TCR Signal Strength Regulates Akt Substrate Specificity to Induce

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! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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. All results are reported, unless otherwise stated, as fold A–coated beads. As a negative control, CD4 T cell lysate not treated with 2DDCT the Akt substrate Ab was incubated with the protein A beads. Beads were change (2 ) relative to resting T cells. washed two times with lysis buffer and two times in PBS. The captured Downloaded from Akt substrates were eluted from the beads with a urea buffer (8 M urea and Small interfering RNA knockdown of hnRNP L and hnRNP A1 0.1 M Tris/HCl [pH 8.5]). To generate tryptic fragments, we used the filter- Small interfering RNA (siRNA) kits (Origene) were used to knock down the aided sample preparation method (12). All of the immunoprecipitates (IP) expression of hnRNPA1 and hnRNP L in mouse primary CD4+ T cells. The were done in duplicate. nucleofector kits for mouse T cells (Lonza) and Amaxa nucleofector were + Bottom-up proteomics experiments for protein identification used to introduce siRNAs into murine CD4 T cells. To measure the knockdown efficiency, we performed both qPCR analysis and Western blot

Bottom-up proteomics experiments were conducted with either a Thermo- analysis in triplicate. http://www.jimmunol.org/ Finnegan Velos-Orbitrap (Velos Orbi) or Q-Exactive (QE) mass spectrometer coupled to a Waters nanoAcquity ultra-HPLC system via a nanoelectrospray Statistics m ionization source. Between 1 and 5 l of a sample containing tryptic All statistics were calculated in the GraphPad Prism 7 software package. All peptides was injected on a Waters Acquity BEH 300 C18 75 mm internal 3 m analyses used the two-way ANOVA analysis with Bonferroni postanalysis diameter 100-mm-long column packed with 1.7- m particles running a correction. linear gradient of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at 1 ml/min as follows: 98:2 for 10 min, 92:8 at 12 min, 65:35 at 65 min, 20:80 at 68 min until 75 min, then return to initial Results conditions at 76 min and held there to 90 min. Mass spectra were acquired TCR signal strength controls Akt phosphorylation status and in the Orbitrap at 60,000 resolving power, and tandem mass spectra were

