CD4+CD25+Foxp3+ regulatory T cells (Tregs) contribute to the maintenance of peripheral tolerance by inhibiting the expansion and function of conventional T cells. Treg development and homeostasis are regulated by the Ag receptor, costimulatory receptors such as CD28 and CTLA-4, and cytokines such as IL-2, IL-10, and TGF-β. Here we show that the proportions of Tregs in the spleen and lymph nodes of mice with inactive p110δ PI3K (p110δD910A/D910A) are reduced despite enhanced Treg selection in the thymus. p110δD910A/D910A CD4+CD25+Foxp3+ Tregs showed attenuated suppressor function in vitro and failed to secrete IL-10. In adoptive transfer experiments, p110δD910A/D910A T cells failed to protect against experimental colitis. The identification of p110δ as an intracellular signaling protein that regulates the activity of CD4+CD25+Foxp3+ Tregs may facilitate the further elucidation of the molecular mechanisms responsible for Treg-mediated suppression.

The role of PI3K in T cell activation and costimulation has been controversial. We previously reported that a kinase-inactivating mutation (D910A) in the p110δ isoform of PI3K results in normal T cell development, but impaired TCR-stimulated cell proliferation in vitro. This proliferative defect can be overcome by providing CD28 costimulation, which raises the question as to whether p110δ activity plays a role in T cell activation in vivo, which occurs primarily in the context of costimulation. In this study, we show that the PI3K signaling pathway in CD28-costimulated p110δD910A/D910A T cells is impaired, but that ERK phosphorylation and NF-κB nuclear translocation are unaffected. Under in vitro conditions of physiological Ag presentation and costimulation, p110δD910A/D910A T cells showed normal survival, but underwent fewer divisions. Differentiation along the Th1 and Th2 lineages was impaired in p110δD910A/D910A T cells and could not be rescued by exogenous cytokines in vitro. Adoptive transfer and immunization experiments in mice revealed that clonal expansion and differentiation in response to Ag and physiological costimulation were also compromised. Thus, p110δ contributes significantly to Th cell expansion and differentiation in vitro and in vivo, also in the context of CD28 costimulation.

We have previously described critical and nonredundant roles for the phosphoinositide 3-kinase p110␦ during the activation and differentiation of naive T cells, and p110␦ inhibitors are currently being developed for clinical use. However, to effectively treat established inflammatory or autoimmune diseases, it is important to be able to inhibit previously activated or memory T cells. In this study, using the isoform-selective inhibitor IC87114, we show that sustained p110␦ activity is required for interferon-␥ production. Moreover, acute inhibition of p110␦ inhibits cytokine production and reduces hypersensitivity responses in mice. Whether p110␦ played a similar role in human T cells was unknown. Here we show that IC87114 potently blocked T-cell receptorinduced phosphoinositide 3-kinase signaling by both naive and effector/memory human T cells. Importantly, IC87114 reduced cytokine production by memory T cells from healthy and allergic donors and from inflammatory arthritis patients. These studies establish that previously activated memory T cells are at least as sensitive to p110␦ inhibition as naive T cells and show that mouse models accurately predict p110␦ function in human T cells. There is therefore a strong rationale for p110␦ inhibitors to be considered for therapeutic use in T-cell-mediated autoimmune and inflammatory diseases. (Blood. 2010;115:2203-2213)

Activation of PI3K is among the earliest signaling events observed in T cells after conjugate formation with antigenpresenting cells (APCs). The relevant PI3K catalytic isoform and relative contribution of the TcR and CD28 to PI3K activity at the immune synapse have not been determined unequivocally. Using a quantitative imaging-based assay, we show that the PI3K activity at the T cell-APC contact area is dependent on the p110␦, but not the p110␥, isoform of PI3K. CD28 enhanced PIP3 production at the T-cell synapse independently of its YMNM PI3K-recruitment motif that instead was required for efficient PKC recruitment. CD28 could partially compensate for the lack of p110␦ activity during T-cell activation, which indicates that CD28 and p110␦ act in parallel and complementary pathways to activate T cells. Consistent with this, CD28 and p110␦ double-deficient mice were severely immune compromised. We therefore suggest that combined pharmaceutic targeting of p110␦ activity and CD28 costimulation has potent therapeutic potential.

G protein-coupled receptor (GPCR) activation elicits neutrophil responses such as chemotaxis and reactive oxygen species (ROS) formation, which depend on the small G protein Rac and are essential for host defense. P-Rex and Vav are two families of guanine-nucleotide exchange factors (GEFs) for Rac, which are activated through distinct mechanisms but can both control GPCR-dependent neutrophil responses. It is currently unknown whether they play specific roles or whether they can compensate for each other in controlling these responses. In this study, we have assessed the function of neutrophils from mice deficient in P-Rex and/or Vav family GEFs. We found that both the P-Rex and the Vav family are important for LPS priming of ROS formation, whereas particle-induced ROS responses and cell spreading are controlled by the Vav family alone. Surprisingly, fMLF-stimulated ROS formation, adhesion, and chemotaxis were synergistically controlled by P-Rex1 and Vav1. These responses were more severely impaired in neutrophils lacking both P-Rex1 and Vav1 than those lacking the entire P-Rex family, the entire Vav family, or both P-Rex1 and Vav3. P-Rex1/Vav1 (P1V1) double-deficient cells also showed the strongest reduction in fMLF-stimulated activation of Rac1 and Rac2. This reduction in Rac activity may be sufficient to cause the defects observed in fMLF-stimulated P1V1 neutrophil responses. Additionally, Mac-1 surface expression was reduced in P1V1 cells, which might contribute further to defects in responses involving integrins, such as GPCR-stimulated adhesion and chemotaxis. We conclude that P-Rex1 and Vav1 together are the major fMLFR -dependent Dbl family Rac-GEFs in neutrophils and cooperate in the control of fMLF-stimulated neutrophil responses.