TCR Signal Strength Regulates Akt Substrate Specificity To Induce Alternate Murine Th and T Regulatory Cell Differentiation Programs This information is current as of September 27, 2021. William F. Hawse, William C. Boggess and Penelope A. Morel J Immunol published online 9 June 2017 http://www.jimmunol.org/content/early/2017/06/09/jimmun ol.1700369 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/06/09/jimmunol.170036 Material 9.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published June 9, 2017, doi:10.4049/jimmunol.1700369 The Journal of Immunology TCR Signal Strength Regulates Akt Substrate Specificity To Induce Alternate Murine Th and T Regulatory Cell Differentiation Programs William F. Hawse,* William C. Boggess,† and Penelope A. Morel* The Akt/mTOR pathway is a key driver of murine CD4+ T cell differentiation, and induction of regulatory T (Treg) cells results from low TCR signal strength and low Akt/mTOR signaling. However, strong TCR signals induce high Akt activity that promotes Th cell induction. Yet, it is unclear how Akt controls alternate T cell fate decisions. We find that the strength of the TCR signal results in differential Akt enzymatic activity. Surprisingly, the Akt substrate networks associated with T cell fate decisions are qualitatively different. Proteomic profiling of Akt signaling networks during Treg versus Th induction demonstrates that Akt differentially regulates RNA processing and splicing factors to drive T cell differentiation. Interestingly, heterogeneous nuclear Downloaded from ribonucleoprotein (hnRNP) L or hnRNP A1 are Akt substrates during Treg induction and have known roles in regulating the stability and splicing of key mRNAs that code for proteins in the canonical TCR signaling pathway, including CD3z and CD45. Functionally, inhibition of Akt enzymatic activity results in the dysregulation of splicing during T cell differentiation, and knockdown of hnRNP L or hnRNP A1 results in the lower induction of Treg cells. Together, this work suggests that a switch in substrate specificity coupled to the phosphorylation status of Akt may lead to alternative cell fates and demonstrates that proteins involved with alternative splicing are important factors in T cell fate decisions. The Journal of Immunology, 2017, 199: 000–000. http://www.jimmunol.org/ D4+ regulatory T (Treg) cells prevent autoimmunity and regulate other key proteins important for the differentiation control immunopathology during immune responses. and function of either Th or Treg cells. There are .100 known C The TCR binds to peptide–MHC complexes, initiating a Akt substrates (11), yet the full network of substrates phosphor- signaling cascade that determines the fate of the T cell. The ylated by Akt during Th or Treg differentiation remains largely Akt/mTOR pathway plays a critical role in determining CD4+ unexplored. T cell fate (1–3). The induction of Treg cells is inversely correlated To better define how Akt functions in T cell differentiation, we with the degree of Akt/mTOR signaling (1–3). A low degree of identified Akt substrates during the induction of Th and Treg cells Akt/mTOR signaling is necessary for Treg stability and function via mass spectrometry. Remarkably, phosphorylated Akt substrates by guest on September 27, 2021 (4–6). Akt/mTOR signaling is tightly regulated in T cells, and differed during Treg versus Th cell induction, and this was asso- several feedback loops control the degree of Akt/mTOR activation, ciated with different patterns of Akt phosphorylation under both one of which involves phosphatase and tensin homolog (PTEN), the conditions. RNA processing factors were major Akt targets during transcription factors Foxp3 and FoxO1, and mTORC2 (7, 8). both Th and Treg induction, including heterogeneous nuclear ri- Akt is a serine threonine kinase that regulates cellular processes bonucleoprotein (hnRNP) L and hnRNP A1. We determined that including cell proliferation and cellular metabolism (9, 10). Akt RNA splicing of TCR signaling proteins, such as CD3z and CD45, activity is controlled by phosphorylation at Ser473 and Thr308. was regulated by Akt and essential for the Th versus Treg cell fate When Treg cells are activated, only Akt Thr308 is phosphorylated, choice. Knockdown of hnRNP L or hnRNP A1 altered the TCR and phosphorylation of both Thr308 and Ser473 sites results in the signal and changed the ratios of Th versus Treg cells induced. loss of Treg suppressive function (4). It appears that Treg function Together, these results reveal that there are distinct Akt