substrate specificity by guest on September 27, 2021 then generated for the top 10 (with the Velos Orbi) or 12 (with the QE) most abundant ions with charge states ranging between two and five. We previously identified the dominant role of Ag dose in the Fragmentation of selected peptide ions was achieved via collisionally in- induction and expansion of Treg cells (3). In these studies, we duced dissociation at normalized collision energy of 35 eV in either the Velos mass analyzer region of the Velos Orbi or in the C-trap section of the showed that low-dose Ag favored the induction/expansion of Treg QE. Proteome Discoverer 1.4 software using either Mascot or Sequest cells in the islet-specific BDC2.5 and in two OVA-specific TCR search engines with a decoy search at a 1% false discovery rate was used to transgenic lines, OTII and DO11.10 (3), and in T cells stimulated identify proteins present in each sample by matching tandem mass spectra by anti-CD3 (3, 8). The cells generated by these cultures had with peptides expected for proteins in the SwissProt database. strong suppressive function in vitro (3) and in vivo (15). Sig- Bioinformatics naling via the Akt/mTOR pathway influences CD4+ Tcelldif- ferentiation; low levels favor Treg induction and high levels Akt substrates identified during Th and Treg induction were analyzed using the Ingenuity software package (Qiagen). A core analysis was performed favor Th induction (1–3). We recently described several im- using default settings to identify pathways that were targeted by Akt, which portant feedback loops that control the degree of Akt/mTOR were used to bin Akt targets into functional categories. Further analysis was activation in CD4 T cells. These involve the lipid phospha- performed using the PANTHER classification system (13). The p values tase PTEN, the transcription factors Foxp3 and FoxO1, and Akt were calculated by the Ingenuity software package using the right-tailed (7). Importantly, phosphorylation of the transcription factor Fisher exact test, and the recommended p value cutoff for significance was ,0.05. FoxO1 by Akt was critical for the observed reduction of PTEN levels seen after high-dose stimulation (7). These studies prompted Western blotting us to examine how TCR signal strength influenced the phos- + Western blot analysis was performed with Abs purchased from Cell Sig- phorylation status of Akt. CD4 T cells isolated from C57BL/6 naling Technology: phosphor-Zap70 (2701), total phospho-tyrosine (p- mice were stimulated with increasing doses of plate-bound Tyr-1000), Akt (C67E7), phospho-308 Akt (D25E6), phospho-473 Akt anti-CD3 in the presence of anti-CD28. Western blot analysis (C67E7), phosphor-Rictor (D30A3; Cell Signaling), Rictor (53A2), Akt of Ser473 phosphorylation revealed a sharp threshold for Ser473 substrate (23C8D2), FoxO1 (C29H4), hnRNP A1 (D21H11), and Actin (13E5). The hnRNP L Ab (H-78) was purchased from Santa Cruz phosphorylation (Fig. 1A, 1B). Western blot analysis revealed Biotechnology. that phosphorylation of Thr308 occurred during both high- (1 mg/ml) and low-dose (0.25 mg/ml) stimulation. Phosphory- Flow cytometry lation of Ser473 was observed only under high-dose conditions CD4+ T cells were stained with the following mAbs: anti–CD3-PE (BD), (Fig. 1C, 1E). anti–CD4-PerCP5.5 (eBioscience), anti–CD25-Pe-Cy7 (eBioscience), anti- The lack of Ser473 phosphorylation on Akt under low-dose con- Foxp3-Pacific Blue (eBioscience), anti-CD45RB FITC (eBioscience), and anti–phospho-S6 (Ser235) Alexa-647 (Cell Signaling Technology) using ditions could be because of reduced mTORC2 activity, which is the buffers from eBioscience. The stained cells were analyzed on an LSR II only known kinase to phosphorylate this site (16). The activity of flow cytometer, and data were analyzed with the FlowJo software package. mTORC2 is decreased by phosphorylation of the Thr1135 residue The Journal of Immunology 3 on the rapamycin-insensitive companion of mTOR (Rictor) sub- pathway analysis using the Ingenuity and KEGG databases dem- unit (17). Western blot analysis revealed that Rictor Thr1135 was onstrated that key processes, including metabolism, TCR signal- constitutively phosphorylated in unstimulated CD4+ T cells ing, and RNA processing, were targeted by Akt during high- and (Fig. 1F, 1G). Thr1135 remained phosphorylated under low-dose low-dose stimulation (Fig. 2C). Pathways including glycolysis, conditions (Fig. 1C, 1D), but it was significantly reduced during Tec kinase signaling, and the TCA cycle were differentially tar- high-dose stimulation (Fig. 1D). geted by Akt during high-dose stimulation (Fig. 2D). Interestingly, To determine whether differential Akt phosphorylation altered RNA processing and splicing were overrepresented biological the substrates targeted by Akt, we performed a time course of processes targeted by Akt during both high- and low-dose stim- low- and high-dose stimulation followed by Western blot analysis ulation (Fig. 2E). with an Ab that specifically recognizes the phosphorylated Akt Specific proteins targeted by Akt under conditions of Treg or Th substrate motif (XRXRXXS/TX) (18). Substrates appeared at induction were distinct (Fig. 3A, Supplemental Tables I, II). Some 15 min and persisted in both high- and low-dose conditions Akt substrates in both Th and Treg induction included hnRNPs (Fig. 1H, 1I). There were clear qualitative differences in Akt A2B1, enolase, and Hspa8 (Fig. 3A). Akt substrates unique to substrates phosphorylated during low- or high-dose stimulation low-dose stimulation included STAT1, aconitase, hnRNP A1, and (Fig. 1H, 1I). hnRNP L. Akt substrates unique to high-dose stimulation included the mitochondrial ADP/ATP carrier (SLC25A5) and LCP-1 (Fig. Th and Treg induction are marked by different Akt substrates 3A). Further work will be needed to validate many of these sub- To identify proteins phosphorylated by Akt during high- and low- strates, and we concentrated our efforts on the RNA processing dose stimulation, we used a mass spectrometric approach (Fig. 2A). factors hnRNP L and hnRNP A1. Western blot analysis confirmed Downloaded from The anti–phospho-(Ser/Thr) Akt substrate Ab (18) was used to IP that both hnRNP L and hnRNP A1 were immunoprecipitated us- + phosphorylated Akt substrates from CD4 T cells stimulated with ing the Akt substrate motif Ab under low-dose TCR stimulation low- or high-dose anti-CD3 for 15 min in the presence or absence and not under high-dose TCR stimulation (Fig. 3B, 3C). Western of an iAkt (7). This analysis was repeated twice, and 99 unique blot analysis for FoxO1 revealed that FoxO1 was immunoprecip- proteins were identified after removal of those that occurred with itated only under high-dose stimulation consistent with previous

the iAkt or control beads, those that were identiﬁed in only one of studies (7) (Fig. 3B, 3C). http://www.jimmunol.org/ the two experiments, and those that did not contain an Akt con- sensus substrate motif as determined by the Scansite server (19). Akt regulates CD45 splicing during T cell differentiation Of the 99 identiﬁed Akt substrates, only 18 were shared between RNA processing and splicing proteins were a predominant sub- the high- and low-dose T cell activation conditions. Thirty-one group of Akt substrates in activated T cells (Fig. 3A). A classic proteins were unique to high-dose stimulation conditions, and example of a protein regulated by alternative splicing in T cells is 50 were unique to low-dose stimulation conditions (Fig. 2B). A the CD45 phosphatase (Fig. 4A) (20). Expression of the CD45RB by guest on September 27, 2021

