Cγ2 Plays a Role in TCR Signal Transduction and T Cell Selection Guoping Fu, Yuhong Chen, James Schuman, Demin Wang and Renren Wen This information is current as of September 25, 2021. J Immunol published online 25 July 2012 http://www.jimmunol.org/content/early/2012/07/25/jimmun ol.1103458 Downloaded from

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision http://www.jimmunol.org/ • No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at:

http://jimmunol.org/subscription by guest on September 25, 2021 Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 25, 2012, doi:10.4049/jimmunol.1103458 The Journal of Immunology

Phospholipase Cg2 Plays a Role in TCR Signal Transduction and T Cell Selection

Guoping Fu,* Yuhong Chen,* James Schuman,†,‡,x Demin Wang,*,{,‖ and Renren Wen*

One of the important signaling events following TCR engagement is activation of phospholipase Cg (PLCg). PLCg has two isoforms, PLCg1 and PLCg2. It is known that PLCg1 is important for TCR signaling and TCR-mediated T cell selection and functions, whereas PLCg2 is critical for BCR signal transduction and BCR-mediated B cell maturation and functions. In this study, we report that PLCg2 was expressed in primary T cells, and became associated with linker for activated T cells and Src homology 2-domain containing leukocyte protein of 76 kDa and activated upon TCR stimulation. PLCg1/PLCg2 double-deficient T cells displayed further block from CD4 and CD8 double-positive to single-positive transition compared with PLCg1 single- deficient T cells. TCR-mediated proliferation was further impaired in PLCg1/PLCg2 double-deficient T cells compared with 2+

PLCg1 single-deficient T cells. TCR-mediated signal transduction, including Ca mobilization and Erk activation, was further Downloaded from impaired in PLCg1/PLCg2 double-deficient relative to PLCg1 single-deficient T cells. In addition, in HY TCR transgenic mouse model, thymic positive and negative selections were reduced in PLCg1 heterozygous- and PLCg2 homozygous-deficient (PLCg1+/2 PLCg22/2) relative to wild-type, PLCg2single-deficient(PLCg22/2), or PLCg1 heterozygous-deficient (PLCg1+/2)mice.Taken together, these data demonstrate that PLCg2 participates in TCR signal transduction and plays a role in T cell selection. The Journal of Immunology, 2012, 189: 000–000. http://www.jimmunol.org/ cell progenitors in the thymus lack CD4 and CD8 ex- affinity between the TCR and the pep/MHC complex will die of pression and are called double-negative (DN) thymocytes. neglect. Thymocytes with high affinity between the TCR and the The DN cells are divided, based on their developmental pep/MHC complex are considered autoreactive and will be deleted T + 2 + + stages, into DN1 (CD44 CD25 ), DN2 (CD44 CD25 ), and DN3 (negative selection). Thymocytes with intermediate affinity be- 2 (CD44 CD25+) cells (1, 2). DN thymocytes develop into CD4 tween the TCR and the pep/MHC complex will further develop and CD8 double-positive (DP) thymocytes, which then differen- into CD4 and CD8 SP thymocytes (positive selection) (5, 7, 8). tiate into CD4 or CD8 single-positive (SP) cells. For ab T cells, The signal emanating from the TCR signaling complex is the TCR b-chain is rearranged at DN3 stage, followed by a-chain single most important driving force for the thymic selection.

rearrangement at DP stage (3, 4). DP thymocytes that have suc- The TCR transduces its signal through the CD3 molecules. by guest on September 25, 2021 cessfully rearranged both the TCR b- and a-chain express Engagement of the TCR by the pep/MHC complex initiates the the a and b heterodimers that are in association with the CD3 activation cascade of protein tyrosine kinases (PTKs), which in molecules on their cell surface to form the TCR complex and are turn mediates the activation of a series of signaling molecules (9). subjected to thymic selection (5). The random rearrangement of Inositol phospholipid-specific PLCg1 is a key signaling molecule the TCR b- and a-chain genes results in a broad spectrum of that is activated at the early stages of TCR signal transduction. affinity between the TCR and its ligand: the peptide/MHC (pep/ Following TCR engagement, PLCg1 is recruited to the TCR sig- MHC). Selection of DP thymocytes is based on the interaction naling complex through two adaptor molecules, linker for activated affinity between the TCR and the pep/MHC complex presented by T cells (LAT) and Src homology 2-domain containing leukocyte APCs in the thymic microenvironments (6). Thymocytes with low protein of 76 kDa (SLP-76) (10–13), and is phosphorylated on tyrosine residues (14–17), an indicator of PLCg activation (18).

*Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI 53226; Activated PLCg hydrolyzes phosphatidylinositol 4,5-bisphosphate †Department of Cell Biology, Medical College of Wisconsin, Milwaukee, WI 53226; to generate diacylglycerol (DAG) and inositol 1,4,5-trisphosphate ‡Department of Neurobiology, Medical College of Wisconsin, Milwaukee, WI x (IP3) (19–21). DAG activates protein kinase C (PKC), and IP3 53226; Department of Anatomy, Medical College of Wisconsin, Milwaukee, WI 2+ 53226; {Department of Microbiology, Medical College of Wisconsin, Milwaukee, mediates calcium (Ca ) mobilization. In T cells, DAG has also WI 53226; and ‖Department of Molecular Genetics, Medical College of Wisconsin, been shown to activate the Ras/ERK pathway through RasGRP Milwaukee, WI 53226 (22–24). Whereas Ca2+ mobilization activates NFAT through cal- Received for publication November 30, 2011. Accepted for publication June 28, cineurin , PKC activates NF-kB and AP-1 through IkB 2012. kinase and JNK, respectively (25, 26). Among the pathways This work was supported in part by National Institutes of Health Grants R56 AI071239 (to R.W.), RO1 AI052327 (to R.W.), RO1 AI079087 (to D.W.), and PO1 HL44612 (to downstream of PLCg, the Ras/Erk signaling cascade has been D.W.), and by a Scholar Award from the Leukemia and Lymphoma Society (to D.W.). shown to be important for thymic positive selection (27–30). Address correspondence and reprint requests to Dr. Renren Wen, Blood Research PLCg has two isoforms, PLCg1 and PLCg2, which share the Institute, BloodCenter of Wisconsin, 8727 Watertown Plank Road, Milwaukee, WI same domain structure (21, 31). PLCg1 is ubiquitously expressed, 53226. E-mail address: [email protected] whereas PLCg2 expression is restricted to the hematopoietic Abbreviations used in this article: DAG, diacylglycerol; DN, double-negative; DP, system (32). Studies showed that PLCg1 is the predominate iso- double-positive; IP3, inositol 1,4,5-trisphosphate; LAT, linker for activated T cells; pep/MHC, peptide/MHC; PI, propidium iodide; PKC, protein kinase C; PLCg, phos- form expressed in both primary T cells and T cell lines (33), and it pholipase Cg; PTK, protein tyrosine kinase; SLP-76, Src homology 2-domain con- is activated following TCR stimulation (14–17). Immunodepletion taining leukocyte protein of 76 kDa; SP, single-positive. of PLCg1 following TCR stimulation essentially depletes PLCg Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 activity from Jurkat T cell protein extracts (16). Moreover, T cell-

