T Cell–Specific Adaptor Protein Regulates Mitochondrial Function

Published September 20, 2019, doi:10.4049/jimmunol.1801604 The Journal of Immunology

T Cell–Speciﬁc Adaptor Protein Regulates Mitochondrial Function and CD4+ T Regulatory Cell Activity In Vivo following Transplantation

Johannes Wedel,*,†,‡ Maria P. Stack,*,†,‡ Tatsuichiro Seto,*,†,‡ Matthew M. Sheehan,*,† Evelyn A. Flynn,*,†,‡ Isaac E. Stillman,x,{ Sek Won Kong,‡,‖ Kaifeng Liu,*,†,‡,# and David M. Briscoe*,†,‡

The T cell–specific adaptor protein (TSAd), encoded by the SH2D2A gene, is an intracellular molecule that binds Lck to elicit signals that result in cytokine production in CD4+ T effector cells (Teff). Nevertheless, using Sh2d2a knockout (KO; also called TSAd2/2) mice, we find that alloimmune CD4+ Teff responses are fully competent in vivo. Furthermore, and contrary to expectations, we find that allograft rejection is accelerated in KO recipients of MHC class II–mismatched B6.C-H-2bm12 heart transplants versus wild-type (WT) recipients. Also, KO recipients of fully MHC-mismatched cardiac allografts are resistant to the graft-prolonging effects of costimulatory blockade. Using adoptive transfer models, we find that KO T regulatory cells (Tregs) are less efficient in suppressing Teff function and they produce IFN-g following mitogenic activation. In addition, pyrosequencing demonstrated higher levels of methylation of CpG regions within the Treg-specific demethylated region of KO versus WT Tregs, suggesting that TSAd, in part, promotes Treg stability. By Western blot, Lck is absent in the mitochondria of KO Tregs, and reactive oxygen species production by mitochondria is reduced in KO versus WT Tregs. Full transcriptomic analysis demonstrated that the key mechanism of TSAd function in Tregs relates to its effects on cellular activation rather than intrinsic effects on mitochondria/metabolism. Nevertheless, KO Tregs compensate for a lack of activation by increasing the number of mitochondria per cell. Thus, TSAd serves as a critical cell- intrinsic molecule in CD4+Foxp3+ Tregs to regulate the translocation of Lck to mitochondria, cellular activation responses, and the development of immunoregulation following solid organ transplantation. The Journal of Immunology, 2019, 203: 000–000.

he T cell–specific adaptor protein (TSAd), encoded by the Lck and elicits intracellular signals resulting in cytokine produc- SH2D2A gene, is an SH2 domain-containing intracellular tion (1–3). As its name suggests, TSAd was initially identified T adaptor molecule that activates the protein tyrosine kinase based on its expression in T cells (1, 3–6), but more recent studies have demonstrated its expression and function in other cell types *Transplant Research Program, Boston Children’s Hospital, Boston, MA 02115; (6–8). Nevertheless, targeted disruption of the TSAd gene in mice †Division of Nephrology, Department of Pediatrics, Boston Children’s Hospital, does not cause any major developmental abnormalities and is Boston, MA 02115; ‡Department of Pediatrics, Harvard Medical School, Boston, MA 02115; xDepartment of Pathology, Beth Israel Deaconess Medical Center, rather associated with defects in immune function (3, 9, 10). This Boston, MA 02215; {Department of Pathology, Harvard Medical School, Boston, finding indicates that its primary function is indeed linked to T cell ‖ MA 02115; Computational Health Informatics Program, Boston Children’s Hos- biology. Furthermore, genetic polymorphisms in the SH2D2A pital, Boston, MA 02115; and #Division of Pulmonary Medicine, Department of Medicine, Boston Children’s Hospital, Boston, MA 02115 gene in humans that result in lower TSAd protein expression ORCIDs: 0000-0002-3490-8477 (J.W.); 0000-0002-4224-5060 (I.E.S.); 0000-0003- levels are associated with the development of multiple sclerosis 4877-7567 (S.W.K.). and juvenile rheumatoid arthritis (2, 11–13). These collective Received for publication December 6, 2018. Accepted for publication August 10, observations are most suggestive that TSAd plays a major role in 2019. immunity and, notably, in human autoimmune disease. However, This work was supported by the National Institutes of Health (NIH) (R21AI92399) the cell-intrinsic function of TSAd in regulatory T cells (Tregs) is and the Casey Lee Ball Foundation (to D.M.B.). J.W. was supported by a fellowship not known, and its in vivo role in the modulation of T effector cell grant from Deutsche Forschungsgemeinschaft and a travel grant from the Medical Faculty Mannheim, Heidelberg University, Germany. M.M.S. was supported by a (Teff) activation has not been reported. supplement to NIH Grant T32DK007726. Mechanistically, TSAd is reported to augment upstream TCR- The transcriptomic data in this article have been submitted to the Gene Expression dependent signaling through an interaction that results in the Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE134515) under amplification of Lck phosphorylation and Lck-dependent re- accession number GSE134515. sponses (4, 14). TSAd also functions to augment synapse for- Address correspondence and reprint requests to Dr. David M. Briscoe, Transplant + Research Program and the Division of Nephrology, Boston Children’s Hospital, 300 mation between CD4 T cells and APCs (10), and it enhances Longwood Avenue, Boston, MA 02115. E-mail address: [email protected] CD28-dependent costimulation (15). In this manner, TSAd de- The online version of this article contains supplemental material. creases the threshold signal required for Ag-dependent activa- + Abbreviations used in this article: Ct, cycle threshold; ECAR, extracellular acidifi- tion of CD4 T cell subsets. In addition, it has been reported to cation rate; eTreg, effector memory Treg; GSEA, Gene Set Enrichment Analysis; function in T cell migration, notably, in response to the che- iTreg, induced Treg; KO, knockout; MST, median survival time; OCR, oxygen consumption rate; pAdj, adjusted p value; qPCR, quantitative PCR; ROS, reactive oxy- mokines SDF-1a and RANTES (5, 16, 17). However, knockout gen species; Teff, T effector cell; Treg, T regulatory cell; Tresp, responder T cell; (KO) of the Sh2d2a gene in mice is not associated with immu- TSAd, T cell–specific adaptor protein; TSDR, Treg-specific demethylated region; nodeficiency but, rather, results in susceptibility for autoimmunity WT, wild-type. (3, 9). Current models suggest that Treg dysfunction in Sh2d2a2/2 2/2 Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 mice (also referred to in the literature as TSAd or TSAd KO mice)

