From Activation-Induced Cell Death Substrate-2, Protects a T Cell

Overexpression of Insulin Receptor Substrate-1, But Not Insulin Receptor Substrate-2, Protects a T Cell Hybridoma from Activation-Induced Cell Death This information is current as of October 1, 2021. Li Li, Xiulan Qi, Mark Williams, Yufang Shi and Achsah D. Keegan J Immunol 2002; 168:6215-6223; ; doi: 10.4049/jimmunol.168.12.6215 http://www.jimmunol.org/content/168/12/6215 Downloaded from

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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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Overexpression of Insulin Receptor Substrate-1, But Not Insulin Receptor Substrate-2, Protects a T Cell Hybridoma from Activation-Induced Cell Death1

Li Li, Xiulan Qi, Mark Williams, Yufang Shi, and Achsah D. Keegan2

The insulin receptor substrate (IRS) family of signaling molecules is expressed in lymphocytes, although their functions in these cells is largely unknown. To investigate the role of IRS in the protection of T cells from activation-induced cell death (AICD), we transfected the T cell hybridoma A1.1, which is IL-4 responsive but lacks expression of IRS family members with cDNA encoding IRS1 or IRS2. Stimulation of these clones with immobilized anti-CD3-induced expression of CD69 to the same level as the parental A1.1 cells. However, the A1.1 IRS1-expressing cells were markedly resistant to AICD, while the A1.1 IRS2-expressing cells were not. Inhibition of phosphatidylinositol 3؅-kinase in the A1.1 IRS1-expressing cells did not abrogate their resistance to AICD. Fas mRNA was induced similarly by anti-CD3 in A1.1, A1.1 IRS1-expressing, and A1.1 IRS2-expressing cells. However, induction of Downloaded from Fas ligand (FasL) mRNA and functional FasL protein was delayed and decreased in IRS1-expressing cells, but not in IRS2- expressing cells. The induction of transcription from a 500-bp FasL promoter and a minimal 16-mer early growth response element linked to luciferase was also impaired in the IRS1-expressing cells. These results suggest that overexpression of IRS1, but not IRS2, protects A1.1 cells from AICD by diminishing FasL transcription through a pathway that is independent of the tyrosine .phosphorylation of IRS1 and phosphatidylinositol 3؅-kinase activity. The Journal of Immunology, 2002, 168: 6215–6223 http://www.jimmunol.org/ lymphocyte homeostasis is critical for normal immune Since IL-4 is a critical factor in the differentiation of T cells to function. One means of regulating T cell homeostasis is the Th2 type, it is possible that an IL-4-regulated cellular response through a process termed activation-induced cell death participates in the development of the AICD-resistant phenotype. T 3 (AICD) (1). Activation of the TCR using speciﬁc Ag or Abs The binding of IL-4 to its cell surface receptor complex activates directed against TCR components induces the expression of a at least two independent signaling pathways (14, 15). One path- number of genes, including IL-2, CD69, and Fas ligand (FasL) (2). way, STAT6, is necessary for maximal Th2 cell differentiation (but The interaction of the induced FasL with cell surface Fas results in not absolutely required) (16, 17). The second pathway, the insulin cell death via apoptosis and the down-modulation of immune re- receptor substrate pathway (IRS), is very strongly up-regulated by guest on October 1, 2021 sponses (3). Aberrant expression or function of Fas or FasL has during the differentiation of Th2 cells, but not Th1 cells (16). been implicated in a number of diseases, including lymphoprolif- IRS family proteins play a central role in signal transduction by erative disease, autoimmunity, and cancer (3Ð8). The cytokine en- receptors for insulin, insulin-like growth factor I, and a growing vironment during T cell activation inﬂuences T cell differentiation number of cytokines, including IL-4 (17). The IRS proteins can and sensitivity to AICD (9). In the presence of IL-12, activated T become tyrosine phosphorylated on multiple tyrosine residues fol- cells differentiate into Th1, which secrete IL-2, TNF-␣, and IFN-␥. lowing ligand stimulation. These phosphorylated motifs then in- Th1 cells have been reported to be sensitive to AICD (10). In the teract with proteins containing Src homology 2 domains such as presence of IL-4, activated T cells differentiate into Th2 cells that the regulatory subunit of phosphatidylinositol 3Ј-kinase (PI-3K), produce IL-4, IL-5, IL-6, and IL-10. Th2 clones have been re- growth factor receptor binding protein 2, Nck, c-Fyn, and the Src ported to express less FasL than Th1 clones after activation (10) homology domain-containing protein tyrosine phosphatase-2 and primary Th2 cultures have been shown to be intrinsically re- (SHP-2) (18). These signaling intermediates stimulate a variety of sistant to Fas signaling (10Ð13). downstream biological effects, including mitogenesis, gene expression, glucose transport, and suppression of apoptosis. In addition to tyrosine phosphorylation sites, the IRS proteins contain numerous Department of Immunology, Jerome Holland Laboratories, American Red Cross, serine/threonine phosphorylation sites. Some of these sites are con- Rockville, MD 20852 stitutively phosphorylated, while others appear to be regulated by Received for publication July 2, 2001. Accepted for publication April 17, 2002. extrinsic signals. It has been shown that Ser307 in IRS1 is phos- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance phorylated by c-Jun NH2-terminal kinase (JNK) in response to with 18 U.S.C. Section 1734 solely to indicate this fact. TNF-␣ or anisomycin treatment and that this phosphorylation neg- 1 This work was supported in part by U.S. Public Health Service Grants AI45662 (to atively modulates insulin signaling (19). While there have been A.D.K.), AI43384 (to Y.S.), the American Heart Association, Scientist Development many studies on the effect of tyrosine phosphorylation on down- Grant 003033N (to M.W.), and the American Red Cross. stream signals mediated by the IRS family of docking proteins, 2 Address correspondence and reprint requests to Dr. Achsah D. Keegan, Department of Immunology, Jerome Holland Laboratories, American Red Cross, 15601 Crabbs very little is known about the function of serine/threonine Branch Way, Rockville, MD 20852. E-mail address: [email protected] phosphorylation. 3 Abbreviations used in this paper: AICD, activation-induced cell death; IRS, insulin The IRS family is made up of four family members, IRS-1, -2, receptor substrate; A1.1-IRS1, A1.1 IRS1-expressing cell; AP-1, activator protein; -3, and -4. Lymphocytes express IRS1 and/or IRS2 (18). These CM, complete medium; Egr, early growth response; FasL, Fas ligand; FLRE, FasL Ј members share similar overall structure, including an NH2-terminal response element; JNK, c-Jun NH2-terminal kinase; PI-3K, phosphatidylinositol 3 - kinase; SHP-2, Src homology domain-containing protein tyrosine phosphatase-2. pleckstrin homology and protein tyrosine kinase binding domain as