FIGURE 1. TCR signal strength controls Akt phosphorylation status and substrate specificity. (A and B) Western blot of p-Ser473 and total Akt in CD4 T cells stimulated with the indicated concentrations of anti-CD3. A representative blot (A) and the mean 6 SEM of three independent experiments (B) are shown. (C) The phosphorylation status of Thr308 and Ser473 on Akt and total Akt was examined by Western blot on induced Treg (low, 0.25 mg/ml) and Th (high, 1 mg/ml) cells activated for the indicated time points. (D and E) Densitometry was performed to quantitate the levels of p-Thr308 and p-Ser473 observed from the Western blot analysis in (C) from three independent experiments. (F) The phosphorylation status of Rictor Thr1135 was monitored during low- (0.25 mg/ml) and high-dose (1 mg/ml) stimulation by Western blot. Total levels of Rictor and Actin are also shown. These experiments are representative of three independent experiments and quantitated by densitometry in (G). Purified CD4+ T cells were activated under Treg (H)orTh(I) induction conditions for the indicated time points, and Western blot analysis was performed with an Ab specific for the phospho-Akt substrate motif. A two-way ANOVA analysis with Bonferroni posttest was performed. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. 4 TCR SIGNAL STRENGTH DETERMINES Akt SUBSTRATE SPECIFICITY isoform has been used to distinguish Th cell subsets such that These results support the role of Akt in the regulation CD45 CD45RBhigh cells are T effector cells (21), whereas low expres- splicing. sion of CD45RB is a marker of Treg cells (22). hnRNP L and hnRNP A1 regulate splicing of CD45 mRNA (23, Akt regulates the splicing of key regulators in the T cell 24) and are phosphorylated by Akt only under low-dose condi- signaling pathway tions (Fig. 3A, 3B). Flow cytometric analysis of CD45RB ex- Previous CLIPseq data demonstrated that, in human T cells, hnRNP pression revealed that nTreg cells had the lowest CD45RB L binds to many mRNA transcripts encoding proteins in the T cell abundance (Fig. 4B). Induced Treg cells predominantly expressed signaling pathway, including CD45, Fyn, Tec, Lck, and CD247 low CD45RB levels. However, induced Th cells were mostly (25). Based on these reports, qPCR-based assays were designed CD45RB high (Fig. 4B). PCR analysis revealed that, initially, (Table I) to monitor the splicing of mRNA encoding key T cell CD45RO was the predominant isoform expressed by unactivated signaling proteins. murine CD4+ T cells (Fig. 4C). Activation under both low- and Alternatively spliced variants of Lck, Tec kinase, and Fyn high-dose conditions resulted in the increase of total CD45 tran- (Supplemental Fig. 1) were examined. A 2-fold increase in the scription and the production of the CD45RB isoform (Fig. 4C). expression of the Lck-B (26) and FynA (27) isoforms was ob- Pretreatment of the cells with iAkt before activation induced served in T cells stimulated with low-dose anti-CD3 for 24 h (Fig. larger isoforms of CD45, including CD45RB (Fig. 4C). In addi- 4D, 4E). The increase in both the Lck-B and FynA isoforms tion, treatment with the iAkt also appeared to increase total CD45. (Supplemental Fig. 1A, 1B) was Akt dependent because the Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Proteomic and pathway analysis of Akt signaling during Treg versus Th induction. (A) An immunoprecipitation workflow was developed to isolate phospho-Akt substrates during high- and low-dose TCR stimulation, and the Akt substrates were identified with mass spectrometry. (B) From the mass spectrometry analysis, we identified 50 Akt substrates unique to Treg (low) induction, 31 Akt substrates unique to Th (high) induction, and 18 shared Akt substrates. (C) Analysis performed with the Ingenuity software package identified that similar processes were targeted by Akt during both low (upper panel) and high dose (lower panel). (D) A pathway analysis of the Akt substrates identified during low-dose (Treg, checkered bars) and high-dose (Th, solid bars) stimulation was performed in the Ingenuity software package. (E) Akt substrates were placed into functional classes overrepresented in the Treg (checkered bars) or Th (solid bars) datasets. p , 0.05. The Journal of Immunology 5 Downloaded from

FIGURE 3. Identification of specific Akt substrates phosphorylated during Th or Treg induction. (A) Some of the Akt substrates identified in low- (left circles) and high-dose (right circles) conditions. Akt substrates that are shared between low- and high-dose conditions are listed at the overlap between the left (low) and right (high) circles. (B) Western blot analysis was performed on the proteins bound to the Akt substrate Ab (beads) versus the amount of protein remaining in solution (Sup.) for hnRNP L, hnRNP A1, and FoxO1 from cells stimulated with high (H) or low (L) dose anti-CD3. (C) Densitometry http://www.jimmunol.org/ was performed to determine the ratio of the proteins bound to the Akt substrate Ab (beads) versus the amount of protein remaining in solution from three independent experiments (hnRNP L and hnRNP A1) or two independent experiments (FoxO1). A two-way ANOVA analysis with Bonferroni posttest was performed. ***p , 0.001, ****p , 0.0001. presence of iAkt inhibited the observed increase (Fig. 4D, 4E). spleen of Foxp3 reporter mice and examined the expression of Tec Tec kinase, which regulates IL-2 and IL-4 gene transcription (28), isoforms. There was no observed difference in expression of Tec also underwent alternative splicing during T cell differentiation. isoforms between naive T cells and nTreg cells (Fig. 4G). The PH domain is critical for Tec activity (29), and splicing Another target for alternative splicing is CD247, which has a key

generates an isoform, Tec_C, that lacks the PH domain function in the TCR/CD3 complex (31, 32). There are four splice by guest on September 27, 2021 (Supplemental Fig. 1C) (30). During low-dose stimulation, the variants of CD247 (33, 34), which include CD3h, CD3z, CD3u, levels of Tec_C increased .20-fold but remained constant during and CD3i. Both CD3h and CD3u lack the third intracellular high-dose stimulation (Fig. 4F). These changes were Akt depen- ITAM, which could modulate signaling (Fig. 5A) (31, 32). The dent because cells pretreated with iAkt failed to increase expres- CD3z, CD3u, and CD3h isoforms of CD247 were detectable sion of the Tec_C isoform under low-dose conditions (Fig. 4F). To during both low- and high-dose stimulation (Fig. 5B, 5D). During determine whether Akt-regulated splicing of Tec kinase induced high-dose stimulation, CD3z mRNA expression increased 20-fold by T cell activation was maintained in fully differentiated Treg over the initial level, and this increase was Akt dependent cells, we sorted naive CD4+ T cells and nTreg cells from the (Fig. 5B–D). Conversely, during low-dose stimulation, CD3h and

