migrating to distal tumor sites but were more effective at clearing tumors actively introduced into the peripheral blood. Collectively, these data identify PTEN as an essential regulator of NK cell localization in vivo during both homeostasis and malignancy.Natural killer (NK) cells are innate lymphoid cells critical for host defense against pathogens and antitumor responses (1–3). In the naïve mouse, NK cells are distributed among a number of hematopoietic and nonhematopoietic organs, including major reservoirs within the spleen, blood, and bone marrow (BM). Factors that orchestrate NK cell trafficking during homeostasis, including chemokine receptors and adhesion molecules, remain largely unknown. The majority of studies have focused on receptors controlling NK cell migration out of the BM, such as CXCR4 and S1P₅ (4–6). During inflammatory states, other receptors have defined roles for specific tissue homing, including CCR1, CCR5, CXCR3, CX3CR1, and CXCR6 (7). Integrin molecules, such as very late antigen-4 (VLA-4), also have specific functions in retaining NK cells within BM sinusoids (8). However, the downstream intracellular signaling pathways important for trafficking remain unclear, especially in light of the complex interplay of multiple chemotactic signals during an immune response.One family of enzymes regulating a number of chemokine receptors includes the phosphoinositide 3-kinase (PI3K) signaling pathway, which plays a broad role in regulating cellular proliferation, gene expression, survival, cytoskeleton rearrangement, and migration (9). In immune cells, the PI3K pathway may be activated downstream of a number of receptors, including cytokine receptors and G protein-coupled receptors (GPCRs), the latter of which include most chemokine receptors. Stimulation of the PI3Ks results in the generation of phosphatidylinositol phosphate lipids, such as PI(3,4,5)P₃, and subsequent recruitment and activation of downstream signaling proteins, including Vav, Akt, and PDK1 (9, 10). Exogenous inhibition of PI3Ks suppresses perforin and granzyme B polarization and NK cell cytotoxicity (11). Additionally, deficiency of the leukocyte-selective PI3K p110γ or p110δ subunit results in defective NK cell development and maturation and alters the production of IFN gamma (IFN-γ) and cytotoxicity (12–14). New evidence has also linked IL-15 to the mammalian target of rapamycin (mTOR) pathway via PI3K activation (15, 16). Thus, PI3K signaling is a critical pathway for NK cell biological processes.Two primary phosphatases oppose PI3K generation of the active secondary messenger PI(3,4,5)P₃: SH2-containing inositol-5′-phosphatase 1 (SHIP1) and phosphatase and tensin homolog (PTEN). SHIP1 specifically dephosphorylates PI(3,4,5)P₃ to PI(3,4)P₂, whereas PTEN dephosphorylates the 3′ inositol phosphate on PI(3,4,5)P₃, PI(3,4)P₂, and PI(3)P, thereby also counteracting other classes of PI3Ks (10). SHIP1^−/− BM-chimeric mice have no overt alterations in NK cell distribution, and its intrinsic role in NK cell development only affects the terminal differentiation of mature NK cells (17). However, there are no reported studies of PTEN function in NK cells to date. The effects of lymphocyte-selective PTEN deficiency in T and B cells result from increased PI3K signaling and include increased cell survival and proliferation, lowered activation threshold through the B-cell receptor (18), and loss of costimulatory requirements in T cells (19). The role of phosphatases, particularly SHIP1 and PTEN, in cellular migration, however, remains controversial and appears to be dependent on both the cell studied and the mode of stimulation (20–22). Furthermore, unique aspects of PTEN function have been reported, including protein phosphatase activity (23) and regulation via intracellular sequestration to the cytoplasmic membrane (24). As PTEN is a major nonredundant regulator of PI3K signaling, we hypothesized that disruption of PI3K inhibition would uniquely impact NK cell developmental and functional pathways.In this study, we generated NK cell-specific PTEN-deficient mice, which have diminished opposing lipid phosphatase activity to all known PI3K family members. PTEN-deficient mice display significant defects in peripheral organ and BM NK cell compartments, with a large proportion of NK cells being inappropriately localized to the blood. These effects were due in part to alterations in NK cell trafficking in vivo, a defect that also prevented their recruitment to a localized tumor challenge. These results identify a previously unidentified role for PTEN in regulating NK cell tissue distribution during both homeostasis and malignancy in vivo.