well as a -COOH-terminal region containing numerous tyrosine phos- tained from BD Biosciences (San Diego, CA). Anti-IRS1, -IRS2, and -p85 phorylation sites. The common structure suggests some similarity of of PI-3K were purchased from Upstate Biotech (Lake Placid, NY). function among the IRS proteins. Indeed, all four IRS family mem- Stable transfections bers are able to associate with the p85 subunit of PI-3K via YXXM motifs, and both IRS1 and IRS2 can mediate anti-apoptotic functions A1.1 cells were washed and resuspended in PBS. For each transfection, ϫ 7 ␮ through the regulation of PI-3ЈK activity in some transfected cell lines 2 10 cells were mixed with 10 g cDNA vector carrying IRS1 or IRS2 coding region (a gift from Dr. J. Pierce, Laboratory of Cellular and Mo- (20). While many studies have focused on the similarities, newer anal- lecular Biology, National Cancer Institute, National Institutes of Health, yses are beginning to reveal differences. Comparisons between IRS1 Bethesda, MD) or an empty neomycin resistance vector and subjected to and IRS2 have found differences in their efficiency at recruiting SH2- electroporation using a Bio-Rad gene-pulsar (Hercules, CA) set at 200 V ␮ domain containing molecules after insulin or IL-4 treatment (18). and 960 F. After transfection, cells were cultured overnight in appropriate medium before selection with G418 (Life Technologies, Grand Island, Moreover, the expression of IRS2 in fibroblasts lacking IRS1 does not NY). Neomycin-resistant lines were tested for expression of IRS1 or IRS2 reconstitute normal insulin or insulin-like growth factor I responses, by Western blotting. suggesting that these two molecules can have important nonredundant function (21). Regions of the IRS molecules between the known mod- Immunoprecipitation and immunoblotting ules are not highly homologous; however, very little is known about Analysis of phosphotyrosine-containing proteins was performed as previ- how they may contribute to signaling. Interestingly, IRS1 lacking all ously described (32). Briefly, cells were starved in RPMI 1640 for2hat ϫ 7 tyrosine residues retains the ability to mediate a mitogenic response to 37¡C. After washing, 2 10 cells were resuspended in RPMI. Where indicated, they were stimulated with murine IL-4 (10 ng/ml) for 10 min or insulin in transfected cells, suggesting the presence of phosphoty- anti-CD3 for various times. The reaction was terminated by 10-fold dilu- rosine-independent mechanisms of signaling by IRS family members ␮ tion in ice-cold PBS containing 100 MNa3VO4. Cell pellets were lysed Downloaded from (22). in HEPES lysis buffer (50 mM HEPES, 50 mM NaCl, 0.5% Nonidet P-40, Normal T cells have been shown to express IRS family mem- 1mMNa3VO4, 50 mM NaF, 10 mM pyrophosphate, 1 mM PMSF, and bers to varying degrees. Human and murine thymocytes and hu- protease inhibitor mixture) and clarified by centrifugation. To detect IRS1, IRS2, and the p85 subunit of PI-3K phosphorylation, the soluble fraction man peripheral T cells express both IRS-1 and IRS-2 (23Ð25). was immunoprecipitated with polyclonal anti-IRS, anti-IRS2, or anti-p85. Murine T cells express IRS2 and low levels of IRS-1 (16, 18, 26). The precipitates were washed in lysis buffer and solubilized in SDS sample Murine Th2 clones express large amounts of IRS2, and during in buffer. The samples were separated on 7.5% SDS-polyacrylamide gels be- vitro differentiation in the presence of IL-4, Th2 cells acquire el- fore transfer to a polyvinylidene difluoride membrane. The membranes http://www.jimmunol.org/ were then probed with a monoclonal antiphosphotyrosine Ab, RC20. The evated expression of IRS2, while in Th1 cells IRS2 expression bound Abs were detected using enhanced chemiluminescence (Amersham, remains low (16). Several murine thymic lymphoma cell lines Arlington Heights, IL). The blots were stripped and reprobed as necessary. demonstrate constitutive tyrosine phosphorylation of both IRS1 and IRS2 (A. D. Keegan, unpublished observation). FACS assay for CD69 and apoptosis The role the IRS family members play in the regulation of T cell CD69 expression was examined by staining the cells with anti-CD69 Ab growth and survival is not clear. There is no major immunological after stimulation with anti-CD3 for various times, followed by FACS anal- change in mice lacking IRS1 or IRS3 (27, 28). In mice lacking ysis (FACScan, BD Biosciences, Mountain View, CA). The percentage of apoptotic cells was determined by analyzing the nuclear DNA content by IRS2 there is no gross change in immune function (29); however, propidium iodide staining as indicated (32). Briefly, after culture cells were by guest on October 1, 2021 there is a modest reduction in IL-5 production by Th2 cells, with resuspended in 0.25 ml propidium iodide solution (50 ␮g/ml propidium no effect on T cell survival (26). Given the many redundant func- iodide, 0.1% sodium citrate, 0.1% Nonidet P-40, and 50 ␮g/ml RNase tions of the IRS family members, it is possible that the function of (Sigma, St. Louis, MO)) and incubated for 30 min at room temperature. one member may be compensated for by that of another during DNA content was then analyzed by flow cytometry. The apoptotic cells were defined as those with Ͻ2 N DNA content. development. Therefore, to address the role of IRS family members in T cell Northern blotting function, we used a T cell hybridoma cell line, A1.1, that lacks Total RNA was isolated with affinity columns (Amersham Pharmacia expression of all IRS family members, but demonstrates IL-4-in- Biotech, Uppsala, Sweden) according to the protocol recommended by duced tyrosine phosphorylation of STAT6. These cells have been the manufacturer. RNA samples were fractionated on 1% agarose/2.2 M extensively used to analyze the signaling pathways activated by formaldehyde denaturing gels and transferred onto Nytran membranes the TCR that lead to regulation of Fas and FasL expression and (Schleicher & Schuell, Keene, NH). The cDNA encoding mouse Fas and FasL were provided by Dr. S. Nagata (Osaka Bioscience Institute, Osaka, AICD (1, 30, 31). To investigate whether IRS proteins can influ- Japan), and cDNA encoding early growth response gene (Egr)-1, -2, and -3 ence AICD, we transfected IRS1 or IRS2 cDNA into A1.1 cells. have been described previously (33, 34). The cDNA probes were labeled We found that overexpression of IRS1, but not IRS2, protected by random priming with [32P]dCTP (Amersham Pharmacia Biotech, Pis- A1.1 cells from AICD primarily through decreased induction of cataway, NJ) according to the manufacturer’s instructions. Prehybridiza- ϫ FasL expression. tion and hybridization were conducted at 42¡C in a solution containing 5 SSC (10ϫ SSC is 1.5 M NaCl and 0.15 M sodium citrate), 2.5 mM EDTA, 0.1% SDS, 5ϫ Denhardt’s solution, 2 mM sodium pyrophosphate, 50 mM sodium phosphate, and 50% formamide. After washing with 0.2ϫ SSC and Materials and Methods 0.1% SDS at 56¡C for 1 h, hybridization signals were detected by Cells, reagents, and Abs autoradiography. The murine T cell hybridoma (A1.1) (1) was maintained in RPMI 1640 Microcytotoxicity assay complete medium (CM; Life Technologies, Gaithersburg, MD) that was 51 supplemented with 2 mM L-glutamine, 50 mM 2-ME, 10% heat-inactivated The Cr release microcytotoxicity assay has been described previously FBS (Sigma, St. Louis, MO), and 1.0% penicillin/streptomycin mixture (35). Briefly, target cells were labeled with 51Cr (NEN, Boston, MA) for (BioWhittaker, Walkersville, MD). The target cell lines L1210 and L1210 1.0hat37¡C. After washing with PBS four times, labeled target cells were expressing Fas (obtained from Dr. P. Golstein, Centre Nationale de la mixed with effector cells at various ratios. The supernatant was harvested Recherche Scientifique-Institut National de la Sante« et de la Recherche 4 h after culturing the mixed cells. The radioactivity was measured with a Me«dicale, Marseille, France) were maintained in RPMI 1640 CM. L cells gamma counter (Wallac, Turku, Finland). The spontaneous release of the and L cells expressing FasL were obtained from Dr. T. A. Ferguson (Wash- radioactivity from the 51Cr-labeled target cells was usually Ͻ15%. The ington University School of Medicine, St. Louis, MO) and were maintained percent specific release was calculated using the following formula: ex- in DMEM-CM. PMA and LY294002 were obtained from Calbiochem (San perimental release Ϫ spontaneous release/total release Ϫ spontaneous Diego, CA), FITC anti-mouse CD69 and anti-phosphotyrosine were ob- release. The Journal of Immunology 6217

