Journal: Journal of Biological Chemistry

Article Title: T cells transduce T-cell receptor signal strength by generating different phosphatidylinositols

doi: 10.1074/jbc.ra118.006524

Figure Lengend Snippet: Figure 1. T cells generate a different landscape of PIPs in response to TCR signal strength. A) A simplified model of T cell activation focusing on AKT activation was constructed. Simulations in Matlab were performed where TCR signal strength was modulated by altering the amount of TCR-pMHC in the simulation. The abundance of (B) PTEN, (C) P-PDK1, (C) mTORC2, (D) phosphorylated AKT, (E) PI(4,5)P2 and (F) PIP3 were plotted as a function of TCR signal strength. Mass ELISA assays were used to measure the amount of (G) PI(4,5)P2, (H) PIP3 and (I) PI(3,4)P2 generated in murine CD4+ T cells isolated by negative selection stimulated using a low (0.25 µg/mL) or high (1.0 µg/mL) dose of plate bound anti-CD3 antibody and a constant amount of soluble anti-CD28 antibody (1 µg/mL) in the presence or absence of 10 µM PTEN inhibitor (SF1670). CD4+ T cells were nucleofected with either scrambled control (SC) or siRNA targeting PTEN (siRNA) for 48 hours and Western blotting was utilized to monitor PTEN levels and actin was utilized as a loding control (J). Mass ELISA assays were used to measure the amount of (K) PI(4,5)P2, (L) PIP3 and (M) PI(3,4)P2 generated in murine CD4+ T cells treated with the scrambled control or siRNA targeting PTEN that were activated for ten minutes with either a low (0.25 µg/mL) or high (1.0 µg/mL) dose of plate bound anti-CD3 antibody and constant amount of soluble anti-CD28 antibody (1 µg/mL). Each experiment was repeated three times and error bars are ± standard deviation. A two-way ANOVA statistical test was performed; <0.0001= ****, <0.001= ***, <0.01= ** and <0.05= *. Symbols over data points are comparisons between the low and high dose groups and symbols in the legend are between the untreated and SF1670 treated groups.

Article Snippet: The standard curve was fit assuming a sigmoidal dose response with variable slope and the level of phosphatidylinositol in each sample was extrapolated in the Graphpad Prism 8 software package. siRNA knockdownA murine PTEN siRNA kit (Origene) was used to knock down PTEN expression.

Techniques: Activation Assay, Construct, Enzyme-linked Immunosorbent Assay, Generated, Isolation, Selection, Control, Western Blot, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: T cells transduce T-cell receptor signal strength by generating different phosphatidylinositols

doi: 10.1074/jbc.ra118.006524

Figure Lengend Snippet: Figure 5. FAK is hyperactivated by a weak TCR signal and is essential for optimal FOXP3 induction. A) FAK activation in murine CD4+ T cells purified by negative selection stimulated with either a low or high dose (0.25 µg/mL and 1.0 µg/mL respectively) of plate bound anti-CD3 antibody and soluble CD28 (1.0 µg/mL) was monitored by tracking phosphorylation of Y397 by Western blotting. B) Densitometry was utilized to quantitate the average P-Y397 FAK from three independent experiments. C and D) CD4+ T cells were activated with a low dose (0.25 µg/mL) of anti-CD3 antibody and soluble anti-CD28 antibody (1.0 µg/mL) with various doses of a FAK inhibitor (FAK Inhibitor 14 Tocris) for four days. Flow cytometry was used to track the expression of Foxp3 and CD25 on CD4+ T cells. The average percentage of CD4+Foxp3+CD25+ T cells were quantitated. E) Murine CD4+ T cells purified by negative selection were nucleofected with scrambled control or siRNA targeting FAK and Western blotting was utilized to monitor FAK levels after 48 Hours following nucleofection. F) T cells were then activated with a low or high dose (0.25 µg/mL and 1.0 µg/mL respectively) of plate bound anti-CD3 antibody and soluble CD28 (1.0 µg/mL) and Foxp3 and CD25 expression was measured by flow cytometry 48 hours after activation to monitor the generation of (G) Foxp3+CD25+ and (H) Foxp3-CD25+ cells. Data are from three independent experiments ± standard deviation. A two-way ANOVA statistical test was performed for data in panel B. A one-way ANOVA statistical test was performed from data in panel C. T tests were performed for data in panels G and H. For all statistical tests, P values were summarized as: <0.0001= ****, <0.001= *** and <0.01= **.

Article Snippet: The standard curve was fit assuming a sigmoidal dose response with variable slope and the level of phosphatidylinositol in each sample was extrapolated in the Graphpad Prism 8 software package. siRNA knockdownA murine PTEN siRNA kit (Origene) was used to knock down PTEN expression.

Techniques: Activation Assay, Purification, Selection, Phospho-proteomics, Western Blot, Flow Cytometry, Expressing, Control, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: T cells transduce T-cell receptor signal strength by generating different phosphatidylinositols

doi: 10.1074/jbc.ra118.006524

Figure Lengend Snippet: Figure 6. The balance of PI(4,5)P2 versus PIP3 is necessary for interpreting TCR signal strength and setting AKT activation thresholds. A) TCR signaling engages PI3K to generate PIP3, which in turn activates both PDK1 and mTORC2. Pdk1 phosphorylates S308 on AKT and MTORC2 phosphorylates T473 on AKT, which are required for AKT activation. B) The abundance of PIP3 generated in CD4+ T cells activated for ten minutes with varying doses of plate bound anti-CD3 antibody indicated in each panel and constant amount of soluble anti-CD28 antibody (1 µg/mL) in the presence or absence of 10 µM PTEN inhibitor, SF1670, was determined by a mass ELISA assay from three independent experiments. C) Murine CD4+ T cells were activated for ten minutes in triplicate with varying doses of plate bound anti-CD3 antibody and 1 µg/mL anti-CD28 antibody in the presence or absence of 10 µM PTEN inhibitor (SF1670). D) The abundance of PTEN as a function of anti-CD3 antibody concentration was determined by densitometry. E and F) P-PDK1, p-RICTOR, p- AKT 308 and p-AKT473 Western blots were quantitated by densitometry where all phosphorylated species were normalized to the total amount of the respective protein. G and H) The normalized abundance of p- PDK1, p-RICTOR, p-AKT 308 and p-AKT 473 determined by Western blotting was plotted versus the level of PIP3 generated.

Article Snippet: The standard curve was fit assuming a sigmoidal dose response with variable slope and the level of phosphatidylinositol in each sample was extrapolated in the Graphpad Prism 8 software package. siRNA knockdownA murine PTEN siRNA kit (Origene) was used to knock down PTEN expression.

Techniques: Activation Assay, Generated, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot