PIK3IP1/TrIP restricts activation of T cells through inhibition of the PI3K/Akt Pathway

Phosphatidylinositide-3-Kinases (PI3Ks) are a family of lipid kinases that play important intracellular signaling roles in cellular functions such as cell proliferation, motility, growth, intracellular trafficking, differentiation and survival. PI3K produces PIP3 which further facilitates the activation of downstream effectors such as Akt and PDK1. These effectors facilitate the cellular processes associated with PI3K activity. Conversely, because of the nature of PI3Ks roles, dysregulation of PI3K, leading to over-activity of the PI3K pathway is implicated in many cancers. PTEN, SHIP and INPP4B are negative regulators of PI3K activity that function downstream of PI3K and have been shown to act as tumor suppressors. Recently, PI3K Interacting Protein 1 (PIK3IP1) or TrIP (Transmembrane Inhibitor of PI3K), a novel negative regulator that functions upstream and proximal to PI3K, has been identified. TrIP, is a transmembrane protein and has been shown to down regulate PI3K activity leading to reduction of phosphorylation on Akt in carcinoma cell lines. Our lab was the first to show that TrIP can inhibit TCR signaling and TCR activation, in T cell lines. Here I have shown that both the extracellular kringle domain and intracellular p85-like domain are required for TrIP to inhibit PI3K and T cell activation. Interestingly, I have also found that cell surface expression of TrIP is acutely down-regulated during T cell activation. Furthermore, my results indicate that TrIP oligomerizes in order to inhibit PI3K signaling. Using knockout mice lacking expression of TrIP in T-cell compartments, I have been able to better define the requirements for TrIP in primary T cells upon TCR activation and in response to infection. Finally, I have developed a TrIP ecto-domain hIgG1 Fc fusion protein. This fusion protein has been used as an immunogen for the development of antibodies against TrIP in mice. This fusion protein has also been used to evaluate a possible ligand for TrIP

Unexpected role for IL-17 in protective immunity against hypervirulent Mycobacterium tuberculosis HN878 infection

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), infects one third of the world's population. Among these infections, clinical isolates belonging to the W-Beijing appear to be emerging, representing about 50% of Mtb isolates in East Asia, and about 13% of all Mtb isolates worldwide. In animal models, infection with W-Beijing strain, Mtb HN878, is considered "hypervirulent" as it results in increased mortality and causes exacerbated immunopathology in infected animals. We had previously shown the Interleukin (IL) -17 pathway is dispensable for primary immunity against infection with the lab adapted Mtb H37Rv strain. However, it is not known whether IL-17 has any role to play in protective immunity against infection with clinical Mtb isolates. We report here that lab adapted Mtb strains, such as H37Rv, or less virulent Mtb clinical isolates, such as Mtb CDC1551, do not require IL-17 for protective immunity against infection while infection with Mtb HN878 requires IL-17 for early protective immunity. Unexpectedly, Mtb HN878 induces robust production of IL-1β through a TLR-2-dependent mechanism, which supports potent IL-17 responses. We also show that the role for IL-17 in mediating protective immunity against Mtb HN878 is through IL-17 Receptor signaling in non-hematopoietic cells, mediating the induction of the chemokine, CXCL-13, which is required for localization of T cells within lung lymphoid follicles. Correct T cell localization within lymphoid follicles in the lung is required for maximal macrophage activation and Mtb control. Since IL-17 has a critical role in vaccine-induced immunity against TB, our results have far reaching implications for the design of vaccines and therapies to prevent and treat emerging Mtb strains. In addition, our data changes the existing paradigm that IL-17 is dispensable for primary immunity against Mtb infection, and instead suggests a differential role for IL-17 in early protective immunity against emerging Mtb strains. © 2014 Gopal et al

Adenoviral IL-17 overexpression reverses the increased susceptibility to <i>Mtb</i> HN878 infection in IL-17^−/− mice.