Luciferase assay phosphorylation that was substantially increased after IL-4 stimu- Luciferase activity was evaluated using a chemiluminescence assay kit lation. Clones expressing IRS2 demonstrated minimal basal phos- (Tropix, Bedford, MA). One million cells were transiently transfected with phorylation of IRS2 that was increased after IL-4 stimulation. In 1 ␮g purified luciferase reporter plasmids containing the 5Ј region of the the parental A1.1 cells or A1.1 expressing the neomycin gene murine FasL gene from Ϫ511 to ϩ1 (36), a 16 mer encoding the critical alone, we did not detect the expression or tyrosine phosphorylation CD3 response element from the FasL promoter containing an Egr binding of IRS1 or IRS2 as expected. To determine whether the expression site (33, 34) (termed FasL response element (FLRE)), three copies of a NFAT site derived from the murine IL-2 promoter, six copies of a con- of IRS1 or IRS2 in A1.1 cells influenced signaling through the sensus AP-1 site, or two copies of a NF-␬B site derived from mouse ␬ light TCR, we analyzed the induction of CD69 in A1.1, A1.1-IRS1, and chain enhancer cDNA (37) by electroporation. The cells were then cultured A1.1-IRS2 before and after anti-CD3 treatment by FACS analysis in CM overnight. After washing, harvested cells were stimulated by im- (Fig. 1B). The A1.1 cells expressing IRS1 or IRS2 responded to mobilized anti-CD3 for various times. Ten microliters of the extract from activated cells was mixed with 100 ␮l luciferase assay reagent, and lucif- anti-CD3 stimulation by the induction of cell surface CD69 to erase activity was measured for5sinaML2250 luminometer (Dynatech, levels similar to the parental A1.1 cells and with similar kinetics. Chantilly, VA). In each experiment 1.5 ␮g vector carrying ␤-galactosidase In addition, all showed similar levels of IL-2 production and stim- driven by the CMV promoter was also transfected as a control for trans- ulation of tyrosine phosphorylation of cellular substrates (data not fection efficiency as previously described (36). Transfection without any plasmid was used to detect background luciferase activity, which was shown), suggesting that global TCR signaling was not grossly af- minimal. fected by the transfected genes. Results Expression of IRS1, but not IRS2, protects A1.1 cells from AICD

