CD25 and Protein Phosphatase 2A Cooperate to Enhance IL-2R Signaling in Human Regulatory T Cells This information is current as Ying Ding, Aixin Yu, George C. Tsokos and Thomas R. of September 30, 2021. Malek J Immunol published online 13 May 2019 http://www.jimmunol.org/content/early/2019/05/10/jimmun ol.1801570 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/05/10/jimmunol.180157 Material 0.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 30, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 13, 2019, doi:10.4049/jimmunol.1801570 The Journal of Immunology CD25 and Protein Phosphatase 2A Cooperate to Enhance IL-2R Signaling in Human Regulatory T Cells Ying Ding,* Aixin Yu,* George C. Tsokos,† and Thomas R. Malek*,‡ Low-dose IL-2 therapy is a direct approach to boost regulatory T cells (Tregs) and promote immune tolerance in autoimmune patients. However, the mechanisms responsible for selective response of Tregs to low-dose IL-2 is not fully understood. In this study we directly assessed the contribution of CD25 and protein phosphatase 2A (PP2A) in promoting IL-2R signaling in Tregs. IL-2– induced tyrosine phosphorylation of STAT5 (pSTAT5) was proportional to CD25 levels on human CD4+ T cells and YT human NK cell line, directly demonstrating that CD25 promotes IL-2R signaling. Overexpression of the PP2A catalytic subunit (PP2Ac) by lentiviral transduction in human Tregs increased the level of IL-2R subunits and promoted tyrosine phosphorylation of Jak3 and STAT5. Interestingly, increased expression of CD25 only partially accounted for this enhanced activation of pSTAT5, indicating that PP2A promotes IL-2R signaling through multiple mechanisms. Consistent with these findings, knockdown of PP2Ac in Downloaded from human Tregs and impaired PP2Ac activity in mouse Tregs significantly reduced IL-2–dependent STAT5 activation. In contrast, overexpression or knockdown of PP2Ac in human T effector cells did not affect IL-2–dependent pSTAT5 activation. Overexpres- sion of PP2Ac in human Tregs also increased the expressions of proteins related to survival, activation, and immunosuppressive function, and upregulated several IL-2–regulated genes. Collectively, these findings suggest that CD25 and PP2A cooperatively enhance the responsiveness of Tregs to IL-2, which provide potential therapeutic targets for low-dose IL-2 therapy. The Journal of Immunology, 2019, 203: 000–000. http://www.jimmunol.org/ nterleukin-2 is a key cytokine that promotes immune re- immune system attacks self-tissues (5). Completed clinical trials sponses and is also essential for immune tolerance through its indicate that low-dose IL-2 therapy is safe, increases Tregs in most I action on Foxp3+ regulatory T cells (Tregs) (1). The reali- patients, and is accompanied by clinical benefit in patients with zation that low IL-2R signaling in mice effectively promotes Treg chronic graft-versus-host disease, hepatitis C virus–induced vas- development and homeostasis, but not T effector (Teff) responses culitis, alopecia areata, and systemic lupus erythematosus (6–9). (2), favors the concept that low amounts of IL-2 may selectively Low-dose IL-2 is in a range of 0.5–3 3 106 IU/m2, administered at boost Treg activity in the context of autoimmune diseases. Pre- various frequencies (from daily to biweekly). These levels of IL-2 clinical studies showed that low doses of IL-2 or agonist IL-2/ are ∼30- to 100-fold lower than used in cancer immunotherapy, by guest on September 30, 2021 anti–IL-2 complexes supported immune tolerance in the context of where the goal has been to boost Teff and NK cells. A critical diabetes-prone NOD mice, experimental autoimmune encephalo- aspect of low-dose IL-2 therapy in autoimmunity is that so far myelitis, and allogenic islet transplantation (3, 4). Low-dose IL-2 there has been no indication of activation of autoreactive Teff is now being advanced as a promising therapeutic approach in cells, although sometimes regulatory CD56hi NK cells and eo- patients with autoimmune diseases or other situations in which the sinophils increase (7, 10). IL-2 signaling is initiated by binding of IL-2 to the IL-2R, which is expressed on the cell surface as either the intermediate-affinity *Department of Microbiology and Immunology, Miller School of Medicine, Univer- IL-2R, a dimer of IL-2Rb (CD122) and gc (CD132), or the high- † sity of Miami, Miami, FL 33136; Department of Medicine, Beth Israel Deaconess affinity IL-2R, a trimer of IL-2Ra (CD25), IL- 2Rb and gc (11). Medical