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801604 2 INTRINSIC FUNCTION OF TSAd IN CD4+ T REGULATORY CELLS isduetoadeficiencyinIL-2productionbyTeffsandthus peroxidase-activity was developed using 0.05% 3-amino-9-ethyl-carbazole IL-2–dependent survival and activity of CD4+Foxp3+ Tregs (9). and 0.015% hydrogen peroxide in 0.05 M acetate buffer (pH 5.5). Finally, However, the autoimmune disease previously reported in TSAd2/2 each slide was counterstained in Gill hematoxylin (Sigma-Aldrich, 2/2 2/2 St. Louis, MO) and mounted in glycerol gelatin (Thermo Fisher Scien- mice is different from that reported in Il2 or Il2r mice (18–20). tific). Images were evaluated on a Nikon Eclipse 80i (Nikon, Mississauga, Also, recent studies are suggestive that TSAd may have a cell-intrinsic ON, Canada) using a Retiga 2000R CCD camera (QImaging, Surrey, function in Tregs (21). These observations indicate that its biology has Canada) equipped with NIS Elements software (version 3.22.15; Nikon, 3 broad implications in Treg-associated disease(s). Melville, NY). Infiltrates were evaluated by standard grid counting at 40 high-power field magnification of 12 different fields per slide by three In this study, we wished to determine the relative function of independent investigators. Data were expressed as the mean infiltration per TSAd in Teffs and Tregs and the development of immunoregu- allograft. lation in vivo following transplantation. Contrary to expectations, Isolation and culture of murine T cells and T cell subsets we find that allopriming and Teff responses are sustained in 2 2 TSAd / recipients of cardiac transplants. Furthermore, we find Murine T cell subsets were isolated from splenocytes using the EasySep + + + + that TSAd2/2 recipients have accelerated graft failure following mouse CD4 , naive CD4 , and CD4 CD25 T Cell Isolation Kits MHC class II–mismatched transplantation, and they are resistant (STEMCELL Technologies, Vancouver, Canada) or FACS sorted using a FACSAria IIU FACS sorter (BD Biosciences, San Diego, CA). Purity was to the graft-prolonging effects of costimulatory blockade. Mech- 2 2 consistently above 95% for bead-isolated cells and up to 98% for FACS- anistically, we find that TSAd / Tregs are less efficient in sup- sorted cells (Supplemental Fig. 1). Graft-infiltrating cells were isolated by pressing Teff responses and they possess effector function as enzymatic (1 mg/ml collagenase I; Worthington, Lakewood, NJ) and evidenced by IFN-g production. Untargeted transcriptomic RNA- mechanical disruption and subsequently enriched over a 67–44% Ficoll density gradient. Isolated CD4+ T cells were cultured in RPMI 1640 sequencing indicates that the major function of TSAd in Tregs (Lonza, Walkersville, MD) supplemented with 10% FBS (Sigma-Aldrich), relates to its effects on cellular activation, suggesting that it is 2mML-glutamine, 1 mM sodium pyruvate, 0.75 g/l sodium bicarbonate, potent in modulating CD4+Foxp3+ Treg biology through cell- 100 U/ml penicillin/streptomycin, 0.1 mM nonessential amino acids (all intrinsic mechanisms. We also find that mitochondria in TSAd2/2 Lonza) and 50 mM 2-ME (Sigma-Aldrich). The cells were stimulated with Tregs lack Lck, and have reduced reactive oxygen species (ROS) plate-bound anti-CD3 (clone 145-2C11; Bio X Cell) in the absence or 2/2 presence of soluble anti-CD28 (clone 37.51; BioLegend), and cytokines production/activation. However, TSAd Tregs compensate by were evaluated as indicated by each experiment. In vitro differentiation of increasing the numbers of mitochondria per cell, and this effect is CD4+ Tregs into induced Tregs (iTregs) was performed by culturing naive 2 not associated with altered Treg metabolism. These collective CD4+CD25 cells for 2 d with plate-bound anti-CD3 (2 mg/ml), anti-CD28 findings have broad implications and suggest that mutations in the (1 mg/ml), anti–IL-4 (500 ng/ml, clone 11B11), anti–IFN-g (2 mg/ml, clone + + XMG1.2), murine TGF-b1 (5 ng/ml), murine IL-2 (50 U/ml) (all Bio- SH2D2A gene in humans will result in generalized CD4 Foxp3 Legend) and 10 nM rapamycin (Wyeth-Ayerst, Philadelphia, PA) or 100 Treg dysfunction and susceptibility to chronic inflammatory/ nM all-trans retinoic acid (Sigma-Aldrich) in DMEM (Lonza) supple- autoimmune disease(s) and to graft loss following solid organ mented with 10% FBS (Sigma-Aldrich), 2 mM L-glutamine, 1 mM sodium transplantation. pyruvate, 0.75 g/l sodium bicarbonate, 100 U/ml penicillin/streptomycin, 0.1 mM nonessential amino acids (all from Lonza) and 50 mM 2-ME (Sigma-Aldrich). On day 2, the cells were transferred to a new cell culture Materials and Methods plate in medium supplemented with 50 U/ml IL-2 in the absence of anti- Mice CD3 for an additional 2 d, after which efficiency was evaluated by CD25 and Foxp3 expression. Male 6–8-wk-old C57BL/6 (H-2b), B6.C-H-2bm12 and BALB/c (H-2d) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Proliferation assays 2/2 2/2 2/2 Sh2d2a mice (also called RIBP and TSAd were generated by 3 Jeffrey A. Bluestone [University of California, San Francisco, CA (3)] and T cell proliferation was assessed by the addition of 1 mCi of [ H]thymidine per well (PerkinElmer, Boston, MA) during the last 16 h of culture. In- gifted to the laboratory by Philip King [University of Michigan, Ann 3 Arbor, MI (9)]. The mice have been backcrossed onto the C57BL/6 (H-2b) corporated [ H]thymidine was assessed in a Wallac 1450 MicroBeta background. TriLux scintillation counter (PerkinElmer). Cardiac transplantation ELISpot assays + 3 4 Intra-abdominal heterotopic heart transplantation was performed as pre- CD4 T cells (3 10 ) were cultured in 96-well polyvinylidene viously described (22, 23), and graft survival was monitored by palpation fluoride plates (Immobilon-P; Millipore, Billerica, MA) and stimulated of the heartbeat. In some experiments, BrdU (1 mg; BioLegend, San with irradiated (1700 rad) splenocytes and/or with anti-CD3/anti- Diego, CA) was injected i.p. every 12 h for 72 h into recipients, and BrdU CD28–coated beads (1:1 ratio beads/cell; Life Technologies, Carlsbad, incorporation in CD4+ T cell subsets was analyzed by flow cytometry CA) for 24 h. Subsequently, the cells were stained according to the using the BD BrdU Flow Kit (BD Pharmingen, San Jose, CA). Anti-mouse ELISpot manufacturers protocol (eBioscience, San Diego, CA and BD CD40L Ab (anti-CD154, clone MR-1), rCTLA4–Ig or control Ig (clone Biosciences). After staining, the plates were scanned and analyzed on human Fc-G1) were administered on days 0, 2, and 4 posttransplantation an ImmunoSpot S6 Ultra ELISpot reader (version 5.0; CTL, Shaker (200 mg/d; all from Bio X Cell, West Lebanon, NH). All studies were Heights, OH). performed according to an approved Institutional Animal Care and Use Committee protocol at Boston Children’s Hospital. Cytosolic, mitochondrial and nuclear protein isolation, and Western blot analysis Immunohistochemistry Nuclear extracts were prepared by lysing cells in hypotonic buffer Following harvest, allografts were divided and either frozen in liquid ni- containing 10 mM HEPES, 10 mM potassium chloride, 1.5 mM mag- trogen in OCT or fixed in 10% formaldehyde in PBS (Thermo Fisher nesium chloride, 1 mM EDTA, 500 mM DTT, and 200 mMPMSF(all Scientific, Kalamazoo, MI) overnight at 4˚C. Formalin-fixed tissue was Sigma-Aldrich) supplemented with protease and phosphatase inhibitors paraffin-embedded, sectioned (∼3-mm thick), and mounted on Poly-L- (Thermo Fisher Scientific, Tewksbury, MA) for 15 min on ice. After Lysine coated slides (Thermo Fisher Scientific, Tewksbury, MA). The the addition of 1% IGEPAL, the cells were vortexed, and cytosolic and slides were deparaffinized, rehydrated, and stained with Harris H&E (all nuclear fractions were separated by centrifugation. Nuclei were resus- from Thermo Fisher Scientific). Frozen tissue was cryosectioned (∼4-mm pended in 20 mM HEPES, 25% glycerol, 4.2 mM sodium chloride, thick), fixed in acetone for 10 min, quenched with 0.3% hydrogen peroxide 1.5 mM magnesium chloride, 200 mM EDTA, 0.5 mM DTT, 200 mM in PBS for 30 min, blocked for 30 min with 2% BSA in PBS and incubated PMSF (all Sigma-Aldrich), and protease and phosphatase inhibitors with primary Ab (diluted in 2% BSA/PBS) overnight at 4˚C. After a wash (Thermo Fisher Scientific) for 20 min on ice; subsequently, DNA was in 0.5% Tween 20 in PBS, sections were incubated with a species-specific removed by centrifugation. Mitochondrial proteins were isolated using peroxidase-labeled secondary Ab (Jackson ImmunoResearch, West Grove, the AMRESCO Mitochondrial Protein Isolation Buffer (VWR, Radnor, PA) for 1 h at room temperature. The slides were washed three times and PA) supplemented with protease and phosphatase inhibitors (Thermo The Journal of Immunology 3