FIGURE 4. Akt regulates the splicing of proteins involved in TCR signaling. (A) Diagram depicting the splicing variants for CD45. (B) Flow cytometric analysis of the expression of the CD45RB isoform on induced Treg cells (iTregs), nTregs, and induced Th cells (iTh). (C) A PCR-based assay was used to monitor the splicing of CD45 under low- or high-dose conditions with and without an iAKT. Results are representative of two independent experiments. (D) The levels of speciﬁc splice isoforms of Lck (LCKA or LCKB), (E) Fyn (FynA or FynB), and (F) Tec (Tec_C or Tec_A) were monitored by qPCR at 12 h of high or low TCR stimulation with (+ I) and without the iAKT inhibitor. (G) Naive CD4+ T cells (white) and nTregs (black) were isolated from a Foxp3-RFP reporter mouse, and the levels of Tec isoforms were measured by qPCR. (D–F) Results represent the mean 6 SEM of three independent experiments. A two-way ANOVA analysis with Bonferroni posttest was performed. *p , 0.05, **p , 0.01, ****p , 0.0001. 6 TCR SIGNAL STRENGTH DETERMINES Akt SUBSTRATE SPECIFICITY

Table I. Primer sequences used in alternative splicing analysis

Isoform Forward Primer Reverse Primer LCK A 59-GCCTCTGAGCTGACGATCT-39 59-CACACGTCAATGTTCTCCATCC-39 LCK B 59-GGCTAGGGAGCATCATGTGAATA-39 59-TCCAGTCATCTTCAGGGTTTG-39 FYN A 59-CTGTCACAAAGGGATGCCA-39 59-TGATCAACTGCAGGGATTCTC-39 FYN B 59-TTCATCAAGTTGTACCCCACAAA-39 59-AGCCACACTTCAGCGAAAC-39 TEK A 59-TCCCAAGTAATTACGTCACAGG-39 59-GCTGTTCTGTTTGCTTCTG-39 TEK C 59-GATGGTGTCATTCCCTGTCAA-39 59-TGCCCAGAATTTAGGATGGTATT-39 CD3z 59-CAGAAAGACAAGATGGCAGAAG-39 59-CATATGCAGGGCATCATAGGT-39 CD3u 59-CAGAAAGACAAGATGGCAGAAG-39 59-TCTTATGTCGGCAGGCTTTG-39 CD3h 59-CAGAAAGACAAGATGGCAGAAG-39 59-ACATCTCCTTCCTCTCCTGTAG-39 Total CD247 59-GGA TCC CAA ACT CTG CTA CTT-39 59-CTG TAG GCT TCT GCC ATC TT-39

CD3u mRNA expression increased by 20- and 4-fold, respectively To determine whether Akt-regulated splicing induced by T cell (Fig. 5D). activation was maintained in fully differentiated Treg cells, we sorted naive CD4+ T cells and nTreg cells from the spleen of Redistribution of splice variant usage during Treg induction Foxp3 reporter mice and examined the expression of CD247 results in dampened signaling isoforms. Naive T cells predominantly express the CD3z isoform, Downloaded from Altered splice variant usage of signaling molecules may be one of whereas nTreg cells preferentially express the CD3u isoform and the factors that alter the threshold required for T cell activation little CD3z (Fig. 5G). between Th cells compared with Treg cells (26, 27). To test whether signaling was altered during Treg differentiation, we hnRNP A1 and hnRNP L are critical for Treg induction activated T cells under Th (1 mg/ml anti-CD3) or Treg (0.25 mg/ml hnRNP proteins inﬂuence alternative splicing in T cells, including anti-CD3) conditions for 36 h; cells were rested for 4 h and hnRNP L and hnRNP A1 (35). In humans, hnRNP A1 synergizes restimulated with a high dose of anti-CD3. During the initial with hnRNP L to block splicing of exons 4, 5, and 6 in CD45 (24). http://www.jimmunol.org/ stimulation, both the Treg and Th induction conditions resulted in hnRNP L and hnRNP A1 were both phosphorylated by Akt during similar tyrosine phosphorylation at early time points, although this low-dose stimulation (Fig. 3A, 3B), suggesting that their activity was poorly sustained with low-dose anti-CD3 (Fig. 5E). Upon re- could be important for Treg differentiation. We used an siRNA stimulation with high dose, anti-CD3 cells destined to become Treg approach to knock down hnRNP A1 and hnRNP L to examine cells exhibited a much-reduced level of total tyrosine phosphoryla- their roles in T cell differentiation. The siRNA knockdown tion compared with cells destined to become Th cells (Fig. 5F). resulted in 1- and 200-fold reduction in the expression of hnRNP by guest on September 27, 2021