Characterization of A1.1 cells expressing IRS1 or IRS2 Downloaded from IRS family members are expressed in T lymphocytes (16, 18, 23Ð In previous studies we showed that overexpression of IRS1 in the 26); however, their function in T cell biology is unclear. Lack of IL-3-dependent myeloid cell line 32D protected from IL-3 with- IRS family member expression in mice does not cause a major drawal-induced death (32). Furthermore, it has been shown that effect on lymphocyte responses (26Ð29). Lack of IRS2 expression elevated expression of IRS1 or IRS2 is able to protect a number of results in a decrease in T cell proliferation and a modest suppres- cell types from apoptosis and is often associated with the onco- sion of Th2 development when analyzed in vitro (26). The lack of genic phenotype (20, 39Ð42). Therefore, we tested the sensitivity http://www.jimmunol.org/ both genes results in embryonic lethality (38). Therefore, to ana- of the IRS-expressing cell lines to AICD by propidium iodide lyze the function of the IRS family members in T cell activation staining of nuclear DNA content (Fig. 2). Interestingly, the cells induced responses, we took advantage of a T cell hybridoma, A1.1, overexpressing IRS1 were resistant to apoptosis induced by culture that lacks expression of all IRS family members, but can respond with plate-bound anti-CD3, while the cells expressing IRS2 were to IL-4 treatment with the tyrosine phosphorylation of STAT6. not. For example, control neomycin-resistant A1.1 cells responded These cells were transfected with cDNA encoding IRS1 or IRS2 to plate-bound anti-CD3 with an increase in the percentage of ap- with a neomycin resistance gene or a neomycin resistance gene optotic cells from 5 to 47%, while the percentage of A1.1IRS1a alone. Cells able to grow in the presence of G418 were selected cells rose from 8 to only 11%. A1.1IRS2a cells responded to anti- and tested for IRS expression by Western blotting using the ap- CD3 with an increase in the percentage of apoptotic cells from 5 by guest on October 1, 2021 propriate Ab. The ability of IL-4 to stimulate tyrosine phosphor- to 47%. The addition of IL-4 during the stimulation did not further ylation of the transfected IRS1 or IRS2 in the selected clones was enhance the protection of AICD in either A1.1-IRS1 or A1.1-IRS2. analyzed by immunoprecipitation, followed by immunoblotting These preliminary studies in A1.1 cells indicate that IRS1 specif- (Fig. 1A). Clones expressing IRS1 demonstrated a basal level of ically signals protection of T cells from AICD. This could be