Fisher Scientific). Samples were subjected to electrophoresis on 4–15% methylated within the TSDR (maximum of 50% demethylation in female gradient SDS-polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA), samples). and separated proteins were transferred to PVDF membranes (Millipore). Membranes were blocked in TBS containing 0.1% Tween 20 and 5% BSA Transcriptomic analysis (Thermo Fisher Scientific) for 1 h and incubated with primary Abs over- RNA-sequencing library preparations were performed as previously night at 4˚C. Membranes were subsequently washed and incubated with described (23, 25). Total RNA was isolated using MyOne SILANE species-specific peroxidase-labeled secondary Abs (Jackson ImmunoResearch) Dynabeads (Thermo Fisher Scientific). RNA was fragmented and bar- for 1 h at room temperature. After washing, the protein of interest coded using 8-bp barcodes with standard Illumina adaptors. Libraries was visualized by chemiluminescence (Thermo Fisher Scientific) in a were enriched using Agencourt AMPure XP bead cleanup (Beckman ChemiDoc MP Imaging System (Bio-Rad Laboratories). The mem- Coulter, Brea, CA), PCR amplified, gel purified, and quantified using a branes were stripped (Thermo Fisher Scientific) and reprobed with Qubit HS (High Sensitivity) DNA Kit (Thermo Fisher Scientific). Libraries Abs to evaluate protein loading. were quality tested using Tapestation High Sensitivity DNA tapes (Agilent Multianalyte profiling of Treg cytokine production Technologies, Santa Clara, CA) and sequenced on a HiSeq 2500 (Illumina, San Diego, CA) using 50-bp single-end reads. Reads were aligned to the Cell culture supernatants were profiled using the Mouse Cytokine/Chemokine mouse reference genome GRCm38.p6 using 2-pass STAR aligner version 25 Plex Magnetic Bead Panel (MiliporeSigma) on a Luminex LX200 2.6.0 (26), and gene expression was quantified using the GENCODE platform equipped with xPONENT software (version 3.1.871.0; Luminex, gene models (Release M14) using HTseq-count method (27). Counts Austin, TX). were normalized, and differential gene expression and principle compo- nents were calculated using the DESeq2 method (version 1.14.1) in the R Flow cytometric analysis Statistical Computing Environment (version 3.3.2) (28). False discovery , Cells were incubated with conjugated mAbs diluted in 0.5% BSA in PBS rates were calculated, and genes with an adjusted p value (Padj) 0.001 for 30 min at 4˚C, washed, and fixed with 4% paraformaldehyde. Foxp3 were considered differentially expressed. Gene Set Enrichment Analysis (GSEA; version 3.0; Broad Institute, Cambridge, MA) was performed using expression was evaluated by fixing and permeabilizing cells using a 2/2 + high buffer set prior to staining using an anti-Foxp3 mAb (clone FJK-16s) or the log2 ratio of TSAd to WT CD4 CD25 Tregs at each time point, isotype (eBR2a) as a control, according to the manufacturer’s instruc- and the immunologic gene sets (C7) of the Molecular Signatures Database tions (all eBioscience). IRF-4 (clone IRF4.3E4), Helios (clone 22F6), with default parameters of GSEA (29). The transcriptomic dataset has EOS (clone ESB7C2; eBioscience), and Blimp-1 (clone 5E7) staining been submitted to the Gene Expression Omnibus database under ac- was performed using the True-Nuclear Transcription Factor Buffer set cession number GSE134515 (https://www.ncbi.nlm.nih.gov/geo/query/ according to supplier’s instructions (all from BioLegend). Cells were acc.cgi?acc=GSE134515). analyzed on a FACSCalibur flow cytometer (BD Biosciences) within Quantitative PCR 24 h of staining, and at least 50,000 gated events were collected per sample. Data were analyzed using FlowJo software (version X.0.7; Total RNA was isolated using the RNeasy Isolation Kit (QIAGEN, Valencia, Tree Star, Ashland, OR). CA) and reverse-transcribed into cDNA using the qScript Supermix from Quanta Biosciences (Gaithersburg, MD). cDNA was diluted 1:10 in RNase- Treg suppression assays free water and stored at 220˚C until use. Quantitative PCR (qPCR) In vitro suppression was performed by coculture of CFSE-labeled (2 mM; was performed on a 7300 Real-Time PCR system (Applied Biosystems, Life Technologies) CD4+CD252 T responder cells with increasing ratios Foster City, CA) using 1 ml cDNA, PerfeCTa SYBR Green (Quanta of CD4+CD25high Tregs or iTregs and either irradiated (1700 rad) APC or Biosciences) and individual primers (Table I) in a final concentration of anti-CD3/anti-CD28 coated beads (1:1 ratio beads/responder cell; Life 5 mM. Samples were run in duplicate, and the relative gene expression was determined with the comparative cycle threshold (Ct) method and Technologies). Responder cell activation was analyzed using the standard 2Ct(target gene)2Ct(GAPDH) CFSE-dilution assay, and expansion indices were calculated using the expressed as 2 . FlowJo proliferation module (version 9.9.3; Tree Star); suppression by KO Statistics cells were normalized to the suppressive function of wild-type (WT) Tregs, as previously described (24). Normalized suppressive function was plotted Statistical analyses were performed using one-way ANOVA or the Student t against the corresponding ratio of Tregs: naive CD4+ responder T cells test (Prism, version 5.03; GraphPad, La Jolla, CA) as appropriate. If the (Tresp). Suppressive function was also evaluated using the IFN-g ELISpot equality test failed, the Kruskal–Wallis test was used. All p values ,0.05 assay. were considered statistically significant. Heatmaps were generated using the heatmap.2 function in the gplots package (version 3.0.1.1). Treg-specific demethylated region methylation analysis Treg-specific demethylated region (TSDR) DNA methylation analysis 2/2 Results was performed on two male and one female TSAd and on three age/ + 2/2 sex-matched WT controls. FACS-sorted CD4+ Treg and Teffs (purity CD4 Teff responses are sustained in vivo in TSAd mice . of 99%) were pelleted, frozen, and processed for pyrosequencing We initially cultured purified populations of CD4+ T cells from WT or by EpigenDx (Hopkinton, MA). Briefly, genomic DNA was isolated, 2/2 bisulfite-modified, and the 14 CpG islands within the promoter and TSAd mice with anti-CD3/anti-CD28, and we compared prolif- intron 1 of the Foxp3 gene were amplified by PCR and pyrose- erative responses as well as IL-2, IL-4, and IFN-g production quenced. Sequences were aligned to the mouse genome, and the by ELISpot. As illustrated in Fig. 1, TSAd2/2 CD4+ T cells, percentage of converted (unmethylated) cytosine to uracil/thymidine including both naive CD4+CD252 as well as CD4+CD25high sub- for each CpG site was evaluated. All 14 CpG regions in each sample were , analyzed, and low DNA methylation in the regions 1–7 was used to indicate sets, are hypoproliferative (p 0.001) and produce lower levels of a comparable purity between KO and WT cells. Only the male samples cytokines (p , 0.001) versus WT cells following activation with are shown, as we observed that the inactivated X chromosome remains mitogen. However, increasing the concentration of mitogen and/or

Table I. Primer sequences

Gene Forward Primer Reverse Primer Il2 59-GTGCTCCTTGTCAACAGCG-39 59-GGGGAGTTTCAGGTTCCTGTA-39 Il4 59-CGTTTGGCACATCCATCTCC-39 59-TCATCGGCATTTTGAACGAG-39 Il5 59-AAAGAGAAGTGTGGCGAGGAGA-39 59-CACCAAGGAACTCTTGCAGGTAA-39 Il12a 59-GTCACCTGCCCAACTGCC-39 59-TATTCTGCTGCCGTGCTTCC-39 Ifng 59-AACGCTACACACTGCATCTTGG-39 59-GCCGTGGCAGTAACAGCC-39 Tnf 59-CTACTCCCAGGTTCTCTTCAA-39 59-GCAGAGAGGGAAAGGTTGACTTTC-39 Tgfb1 59-GGTTCATGTCATGGATGGTG-39 59-TGAGTGGCTGTCTTTTGACG-39 4 INTRINSIC FUNCTION OF TSAd IN CD4+ T REGULATORY CELLS

FIGURE 1. Effect of TSAd on CD4+ T cell activation responses in vitro. (A) Purified CD4+ T cells from WT or TSAd2/2 mice (DTSAd) were cultured in media with/without plate-bound anti-CD3 and soluble anti-CD28 (1 mg/ml each) for 24 h, and IFN-g production was evaluated by ELISpot. (B) Bead-sorted CD4+CD252 naive T cells or (C) CD4+CD25high Tregs from WT or TSAd2/2 mice were stimulated with increasing concentrations of anti-CD3 alone (0.1–3 mg/ml) or anti-CD3 (1 mg/ml) with increasing concentrations of IL-2. Proliferation was assessed after 72 h by [3H]thymidine incorporation. Data is expressed as mean cpm 6 SD of a representative experiment performed in triplicate. Each figure is representative of five independent experiments. Statistical analyses were performed using the Student t test (A) or one-way ANOVA (B and C). the addition of IL-2 into cultures overcomes this threshold effect found no differences versus WT recipients (Fig. 3I). In contrast, and readily induces activation responses in KO cell subsets TSAd2/2 Teff proliferation was significantly increased (Fig. 3I), and (Fig. 1B, 1C). These results confirm previous observations (3, 5, allopriming of recipient CD4+ Teffs was greater in TSAd2/2 versus 6, 14) and support a model linking TSAd biology to CD4+ Teff WT recipients (Fig. 3J). Collectively, these findings are most sug- function.However,TSAd2/2 mice fail to develop symptoms of im- gestive that TSAd is primarily functional in the suppressive activity munodeficiency, and the phenotypes of TSAd2/2 CD4+ T cells and of CD4+Foxp3+ Tregs. CD4+Foxp3+ Tregs are similar to WT mice (Supplemental Fig. 2) TSAd2/2 recipients of fully MHC-mismatched cardiac even following exposure to pathogens [data not shown and (6)]. Thus, allografts are resistant to costimulation blockade we postulated that these in vitro findings may not be of significance in vivo. To further determine the effects of TSAd on alloimmune CD4+ To next evaluate CD4+ Teff function in vivo, we used TSAd2/2 T cell responses, we examined the effect of costimulatory 2 2 mice (H-2b background) as recipients of fully MHC-mismatched blockade on graft survival using either TSAd / or WT mice as BALB/c (H-2d) donor heart transplants. Contrary to expectations, recipients of fully MHC-mismatched BALB/c cardiac allografts we found that graft survival was identical in TSAd2/2 and WT (Fig. 4A, 4B). Peritransplant administration of anti-CD40L (anti- recipients (Fig. 2A). Because this mismatch combination results CD154, on days 0, 2, and 4) resulted in prolonged graft survival in in the clonal expansion, differentiation, and function of CD4+ WT recipients (MST . 40 d) versus untreated or control Ig-treated Teffs (30), our findings indicate that TSAd is redundant for recipients (MST of 8 d; p , 0.01, Fig. 4A), as previously reported alloimmune effector CD4+ T cell activation and acute rejection (23, 33). However, anti-CD40L was ineffective in prolonging sur- 2 2 in vivo. vival in TSAd / recipients (MST 19 d, p , 0.01, Fig. 4A), and all grafts failed (n = 9) by day 22 posttransplant (Fig. 4A). Similarly, TSAd2/2 mice reject cardiac allografts following MHC class CTLA4–Ig treatment prolonged graft survival in WT recipients II–mismatched transplantation 2 2 (MST . 40 d; Fig. 4B) but was ineffective in TSAd / recipient We next evaluated graft survival following the transplantation of mice (MST = 10 d; p , 0.05). MHC class II–mismatched B6.C-H-2bm12 donor hearts into either Our collective findings are suggestive that CD4+ Treg activity is 2 2 TSAd / or WT mice (both H-2b). In this model, CD4+ Teff reduced in TSAd2/2 mice in vivo, resulting in a failure to suppress activation and rejection is more insidious because of the relative allopriming and graft rejection. To confirm this interpretation, we expansion and function of CD4+ Tregs (31, 32). As expected, next transplanted fully MHC-mismatched BALB/c hearts into median survival time (MST) was .45 d (Fig. 2B). Consistently, C57BL/6 Rag22/2 Il2rg2/2 recipients and adoptively transferred however, we observed that graft failure was markedly accelerated either TSAd2/2 or WT Tregs on day 2 posttransplantation. On day 2 2 in TSAd / recipients (MST = 22 d) versus WT recipients 18 posttransplantation, recipient mice received CD4+CD252 Teffs (p , 0.01, Fig. 2B). Two weeks posttransplantation, inflam- to challenge Treg-based immunoregulation. In control mice matory cell infiltrates were markedly increased within allo- (withoutTregtransferonday2),wefindthatday18transferofWT 2 2 grafts harvested from TSAd / recipients (Fig. 2C–G and data Teffs elicits graft failure within 16 d posttransfer. In contrast, transfer not shown) and the mRNA expression of several proinflammatory into mice that received WT Tregs (on day 2) resulted in a prolongation 2 2 cytokines were increased in allografts harvested from TSAd / of graft survival to a median of 50 d posttransfer (Fig. 4C). However, versus WT recipients (Fig. 2H). Phenotyping demonstrated ex- graft survival was not as prolonged when TSAd2/2 Tregs were used in panded numbers of CD3+ T cells, lower numbers of CD4+Foxp3+ the initial day 2 adoptive transfer (MST = 30 d; p , 0.05 versus WT subsets (p , 0.05), and a higher Teff/Treg ratio (p , 0.01) in Tregs, Fig. 4C). 2/2 TSAd versus WT recipients (Fig. 3A–E, Supplemental Fig. 3). + + 2/2 On day 18 posttransplantation, there was no difference in the CD4 Foxp3 Tregs from TSAd mice have attenuated frequency of CD44highCD62Llow effector memory Treg (eTreg) activation responses and possess proinflammatory subsets or NRP-1+ Tregs (Fig. 3F). However, TSAd2/2 Tregs had effector function a trend for lower levels of expression of Helios, EOS, and Blimp-1 To understand the molecular basis for the function of TSAd in Tregs, (Fig. 3G) as well as cell surface GITR, CTLA4, and PD-1 (Fig. 3H), we performed untargeted transcriptomic profiling of FACS-sorted and suggestive of an overall reduced immunoregulatory phenotype. We mitogen-activated TSAd2/2 and WT CD4+CD25high cells using RNA- also examined the expansion and turnover of Tregs in TSAd2/2 sequencing (Fig. 5A–D). We find that TSAd regulates (Padj , 0.001) transplant recipients using the in vivo BrdU incorporation assay and the expression of only a few genes in resting Tregs (no anti-CD3 The Journal of Immunology 5