FIGURE 5. Altered CD247 splicing regulates TCR signaling. (A) CD247, more commonly referred to as CD3z, has four splice variants. (B–D) Purified CD4+ T cells were activated under high- and low-dose conditions, and qPCR was used to monitor the levels of CD247 isoforms [h (B), u (C), and z (D)] during differentiation. (D) Data are also presented for CD4+ T cells skewed under Th conditions and treated with iAkt (Th + I). Results shown are normalized to the total level of CD247 mRNA and represent the mean 6 SEM of three independent experiments. A two-way ANOVA analysis with Bonferroni posttest was performed. *p , 0.05, **p , 0.01, ****p , 0.0001. (E) Purified CD4+ T cells were stimulated with low- (L) or high (H)-dose stimulation for the indicated times. Western blotting was performed for total tyrosine phosphorylation. (F) Purified CD4+ T cells were activated for 38 h under Treg (0.25 mg/ml anti-CD3) or Th (1 mg/ml anti-CD3) skewing conditions, rested for 4 h, and restimulated with 1 mg/ml of plate-bound anti-CD3 Ab and 1 mg/ml soluble anti-CD28 Ab. Western blot analysis was performed for total tyrosine phosphorylation and actin at the indicated time points. The blot shown is representative of three independent experiments. (G) Naive CD4+ T cells and nTregs were isolated from a Foxp3-RFP reporter mouse, and the levels of the CD247 isoforms were measured by qPCR. The results shown are representative of two independent experiments. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 6. hnRNP A1 or hnRNP L are required for optimal Treg induction. (A) The mRNA expression levels of hnRNP L and hnRNP A1 were measured by qPCR 24 h after electroporation with the respective siRNAs. (B and C) The protein abundance was measured by Western blotting (B), and the protein levels were quantiﬁed by densitometry from two independent experiments (C). (D) Knockdown of hnRNP A1 resulted in altered CD45 splicing in by guest on September 27, 2021 resting CD4+ T cells relative to a scrambled control, as monitored by PCR analysis of exons 4–6 and gel electrophoresis 24 h after electroporation. (E–G) CD4+ T cells were electroporated with siRNA targeting hnRNP A1 or hnRNP L, rested for 24 h, and activated with either a low or high TCR signal. (E)At 12 h postactivation, ﬂow cytometry was used to monitor the expression of CD45RB and phosphorylation of S6 under Th and Treg induction conditions in cells treated with the scrambled or hnRNP A1 targeting siRNA. (F and G) Cells treated with scrambled, hnRNP A1 (F), or hnRNP L (G) siRNA were gated on CD3+CD4+ T cells. The CD25 and Foxp3 markers were used to track the induction of Th (CD3+CD4+Foxp32CD25+) and Treg (CD3+CD4+Foxp3+ CD25+) cells. (H) The percentage of CD4+ Treg or Th cells was plotted averaged over four independent experiments. A two-way ANOVA analysis with Tukey posttest was performed. *p , 0.05, **p , 0.01, ***p , 0.001.

A1 and hnRNP L mRNAs (Fig. 6A) and a 7- to 15-fold reduction 6H). Under low-dose conditions, hnRNP A1 knockdown resulted in hnRNP A1 and hnRNP L protein (Fig. 6B, 6C). Knockdown of in .60% of the cells becoming Th versus 21% in the control hnRNP A1 in resting CD4+ T cells resulted in the production of sample. In addition, hnRNP A1 knockdown resulted in signifi- larger CD45 isoforms compared with the scrambled control cantly decreased induction of Treg cells relative to the control. (Fig. 6D), similar to that observed when the enzymatic activity of Similarly, hnRNP L knockdown also resulted in significantly in- Akt was inhibited (Fig. 4C). Notably, the CD45RB isoform is creased Th cell generation and decreased Treg cell induction more abundant with knockdown of hnRNP A1. (Fig. 6G, 6H). To determine the function of hnRNP A1 or hnRNP L in CD4+ T cell differentiation, we treated cells with hnRNP A1 or Discussion hnRNP L siRNA for 24 h and activated them under low- and In this article, we identified distinct Akt substrates during the high-dose conditions. At 12 h postactivation in cells treated differentiation of CD4+ T cells into Th or Treg cell subsets. Al- with hnRNP A1 siRNA, CD45RB was elevated in both high- ternate signaling networks were associated with differential reg- and low-dose stimulation (Fig. 6E). In addition, knockdown ulation of the phosphorylation status of Akt. Of interest was the of hnRNP A1 resulted in increased phosphorylation of S6 un- regulation of splicing factors by Akt, and we observed that the der both high- and low-dose stimulation (Fig. 6E). Phospho- splicing of multiple mRNAs encoding key TCR signaling mole- S6 abundance inversely correlated to the induction of Treg cules was Akt dependent. Two proteins known to regulate splicing cells (3, 15), suggesting that hnRNP A1 is important for Treg in T cells, hnRNP A1 and hnRNP L, were Akt substrates during induction. Treg induction. Knockdown of either hnRNP A1 or hnRNP L The impact of hnRNP A1 or hnRNP L knockdown on CD4+ suppressed Treg induction by elevating Akt/mTOR signaling. T cell differentiation was analyzed. At 48 h postactivation in high- Together, this work provides mechanistic insight into how Akt dose conditions, similar numbers of Th cells were observed in initiates different differentiation programs to drive alternate CD4+ cells treated with hnRNP A1 and the scrambled siRNA (Fig. 6F, T cell fate decisions. 8 TCR SIGNAL STRENGTH DETERMINES Akt SUBSTRATE SPECIFICITY