FIGURE 1. Characterization of A1.1 cells expressing IRS1 and IRS2. A, A1.1 cells were transfected with vector containing IRS1 or IRS2 cDNA or a neomycin resistance plasmid alone as indicated. Parental A1.1, neo-resistant A1.1, and several IRS- positive cell lines (identiﬁed with lowercase letters) were stimulated with IL-4 (10 ng/ml) at room temperature for 10 min. The cell lysates were immunoprecipitated with Abs against IRS1 or IRS2, followed by immunoblotting with anti-phosphotyrosine (1st IB). The blots were then probed with either anti- IRS1 or anti-IRS2 (2nd IB) as appropriate. B, A1.1, A1.1-IRS1, and A1.1-IRS2 cell lines were incubated on anti-CD3-precoated plates for 0, 3, or 6 h. The cells were then harvested, washed, and stained with FITC-con- jugated anti-CD69 Ab. Expression of CD69 was analyzed by FACS. 6218 PROTECTION FROM AICD BY IRS1

FIGURE 2. Sensitivity of A1.1, A1.1-IRS1, and A1.1-IRS2 cells to AICD. Parental A1.1, neo-resistant A1.1, A1.1-IRS1, and A1.1-IRS2 cell lines were incubated in anti-CD3-precoated plates for8hinthe presence or the absence of IL-4 (10 ng/ml). The percentage of apoptotic cells was measured by propidium iodide staining of nuclear DNA content, followed by FACS analysis. The percentage of cells expressing a hypodiploid DNA content is shown.

through the regulation of molecules that activate the apoptotic sig- tion of IRS1 or on its association with p85. These results demon- nal (i.e., Fas or FasL) or by suppression of the apoptotic program strate that the signal transduction for the prevention of cell death itself. by overexpressing IRS1 in A1.1 cells is not dependent upon tyrosine phosphorylation of IRS1 and the downstream activation of Downloaded from Signal transduction for the protection of cell death by PI-3K. overexpressing IRS1 is through a PI-3K independent pathway Previous studies in the 32D cell model demonstrated that the IRS1- Effect of IRS1 on expression of Fas and FasL after TCR dependent prevention of apoptosis was associated with the activa- stimulation tion of PI-3K (20, 32). To examine whether the ability of IRS1 A1.1 cells commit to AICD in a Fas- and FasL-dependent manner expression to protect A1.1 cells from AICD was dependent upon (1, 30). To directly examine the effect of IRS1 expression on the http://www.jimmunol.org/ PI3K, we performed the anti-CD3 stimulation in the presence or levels of Fas and FasL induction, we performed Northern blot the absence of the inhibitor of PI-3K, LY294002 (Fig. 3A). Inhi- analysis (Fig. 4A). mRNA encoding Fas was detected in untreated bition of PI3K with this agent did not reverse the protection from cells and was increased after anti-CD3 treatment in A1.1 cells and AICD seen in the IRS1-expressing cells. In fact, at concentrations in IRS1- and IRS2-expressing cells. The expression of Fas-L Ͼ3 ␮M, LY294002 suppressed AICD in all A1.1 cells, including mRNA was undetectable in untreated cells, induced within 2 h, parental cells. Similar results were obtained using wortmannin and induced to high levels within 4Ð5 h after anti-CD3 treatment (data not shown). We further analyzed the association of IRS1 with in the parental A1.1 and the IRS2-expressing cells. Interestingly, the p85 subunit of PI-3K (Fig. 3B). The coprecipitation of p85 with the induction of FasL mRNA in the IRS1-expressing cells was not by guest on October 1, 2021 IRS1 was increased signiﬁcantly by IL-4 treatment, but not by observed until8hofstimulation. anti-CD3; the ability of IRS1 and p85 to coprecipitate was corre- To assess the level of functional cell surface FasL, we per- lated with the IL-4-induced tyrosine phosphorylation of IRS1 as formed 51Cr release assays using the L1210 T cell line expressing shown previously (20), indicating that the IL-4 activated IRS1/p85 Fas as a result of transfection as a target cell (Fig. 4B). As a control, signaling pathway is present in these cells. However, stimulation L1210 cells lacking Fas expression were also used. The parental A1.1, through the TCR did not have an effect on tyrosine phosphoryla- A1.1 IRS1-expressing (A1.1-IRS1), and A1.1 IRS2-expressing

FIGURE 3. Role of PI-3K in the IRS1-mediated protection from AICD. A, Neo-resistant A1.1 and A1.1-IRS cell lines were incubated in uncoated or anti-CD3-precoated plates in the presence or the absence of various concentrations of LY294002 for 8 h. The percentage of apoptotic cells was measured as described in Fig. 2. The percentage of cells expressing hypodiploid DNA content is shown. B, Parental A1.1, neo-resistant A1.1, and A1.1-IRS1 cell lines were incubated in anti-CD3-precoated plates for3horwere stimulated with IL-4 (10 ng/ml) for 10 min. Lysates from these stimulated cells were immunoprecipitated with anti-IRS1 Ab, followed by immunoblotting with anti-P85 (1st IB), anti-PY (2nd IB), and anti-IRS1 sequentially. The positions of the m.w. markers are shown. The Journal of Immunology 6219 Downloaded from http://www.jimmunol.org/