Center, Harvard Medical School, Boston, MA 02215; and ‡Diabetes Re- search Institute, Miller School of Medicine, University of Miami, Miami, FL 33136 Because IL-2 can stimulate both Tregs and autoreactive T cells, ORCIDs: 0000-0002-0557-8077 (A.Y.); 0000-0001-9589-2360 (G.C.T.). important considerations to advance this therapy are related to Received for publication November 30, 2018. Accepted for publication April 20, the window of selectivity of low-dose IL-2 toward Tregs and 2019. the mechanisms that impose this selectivity. In this regard, we This work was supported by National Institutes of Health (NIH) Grant R01 AI131648 previously showed that IL-2–dependent STAT5 activation and and the Diabetes Research Institute Foundation (to T.R.M.), and NIH Grants R01 downstream gene activation in human Tregs occurred at ∼10–15- AI068787 and R01 AI136924 (to G.C.T.). and 100-fold lower concentrations of IL-2, respectively, than in Y.D. designed, performed, and analyzed most of the experiments and wrote the CD4+ CD45RO+ T memory (Tm) cells (12), where the latter manuscript; A.Y. performed and analyzed experiments on CD4+ T cells and YT cell line; G.C.T. provided the Ppp2r1aflox/flox mice and edited the manuscript; represents a viable pharmacologic range to target Tregs. These T.R.M. conceived the project, designed the overall study, and wrote the paper. selective responses by human Tregs correlated with their higher Address correspondence and reprint requests to Dr. Thomas R. Malek, Department of expression of CD25 than CD4+ Tm cells (13). Indeed, in vitro Microbiology and Immunology, Miller School of Medicine, University of Miami, fully activated T cells exhibited over a 1000-fold range of re- 1600 NW 10th Avenue, Miami, FL 33136. E-mail address: [email protected] sponse to IL-2 as measured by pSTAT5 activation (13), supporting The online version of this article contains supplemental material. the notion that CD25 levels dictate the sensitivity of their re- Abbreviations used in this article: MFI, mean fluorescence intensity; PP2A, protein phosphatase 2A; PP2Aa, PP2A scaffold subunit; PP2Ac, PP2A catalytic subunit; sponses to IL-2. Nevertheless, in vitro activated human T cells SFM, OpTmizer CTS T cell expansion medium; shRNA, short hairpin RNA; Teff, remain less responsive to IL-2 than human Tregs, even though T effector; TEM, Teff memory; Tm, T memory; Treg, regulatory T cell; YFP, yellow the former expressed higher levels of all IL-2R subunits (12). fluorescent protein. These latter data suggest that other cell intrinsic factors, separate Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 from CD25 levels, contribute to the high IL-2 sensitivity of Tregs www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801570 2 CD25 AND PP2A IN IL-2R SIGNALING SENSITIVITY and that assessment of IL-2 responsiveness by a heterogeneous Human CD4+ T cells were enriched by negative selection with the MACS + population of activated T cells may not directly relate to differ- CD4 T Cell Isolation Kit II (Miltenyi Biotec, Auburn, CA). The purified CD4+ T cells were stained and sorted using a BD FACSAria into Tregs ential responses by Tregs and Teff cells. + hi lo + lo hi (CD4 CD25 CD127 ) and Teff memory (TEM) (CD4 CD25 CD127 Protein phosphatase 2A (PP2A) is a ubiquitously expressed, CD45RA2) cells. Sorted cells were typically .90% pure. Tregs were + 2 highly conserved serine/threonine phosphatase that contributes to $85% Foxp3 and TEM cells were $93% Foxp3 . Purified Tregs and Treg function as assessed by Treg-specific knockout of PP2A TEM cells were initially cultured with anti-CD3/CD28 beads (Dynabeads activity (14). PP2A consists of three subunits: a scaffold subunit Human Treg Expander; ThermoFisher Scientific, Vilnius, LT) and human IL-2 (500 U/ml) and then were subcultured with IL-2 (500 U/ml) in (PP2Aa), a catalytic subunit (PP2Ac), and a regulatory subunit OpTmizer CTS T cell expansion medium (designated as SFM) (Life (PP2Ab). The scaffold (a, PPP2R1A and b, PPP2R1B) and cat- Technologies, Grand Island, NY). The anti-CD3/anti-C28/IL-2–activated alytic (a, PPP2CA, and b, PPP2CB) subunits are each encoded by and expanded TEM cells are referred to as Teff cells. two homologous genes, with the a isoform being ∼10-fold more Plasmid construction abundant than the b isoform (15). In contrast, the regulatory subunits are coded by a large variety of genes that are grouped into The lentiviral vector pRRL-sin-cPPT-MCS-IRES-emdGFP (a gift from four families (B, B’, B’’, B’’’) (16).