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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 (Treg cells). Compromised suppressive activity of Treg 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 Treg cells. Treg cells exhibited high PP2A activity, and Treg 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, Treg cells altered their metabolic and cytokine profile and were unable to suppress effector immune responses. Therefore, PP2A is required for the function of Treg cells and the prevention of autoimmunity.

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Figure 1: PP2Aflox mice develop multi-organ autoimmunity.
Figure 2: Spontaneous activation of T cells and B cells in PP2Aflox mice.
Figure 3: TCR activation induces SET-mediated phosphorylation of PP2AC at Tyr307 in Tconv cells but not in Treg cells.
Figure 4: Treg cells have a greater ceramide content than that of Tconv cells, through Foxp3-mediated inhibition of Sgms1.
Figure 5: PP2A inhibits the mTORC1 pathway in Treg cells.
Figure 6: PP2Aflox Treg cells exhibit metabolic, proliferation and cytokine-production abnormalities.
Figure 7: Rapamycin reverses the abnormal profile of PP2Aflox Treg cells.

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Acknowledgements

We thank P. Seth for advice on the use of the Seahorse XF24 flux analyzer; J. Bielawski and J. Pierce for advice and technical assistance in studies of ceramide species by mass spectrometry; and A. Rao (La Jolla Institute for Allergy and Immunology) for the MSCV-FOXP3-IRES-Thy1.1 plasmid (initially generated at Harvard Medical School and the University of Colorado at Boulder). Supported by the US National Institutes of Health (R01 AI068787 to G.C.T.) and the Arthritis Foundation (S.A.A.).

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S.A.A. designed, performed and analyzed experiments and wrote the manuscript; N.R.-R., A.S.-F., N.D., E.O. and J.C.C. performed and analyzed experiments; M.G.T. analyzed the histopathology samples; and G.C.T. designed the overall study, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to George C Tsokos.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Treg cells exhibit greater PP2A activity than that of Tconv cells, and PP2Aflox mice develop multi-organ autoimmunity.

(a) The enzymatic activity of PP2AC was quantified in CD4+CD25- Tconv cells and CD4+CD25+ Treg cells (n=5 per group). Data are from one experiment representative of two independent experiments with similar results. (b) Left: Real-time PCR of the Ppp2r1a and Ppp2r1b genes in unstimulated and stimulated naïve CD4+ T cells with CD3 plus CD28 antibodies (2μg/mL) for 24 hours. Each isoform was multiplexed separately in Taqman assays with the housekeeping gene Actb (n=3 per group). Right: Primer efficiency for the two isoforms with serial dilutions of the input DNA, demonstrating that the two PCRs retain efficiency over a wide range of DNA concentrations. (c) The appearance of PP2Awt (left) and PP2Aflox (right) mice at 12 weeks of age. (d) Macroscopic and histologic image of a representative skin lesion of PP2Aflox mice. (e) Scaliness and crusting of the tail of PP2Aflox mice. (f) PP2Aflox mice that survived beyond 15 weeks of age (shown here at 19 weeks of age) developed significant skin disease with secondary ulceration. (g) The secondary lymphoid organs (upper: spleen, lower: peripheral lymph nodes) of PP2Awt and PP2Aflox mice at 12 weeks of age. (h) Overt cervical lymphadenopathy in PP2Aflox mice. Images for (c-h) are from one experiment representative of at least three independent experiments with similar results. Mean ± s.e.m., *P<0.01 (unpaired, two-tailed t-test).

Supplementary Figure 2 Lymphoproliferation in PP2Aflox mice.

PP2Awt and PP2Aflox mice were injected i.p. with EdU (500μg/mouse). The dividing cells were allowed to incorporate in vivo the EdU for 18 hours and the mice were then euthanized and the splenocytes stained for EdU. The EdU staining for the CD4+ T cells is shown. A representative histogram (left) and quantification of the results (right) is shown (n=3 per group). Data are from one experiment representative of two experiments with similar results. Mean ± s.e.m., *P<0.01 (unpaired, two-tailed t-test).

Supplementary Figure 3 Treg cells have decreased phosphorylation of PP2Ac at Tyr307 upon activation through modulation of the ceramide-SET-PP2A pathway.