Previous work correlated the phosphorylation status of Akt with favored Th induction. A recent study of LckCre+hnRNP Lfl/fl mice regulating its enzymatic activity (36). Thr308 is phosphorylated by demonstrated a striking defect in peripheral T cell numbers that PDK1 (9), which is directly activated by PIP3 downstream of TCR was associated with defects in thymocyte migration to the pe- signaling, and is dephosphorylated by protein phosphatase 2A riphery (46). This study identified multiple changes in alternative (37). Ser473 is phosphorylated by mTORC2 (16) and dephos- splicing of proteins involved in T cell signaling and chemotaxis phorylated by PHLPP (38). For maximal enzymatic activity, it is (46), and showed an increase in splicing to the CD45RB isoform, thought that Akt should be phosphorylated at both Thr308 and which we also observed in our study. Although it is well accepted Ser473, and that if only one of the sites is phosphorylated, enzy- that induction of transcription factors including Foxp3 drives matic activity is much reduced (36). The phosphorylation of T cell differentiation programs, our results suggest that splicing is FoxO1 by Akt requires that both Thr308 and Ser473 be phosphor- a key driver of differentiation. HnRNP L binds .2000 mRNAs, ylated on Akt (4, 7, 39). Intriguingly, human CD4+ CD25+ T cells and it is likely that regulation of splicing factors by Akt results in stimulated with anti-CD3 are defective in phosphorylation of the different complements of protein isoforms during Th and Treg Ser473 site on Akt, whereas Thr308 is robustly phosphorylated. differentiation. Introduction of an acidic residue at the Ser473 site resulted in Work presented in this article identified novel Akt substrates constitutive activation of Akt in human Treg cells, which abro- differentially phosphorylated during the induction of Th and Treg gated their suppressive activity and restored the ability of Akt to cells from naive CD4+ cells and demonstrated that Akt activity exclude FoxO1 from the nucleus (4). was functionally important for regulating splicing programs. Fu- Low-dose stimulation was marked with suppressed phosphor- ture work will be aimed at validating the novel Akt substrates ylation of Ser473 on Akt and normal levels of Thr308 phosphoryla- identified in this study. The phosphorylation status of Akt was Downloaded from tion, whereas high-dose stimulation induced robust phosphorylation different based on whether T cells were stimulated with high or of both Ser473 and Thr308. The ability of mTORC2 to phosphorylate low dose, and this influenced Akt activity and substrate specificity. Ser473 is controlled by Thr1135. This inhibitory site, Thr1135,onthe In conclusion, this work provides novel insight into CD4+ T cell Rictor subunit of mTORC2 remained phosphorylated under low- differentiation by identifying unique Akt substrates during Treg dose stimulation. Conversely, high-dose stimulation resulted in and Th induction, defining how the phosphorylation status of Akt 1135 dephosphorylation of Thr , which correlated with elevated levels regulates substrate specificity, and identifying that Akt-mediated http://www.jimmunol.org/ of Ser473 phosphorylation. The mechanism of mTORC2 activation phosphorylation of RNA processing factors plays a major role in in T cells is not well established. One possibility is that TCR sig- determining CD4+ T cell fate. naling generates PIP3 to activate mTORC2, which was observed in HEK293 T cells (40). In this model, PIP3 would be higher during Acknowledgments high-dose stimulation to activate mTORC2 and lower in low-dose We thank Matt Gable for technical assistance. We thank Dr. Greg Delgoffe conditions to weakly activate mTORC2. This would allow for el- and Dr. Larry Kane for careful reading of the manuscript. evated PIP3 levels, heightened mTORC2 activity, and increased phosphorylation of Akt Ser473.

Disclosures by guest on September 27, 2021 A surprising result is the essential role of Akt-dependent mRNA The authors have no financial conflicts of interest. splicing in CD4+ T cell differentiation. Notably, Treg induction resulted in the reduction of the CD3z isoform of CD247 and the emergence of the CD3h and CD3u isoforms. Both the CD3h and References CD3u isoforms lack the third ITAM domain, which could impact 1. Haxhinasto, S., D. Mathis, and C. Benoist. 2008. The AKT-mTOR axis regulates downstream signaling events. T cells with more of the CD3h and de novo differentiation of CD4+Foxp3+ cells. J. Exp. Med. 205: 565–574. u 2. Sauer, S., L. Bruno, A. Hertweck, D. Finlay, M. Leleu, M. Spivakov, CD3 isoforms exhibited reduced early TCR signaling, suggesting Z. A. Knight, B. S. Cobb, D. Cantrell, E. O’Connor, et al. 2008. T cell receptor that splicing of CD247 is used to tune the level of TCR signaling signaling controls Foxp3 expression via PI3K, Akt, and mTOR. Proc. Natl. during T cell differentiation. Previous studies demonstrated that Acad. Sci. USA 105: 7797–7802. the three CD3z ITAMs are critical for optimal T cell activation 3. Turner, M. S., L. P. Kane, and P. A. Morel. 2009. Dominant role of antigen dose in CD4+Foxp3+ regulatory T cell induction and expansion. J. Immunol. 183: 4895–4903. (41). In addition, defects in CD3z signaling correlated with dis- 4. Crellin, N. K., R. V. Garcia, and M. K. Levings. 2007. Altered activation of AKT ease pathogenesis in patients with systemic lupus erythematosus is required for the suppressive function of human CD4+CD25+ T regulatory (42). In nTreg cells, the CD3u isoform dominated, suggesting that cells. Blood 109: 2014–2022. 5. Huynh, A., M. DuPage, B. Priyadharshini, P. T. Sage, J. Quiros, C. M. Borges, the alternative splicing observed during Treg induction remains N. Townamchai, V. A. Gerriets, J. C. Rathmell, A. H. Sharpe, et al. 2015. Control after differentiation. This may provide an explanation for the re- of PI(3) kinase in Treg cells maintains homeostasis and lineage stability. Nat. duced TCR signaling in Treg cells, which was recently investi- Immunol. 16: 188–196. 6. Shrestha, S., K. Yang, C. Guy, P. Vogel, G. Neale, and H. Chi. 2015. Treg cells gated (43). This study examined the role of several Treg-specific require the phosphatase PTEN to restrain TH1 and TFH cell responses. Nat. negative regulators, such as CTLA-4, PD-1, CD5, and DUSP4, but Immunol. 16: 178–187. these were not found to be responsible for the reduced signaling in 7. Hawse, W. F., R. P. Sheehan, N. Miskov-Zivanov, A. V. Menk, L. P. Kane, J. R. Faeder, and P. A. Morel. 2015. Cutting edge: differential regulation of Treg cells (43). Together, these results suggest that splicing reg- PTEN by TCR, Akt, and FoxO1 controls CD4+ T cell fate decisions. J. Immunol. ulated by Akt functions to rewire the canonical TCR signaling 194: 4615–4619. pathway to drive alternate CD4+ T cell differentiation programs. 8. Miskov-Zivanov, N., M. S. Turner, L. P. Kane, P. A. Morel, and J. R. Faeder. During low-dose stimulation, both hnRNP A1 and hnRNP L 2013. The duration of T cell stimulation is a critical determinant of cell fate and plasticity. Sci. Signal. 6: ra97. were phosphorylated by Akt (44, 45). There is a defined Akt 9. Alessi, D. R., S. R. James, C. P. Downes, A. B. Holmes, P. R. Gaffney, phosphorylation site on hnRNP A1 at Ser199, and this has been C. B. Reese, and P. Cohen. 1997. Characterization of a 3-phosphoinositide- shown to influence hnRNP A1 function in cell lines (44). In the dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr. Biol. 7: 261–269. case of hnRNP L, there are two potential Akt phosphorylation 10. Sarbassov, D. D., S. M. Ali, and D. M. Sabatini. 2005. Growing roles for the sites, and a recent study showed that Akt phosphorylation of mTOR pathway. Curr. Opin. Cell Biol. 17: 596–603. hnRNP L influenced the alternative splicing of caspase 9 in cell 11. Vasudevan, K. M., and L. A. Garraway. 2010. AKT signaling in physiology and disease. Curr. Top. Microbiol. Immunol. 347: 105–133. lines (45). Knockdown of either hnRNP A1 or hnRNP L reduced 12. Wisniewski, J. R., A. Zougman, N. Nagaraj, and M. Mann. 2009. Universal + the capacity of CD4 T cells to differentiate into Treg cells and sample preparation method for proteome analysis. Nat. Methods 6: 359–362. The Journal of Immunology 9

