Phosphatase PP2A is requisite for the function of regulatory T cells

Abstract

Homeostasis of the immune system depends on the proper function of regulatory T cells (T(reg) cells). Compromised suppressive activity of T(reg) cells leads to autoimmune disease and graft rejection and promotes anti-tumor immunity. Here we report a previously unrecognized requirement for the serine-threonine phosphatase PP2A in the function of T(reg) cells. T(reg) cells exhibited high PP2A activity, and T(reg) cell-specific ablation of the PP2A complex resulted in a severe, multi-organ, lymphoproliferative autoimmune disorder. Mass spectrometry revealed that PP2A associated with components of the mTOR metabolic-checkpoint kinase pathway and suppressed the activity of the mTORC1 complex. In the absence of PP2A, T(reg) cells altered their metabolic and cytokine profile and were unable to suppress effector immune responses. Therefore, PP2A is required for the function of T(reg) cells and the prevention of autoimmunity.

Figure 1. PP2A^flox mice develop multi-organ autoimmunity

(a, b) The body weight (a, n=17 for PP2A^wt and n=9 for PP2A^flox) and the spleen weight (b, n=5 per group) of 10–14 week-old PP2A^wt and PP2A^flox mice were quantified. (c) H&E staining of the lungs, the skin, the pancreas, the salivary glands and the stomach of PP2A^wt and PP2A^flox mice (scale bar represents 200 μm). Images are from one experiment representative of three independent experiments with similar results (n=3 mice per group per experiment). Mean ± s.e.m. is shown, *P<0.001 (unpaired, two-tailed t-test).

Figure 2. Spontaneous T cell and B cell activation in PP2A^flox mice

(a, b) CD4⁺ and CD8⁺ T cells of 10–14 week-old PP2A^wt and PP2A^flox mice were stained for CD62L and CD44 (a, spleen, peripheral and mesenteric lymph nodes) and for the production of the indicated cytokines (b, spleen). Data shown are from one experiment representative of three independent experiments (n=3 mice per group) (c) The levels of serum IgM, IgG, IgA and IgE in PP2A^wt and PP2A^flox mice were quantified (n=8 for PP2A^wt and n=5 for PP2A^flox mice). (d) The percentages of T follicular helper (T_fh) cells and germinal center (GC) B cells in the spleen from PP2A^wt and PP2A^flox mice are shown. Data shown are from one experiment representative of two independent experiments (n=3 mice per group per experiment). (e) Sera from PP2A^wt and PP2A^flox mice were analyzed for the presence of autoantibodies. Each individual column of the heatmap represents an individual mouse (total 4 mice per group). Mean ± s.e.m is shown. *P<0.01 (unpaired, two-tailed t-test).

Figure 3. TCR activation induces SET-mediated phosphorylation of PP2A_C at Y307 in T_conv cells but not T_reg cells

(a, b) Naïve CD4⁺ T cells (a) or T_conv and T_reg cells (b) were stimulated with CD3 plus CD28 antibodies for the indicated time periods. Immunoblotting for p-PP2A_C (Y307) was then performed. Data are from one experiment representative of two independent experiments with similar results (n=3 mice per experiment). (c, f) Splenocytes isolated from Foxp3^IRES-GFP mice were stimulated with CD3 plus CD28 antibodies for 24 hours or left unstimulated. Intracellular staining was then performed for PP2A_C (c) or SET (f). Data are from one experiment representative of three independent experiments with similar results (n=3 mice per experiment). (d) Naïve CD4⁺ T cells were stimulated with CD3 plus CD28 for 0, 0.5, 1, 3, 6, 18 and 24 hours. Intracellular staining was then performed for SET and p-PP2A_C (Y307). A representative histogram (left) and quantification of the results (right) is shown from one of two independent experiments with similar results (n=3 per group). (e) Intracellular staining for p-PP2A_C (Y307) of naïve CD4⁺ T cells activated with CD3 plus CD28 antibodies for 24 hours and then spin-infected with an empty or a Set-expressing mCherry lentiviral vector. The analysis was done on mCherry⁺ T cells (n=5 per group, one of two experiments with similar results is shown). Mean ± s.e.m. is shown, MFI: Mean fluorescent intensity, *P<0.01 (unpaired, two-tailed t-test).

Figure 4. T_reg cells display higher ceramide content through Foxp3-mediated inhibition of Sgms1

(a, b) Naïve CD4⁺ T cells were stimulated with anti-CD3 plus anti-CD28 for 24h and then treated with sphingomyelinase (SMase, 0.5 units/mL) or vehicle (50% glycerol in PBS) for 1h. The cells were then stained for ceramide (a) or p-PP2A_C (Y307) (b). A representative histogram (left) and quantification of the results (right) is shown (n=3, one of two experiments with similar results is shown). (c) T_conv and T_reg cells were stimulated for 24h and then subjected to ESI-MS/MS for the quantification of ceramide species. (d) Ceramide content of CD3⁺CD4⁺Foxp3⁻ and CD3⁺CD4⁺Foxp3⁺ was quantified using flow cytometry (n=3 mice). Data are from one of three experiments with similar results. (e) T_conv and T_reg cells were subjected to chromatin immunoprecipitation using a Foxp3-specific antibody or an IgG control antibody. Shown is binding enrichment at the mouse Sgms1 gene, normalized to the input (n=3 per group, one representative of two independent experiments with similar results is shown). (f) Naïve CD4⁺ T cells were stimulated for 24h and then spin-infected with an mCherry-expressing lentivirus that harbored or not the murine Sgms1 coding sequence. CD4⁺mCherry⁺ cells were analyzed for ceramide content using flow cytometry. A representative histogram (left) and quantification of the results (right) is shown. Data are from one experiment representative of two independent experiments with similar results. Mean ± s.e.m. is shown, MFI: Mean fluorescent intensity, *P<0.05, **P<0.01, ***P<0.001 (unpaired, two-tailed t-test).

