Phosphatidylinositol (4,5) bisphosphate controls T cell activation by regulating T cell rigidity and organization

Abstract

Here we investigate the role of Phosphatidylinositol (4,5) bisphosphate (PIP(2)) in the physiological activation of primary murine T cells by antigen presenting cells (APC) by addressing two principal challenges in PIP(2) biology. First, PIP(2) is a regulator of cytoskeletal dynamics and a substrate for second messenger generation. The relative importance of these two processes needs to be determined. Second, PIP(2) is turned over by multiple biosynthetic and metabolizing enzymes. The joint effect of these enzymes on PIP(2) distributions needs to be determined with resolution in time and space. We found that T cells express four isoforms of the principal PIP(2)-generating enzyme phosphatidylinositol 4-phosphate 5-kinase (PIP5K) with distinct spatial and temporal characteristics. In the context of a larger systems analysis of T cell signaling, these data identify the T cell/APC interface and the T cell distal pole as sites of differential PIP(2) turnover. Overexpression of different PIP5K isoforms, as corroborated by knock down and PIP(2) blockade, yielded an increase in PIP(2) levels combined with isoform-specific changes in the spatiotemporal distributions of accessible PIP(2). It rigidified the T cell, likely by impairing the inactivation of Ezrin Moesin Radixin, delayed and diminished the clustering of the T cell receptor at the cellular interface, reduced the efficiency of T cell proximal signaling and IL-2 secretion. These effects were consistently more severe for distal PIP5K isoforms. Thus spatially constrained cytoskeletal roles of PIP(2) in the control of T cell rigidity and spatiotemporal organization dominate the effects of PIP(2) on T cell activation.

Figure 1. Different PIP5K isoforms display distinct spatiotemporal patterns.

5C.C7 T cells were transduced to express GFP-tagged PIP5K isoforms and activated with CH27 APCs and 10 µM MCC agonist peptide. The graphs show the percentage of cell couples with standard errors that displayed accumulation of PIP5K γ87-GFP (A), α-GFP (B), β-GFP (C), and γ90-GFP (D) with the indicated patterns relative to tight cell coupling. Representative images are given in Fig. S1B–E. Representative movies have been published (γ87 [6]) or are given as Movies S1, S2, S3. 34–59 cell couples were analyzed per condition.

Figure 2. PIP5K overexpression alters PIP₂ levels and localization.

A. PIP₂ levels were determined by immunofluorescence staining of 5C.C7 T cells transduced with different GFP-tagged PIP5K isoforms as indicated. PIP₂ levels of T cells with PIP5K overexpression (grey bars) and control (black bars) are normalized to control and are given with standard errors for two independent experiments each. An asterisks indicates statistical significance versus control with p<0.005. 100–200 cells were analyzed per experiment. A representative PIP₂ staining experiment is shown as Fig. S2D. B. 5C.C7 T cells were transduced to express PLCδ PH-GFP and activated with CH27 APCs and 10 µM MCC agonist peptide. The graph shows the percentage of cell couples with standard errors that displayed accumulation of PLCδ PH-GFP with the indicated patterns relative to tight cell coupling. 60 cell couples were analyzed. C, D. Similar to B, the panels show patterning data for 5C.C7 T cells expressing PLCδ PH-GFP with concurrent overexpression of PIP5K β (C) or γ90 (D). 41, 26 cell couples were analyzed per condition. Representative images for panels B–D are given in Fig. S2E–G. Representative movies have been published (γ87 [6]) or are given as Movies S4, S5, S6.

Figure 3. Manipulation of PIP5K expression affects IL-2 secretion and proximal signal transduction.