FIGURE 2. Accelerated rejection of cardiac allografts in TSAd2/2 recipient mice (DTSAd). (A) Graft survival following transplantation of fully MHC-mismatched BALB/c hearts into C57BL/6 WT or TSAd2/2 recipient mice. (B) Graft survival following transplantation of MHC class II– mismatched B6.C-H-2bm12 hearts into WT or TSAd2/2 recipients (Gehan–Breslow–Wilcoxon test). (C) Histology of B6.C-H-2bm12 cardiac allografts harvested on day 18 posttransplantation from WT and TSAd2/2 recipients; representative photomicrographs and infiltration as identified by standard grid counting in three allografts per condition (lower graph plots, Student t test). Scale bar, 25 mm. (D–G) Intragraft infiltrates were evaluated on day 18 posttransplantation in allografts harvested from WT or TSAd2/2 recipients; (D) Representative flow cytometry analysis, (E) the mean frequency of CD3+, CD4+, and CD8+ cells, (F) the CD4+/CD8+ ratio 6 SD of (n = 3) animals per group, and (G) representative flow cytometry and bar graphs of the frequency of Foxp3+ cells within the CD4+ population 6 SD in (n = 3) mice per group. *p , 0.05, **p , 0.01, ****p , 0.0001. (H) Intragraft cytokine mRNA expression on day 18 posttransplantation by qPCR. Bar graphs represent the relative mRNA expression 6 SD of (n = 3) per condition (one-sample t test). stimulation; Supplemental Fig. 4A). Whereas WT Tregs respond effector function. Collectively, untargeted transcriptomic analysis to anti-CD3 stimulation with the differential expression of .300 indicates that TSAd2/2 Tregs are generally unresponsive to anti-CD3 genes (Supplemental Fig. 4B), we find that anti-CD3–activated stimulation, further supporting a critical role for TCR/TSAd–induced TSAd2/2 Tregs have a minimal response, with the differential signaling in active immunoregulation and/or in Treg phenotype(s). expression of only four genes (Supplemental Fig. 4B). Principal To next evaluate the effect of TSAd on CD4+ Treg stability, we component analyses of WT transcriptomes showed activation- performed DNA methylation assays of CpG motifs within the induced changes (Fig. 5B). In contrast, TSAd2/2 transcriptomes TSDR of the Foxp3 gene. CD4+Foxp3+ Tregs were FACS-sorted did not respond to mitogen activation (Fig. 5B). GSEA comparing from the spleens of WT or TSAd2/2 mice, DNA was isolated, and activated TSAd2/2 to WT transcriptomes showed a high nor- methylation was analyzed, as we described (23). In contrast to WT malized enrichment score for genes expressed in resting Tregs Tregs, which are heavily demethylated, pyrosequencing demon- (Fig. 5C). Independent of their activation status, we also noted that strated increased methylation of CpG regions 9–14 of the TSDR in TSAd2/2 Treg transcriptomes were enriched for genes that are TSAd2/2 Tregs (Fig. 5E, Supplemental Fig. 4D). This observation expressed in Teffs (Fig. 5D, Supplemental Fig. 4C), suggesting is further suggestive that TSAd2/2 Tregs are not fully activated and/or that TSAd2/2 Tregs have potential to be unstable and/or possess may lack suppressive function. In standard in vitro suppression assays 6 INTRINSIC FUNCTION OF TSAd IN CD4+ T REGULATORY CELLS

FIGURE 3. CD4+ Teff/Treg phenotype and expansion in TSAd2/2 recipients of cardiac allografts. Splenocytes from either C57BL/6 WT or TSAd2/2 (DTSAd) recipients of B6.C-H-2bm12 donor allografts were harvested on day 18 posttransplantation and were analyzed by flow cytometry and by ELISpot. (A) Representative flow cytometry analysis, (B) the mean frequency of CD3+, CD4+, and CD8+ cells and (C) the CD4+/CD8+ ratio 6 SD of (n = 4) animals per group. (D) Representative flow cytometry and bar graphs of the frequency of Foxp3+ cells within the CD4+ population 6 SD in (n = 4) mice. (E) The ratio of CD4+CD44highCD62Llow Teffs to CD4+Foxp3+ Tregs 6 SD in (n = 4) mice. (F) The frequency or mean fluorescence intensity (MFI) of CD4+Foxp3+ Treg subsets expressing CD44, CD62L, and NRP-1, (G) the transcription factors IRF-4, Helios, EOS, and Blimp-1 and (H) the immuno- modulatory proteins Lag3, CTLA4, PD-1, and GITR. (I) On day 15 posttransplantation, recipients were pulsed with BrdU i.p. (every 12 h for a total of 3 d), and splenocytes were harvested on day 18. A representative dot plot of BrdU incorporation within CD4+Foxp32 Teffs (lower quadrants) and within CD4+Foxp3+ Tregs (upper quadrants) by flow cytometry. The bar graph illustrates the mean percentage of BrdU+ cells 6 SD within the Teff or Treg populations in (n = 4) animals per group. (J) Recipient splenocytes were cocultured with irradiated (1700 rad) donor APCs (B6.C-H-2bm12) in a mixed lymphocyte reaction, and allopriming was evaluated by the analysis of IFN-g (upper panel) and IL-2 (lower panel) production by ELISpot. Assays were performed in triplicate, and are depicted as mean spots per well 6 SD of six independent experiments. In each panel, statistical analysis and p values were calculated using the Student t test.

(using purified FACS-sorted populations of WT or TSAd2/2 Tregs), TSAd2/2 iTregs (CD4+CD25highFoxp3+ cells) expand in a similar we find no major difference in the percentage inhibition of WT manner as that observed using WT cells. Collectively, these ob- CD4+CD252 responderproliferationbyeachTregpopulation(Fig.5F, servations are suggestive that TSAd promotes activation responses 5G). However, TSAd2/2 Tregs were found to produce high within CD4+ Treg subsets and that KO cells have the potential to levels of IFN-g in suppression assays (Fig. 5H). Furthermore, dedifferentiate and/or to possess effector function. purified populations of CD4+CD25high Tregs isolated from the 2/2 TSAd regulates mitochondrial Lck activity as well as spleens of TSAd mice produced significantly higher levels + of IFN-g (p , 0.01), and there was a trend for higher secretion mitochondrial mass in CD4 Tregs of IL-17 as compared with WT Tregs (Supplemental Fig. By Western blot analysis, we find that TSAd is expressed in the 4E). Finally, we evaluated the effect of TSAd on Treg ex- cytosol but not within the mitochondria of Tregs (Fig. 6A). We also pansion and differentiation by culturing naive TSAd2/2 CD4+ find that Lck is expressed in both the cytosol and the mitochondria T cells in iTreg-inducing conditions in vitro. As illustrated in of WT CD4+CD25high Tregs (Fig. 6B) but is notably lacking Supplemental Fig. 4F (and data not shown), we find that on the mitochondria of TSAd2/2 Tregs (Fig. 6B). This striking The Journal of Immunology 7

FIGURE 4. TSAd2/2 recipients are resistant to the graft-prolonging effects of costimulatory blockade. Fully MHC-mismatched BALB/c hearts were transplanted into WT or TSAd2/2 (DTSAd) and were treated with (A) anti-CD40L or (B) CTLA4–Ig i.p. on days 0, 2, and 4 posttransplantation. Graft survival was monitored by palpation. Statistics were performed by comparing outcomes in treated TSAd2/2 versus treated WT recipients. (C) Fully MHC- mismatched BALB/c hearts were transplanted into C57BL/6 Rag2 Il2rg double-KO recipients. On day 2 posttransplantation, recipients received FACS- sorted CD4+Foxp3+ Tregs (2 3 105) from either WT or TSAd2/2 mice by tail vein injection. On day 18 posttransplantation, recipients were challenged with WT CD4+CD252 Teffs (3 3 106 cells) by tail vein injection. Control recipients did not receive Tregs on day 2. Graft survival following Teff transfer was evaluated by palpation. Statistics in (A)–(C) were performed using the Gehan–Breslow–Wilcoxon test.