Figure 5. PP2A inhibits the mTORC1 pathway in T_reg cells

(a) Jurkat T cells were treated with SMase (0.5 units/mL) or vehicle (50% glycerol in PBS) for 1 hour and then stained for p-AKT^T308, p-AKT^S473 and p-S6. Data are from one experiment representative of two independent experiments with similar results. (b) Naïve CD4⁺ T cells were stimulated with CD3 plus CD28 antibodies (2 μg/mL) for 24h and then incubated with okadaic acid or DMSO for 3 hours. During the last hour the cells were treated with SMase or vehicle and then stained for p-AKT^T308, p-AKT^S473 and p-S6 (n=3 per treatment group). Data are from one experiment representative of two independent experiments with similar results. (c) Foxp3⁺ T_reg cells were isolated from PP2A^wt and PP2A^flox mice and stained for p-S6 (n=3 mice per group). A representative histogram (left) and quantification of the results (right) is shown. Data are from one experiment representative of three independent experiments with similar results. Mean ± s.e.m., MFI: Mean fluorescent intensity, ns P>0.05, *P<0.05, **P<0.01, ***P<0.001 (unpaired, two-tailed t-test).

Figure 6. PP2A^flox T_regs exhibit metabolic, proliferation and cytokine production abnormalities

(a, b) The extracellular acidification rate (a, ECAR) and the oxygen consumption rate (b, OCR) of PP2A^wt and PP2A^flox T_reg cells are shown. Left: Representative stress tests (a: glycostress test, b: mitostress test). Right: quantification of the results is shown (n=4). Data are from one experiment representative of three independent experiments with similar results. (c) The proliferation rate of PP2A^wt and PP2A^flox T_reg cells was quantified in vivo after i.p. injection of EdU (n=3, one experiment is shown representative of two independent experiments). (d) The percentage of CD25⁺Foxp3-YFP⁺ T cells in the spleens, peripheral and mesenteric lymph nodes of 10–14 week-old PP2A^wt and PP2A^flox mice is shown (n=4 mice per group, one experiment is shown representative of two independent experiments). (e) T_reg cells from the spleens of 10–14 week-old PP2A^wt and PP2A^flox mice were stimulated with PMA/Ionomycin for 6h and then stained intracellularly for the indicated cytokines (n=3 mice per group). Data are from one experiment representative of three independent experiments with similar results. (f, g) PP2A^wt and PP2A^flox T_reg cells were stained for CD25, CTLA-4, PD-1 and LAP (f) or CD98 (g, representative histogram and quantification of the results is shown). Data are from one experiment representative of two independent experiments with similar results (n=3 mice per group) (h) In vitro suppression assay using effector CD45.1⁺CD4⁺ (T_eff) T cells and PP2A^wt or PP2A^flox T_reg cells at the indicated ratios (T_eff:T_reg cells). Mean ± s.e.m., MFI: Mean fluorescent intensity, *P<0.001 (unpaired, two-tailed t-test).

Figure 7. Rapamycin reverses the abnormal profile of the PP2A^flox T_reg cells

(a, b) FACS-sorted PP2A^wt and PP2A^flox T_reg cells were stimulated with CD3 plus CD28 and then expanded for 4 days in the presence of 100 IU/mL of IL-2. During the final 24 hours, they were treated with 100nM of Rapamycin or DMSO. (a) The T_reg cells (gated on FoxP3-YFP⁺CD4⁺ T cells) were re-stimulated with PMA/Ionomycin for 6 hours and stained for IL-17 and IL-2. (b) The ECAR and OCR of PP2A^wt and PP2A^flox T_reg cells (sorted again as Foxp3-YFP⁺CD4⁺ T cells at the end of the culture) were measured (n=3 mice per group, unpaired, two-tailed t-test). Data are from one experiment representative of two independent experiments with similar results. (c) Left: H&E staining of the lungs and the salivary glands of PP2A^wt and PP2A^flox mice treated or not with rapamycin (scale bar represents 100 μm). Images are from one experiment representative of two independent experiments with similar results. Right: combined clinical score of inflammation in the liver, skin, stomach, salivary glands, lungs and pancreas of PP2A^wt and PP2A^flox mice treated or not with rapamycin (one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test). (d, e) Ex vivo amounts of p-S6 (d) and CD98 expression (e) in T_regs (FoxP3-YFP⁺) from mice in (c). (f) In vitro suppression assay using effector CD45.1⁺CD4⁺ (T_eff) T cells and T_reg cells from PP2A^wt or PP2A^flox rapamycin-treated and vehicle-treated mice. Representative histograms showing CFSE dilution and percentage of divided cells (ratio T_reg/T_eff: 1/1). Data are from one experiment representative of two independent experiments with similar results. Mean ± s.e.m., *P<0.05, **P<0.01, ***P<0.001.