(a) Splenocytes isolated from Foxp3IRES-GFP mice were stimulated with CD3 plus CD28 (2μg/mL) antibodies for 24 hours. Intracellular staining was then performed for p-PP2AC (Y307) in CD4+Foxp3- and CD4+Foxp3+ T cells (n=3 per group). Data are from one experiment representative of three independent experiments with similar results. (b) Intracellular staining for p-PP2AC (Y307) of naïve CD4+ T cells transfected with a scramble shRNA or Set-specific shRNA mCherry-expressing plasmid and then activated with CD3 plus CD28 (2μg/mL) antibodies for 24 hours. The analysis was done on mCherry+ T cells (n=3 per group). Data are from one experiment representative of two independent experiments with similar results. (c) Jurkat T cells were treated with SMase or vehicle for 1h and then lysed for protein immunoprecipitation with a PP2AC-specific antibody or a mouse IgG control antibody. The blots were then probed for SET and PP2AC. Image is from one experiment representative of two independent experiments with similar results. (d) Naïve CD4+ T cells were stimulated with CD3 plus CD28 (2μg/mL) antibodies for 24h and then treated with vehicle or SMase for 1 or 2 hours. The cells were then lysed for immunoblot and probed for p-PP2AC (Y307) and total PP2AC. The relative ratio of p-PP2AC/total PP2AC band densities for each lane is also shown. Image is from one experiment representative of two independent experiments with similar results. Mean ± s.e.m., MFI: Mean fluorescent intensity, *P<0.05, **P<0.01 (unpaired, two-tailed t-test).

Supplementary Figure 4 Foxp3 inhibits the expression of SMS1.

(a) Jurkat T cells were spin-infected with a Thy1.1 expressing retrovirus that harbored or not the human FOXP3 coding sequence. Thy1.1+ cells were FACS-sorted 24 hours after infection and analyzed for FOXP3 and SGMS1 mRNA levels by real time PCR. (b) Immunoblot of protein extracts from control (GFP) or FOXP3 infected Jurkat T cells were probed for FOXP3 and SGMS1. Immunoblot (left) and relative quantification (right) of two distinct GFP-FOXP3 replicate pairs are shown. Results (a-b) are from one experiment representative of three independent experiments.

Supplementary Figure 5 Activated PP2A interacts with the mTOR pathway in Treg cells.

(a) Jurkat T cells were treated with SMase (0.5 units/mL) or vehicle (50% glycerol in PBS) for 1h and then lysed for protein immunoprecipitation with a PP2AC-specific antibody. The indicated bands (arrows) were subjected to mass spectrometry. (b) The Ingenuity Pathway Analysis (IPA) of 269 proteins identified in (a) is shown. (c) Jurkat T cells treated as in (a) were lysed for protein immunoprecipitation with a PP2AC-specific antibody (upper) or a Raptor-specific antibody (lower). The immunoblots were then probed for PP2AC and Raptor. Image is from one experiment representative of two independent experiments with similar results.

Supplementary Figure 6 Treg cell phenotype of PP2Awt and PP2Aflox mice.

(a) The glycolytic (ECAR) and oxidative phosphorylation (OCR) rate of PP2Awt Tconv and PP2Aflox Treg cells were measured before and after 24 hours of stimulation with CD3 plus CD28 antibodies. Results are normalized to PP2Awt Tconv cells for each condition. (b) PP2Awt and PP2Aflox Tregs were stained for CD73, GITR and FR4. Data (a-b) are from one experiment representative of two independent experiments with similar results. Mean ± s.e.m. is shown.

Supplementary Figure 7 Rapamycin normalizes the proportion of effector lymphocytes in PP2Aflox mice.

(a) The proportion of effector memory (CD44+CD62L-) CD4+ T cells in blood was assessed before rapamycin treatment and in a weekly basis after treatment initiation. Progression in the percentage of cells is depicted (two-way ANOVA followed by Bonferroni’s test). (b) Numbers of effector memory CD4+ and CD8+ T cells in spleen from rapamycin-treated and untreated PP2Awt and PP2Aflox mice after 4 weeks of treatment (ANOVA followed by Tukey’s multiple comparison test). (c, d) Percentage of GC (GL-7+FAS+) B cells (c) and Tfh cells (PD-1+CXCR5+) within CD4+ T cells (d) in spleens from (b) are shown. Cumulative (a and b) and representative (c and d) data from two experiments with similar results are demonstrated. Mean ± s.e.m. is shown, *P<0.05, **P<0.01, ***P<0.001.

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Apostolidis, S., Rodríguez-Rodríguez, N., Suárez-Fueyo, A. et al. Phosphatase PP2A is requisite for the function of regulatory T cells. Nat Immunol 17, 556–564 (2016). https://doi.org/10.1038/ni.3390

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