finding is suggestive that TSAd regulates Lck kinase activity MHC class II–mismatched cardiac transplants and that TSAd2/2 within mitochondria. Because the Lck kinase is reported to be recipients are resistant to the graft-prolonging effects of costim- functional within the mitochondria of CD4+ T cells (34), we next ulatory blockade. Furthermore, in adoptive transfer studies, assessed depolarization of mitochondria following treatment with we find that TSAd2/2 Tregs are less efficient than WT Tregs in anti-CD3 and found that it is markedly absent in TSAd2/2 versus the suppression of allograft rejection. Untargeted transcriptomic WT cells (Fig. 6C). We also used a ROS-sensitive probe (CM- analysis indicates that the major phenotype of TSAd2/2 Tregs is a H2DCFDA) to evaluate function and found that activation-induced lack of TCR-dependent activation, and mechanistic studies dem- ROS production is lacking in mitochondria from TSAd2/2 Tregs onstrate that TSAd2/2 Tregs lack mitochondrial Lck expression as following stimulation (Fig. 6D). These findings indicate that TSAd well as ROS production. Collectively, these findings indicate that is associated with mitochondrial Lck activity and is functional in TSAd has multiple mechanisms of function in Tregs. Because mitochondrial activation/depolarization within Tregs. mutations in the SH2D2A gene (encoding TSAd) are associated We next isolated CD4+CD25high Tregs from WT and TSAd2/2 with autoimmunity in humans, these observations have broad mice, and compared metabolic activity using extracellular flux clinical implications. assays in a Seahorse XFe96 bioanalyzer. The measurement of Previous studies (3, 5, 14) have demonstrated that TSAd reg- glycolytic activity (as assessed by the extracellular acidification ulates the threshold for TCR-dependent activation in CD4+ Teffs rate [ECAR]) (Fig. 6E, left panel), was identical in each cell type. in vitro. Similarly, we find that mitogen-induced activation of Also, oxidative phosphorylation, as measured by the oxygen naive TSAd2/2 CD4+ T cells in vitro is attenuated as compared consumption rate (OCR), was identical at basal levels as well as with WT controls. Because TSAd is rapidly induced upon T cell under maximal stress conditions (Fig. 6E, right panel). Thus, activation, this finding is suggestive that its biological effects are metabolism appears to be intact despite the absence of mitchon- important to sustain the continuous activation of effector cells. drial Lck in TSAd2/2 Tregs. However, the differentiation of T helper cells is normal in the Because Lck-dependent events also regulate mitochondrial absence of TSAd (6), and TSAd2/2 mice are not immunodeficient morphology (34), we finally performed electron microscopy (3, 9) and have an improved tumor clearance compared with their on WT and TSAd2/2 Tregs. As illustrated in Fig. 6F, we find a WT littermates (21). In our studies, we find that acute rejection and prominent increase in the number of mitochondria per TSAd2/2 graft failure is not delayed following fully MHC-mismatched trans- cell (Fig. 6F; p , 0.0001 versus WT Tregs), but there was no plantation into TSAd2/2 mice, and alloimmune priming and rejection demonstrable difference in morphology. We also used MitoTracker is enhanced following MHC class II–mismatched cardiac transplan- Green uptake and qPCR to confirm the significant increase in tation. Thus, despite in vitro findings suggesting that TSAd promotes mitochondrial mass in TSAd2/2 TregsversusWTcells(Fig.6G,6H, and/or sustains effector responses, we find that alloimmune CD4+ p , 0.05 and p , 0.0001, respectively). We interpret these col- T cell activation is fully functional in vivo in the absence of TSAd. lective observations to suggest that the increase in mitochondrial In this study, our key findings demonstrated that the primary mass in TSAd2/2 cells is sufficient to sustain metabolic function biological effects of TSAd relate to its function in CD4+Foxp3+ at baseline and may account for the normal phenotype of Tregs in Tregs. For example, the absence of Lck protein expression on unmanipulated mice. Furthermore, our findings demonstrate that mitochondria within TSAd2/2 Tregs is suggestive that TSAd the additional effects of TSAd on eTreg activation responses and serves (in part) to shuttle Lck from the cytoplasm to the mito- Foxp3 stability are critical to sustain immunoregulation following chondria of Tregs. It has however been challenging to link this transplantation. biological response to a generalized defect in immunometabolism. Whereas activation-induced ROS production is absent in Tregs Discussion from TSAd2/2 mice, ECAR and OCR is similar to that observed In these studies, we show that the Lck adaptor protein TSAd elicits in WT mice. Because there are increased numbers of mitochondria cell-intrinsic signals in CD4+Foxp3+ Tregs that are associated with in TSAd2/2 Tregs, it is possible that there is some compensation enhanced activation and immunoregulatory function. Specifically, to sustain immunometabolism. However, it is also possible that these we find that graft failure is accelerated in TSAd2/2 recipients of effects are independent. 8 INTRINSIC FUNCTION OF TSAd IN CD4+ T REGULATORY CELLS

FIGURE 5. Cell-intrinsic function of TSAd in CD4+Foxp3+ Tregs. (A–D) Transcriptomic analysis of FACS-sorted WT and TSAd2/2 (DTSAd) CD4+CD25high Tregs either unactivated or following activation with anti-CD3 (1 mg/ml) for 2–24 h. (A) Heatmaps illustrate 222 differentially expressed genes (KO versus WT; Padj , 0.001 at each time point). (B) Principal component analysis of differentially expressed genes (Padj , 0.001) following activation of WT (black line/arrow) and TSAd2/2 Tregs with anti-CD3 for 2–24 h (each time point is color coded). (C) GSEA of TSAd2/2 transcriptomes (24 h activation) against genes that are upregulated in resting and activated Tregs (GSE15659: resting Treg versus activated Treg up). (D) GSEA of TSAd2/2 transcriptomes (0–24 h activation) against genes that are upregulated in Teffs or Tregs (each time point is color coded; GSE20366: TregLP versus TconvLP up). (E)CD4+Foxp3+ Tregs and CD4+Foxp32 Teffs were FACS-sorted from the spleens of male WT and TSAd2/2 mice, and DNA methylation was assessed by bisulﬁte conversion and pyrosequencing. Heat maps represent the mean level of methylation of 14 CpG islands within the TSDR region of the Foxp3 gene in two independent experiments. (F–H) In vitro Treg suppression assays were performed using Tresp in combination with increasing ratios of FACS-sorted WT or TSAd2/2 CD4+CD25high Tregs. Suppression was assessed by the evaluation of Tresp proliferation by CFSE dilution in (F)and(G)orIFN-g production by ELISpot in (H). (G) Expansion indices were calculated from (n = 3) independent experiments normalized for Treg-suppressive capacity 6 SD. p = n.s. by two- way ANOVA. (H) The bar graphs illustrating Treg-mediated suppression of responder IFN-g production 6 SD are representative of (n = 2) experiments performed in triplicate.

Another major mechanistic observation from untargeted We also questioned whether the effector phenotype (IFN-g transcriptomic profiling of Tregs indicates that the key phe- production) of KO Tregs is interrelated with a lack of Lck and notype of KO Tregs is a profound defect in TCR-dependent ROS production by mitochondria. In pilot studies (data not shown), activation responses. Thus, it is most likely that TSAd functions we performed iTreg induction assays in the absence or presence of to elicit eTreg activation responses and these effects are associated mitochondrial-specific ROS inhibitors or mitochondrial-specific with TCR-independent translocation of Lck to mitochondria. ROS inducers. Both increased and decreased ROS generation The Journal of Immunology 9