13. Mi, H., A. Muruganujan, J. T. Casagrande, and P. D. Thomas. 2013. Large-scale 30. Mano, H., Y. Yamashita, K. Sato, Y. Yazaki, and H. Hirai. 1995. Tec protein- gene function analysis with the PANTHER classification system. Nat. Protoc. 8: tyrosine kinase is involved in interleukin-3 signaling pathway. Blood 85: 343– 1551–1566. 350. 14. Wu, Z., X. Jia, L. de la Cruz, X.-C. Su, B. Marzolf, P. Troisch, D. Zak, 31. Clayton, L. K., A. C. Diener, A. Lerner, A. G. Tse, S. Koyasu, and A. Hamilton, B. Whittle, D. Yu, et al. 2008. Memory T cell RNA rearrangement E. L. Reinherz. 1992. Differential regulation of T-cell receptor processing and programmed by heterogeneous nuclear ribonucleoprotein hnRNPLL. Immunity surface expression affected by CD3 theta, an alternatively spliced product of the 29: 863–875. CD3 zeta/eta gene locus. J. Biol. Chem. 267: 26023–26030. 15. Turner, M. S., K. Isse, D. K. Fischer, H. R. Turnquist, and P. A. Morel. 2014. 32. Moulton, V. R., and G. C. Tsokos. 2010. Alternative splicing factor/splicing Low TCR signal strength induces combined expansion of Th2 and regulatory factor 2 regulates the expression of the z subunit of the human T cell receptor- T cell populations that protect mice from the development of type 1 diabetes. associated CD3 complex. J. Biol. Chem. 285: 12490–12496. Diabetologia 57: 1428–1436. 33. Lerner, A., L. D’Adamio, A. C. Diener, L. K. Clayton, and E. L. Reinherz. 1993. 16. Sarbassov, D. D., D. A. Guertin, S. M. Ali, and D. M. Sabatini. 2005. Phos- CD3 zeta/eta/theta locus is colinear with and transcribed antisense to the gene phorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science encoding the transcription factor Oct-1. J. Immunol. 151: 3152–3162. 307: 1098–1101. 34. Nocentini, G., S. Ronchetti, A. Bartoli, G. Testa, F. D’Adamio, C. Riccardi, and 17. Dibble, C. C., J. M. Asara, and B. D. Manning. 2009. Characterization of Rictor G. Migliorati. 1995. T cell receptor iota an alternatively spliced product of the phosphorylation sites reveals direct regulation of mTOR complex 2 by S6K1. T cell receptor zeta gene. Eur. J. Immunol. 25: 1405–1409. Mol. Cell. Biol. 29: 5657–5670. 35. Rothrock, C. R., A. E. House, and K. W. Lynch. 2005. HnRNP L represses exon 18. Moritz, A., Y. Li, A. Guo, J. Villeń, Y. Wang, J. MacNeill, J. Kornhauser, splicing via a regulated exonic splicing silencer. EMBO J. 24: 2792–2802. K. Sprott, J. Zhou, A. Possemato, et al. 2010. Akt-RSK-S6 kinase signaling 36. Alessi, D. R., M. Andjelkovic, B. Caudwell, P. Cron, N. Morrice, P. Cohen, and networks activated by oncogenic receptor tyrosine kinases. Sci. Signal. 3: ra64. B. A. Hemmings. 1996. Mechanism of activation of protein kinase B by insulin 19. Obenauer, J. C., L. C. Cantley, and M. B. Yaffe. 2003. Scansite 2.0: proteome- and IGF-1. EMBO J. 15: 6541–6551. wide prediction of cell signaling interactions using short sequence motifs. 37. Resjo¨, S., O. Go¨ransson, L. Ha¨rndahl, S. Zolnierowicz, V. Manganiello, and Nucleic Acids Res. 31: 3635–3641. E. Degerman. 2002. Protein phosphatase 2A is the main phosphatase involved 20. Hermiston, M. L., Z. Xu, and A. Weiss. 2003. CD45: a critical regulator of in the regulation of protein kinase B in rat adipocytes. Cell. Signal. 14: 231– signaling thresholds in immune cells. Annu. Rev. Immunol. 21: 107–137. 238. 21. Morrissey, P. J., K. Charrier, S. Braddy, D. Liggitt, and J. D. Watson. 1993. CD4+ 38. Gao, T., F. Furnari, and A. C. Newton. 2005. PHLPP: a phosphatase that directly Downloaded from T cells that express high levels of CD45RB induce wasting disease when trans- dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth. Mol. ferred into congenic severe combined immunodeficient mice. Disease development Cell 18: 13–24. is prevented by cotransfer of purified CD4+ T cells. J. Exp. Med. 178: 237–244. 39. Bhaskar, P. T., and N. Hay. 2007. The two TORCs and Akt. Dev. Cell 12: 487– 22. Powrie, F., J. Carlino, M. W. Leach, S. Mauze, and R. L. Coffman. 1996. A 502. critical role for transforming growth factor-beta but not interleukin 4 in the 40. Gan, X., J. Wang, B. Su, and D. Wu. 2011. Evidence for direct activation of suppression of T helper type 1-mediated colitis by CD45RB(low) CD4+ T cells. mTORC2 kinase activity by phosphatidylinositol 3,4,5-trisphosphate. J. Biol. J. Exp. Med. 183: 2669–2674. Chem. 286: 10998–11002.