signaling depends on the ability to modulate Akt activity and that Akt may networks that drive Th versus Treg induction, and identify RNA splicing factors as determinants of CD4+ T cell fate decisions. *Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15261; and †Department of Chemistry and Biochemistry, University of Notre Dame, Notre Materials and Methods Dame, IN 46556 Mice ORCIDs: 0000-0001-8371-3345 (W.F.H.); 0000-0002-1743-3676 (P.A.M.). C57BL/6 mice were purchased from The Jackson Laboratory. C57BL/6- Received for publication March 13, 2017. Accepted for publication May 19, 2017. Foxp3tm1Flv/J (Foxp3-RFP reporter) mice were a kind gift from Dr. D. This work was supported by National Institutes of Health Training Grant T32 Vignali (University of Pittsburgh). All of the mice were housed in a AI089443 (to W.F.H.) and by a Competitive Medical Research Fund grant from pathogen-free facility at the University of Pittsburgh. Mice were handled the University of Pittsburgh Medical Center (to W.F.H.). under Institutional Animal Care and Use Committee–approved guidelines in accordance with approved protocols. Address correspondence and reprint requests to Dr. Penelope A. Morel, University of Pittsburgh, 200 Lothrop Street, BST E1055, Pittsburgh, PA 15261. E-mail address: CD4+ T cell isolation and activation [email protected] + The online version of this article contains supplemental material. CD4 T cells were isolated as previously described (7). For the mass spectrometry and biochemical experiments, CD4+ T cells were isolated Abbreviations used in this article: hnRNP, heterogeneous nuclear ribonucleoprotein; + iAkt, Akt inhibitor; IP, immunoprecipitate; nTreg, natural Treg; PTEN, phosphatase from C57BL/6 spleens using a CD4 negative selection kit (Miltenyi and tensin homolog; QE, Q-Exactive; qPCR, quantitative PCR; siRNA, small interfering Biotec). Induction of Treg and Th cells was performed as previously de- + RNA; Treg, regulatory T. scribed (7). To generate Treg cells, we activated freshly isolated CD4 T cells with 0.25 mg/ml plate-bound anti-CD3 mAb with 1 mg/ml soluble Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 anti-CD28 mAb. To generate Th cells, we activated freshly isolated CD4+ www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700369 2 TCR SIGNAL STRENGTH DETERMINES Akt SUBSTRATE SPECIFICITY T cells with 1.0 mg/ml plate-bound anti-CD3 mAb with 1 mg/ml soluble CD45 splicing assay + anti-CD28 mAb. In some experiments, natural Treg (nTreg; CD4 Foxp3- + RFP+) and naive T (CD4+ Foxp3-RFP2) cells were sorted using a FAC- Total RNA was isolated from CD4 T cells with the RNeasy Plus Mini Kit SAria from the spleen of Foxp3-RFP reporter mice. (Qiagen). Reverse transcription reactions were performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). A CD45 Immunoprecipitation of Akt substrates and generation of Akt splicing assay was performed as previously described (14). In brief, prim- 9 substrate tryptic fragments ers were designed to flank exons 4–6 of CD45 (primer 1: 5 -GGCAA- CACCTACACCCA-39, primer 2: 59-GCTTGCAGGCCCAGA-39). PCRs Five million CD4+ T cells were activated in Treg or Th conditions in the (30 cycles) were performed using the goTaq DNA polymerase (Promega). presence or absence of an Akt inhibitor (iAkt; Akt1/2 10 mM). After PCRs were resolved on a 1% agarose gel, SeaKem LE Agarose (Lonza), 15 min of activation, cells were lysed in 500 ml of ice-cold buffer con- stained with ethidium bromide (Sigma) and imaged on a Red imaging taining 0.5% (v/v) Nonidet P-40, 1% (v/v) Triton X-100, 150 mM NaCl, system (Protein system). 50 mM Tris-HCl [pH 7.4], 1 mM EDTA, and 1 mM EGTA. The lysis buffer also contained protease and phosphatase inhibitors (1 mM Na3VO4, Splice variant isoform quantitative PCR assays 1 mM aprotinin, 1 mM leupeptin, 1 mM pepstatin, 10 mM NaF, 1 mM PMSF, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate). The Total RNA was isolated and reverse transcribed as described above. Primer pairs were designed to look at the splice variant usage for Lck, Fyn Tec, and lysate was sonicated, centrifuged, and incubated with an Ab specific to + phosphorylated Akt substrate motif RXRXX (phospho-Thr/phospho-Ser) CD247 in resting and activated CD4 T cells (Table I). Quantitative PCR (Cell Signaling Technologies) at a ratio of 1 mlofAbto50ml of cell lysate (qPCR) was performed on a StepOne Plus real-time PCR system with the at 4˚C for 12 h. The Ab–Akt substrate complexes were captured by protein SYBR Green Master Mix (Applied Biosystems) from three independent + experiments.