FIGURE 6. TSAd regulates Lck activity and functional responses within mitochondria of Tregs. (A) Western blot analysis of cytosolic (C), mitochondrial (M) and nuclear (N) TSAd expression in bead-sorted WT CD4+CD25high Tregs. Each blot is representative of (n = 3) independent experiments. (B) Lck expression in cytosolic (C) and mitochondrial (M) extracts from bead-sorted WT or TSAd2/2 (DTSAd) CD4+CD25high Tregs by Western blot analysis. Data is representative of (n = 2) experiments. (C) Mitochondrial membrane potential of WT and TSAd2/2 CD4+CD25high Tregs (gated) as analyzed by flow cytometry using JC-1. The bar graph represents the relative ratio of the mean fluorescence intensity (MFI) of the dimeric to monomeric JC-1 probe 6 SD in (n = 7) experiments (Kruskal–Wallis test). (D) Activation-induced ROS generation by WT or TSAd2/2 CD4+CD25high Tregs (gated) following stimulation with 1 mg/ml anti-CD3 for 30 min. CM-H2-DCFDA was analyzed by flow cytometry, and the bar graph illustrates the relative MFI 6 SD of (n =3) independent experiments (Kruskal–Wallis test). (E) Bead-sorted WT and TSAd2/2 CD4+CD25high Tregs were stimulated with anti-CD3/anti-CD28 (both at 1 mg/ml) and IL-2 (10 ng/ml) for 24 h. Left panel, Glycolysis was evaluated by measuring the ECAR following the sequential addition of 10 mM glucose (Gluc), 1 mM oligomycin (Oligo), and 100 mM 2-deoxy glucose (2-DG). Right panel, Oxidative phosphorylation was evaluated by measuring the OCR following the sequential addition of 2 mM oligomycin, 1.5 mM carbonyl cyanide-4-trifluoromethoxy-phenylhydrazone (FCCP), and 1 mM antimycin A and 500 nM rotenone into cultures. One representative of (n = 2) identical experiments is illustrated (mean ECAR/OCR 6 SD of triplicate conditions). p = n.s. by two-way ANOVA. (F) Transmission electron microscopy of bead-sorted CD4+C25high WT and TSAd2/2 Tregs. Mitochondria are highlighted with an asterisk (*). Upper panel, Representative cross sections (scale bar, 1 mm). Lower panel, Box insert from the upper panels (scale bar, 250 nm). The scatter graph represents the mean number of mitochondria per cell 6 SD; one cross section per cell and a total of 40 cells per group (Student t test). (G) Mitochondrial mass of WT or TSAd2/2 CD4+CD25high Tregs (gated) was analyzed by flow cytometry using MitoTracker Green. Bars represent the relative MFI 6 SD of (n = 6) independent experiments (one-sample t test). (H) DNA was isolated from bead-sorted WT or TSAd2/2 CD4+CD25high Tregs, and the mitochondrial mass was evaluated using the ratio of mitochondrial versus nuclear DNA content by qPCR. Bars represent mean mitochondrial DNA copies per cell 6 SD of (n = 3) experiments (Student t test). Nuc, nucleus. during iTreg induction resulted in lower Foxp3 positivity, but proof will require additional studies, and, perhaps, the gener- activation responses and IFN-g production did not change in ation of Lck transgenic mice, which are beyond the scope of the presence of ROS inhibitors or inducers. Thus, we believe this report. that the effects of TSAd are not linked but are related to its Classically, IFN-g is associated with Teff responses, allograft generalized function on eTreg activation. Nevertheless, definitive rejection, and CD4+ T helper 1– and CD8+-mediated immune 10 INTRINSIC FUNCTION OF TSAd IN CD4+ T REGULATORY CELLS responses. However, IFN-g expression has multiple intrinsic and gene SH2D2A contributes to the genetic susceptibility of multiple sclerosis. Genes Immun. 2: 263–268. extrinsic functions that are context dependent in terms of the local 12. Smerdel, A., K. Z. Dai, A. R. Lorentzen, B. Flatø, S. Maslinski, E. Thorsby, tissue microenvironment (35–38). For example, coexpression of Ø. Førre, and A. Spurkland. 2004. Genetic association between juvenile rheu- the Th1 master transcription factor Tbet in Foxp3+ Tregs is im- matoid arthritis and polymorphism in the SH2D2A gene. Genes Immun. 5: 310–312. portant for controlling Th1 and CD8 effector responses (38), but 13. Lorentzen, A. R., C. Smestad, B. A. Lie, A. B. Oturai, E. Akesson, J. Saarela, IFN-g production by unstable Tregs is reported to break immu- K. M. Myhr, F. Vartdal, E. G. Celius, P. S. Sørensen, et al. 2008. The SH2D2A nological tolerance (35, 39). We observed that TSAd2/2 Tregs gene and susceptibility to multiple sclerosis. J. Neuroimmunol. 197: 152–158. + 14. Marti, F., G. G. Garcia, P. E. Lapinski, J. N. MacGregor, and P. D. King. 2006. were efficient in the suppression of CD4 Teff proliferation de- Essential role of the T cell-specific adapter protein in the activation of LCK in spite high levels of IFN-g production. However, analysis of CpG peripheral T cells. [Published erratum appears in 2006 J. Exp. Med. 203: 479.] motifs within the TSDR of the Foxp3 gene demonstrated enhanced J. Exp. Med. 203: 281–287. 15. Tai, X., M. Cowan, L. Feigenbaum, and A. Singer. 2005. CD28 costimulation of methylation, suggesting that the absence of TSAd is associated developing thymocytes induces Foxp3 expression and regulatory T cell differ- with reduced lineage stability. entiation independently of interleukin 2. Nat. Immunol. 6: 152–162. In summary, our findings in this report identify novel cell-intrinsic 16. Park, D., I. Park, D. Lee, Y. B. Choi, H. Lee, and Y. Yun. 2007. The adaptor + + protein Lad associates with the G protein beta subunit and mediates chemokine- mechanisms whereby TSAd functions to enhance CD4 Foxp3 Treg dependent T-cell migration. Blood 109: 5122–5128. activity. Our observations are most significant for the understanding 17. Berge, T., V. Sundvold-Gjerstad, S. Granum, T. C. Andersen, G. B. Holthe, L. Claesson-Welsh, A. H. Andreotti, M. Inngjerdingen, and A. Spurkland. 2010. of immunoregulation following transplantation, especially follow- T cell specific adapter protein (TSAd) interacts with Tec kinase ITK to promote ing treatment with costimulatory blockade (40). In addition, our CXCL12 induced migration of human and murine T cells. PLoS One 5: e9761. findings have broad implications (beyond transplantation) because 18. Sadlack, B., H. Merz, H. Schorle, A. Schimpl, A. C. Feller, and I. Horak. 1993. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell polymorphisms within the SH2D2A gene in humans have been 75: 253–261. linked to autoimmunity. 19. Malek, T. R., A. Yu, V. Vincek, P. Scibelli, and L. Kong. 2002. CD4 regulatory T cells prevent lethal autoimmunity in IL-2Rbeta-deficient mice. Implications for the nonredundant function of IL-2. Immunity 17: 167–178. Acknowledgments 20. Nelson, B. H. 2004. IL-2, regulatory T cells, and tolerance. J. Immunol. 172: We thank Megan Cooper, Kayla MacLeod, and Francesca D’Addio (all 3983–3988. 21. Berge, T., I. H. Grønningsæter, K. B. Lorvik, G. Abrahamsen, S. Granum, from Boston Children’s Hospital) for technical support and Dr. Gary V. Sundvold-Gjerstad, A. Corthay, B. Bogen, and A. Spurkland. 2012. SH2D2A Visner for supervision of this work within the mouse surgical facility. modulates T cell mediated protection to a B cell derived tumor in transgenic We also thank Dr. Peter Sage (Brigham and Women’s Hospital) for mice. PLoS One 7: e48239. guidance on RNA-sequencing, Daniel Brown (Beth Israel Deaconess 22. Corry, R. J., H. J. Winn, and P. S. Russell. 1973. Primarily vascularized allografts of hearts in mice. The role of H-2D, H-2K, and non-H-2 antigens in rejection. Medical Center), Dr. Stephen Alexander (The Children’s Hospital at Transplantation 16: 343–350. Westmead, Sydney, Australia), and Dr. Anne Spurkland (University of 23. Wedel, J., S. Bruneau, K. Liu, S. W. Kong, P. T. Sage, D. M. Sabatini, Oslo, Norway) for helpful discussions. M. Laplante, and D. M. Briscoe. 2019. DEPTOR modulates activation responses in CD4+ T cells and enhances immunoregulation following transplantation. Am. J. Transplant. 19: 77–88. Disclosures 24. Akimova, T., M. H. Levine, U. H. Beier, and W. W. Hancock. 2016. Standard- The authors have no financial conflicts of interest. ization, evaluation, and area-under-curve analysis of human and murine Treg suppressive function. Methods Mol. Biol. 1371: 43–78. 25. Kadoki, M., A. Patil, C. C. Thaiss, D. J. Brooks, S. Pandey, D. Deep, D. Alvarez, U. H. von Andrian, A. J. Wagers, K. Nakai, et al. 2017. Organism-level analysis References of vaccination reveals networks of protection across tissues. Cell 171: 398– 1. Spurkland, A., J. E. Brinchmann, G. Markussen, F. Pedeutour, E. Munthe, 413.e21. T. Lea, F. Vartdal, and H. C. Aasheim. 1998. Molecular cloning of a T cell- 26. Dobin, A., C. A. Davis, F. Schlesinger, J. Drenkow, C. Zaleski, S. Jha, P. Batut, specific adapter protein (TSAd) containing an Src homology (SH) 2 domain and M. Chaisson, and T. R. Gingeras. 2013. STAR: ultrafast universal RNA-seq putative SH3 and phosphotyrosine binding sites. J. Biol. Chem. 273: 4539–4546. aligner. Bioinformatics 29: 15–21. 2. Dai, K. Z., G. Vergnaud, A. Ando, H. Inoko, and A. Spurkland. 2000. The 27. Anders, S., P. T. Pyl, and W. Huber. 2015. HTSeq--a Python framework to work SH2D2A gene encoding the T-cell-specific adapter protein (TSAd) is localized with high-throughput sequencing data. Bioinformatics 31: 166–169. centromeric to the CD1 gene cluster on human Chromosome 1. Immunogenetics 28. Love, M. I., W. Huber, and S. Anders. 2014. Moderated estimation of fold 51: 179–185. change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15: 550. 3. Rajagopal, K., C. L. Sommers, D. C. Decker, E. O. Mitchell, U. Korthauer, 29. Tamayo, P., D. Scanfeld, B. L. Ebert, M. A. Gillette, C. W. Roberts, and A. I. Sperling, C. A. Kozak, P. E. Love, and J. A. Bluestone. 1999. RIBP, a novel J. P. Mesirov. 2007. Metagene projection for cross-platform, cross-species Rlk/Txk- and itk-binding adaptor protein that regulates T cell activation. J. Exp. characterization of global transcriptional states. Proc. Natl. Acad. Sci. USA Med. 190: 1657–1668. 104: 5959–5964. 4. Choi, Y. B., C. K. Kim, and Y. Yun. 1999. Lad, an adapter protein interacting 30. Li, Y., X. C. Li, X. X. Zheng, A. D. Wells, L. A. Turka, and T. B. Strom. 1999. with the SH2 domain of p56lck, is required for T cell activation. J. Immunol. Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of 163: 5242–5249. alloreactive T cells and induction of peripheral allograft tolerance. Nat. Med. 5: 5. Lapinski, P. E., J. A. Oliver, J. N. Bodie, F. Marti, and P. D. King. 2009. The 1298–1302. T-cell-specific adapter protein family: TSAd, ALX, and SH2D4A/SH2D4B. 31. Nagano, H., R. N. Mitchell, M. K. Taylor, S. Hasegawa, N. L. Tilney, and Immunol. Rev. 232: 240–254. P. Libby. 1997. Interferon-gamma deficiency prevents coronary arteriosclerosis 6. Perchonock, C. E., A. G. Pajerowski, C. Nguyen, M. J. Shapiro, and but not myocardial rejection in transplanted mouse hearts. J. Clin. Invest. 100: V. S. Shapiro. 2007. The related adaptors, adaptor in lymphocytes of unknown 550–557. function X and Rlk/Itk-binding protein, have nonredundant functions 32. Schenk, S., D. D. Kish, C. He, T. El-Sawy, E. Chiffoleau, C. Chen, Z. Wu, in lymphocytes. J. Immunol. 179: 1768–1775. S. Sandner, A. V. Gorbachev, K. Fukamachi, et al. 2005. Alloreactive T cell 7. Zeng, H., S. Sanyal, and D. Mukhopadhyay. 2001. Tyrosine residues 951 and responses and acute rejection of single class II MHC-disparate heart allografts 1059 of vascular endothelial growth factor receptor-2 (KDR) are essential for are under strict regulation by CD4+ CD25+ T cells. [Published erratum appears vascular permeability factor/vascular endothelial growth factor-induced endo- in 2005 J. Immunol. 174: 5135.] J. Immunol. 174: 3741–3748. thelium migration and proliferation, respectively. J. Biol. Chem. 276: 32714– 33. Hancock, W. W., M. H. Sayegh, X. G. Zheng, R. Peach, P. S. Linsley, and 32719. L. A. Turka. 1996. Costimulatory function and expression of CD40 ligand, 8. Matsumoto, T., S. Bohman, J. Dixelius, T. Berge, A. Dimberg, P. Magnusson, CD80, and CD86 in vascularized murine cardiac allograft rejection. Proc. Natl. L. Wang, C. Wikner, J. H. Qi, C. Wernstedt, et al. 2005. VEGF receptor-2 Y951 Acad. Sci. USA 93: 13967–13972. signaling and a role for the adapter molecule TSAd in tumor angiogenesis. 34. Vahedi, S., F. Y. Chueh, B. Chandran, and C. L. Yu. 2015. Lymphocyte-specific EMBO J. 24: 2342–2353. protein tyrosine kinase (Lck) interacts with CR6-interacting factor 1 (CRIF1) in 9. Drappa, J., L. A. Kamen, E. Chan, M. Georgiev, D. Ashany, F. Marti, and mitochondria to repress oxidative phosphorylation. BMC Cancer 15: 551. P. D. King. 2003. Impaired T cell death and lupus-like autoimmunity in T cell- 35. Overacre-Delgoffe, A. E., M. Chikina, R. E. Dadey, H. Yano, E. A. Brunazzi, specific adapter protein-deficient mice. J. Exp. Med. 198: 809–821. G. Shayan, W. Horne, J. M. Moskovitz, J. K. Kolls, C. Sander, et al. 2017. Interferon- 10. Abrahamsen, G., V. Sundvold-Gjerstad, M. Habtamu, B. Bogen, and g drives Treg fragility to promote anti-tumor immunity. Cell 169: 1130–1141.e11. A. Spurkland. 2018. Polarity of CD4+ T cells towards the antigen presenting cell 36. Koenecke, C., C. W. Lee, K. Thamm, L. Fo¨hse, M. Schafferus, H. W. Mittru¨cker, is regulated by the Lck adapter TSAd. Sci. Rep. 8: 13319. S. Floess, J. Huehn, A. Ganser, R. Fo¨rster, and I. Prinz. 2012. IFN-g production 11. Dai, K. Z., H. F. Harbo, E. G. Celius, A. Oturai, P. S. Sørensen, L. P. Ryder, by allogeneic Foxp3+ regulatory T cells is essential for preventing experimental P. Datta, A. Svejgaard, J. Hillert, S. Fredrikson, et al. 2001. The T cell regulator graft-versus-host disease. J. Immunol. 189: 2890–2896. The Journal of Immunology 11

