PI3Kδ hyper-activation promotes the development of B cells that exacerbateStreptococcus pneumoniaeinfection in an antibody-independent manner

Abstract

Streptococcus pneumoniae is a major cause of pneumonia and a leading cause of death 1world-wide. Antibody-mediated immune responses can offer protection against repeated 2 exposure to S. pneumoniae, yet vaccines only offer partial protection. Patients with 3 Activated PI3Kδ Syndrome (APDS) are highly susceptible to S. pneumoniae. We generated 4 a conditional knockin mouse model of this disease and identified a CD19 + B220 -B cell 5 subset that is induced by PI3Kδ signaling, is resident in the lungs, and which promotes 6 increased susceptibility to S. pneumoniae during the early phase of infection via an 7 antibody-independent mechanism. We show that an inhaled PI3Kδ inhibitor improves 8 survival rates following S. pneumoniae infection in wild-type mice and in mice with 9 activated PI3Kδ. These results suggest that a subset of B cells in the lung can promote the 10 severity of S. pneumoniae infection, representing a novel therapeutic target. 11 100 2B). This pattern resembles results found in patients with APDS 10 . In wild-type and 101 p110δ E1020K-GL cells, the PI3Kδ-selective inhibitor nemiralisib reduced PIP3 to the background 102 level observed in p110δ D910A cells, which, as expected, were insensitive to nemiralisib ( Fig 103 2A, B). 104 105 PIP3 binds to the protein kinase AKT, supporting its phosphorylation on Thr308 and 106 subsequent activation. Western blotting of purified p110δ E1020K-GL T cells showed increased 107AKT phosphorylation following stimulation with anti-CD3 and anti-CD28 antibodies 108 compared to wild-type cells, whereas AKT phosphorylation in p110δ D910A T cells was below 109 the limit of detection ( Fig 2C). In B cells, both basal and anti-IgM-induced phosphorylation of 110 AKT were elevated in p110δ E1020K-GL cells, while strongly diminished in p110δ D910A B cells (Fig 111 2D). The phosphorylation of ERK and the AKT effector proteins, FOXO and S6, were similarly 112 affected. All phosphorylation events in wild-type and p110δ E1020K-GL cells were reduced to 113 the levels observed in p110δ D910A cells by inhibition with nemiralisib. As expected, p110δ 114 protein expression was not affected by the E1020K or D910A mutations (Figs 2C, D). 115 116

T and B‐cell signaling in activated PI3K delta syndrome: From immunodeficiency to autoimmunity

“…In line with the human data, Pik3cd E1020K/+ mice showed a partial B‐cell developmental block in the BM and expansion of transitional B cells in the periphery, although, one study did not observe increased transitional B cells, possibly because of different gating strategies or the use of a conditional mouse model that expressed mutant PI3Kδ at later stages in mature B cells . These changes were accompanied by outgrowth of marginal zone B cells at the expense of mature follicular B cells.…”

“…As observed in APDS patients, IgM levels were higher in mutant mice than WT littermates . However, in contrast to the variable levels of serum IgG in patients, serum IgG subsets were consistently increased in Pik3cd E1020K/+ mice, possibly as result of greater PC differentiation. Notably, Pik3cd E1020K/+ B cells had intrinsic defects in switching to IgG1, IgG2, and IgG3 in culture .…”

“…Strikingly, we also detected a strong peripheral immune reaction toward gut bacteria in Pik3cd E1020K/+ mice, with more serum IgG that bound autologous bacteria, as detected by flow cytometry. Nonetheless, we did not find evidence for “sticky” polyreactive antibodies in these animals, either through the analysis of serum antibodies binding plastic, or phosphatidyl‐choline (SP unpublished observations), although Okkenhaug and colleagues observed increased reactivity to phosphatidyl‐choline in their mouse model . Such antibodies could be generated in an antigen‐specific manner from BCR‐mediated recognition of microbial proteins or products/metabolites coming from commensal bacteria, either in periphery or in the GALT, followed by B‐cell migration in the periphery.…”

“…A crucial metabolic regulator in B cells is mTORC1, which helps promote glycolytic and anabolic metabolism . B cells expressing active PI3Kδ show greater phosphorylation of S6, indicative of increased mTORC1 activity . This may contribute to the greater B‐cell proliferation and GC differentiation in Pik3cd E1020K/+ mice, as supported by a recent publication demonstrating a requirement for mTORC1 activity for B‐cell expansion, GC formation and the generation of antigen‐specific memory B cells and plasma cells; mTORC1 was implicated in the induction of BCL‐6 and IRF‐4, critical transcriptional regulators of GC B cell and plasma cells, respectively .…”

“…Recently, four independent groups, including ours, generated heterozygous mice expressing the E1020K mutant of PI3Kδ, equivalent to the most common APDS mutation . Pik3cd E1020K/+ mice recapitulate many phenotypes of APDS, including peripheral blood lymphopenia yet expansion of lymphocytes in the spleen, peripheral lymph nodes (LN), and gut‐associated lymphoid tissues (GALT), lymphoid nodules in lung and gut, elevated IgM, and autoimmune manifestations .…”