FIGURE 4. Expression of Fas and Fas ligand. Parental A1.1 (square), A1.1-IRS1 (diamond), and A1.1-IRS2 (circle) cell lines were incubated in anti-CD3-precoated plates for various times as noted. The mRNA was extracted from the cells and analyzed by Northern blotting. The blots were probed sequentially with 32P-labeled probes for Fas, FasL, and GAPDH. B, Parental A1.1, A1.1-IRS1, and A1.1-IRS2 cell lines were stimulated with immobilized anti-CD3 for 0, 2.5, or5hasindicated and used as effectors in a cytotoxicity assay. L1210 cells lacking or expressing Fas were labeled with 51Cr and used as targets. The effector and target cells were mixed at various E:T cell ratios. Four hours later, the supernatant was collected, and 51Cr release into the supernatant was measured by gamma counting. The speciﬁc cytotoxic activity was calculated as described in Materials and Methods. Spontaneous release was Ͻ15% for all experiments. by guest on October 1, 2021

(A1.1-IRS2) cells were treated with anti-CD3 for 0, 2.5, or 5 h the protection from AICD by overexpressing IRS1, but not IRS2, before adding the labeled target cells. After2hofstimulation, the is predominantly through delayed expression of FasL, rather than A1.1 and A1.1-IRS2 cells showed cytotoxicity against L1210 Fas through suppression of Fas signaling. targets (ϳ20% specific lysis), but not against L1210. However, the A1.1-IRS1 showed no cytotoxicity against Fas-expressing target Effect of IRS expression on transcription factor activation cells at this time point, which is consistent with the Northern blot by TCR results. After 5 h of anti-CD3 stimulation, the parental cells and the To determine whether the IRS1-specific effect on the induction of IRS2-expressing cells demonstrated ϳ50Ð60% specific cytotox- FasL after TCR stimulation was acting at the level of transcription, icity, while the A1.1-IRS1 cells demonstrated ϳ20%. Inhibition of we tested the inducibility of FasL promoter-luciferase constructs in cytotoxic activity by IRS1 expression was also apparent after 8 h transiently transfected A1.1 cells (Fig. 6). The FasL-luciferase of activation (data not shown). construct consists of 511 bp of the murine FasL promoter (36). Since overexpression of IRS1 has been correlated with resis- Stimulation of A1.1 and A1.1-IRS2 cells with anti-CD3 resulted in tance to factor withdrawal-induced apoptosis in IL-3-dependent an increase in luciferase activity over time; there was an increase cells (32), we next tested whether IRS expression affected the sen- of ϳ5- to 7-fold over background by3hoftreatment. By contrast, sitivity of A1.1 cells to be killed via cell surface Fas (Fig. 5). The the A1.1-IRS1 cells showed only an ϳ2-fold increase over back- parental A1.1, A1.1-IRS1, A1.1-IRS2 cells were treated with PMA ground with this promoter construct. to induce Fas expression and render them sensitive to FasL-medi- We further tested the regulation of a minimal TCR-regulated ated killing without inducing FasL itself (30). Such treatment in- response element derived from the FasL promoter that contains an duced similar levels of Fas mRNA in these three cell types (data Egr binding site, the FLRE (33, 34). Stimulation of the A1.1 and not shown). The ability of L cells expressing sense or antisense A1.1-IRS2 cells with anti-CD3 resulted in an increase in FLRE- constructs for FasL to induce apoptosis in these cells was analyzed. driven luciferase activity comparable to the activity driven by the We found that all three cell types were able to be killed by FasL- FasL promoter. By contrast, IRS1-expressing cells showed only a expressing L cells, but not by FasL-negative cells. At a 1.5:1 E:T minor increase over background. These results are consistent with cell ratio the parental cells, A1.1-IRS1, and A1.1-IRS2 cells the Northern blotting results (Fig. 4A) and suggest that the mech- showed 29, 18, and 18% apoptosis, respectively, suggesting a anism of the IRS1 effect is at least in part via interference of the modest effect of the IRS proteins on sensitivity to Fas-mediated TCR-induced transcription from the minimal FLRE element of the apoptosis. This effect was not observed at a 4.5:1 E:T cell ratio; FasL promoter. under these conditions all three targets demonstrated a high level There are a number of cis-acting elements in the FasL promoter of apoptosis (37Ð45%). Taken together these results indicate that that have been shown to interact with trans-acting transcription 6220 PROTECTION FROM AICD BY IRS1

