Search

Interleukin-7 (IL-7) is essential for the development of T cells in humans and mice where deficiencies in IL-7 signaling result in severe immunodeficiency. T cells require IL-7 at multiple points during development; however, it is unclear when IL-7 is first necessary. We observed that mice with impaired IL-7 signaling had a large reduction in the number of early thymic progenitors (ETPs) while mice that overexpress IL-7 had greatly increased numbers of ETPs. These results indicated that the development of ETPs is sensitive to IL-7. Bone marrow progenitors of ETP are present in normal numbers in mice with impaired IL-7 signaling (IL-7Rα449F) and were efficiently recruited to the thymus. Furthermore, ETPs and their progenitors from IL-7Rα449F mice did not undergo increased apoptosis and proliferate normally compared to WT cells. Mixed bone marrow chimeras demonstrated that IL-7 signaling has a cell-intrinsic role in ETP development but was not required for development of bone marrow progenitors. We have shown a novel role for IL-7 signaling in the development of ETPs that is distinct from classic mechanisms of IL-7 regulating survival and proliferation.

The PI3K pathway has emerged as a key regulator of regulatory T cell (Treg) development and homeostasis and is required for full Treg-mediated suppression. To identify new genes involved in PI3K-dependent suppression, we compared the transcriptome of WT and p110δ(D910A) Tregs. Among the genes that were differentially expressed was the gene for the transmembrane cyclic ADP ribose hydrolase CD38. Here we show that CD38 is expressed mainly by a subset of Foxp3(+)CD25(+)CD4(+) T cells originating in the thymus and on Tregs in the spleen. CD38(high) WT Tregs showed superior suppressive activity to CD38(low) Tregs, which failed to upregulate CD73, a surface protein which is important for suppression. However, Tregs from heterozygous CD38(+/-) mice were unimpaired despite lower levels of CD38 expression. Therefore, CD38 can be used as a marker for Tregs with high suppressive activity and the impaired Treg function in p110δ(D910A) mice can in part be explained by the failure of CD38(high) cells to develop.

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.

Lymphopenia-induced proliferation (LIP) is a proliferative program initiated in response to T cell insufficiency caused by acute or chronic immunodepletion. Studies of lymphopenic mice have demonstrated that the cytokine IL-7 and TCR signaling are critical for LIP. We examined how these two factors impact T cell proliferation following transfer into moderately lymphopenic mice. In this study, we show that moderate lymphopenia (∼25% of wild-type lymphocytes) of IL-7Rα knock-in mutant (IL-7Rα449F) mice supports T cell proliferation, although with decreased frequency and kinetics compared with cells transferred to severely lymphopenic (5% of wild-type lymphocytes) IL-7Rα−/− hosts. Although previous studies have demonstrated that elevated IL-7 levels play an important role in LIP, IL-7 availability was not elevated in IL-7Rα449F mice. However, moderate lymphopenia increased access of transferred T cells to self-peptide presented on APCs that can trigger TCR signaling and proliferation. Importantly, we did not detect significant changes in TCR Vβ usage of proliferated T cells recovered from either moderately or severely lymphopenic hosts. Our work demonstrates that polyclonal T cells retain a diverse TCR repertoire following proliferation mediated by either self-peptide–MHC interaction alone or in combination with IL-7, and that T cell reconstitution is most efficient in the presence of increased IL-7 availability.

We have previously described critical and nonredundant roles for the phosphoinositide 3-kinase p110δ during the activation and differentiation of naive T cells, and p110δ 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 p110δ activity is required for interferon-γ production. Moreover, acute inhibition of p110δ inhibits cytokine production and reduces hypersensitivity responses in mice. Whether p110δ played a similar role in human T cells was unknown. Here we show that IC87114 potently blocked T-cell receptor–induced 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 p110δ inhibition as naive T cells and show that mouse models accurately predict p110δ function in human T cells. There is therefore a strong rationale for p110δ inhibitors to be considered for therapeutic use in T-cell–mediated autoimmune and inflammatory diseases.

Activation of PI3K is among the earliest signaling events observed in T cells after conjugate formation with antigen-presenting 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 p110δ, but not the p110γ, 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 PKCθ recruitment. CD28 could partially compensate for the lack of p110δ activity during T-cell activation, which indicates that CD28 and p110δ act in parallel and complementary pathways to activate T cells. Consistent with this, CD28 and p110δ double-deficient mice were severely immune compromised. We therefore suggest that combined pharmaceutic targeting of p110δ activity and CD28 costimulation has potent therapeutic potential.

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.