A, B. 5C.C7 T cells were activated by CH27 APCs and 10 µM MCC agonist peptide for 16 h upon manipulation of PIP5K expression and PIP₂ blockade as indicated. Cell culture supernatants were analyzed for IL-2 by ELISA. A representative IL-2 ELISA is given in (A). Changes in IL-2 secretion upon manipulation of PIP5K expression and PIP₂ blockade relative to non-transduced T cells are given with standard errors in (B) on a logarithmic scale to comparably display reduction and enhancement. ‘GFP’ indicates retroviral expression of GFP as a control. One/two asterisks indicate significance versus non-transduced control with p<0.05/0.005, respectively. Data from 3–6 independent experiments are given. When the agonist peptide concentration during T cell activation was reduced to a limiting concentration, 0.1 µM, knockdown of PIP5K γ still did not result in impaired IL-2 secretion (Fig. S3). C, D. 5C.C7 T cells were activated by CH27 APCs and 10 µM MCC agonist peptide for 2 min upon manipulation of PIP5K expression as indicated. T cell/APC extracts were blotted for LAT Y191 and PLCγ Y783. A representative blot is given in (C). Changes in LAT and PLCγ phosphorylation upon manipulation of PIP5K expression relative to non-transduced T cells are given with standard errors in (D) on a logarithmic scale to comparably display reduction and enhancement. Data from 3–4 independent experiments are given.

Figure 4. PIP5K overexpression rigidifies T cells.

A. 5C.C7 T cells were activated with CH27 APCs and 10 µM MCC agonist peptide. The diameter of the T cell/APC interface at the time of tight cell coupling is given with standard errors relative to that of the T cell body upon manipulation of PIP5K expression as indicated. An asterisk indicates statistical significance with p<0.01 relative to control. 28–54 cell couples were analyzed per condition. B. 5C.C7 T cells were transduced to express TCRζ-GFP and were activated with CH27 APCs and 10 µM MCC agonist peptide. For all cell couples with persistent interface accumulation of TCRζ-GFP it was determined whether preceding cell coupling occurred with (e.g. Figs. S1C, S2E, and S6A) or without (Fig. S4A, Movie S7) the formation of a visible lamellum. Of all T cell/APC couples with persistent TCRζ-GFP interface accumulation, the percentage of cell couples without visible lamellum upon cell coupling is given with standard errors upon manipulation of PIP5K expression, as indicated. An asterisk indicates statistical significance with p<0.001 relative to control. 25–56 cell couples were analyzed per condition. C. 5C.C7 T cells were activated with CH27 APCs and 10 µM MCC agonist peptide. The percentage of cell couples with a visible uropod is given with standard errors relative to tight cell coupling upon manipulation of PIP5K expression and PIP₂ blockade, as indicated. 17–68 cell couples were analyzed per condition.

Figure 5. PIP5K overexpression interferes with ERM dephosphorylation.

A, B. 5C.C7 T cells were activated with α-CD3 and α-CD28 antibodies for 1 or 2 min upon manipulation of PIP5K expression as indicated. T cell extracts were blotted for phospho-Ezrin T567/Radixin T564/Moesin T558. Representative blots are given in (A). Phospho-ERM levels after two minutes of T cell activation are given with standard errors relative to those of T cell extracts from non-stimulated, non-transduced T cells in (B). Modest differences in ERM phosphorylation prior to T cell stimulation were not significant. Data from 3–10 independent experiments are given.

Figure 6. PIP5K overexpression interferes with accumulation of the TCR at the center of the T cell/APC interface.

A. 5C.C7 T cells were transduced to express TCRζ-GFP and activated with CH27 APCs and 10 µM MCC agonist peptide. The graph shows the percentage of cell couples with standard errors that displayed accumulation of TCRζ-GFP with the indicated patterns relative to tight cell coupling. 45 cell couples were analyzed. B, C. Patterning data for 5C.C7 T cells expressing TCRζ-GFP upon concurrent overexpression of PIP5K γ90 (B) or β (C) are displayed as in (A). 36, 25 cell couples were analyzed per condition. Representative images for all three panels are given in Fig. S6A–C. Representative movies are given as Movies S12, S13, S14.