<p>B6 or IL-17^−/− mice were aerosol infected with ∼100 cfu <i>Mtb</i> HN878 and were either infected with a vector control adenovirus expressing luciferase (Adluc) or with an adenoviral vector overexpressing IL-17 (AdIL-17) on day 9. Lung bacterial burden was determined on D30 post-<i>Mtb</i> infection (a). Pulmonary histology was assessed on formalin-fixed, paraffin embedded lung sections that were stained with H&E (b). 100× magnification for H&E sections. The average size of T cell perivascular cuffing (c) and B cell lymphoid follicles (d) was calculated using the morphometric tool of the Zeiss Axioplan microscope. CXCL13 mRNA expression in formalin fixed, paraffin embedded lung sections was studied by in situ hybridization (e). 100× magnification, arrows indicate areas of CXCL-13 mRNA expression. The area occupied by CXCL13 signal by in situ hybridization per 200× field was calculated using the morphometric tool of the Zeiss Axioplan microscope. Serial sections from infected lungs were also processed for immunofluorescence using antibodies specific for CXCL13 and B220 (f). 400× magnification. Immunofluorescence staining was also performed using antibodies specific for or iNOS and F4/80 and the numbers of iNOS⁺ cells were counted (g). 400× magnification. The data points represent values from n = 5 mice per group. *p≤0.05, **p≤0.005, ***p≤0.0005, ns-not significant.</p

<i>Mtb</i> HN878 induces potent IL-17 responses in infected mice.

<p>B6 mice were either aerosol infected with ∼100 cfu <i>Mtb</i> H37Rv or <i>Mtb</i> HN878. Lungs were homogenized in saline on D30 post-infection and IL-17 protein levels were determined by Luminex (a). IL-17 GFP reporter mice were aerosol infected with ∼100 cfu <i>Mtb</i> H37Rv or <i>Mtb</i> HN878. The frequency (b) and absolute number (c) of lung IL-17-producing cells (GFP⁺) was determined by flow cytometry at D25 post-infection. Representative flow cytometry histograms of lung cell suspensions from uninfected (Un), <i>Mtb</i> H37Rv or <i>Mtb</i> HN878-infected IL-17 GFP reporter mice is shown (d). The number of CD3⁺IL-17⁺ lung cells was determined by flow cytometry (e). The percentage of ESAT-6_1–20-specific, IL-17 (f) and IFN-γ (g) producing cells in the lungs of these mice was determined by ELISpot assay. 1×10⁶ lung CD11c⁺ cells from B6 mice and infected in vitro with <i>Mtb</i> H37Rv or <i>Mtb</i> HN878 (MOI 0.01). Subsequently, naïve B6 total lung cell suspensions were added to the culture, and 6 days later, IL-17 levels were determined in supernatants by ELISA (h). The data points represent the mean (±SD) of values from 3–5 samples. *p≤0.05, **p≤0.005, ***p≤0.0005. ns-not significant.</p

CXCR5 deficiency increases susceptibility to <i>Mtb</i> HN878 infection.

<p>B6 and CXCR5^−/− mice were aerosol infected with ∼100 cfu <i>Mtb</i> HN878 and lung bacterial burden was determined on D30 post-infection (a). Pulmonary histology was assessed on formalin-fixed, paraffin embedded lung sections. The average size of T cell perivascular cuffing (b) and B cell lymphoid follicles (c) was calculated using the morphometric tool of the Zeiss Axioplan microscope. Serial sections from infected lungs were also processed for immunofluorescence using antibodies specific for CD3 and B220 (c) or iNOS and F4/80 (d). The number of iNOS-expressing cells per field (d) was counted and shown. 400× magnification. Wild type or CXCR5^−/− ESAT-6 TCR Tg CD4⁺ T cells were in vitro differentiated to the Th17 subset and 2×10⁶ Th17 cells were adoptively transferred into IL-17^−/− mice and then infected with low doses of <i>Mtb</i> HN878. Lung bacterial burden was determined on D30 post-infection (e). The data points represent values from n = 5 mice per group. **p≤0.005, ***p≤0.0005.</p

IL-17 is required for protective immunity against <i>Mtb</i> HN878 infection.

<p>B6 or IL-17^−/− mice were aerosol infected with ∼100 cfu <i>Mtb</i> H37Rv (a), <i>Mtb</i> HN878 (b) or <i>Mtb</i> CDC1551 (c). Lung bacterial burden was determined by plating on D30 or D60 post-infection. The data points represent the mean (±SD) of values from 3–5 samples. *p≤0.05, ***p≤0.0005, ns-not significant.</p

IL-17 mediates control of <i>Mtb</i> HN878 through CXCL13 induction, lymphoid follicle formation and macrophage activation.