FIGURE 5. IRS-expressing A1.1 cells are partially resistant to FAS-mediated killing. Parental A1.1, A1.1-IRS1, and A1.1- IRS2 cell lines were incubated with PMA (10 ng/ml) for 16 h to up-regulate Fas expression. After washing, these treated cells were added to plates containing L cells expressing sense or antisense FasL constructs and incubated for 8 h. The nonadherent A1.1 cells were harvested and analyzed for apoptosis as described in Fig. 2. Downloaded from

factors. The relative importance of NFAT, AP-1, NF-␬B, and Egr these cells. These results indicate that the suppression of transcrip- http://www.jimmunol.org/ genes in the regulation of the FasL promoter in T cell hybridomas tion of the FasL promoter by IRS1 expression is not caused by and primary T cells has been investigated extensively (33, 34, 36, simple suppression of expression of important trans-acting factors 43Ð49). Therefore, we tested the ability of TCR stimulation to such as NFAT, AP-1, NF-␬B, and Egr. activate these transcription factors in the A1.1 cells using multimerized cis-elements linked to luciferase (NFAT, AP-1, NF-␬B) or by Northern blotting (Egr-1, Egr-2, Egr-3). All three cell types Discussion demonstrated similar patterns of induction of NFAT and NF-␬B The regulation of T cell homeostasis is critical for the maintenance activity as measured by luciferase assay after anti-CD3 treatment of self-tolerance and immune surveillance. Alterations in T cell (Fig. 7A). This result is consistent with our observation that these homeostasis have been shown to lead to the development of au- by guest on October 1, 2021 three cell types demonstrated similar levels of CD69 expression toimmunity, lymphoproliferative disease, and cancer (3Ð8). Ele- and IL-2 production after anti-CD3 stimulation. A1.1-IRS1 cells vation of IRS1 and IRS2 protein expression and tyrosine phos- showed elevated activity of AP-1-luciferase compared with paren- phorylation have been linked to the oncogenic potential of several tal cells and A1.1-IRS2 cells at later time points, but not a deﬁcit. cell types and are anti-apoptotic in transfected cell lines (32, 39, It is not clear whether this increase is functionally important, since 42). However, we have found that IRS1 expression in the A1.1 T pharmacologic activation of AP-1 by PMA did not mimic the sup- cell hybridoma renders them resistant to AICD by a mechanism pression of FasL induction mediated by IRS1 expression (data not that is probably independent of tyrosine phosphorylation. shown). All three cell lines demonstrated some level of induction While IRS family proteins are expressed in resting and activated of mRNA encoding Egr-1, -2, and -3. The increase in the level and thymocytes and peripheral T cells to varying degrees, their func- kinetics of Egr by anti-CD3 varied somewhat for the three different tions in T cell biology are unclear (16, 18, 23Ð26). There are no types. However, we did not see a deﬁciency in Egr induction in major immunological defects in the IRS1 or IRS2 single knockout

FIGURE 6. FasL promoter activity after anti-CD3 stimulation. Parental A1.1 (ᮀ), A1.1-IRS1 ({), and A1.1-IRS2 (E) cell lines were transfected with a vector containing the promoter for FasL linked to luciferase or with FLRE-luciferase by electroporation and were cultured overnight. The cells were then stimulated with immobilized anti-CD3 for various times. The luciferase activity in cell lysates was measured using the Tropix kit and a luminometer. The data represented show the relative light units (RLU) Ϯ SEM and are representative of at least three experiments. The Journal of Immunology 6221 Downloaded from http://www.jimmunol.org/