23. Tong, A., J. Nguyen, and K. W. Lynch. 2005. Differential expression of CD45 41. van Oers, N. S. C., P. E. Love, E. W. Shores, and A. Weiss. 1998. Regulation of http://www.jimmunol.org/ isoforms is controlled by the combined activity of basal and inducible splicing- TCR signal transduction in murine thymocytes by multiple TCR z-chain sig- regulatory elements in each of the variable exons. J. Biol. Chem. 280: 38297– naling motifs. J. Immunol. 160: 163–170. 38304. 42. Moulton, V. R., and G. C. Tsokos. 2015. T cell signaling abnormalities con- 24. Chiou, N. T., G. Shankarling, and K. W. Lynch. 2013. hnRNP L and hnRNP A1 tribute to aberrant immune cell function and autoimmunity. J. Clin. Invest. 125: induce extended U1 snRNA interactions with an exon to repress spliceosome 2220–2227. assembly. Mol. Cell 49: 972–982. 43. Yan, D., J. Farache, M. Mingueneau, D. Mathis, and C. Benoist. 2015. Imbal- 25. Shankarling, G., B. S. Cole, M. J. Mallory, and K. W. Lynch. 2014. anced signal transduction in regulatory T cells expressing the transcription factor Transcriptome-wide RNA interaction proﬁling reveals physical and functional FoxP3. [Published erratum appears in 2016 Proc. Natl. Acad. Sci. USA 113: targets of hnRNP L in human T cells. Mol. Cell. Biol. 34: 71–83. E256.]. Proc. Natl. Acad. Sci. USA 112: 14942–14947. 26. Rouer, E., and R. Benarous. 1992. Alternative splicing in the human lck gene 44. Jo, O. D., J. Martin, A. Bernath, J. Masri, A. Lichtenstein, and J. Gera. 2008. leads to the deletion of exon 19 and results in a new type II lck transcript. Heterogeneous nuclear ribonucleoprotein A1 regulates cyclin D1 and c-myc

Oncogene 7: 2535–2538. internal ribosome entry site function through Akt signaling. J. Biol. Chem. by guest on September 27, 2021 27. Brignatz, C., M. P. Paronetto, S. Opi, M. Cappellari, S. Audebert, V. Feuillet, 283: 23274–23287. G. Bismuth, S. Roche, S. T. Arold, C. Sette, and Y. Collette. 2009. Alternative 45. Vu, N. T., M. A. Park, J. C. Shultz, R. W. Goehe, L. A. Hoeferlin, M. D. Shultz, splicing modulates autoinhibition and SH3 accessibility in the Src kinase Fyn. S. A. Smith, K. W. Lynch, and C. E. Chalfant. 2013. hnRNP U enhances caspase- Mol. Cell. Biol. 29: 6438–6448. 9 splicing and is modulated by AKT-dependent phosphorylation of hnRNP L. 28. Yang, W. C., and D. Olive. 1999. Tec kinase is involved in transcriptional reg- J. Biol. Chem. 288: 8575–8584. ulation of IL-2 and IL-4 in the CD28 pathway. Eur. J. Immunol. 29: 1842–1849. 46. Gaudreau, M.-C., F. Heyd, R. Bastien, B. Wilhelm, and T. Mo¨ro¨y. 2012. Al- 29. Yang, W.-C., K. A. Ching, C. D. Tsoukas, and L. J. Berg. 2001. Tec kinase ternative splicing controlled by heterogeneous nuclear ribonucleoprotein L signaling in T cells is regulated by phosphatidylinositol 3-kinase and the Tec regulates development, proliferation, and migration of thymic pre-T cells. pleckstrin homology domain. J. Immunol. 166: 387–395. J. Immunol. 188: 5377–5388.