37. Chang, C. H., J. D. Curtis, L. B. Maggi, Jr., B. Faubert, A. V. Villarino, 39. Delgoffe, G. M., S. R. Woo, M. E. Turnis, D. M. Gravano, C. Guy, D. O’Sullivan, S. C. Huang, G. J. van der Windt, J. Blagih, J. Qiu, et al. 2013. A. E. Overacre, M. L. Bettini, P. Vogel, D. Finkelstein, J. Bonnevier, et al. 2013. Posttranscriptional control of T cell effector function by aerobic glycolysis. Cell Stability and function of regulatory T cells is maintained by a neuropilin-1- 153: 1239–1251. semaphorin-4a axis. Nature 501: 252–256. 38. Levine, A. G., A. Mendoza, S. Hemmers, B. Moltedo, R. E. Niec, M. Schizas, 40. Vincenti, F., C. Larsen, A. Durrbach, T. Wekerle, B. Nashan, G. Blancho, B. E. Hoyos, E. V. Putintseva, A. Chaudhry, S. Dikiy, et al. 2017. Stability and P. Lang, J. Grinyo, P. F. Halloran, K. Solez, et al; Belatacept Study Group. 2005. function of regulatory T cells expressing the transcription factor T-bet. [Pub- Costimulation blockade with belatacept in renal transplantation. N. Engl. J. Med. lished erratum appears in 2017 Nature 550: 142.] Nature 546: 421–425. 353: 770–781.