<p>B6 and IL-17^−/− mice were aerosol infected with ∼100 cfu <i>Mtb</i> HN878 and lungs were harvested on D60 post-infection. Pulmonary histology was assessed on formalin-fixed, paraffin embedded lung sections that were stained with H&E (a). Arrows depict T cell perivascular cuffing. Serial sections from infected lungs were also processed for immunofluorescence using antibodies specific for CD3 and B220 (b) or iNOS and F4/80 (c), and stained for CXCL13 mRNA expression by in situ hybridization (d). 400× magnification for immunofluorescence images. Arrows within (d) indicate CXCL-13 mRNA expression. The average size of T cell perivascular cuffing (a) and B cell lymphoid follicles (b) was calculated using the morphometric tool of the Zeiss Axioplan microscope. The number of iNOS-expressing cells per field (c) was counted and shown. n = 5–9 mice per group. ***p≤0.005.</p

<i>Mtb</i> HN878-driven IL-17 production is dependent on TLR-2 and IL-1β.

<p>1×10⁶ lung DCs from B6 mice were infected in vitro with <i>Mtb</i> H37Rv or <i>Mtb</i> HN878 (MOI 0.01) for 48 h and IL-1β production was determined in the supernatants (a). 1×10⁶ B6 DCs were infected with <i>Mtb</i> H37Rv or <i>Mtb</i> HN878 (MOI 0.01), following which naïve B6 or IL-1R−/− total lung cell suspensions were added to the culture, and IL-17 levels were determined by ELISA on day 6 of the co-culture (b). B6 or IL-1R^−/− mice were aerosol infected with ∼100 cfu <i>Mtb</i> HN878 and lung bacterial burden was determined on D30 post-infection (c). The number of ESAT-6_1–20-specific, IL-17-producing cells in the lungs of uninfected (Un), <i>Mtb</i> HN878-infected B6 and IL-1R^−/− mice was determined by ELISpot assay (d). 1×10⁶ BMDCs were treated in vitro with whole cell lysate, cell wall extract or a lipid extract (20 µg/ml) of <i>Mtb</i> H37Rv or <i>Mtb</i> HN878, and IL-1β production in cell culture supernatants was determined by ELISA (e). 1×10⁶ lung CD11c⁺ cells from B6 or TLR-2^−/− mice and infected in vitro with <i>Mtb</i> H37Rv or <i>Mtb</i> HN878. Subsequently, naïve B6 or TLR-2^−/− total lung cell suspensions were added to the culture, and IL-1β (f) and IL-17 (g) levels were determined by ELISA. The data points represent the mean (±SD) of values from 3–5 samples. *p≤0.05, **p≤0.005, ***p≤0.0005. nd-not detected.</p

IL-17R expression on non-hematopoietic cells is required for protective immunity against <i>Mtb</i> HN878.

<p>Hematopoietic IL-17R^−/− BMC mice (B6 host/−/− BM), non-hematopoietic IL-17R^−/− BMC mice (−/− host/B6 BM), complete IL-17R^−/− BMC mice (−/− host/−/−BM) and complete B6 BMC mice (B6 host/B6 BM) were generated as described under methods. BMC mice were aerosol infected with ∼100 cfu <i>Mtb</i> HN878 and lung bacterial burden was determined on D30 post-infection (a). Pulmonary histology was assessed on formalin-fixed, paraffin embedded lung sections that were stained with H&E and the average size of T cell perivascular cuffing was calculated using the morphometric tool of the Zeiss Axioplan microscope (b). T cell perivascular cuffing is indicated by the arrows. CXCL13 mRNA expression in formalin fixed, paraffin embedded lung sections was studied by in situ hybridization (c). 100× magnification, arrows point to typical areas of CXCL13 mRNA expression. The area occupied by CXCL13 signal by in situ hybridization per 200× field was calculated using the morphometric tool of the Zeiss Axioplan microscope. Serial sections from infected lungs were also processed for immunofluorescence using antibodies specific for CXCL13 and B220 (d). 400× magnification. The data points represent values from n = 3–5 mice per group. *p≤0.05, **p≤0.005.</p