FIGURE 7. Transcription factor activation. A, Parental A1.1 (ᮀ), A1.1-IRS1 ({), and A1.1-IRS2 (E) cell lines were transfected with vectors containing multimerized elements for NFAT, AP-1, and NF-␬B linked to luciferase by electroporation and were cultured overnight. Transfection with ␤-galactosidase was also performed to ensure equivalent levels of transfection. The luciferase activity in cell lysates was measured using the Tropix kit and a luminometer. The data represented show the relative light units (RLU) Ϯ SEM. In some cases the SEM is too small to be observed in the graph. B, Parental A1.1 (ᮀ), A1.1-IRS1 ({), and A1.1-IRS2 (E) cell lines were incubated in anti-CD3-coated plates for various times. Expression of mRNA for Egr-1, Egr-2, Egr-3, and GAPDH was examined by Northern blotting using specific probes. The relative intensities of the bands on the autoradiogram were determined using the public domain software National Institutes of Health Image. The relative ratio of Egr to GAPDH expression is shown. by guest on October 1, 2021 animals (27, 29). Detailed immunological studies have been ham- itor, LY294002, did not reverse the protection from AICD ob- pered by the severe diabetes and infertility of the IRS2 knockout served in the IRS1-expressing cells. mice (29, 50). Furthermore, double-deficient animals die in utero A striking finding is that IRS1 mediates the protection from (38). Therefore, to address the function of the IRS family in T AICD, while IRS2 does not. In vivo and in vitro experiments have cells, we took advantage of the observation that the T cell hybrid- revealed important differences in the signaling capacities of IRS1 oma A1.1 lacks expression of IRS family members (Fig. 1). A1.1 and IRS2. Tyrosine-phosphorylated IRS1 and IRS2 display differ- cells have been used extensively as an in vitro model of AICD (1, ential abilities to associate with the various Src homology 2 do- 30, 31) and have provided valuable information on the regulation main-containing signaling molecules, including p85, growth factor of Fas and FasL expression. This cell line was derived from the ϩ receptor binding protein 2, SHP-2, Fyn, Crk, and phospholipase fusion of a CD4 T cell and BW5147 and undergoes rapid apo- C␥ (18). However, the protective effect of IRS1 was observed in ptosis after anti-CD3 stimulation via a Fas-/FasL-dependent path- the absence of anti-CD3- or IL-4-induced tyrosine phosphoryla- way (30). After anti-CD3 stimulation, expression of Fas mRNA is tion, suggesting that it may act via a phosphotyrosine-independent rapidly induced by a protein kinase C-dependent mechanism. FasL mechanism. Phosphotyrosine-independent effects of IRS1 have mRNA is dramatically induced and is dependent on both the pro- been reported in other systems (22). Both IRS1 and IRS2 contain tein kinase C pathway and the cyclosporin A-sensitive pathway. Ͼ70 potential Ser/Thr phosphorylation sites that can be phosphor- Using this model system we found that IRS1 expression sup- ylated by a variety of kinases (JNK, ERK, casein kinase II, c-Akt, presses AICD, while IRS2 expression does not. Due to the ability of IRS family members to link to a PI-3K/PKB pathway (27), we protein kinase C). In the mid-region of the IRS molecules (aa expected the IRS-expressing cells to be intrinsically resistant to 555Ð898) there are a number of potential phosphorylation sites apoptosis. Using the A1.1 cells as targets for FasL-expressing and two potential JNK binding motifs (19). However, three of five L1210 cells, we found that both the IRS1- and IRS2-expressing Ser sites in this region known to be phosphorylated in IRS1 are cells were modestly protected from Fas-mediated apoptosis at low absent from IRS2 (17). One of the putative JNK binding sites is E:T cell ratios. However, this modest level of protection cannot conserved, but the other is not well conserved. Strikingly, IRS2 explain the specific resistance of IRS1-expressing A1.1 cells to does not have a Ser307 equivalent, the residue in IRS1 that is phos- anti-CD3-induced apoptosis. Addition of IL-4 did not enhance the phorylated by JNK and whose phosphorylation regulates insulin resistance to AICD, while it clearly induced the association of receptor kinase activity (19). It is interesting to speculate that the IRS1 with PI-3K (Fig. 3B) and induced PI-3K activity in the IRS1- IRS1-specific effect is mediated via phosphorylation of these expressing cells (data not shown). Furthermore, the PI-3K inhib- serine residues after TCR stimulation. 6222 PROTECTION FROM AICD BY IRS1

The IRS1-specific suppression of AICD in this hybridoma T cell does not explain inhibition of the FasL promoter. It is also possible model system is most likely due to its effect on the induction of that the presence of IRS1 could support the TCR-stimulated in- FasL itself. Strikingly, we found that significant levels of FasL duction of an as yet unknown factor that could act as a transcrip- mRNA were not detected until after8hofanti-CD3 stimulation in tional repressor. Characterization of the specific compositions of the IRS1-expressing cells. This delay in FasL mRNA was corre- the FLRE-binding complexes in IRS1- vs IRS2-expressing cells lated with a delay in the expression of functional FasL on the cell will require further detailed analysis. surface. This substantial delay in functional FasL expression could The studies reported herein have examined the mechanisms by be important in vivo where cellular interactions frequently occur which IRS family members regulate AICD in a T cell hybridoma transiently. model system. It is not yet clear how these findings relate to FasL The effect of IRS1 on FasL expression appears to be at the level expression in normal T cells. To examine their roles, we are cur- of the FasL promoter, since the induction of a FasL-luciferase rently developing retrovirus-based technology to overexpress/in- construct containing 511 bp of the promoter and a 16 mer derived hibit IRS1 or IRS2 expression in developing primary T cell cul- from the promoter called FLRE linked to luciferase was impaired tures. Such studies could aid in the understanding of the regulation in the IRS1-expressing cells. To date, FasL is the only TCR-in- of T cell homeostasis and potentially provide a novel therapeutic duced gene whose expression is diminished by IRS1. The induc- target for the treatment of diseases such as cancer. tion of CD69, Fas, and IL-2 is unaffected by IRS1 expression (Figs. 1 and 4 and data not shown). These results suggest that the Acknowledgments IRS1 signaling molecule alters the ability of TCR to activate tran- We acknowledge Dr. Jonathan Ashwell for providing Egr and FasL DNA scription factors that specifically regulate the FasL promoter. constructs and helpful suggestions; Dr. Charles Zaharchuk for providing ␬ Downloaded from Multiple transcription factors have been shown to regulate the NFAT, NF- B, and AP-1 constructs; and Dr. Sean Zhang for critical read- expression of FasL (33, 34, 36, 43Ð49). These include NF-␬B, ing of the manuscript. NFAT, Egr, and AP-1. The relative contributions of these factors References to FasL expression vary in the literature and can depend on the 1. Shi, Y. F., M. G., Szalay, M. Boyer, B. Singh, and D. R. Green. 1990. Activation- T-cell line analyzed, the precise FasL construct tested, and the induced cell death (AICD) in T cell hybridomas is due to apoptosis: morpholog- nature of the T cell stimulus. Recent studies indicate that specific ical aspects and DNA fragmentation. J. Immunol. 144:3326.

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