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103458 2 PHOSPHOLIPASE Cg2 IN TCR SIGNALING specific deletion of PLCg1 at DP stage impairs TCR signaling Calcium flux analysis transduction, affects both positive and negative selection, causes Thymocytes (2 3 106) were resuspended in 1 ml PBS plus 2% FBS in the severe reduction of SP thymocytes, and results in peripheral T cell presence of 10 mg/ml indo-1AM (Invitrogen) at room temperature in the lymphopenia (34), whereas PLCg2 deficiency has no effect on dark for 30 min. The cells were then incubated in 200 ml PBS plus 2% FBS T cell development and function (35). Based on these findings, it in the presence of FITC anti-CD4, PE anti-CD8 Abs at room temperature is generally thought that PLCg1 is responsible for PLCg activity in the dark for 15 min. After being washed, the cells were resuspended in 1 ml primary T cell culture medium at room temperature. The cells were downstream of TCR in T cells. run on a LSRII (BD Biosciences), and data were collected for 2 min. However, the observation that PLCg1-deficient T cells exhibit Biotin anti-CD3 Ab was then added to a final concentration of 20 mg/ml, residual Ca2+ influx in response to TCR stimulation implicates the and the cells were run and data were collected for 30 s. Following addition involvement of other PLCg isoform in TCR signal transduction (34). of streptavidin (Pierce) to a final concentration of 8 mg/ml to cross-link the TCR, the cells were run and data were collected for another 9 min. In this study, we found that PLCg2, the other PLCg isoform, was expressed in T cells and activated upon TCR stimulation. Impor- In vitro negative selection tantly, PLCg1/PLCg2 double deficiency further blocked the transi- 2 DP thymocytes were purified from 6-wk-old wild-type and PLCg1+/ tion of DP thymocytes to the SP stage compared with PLCg1 single PLCg22/2 mice by sorting. A total of 1 3 106 DP thymocytes was cul- 2+ deficiency. TCR-mediated Ca mobilization and Erk activation were tured in 500 ml primary lymphocyte culture medium (RPMI 1640 sup- completely abolished in PLCg1/PLCg2 double-deficient relative to plemented with 10% FBS, 2 mM L-glutamine, 10 mM nonessential amino PLCg1 single-deficient T cells. Moreover, in the HY TCR trans- acid, 1 mM sodium pyruvate, 50 mM 2-ME) in a 24-well plate coated overnight with anti-CD3 (10 mg/ml) and anti-CD28 (50 mg/ml) for 20 h. genic mice, thymic positive and negative selections were reduced in The cells were then harvested and stained with propidium iodide (PI; 20 PLCg1 heterozygous- and PLCg2 homozygous-deficient relative to mg/ml) and analyzed by FACS. Downloaded from wild-type, PLCg2single-deficient,orPLCg1 heterozygous-deficient T cells. Taken together, these results demonstrate an unappreciated Cell proliferation assay role of PLCg2 in TCR signal transduction and TCR-mediated thy- Total splenocytes were enumerated and resuspended in PBS (2 3 107/ml). mic selection. The cells were then labeled with 1 mM PKH Fluorescent Cell Linker Dye (PKH26; Sigma-Aldrich), according to the manufacturer’s recommendation. The cells were then washed twice and resuspended at 2 3 106/ml. The cells Materials and Methods (0.5 ml) were cultured in a 48-well plate in medium alone, or stimulated with http://www.jimmunol.org/ plate-bound anti-CD3 (2 mg/ml), plate-bound anti-CD3 (2 mg/ml) plus anti- Mice CD28 (2 mg/ml), plate-bound anti-CD3 (2 mg/ml) plus IL-2 (10 U/ml), and HY TCR and CD4Cre transgenic mice were purchased from Taconic PMA (10 ng/ml) plus ionomycin (500 ng/ml). Seventy-two hours later, the Laboratory. PLCg1+/2 and Rosa-26-YFP mice were provided by G. Car- cells were harvested and stained with PE anti-CD4 and allophycocyanin anti- penter (Vanderbilt University, Nashville, TN) (36) and F. Costantini (Co- CD8, followed by FACS analysis for cell proliferation. lumbia University, New York, NY) (37), respectively. CD4Cre/PLCg1fl/2 and PLCg22/2 mice have been described previously (34, 35). Due to in- 2 2 Results creased lethality of CD4Cre/PLCg1fl/fl and PLCg2 / mice when they were backcrossed to B6 background, the mice in the study were back- PLCg2 is expressed in primary T cells and activated following crossed to B6 mice for only three to four generations before intercrossing. TCR stimulation by guest on September 25, 2021 Mice were bred and maintained in the Biological Research Center of the 2+ Medical College of Wisconsin. All animal protocols were approved by the PLCg1-deficient T cells exhibit residual Ca influx following TCR Medical College of Wisconsin Institutional Animal Care and Use Com- ligation, implicating that the other PLCg isoform, PLCg2, may also mittee. be involved in TCR signaling (34) (data not shown). To explore a potential role of PLCg2 in TCR signaling, we first examined the Western blotting analysis expression levels of PLCg2 in T cells at different developmental For detecting PLCg1 and PLCg2, DN, DP, CD4SP, and CD8SP thymo- stages. PLCg2 was expressed in DN, DP, CD4SP, CD8SP thymo- cytes, and CD4+ and CD8+ splenic T cells were sorted using FACSAria cytes, and CD4, CD8 splenic T cells, and the expression was highest (BD Biosciences). Cells were lysed, and 20 mg of each sample was sub- in DN thymocytes (Fig. 1A). Of note, PLCg1 expression level was jected to direct Western blotting analysis with Abs against PLCg1 (sc-81; consistent throughout T cell development (Fig. 1A). To determine Santa Cruz Biotechnology) or PLCg2 (sc-407; Santa Cruz Biotechnology). For detecting tyrosine phosphorylation of PLCg, splenic T cells were whether PLCg2 expressed in T cells is functional during TCR sig- purified by negative selection using B220-conjugated magnetic beads and naling, we assessed TCR-induced activation of PLCg2. Upon anti- MACS columns (Miltenyi Biotec). Purified T cells were stimulated for 2 CD3 stimulation of purified splenic T cells, PLCg2 was markedly min on tissue culture plate coated with 10 mg/ml anti-CD3 Abs, and cells phosphorylated on tyrosine residues (Fig. 1B), which is an indicator were directly lysed on the plate. PLCg1 or PLCg2 proteins in the cell lysates were immunoprecipitated with anti-PLCg1 or anti-PLCg2 Abs, of PLCg activation (18, 21). As expected, PLCg1 was also phos- respectively, and then subjected to Western blotting analysis using anti- phorylated on tyrosine residue following anti-CD3 stimulation (Fig. phosphorylated tyrosine Ab 4G10 (Millipore). For detecting PLCg1 1B). Phosphorylation of PLCg2 at Y759, which is an activation- 7 phosphorylation, 1 3 10 purified splenic T cells were used. For detecting dependent phosphorylation site (38, 39), was also observed fol- 3 7 PLCg2 phsophorylation, 5 10 purified splenic T cells were used. For lowing TCR stimulation (Fig. 1C). In addition, TCR engagement detecting phosphorylation of PLCg2 at Y759 and interaction of PLCg2 with SLP-76 and LAT, purified splenic T cells (3 3 107 cells at 5 3 107/ml) induced the association of PLCg2 with LAT and SLP-76 (Fig. 1D), were coated with biotin anti-CD3 (10 mg/ml), followed by cross-linking suggesting that PLCg2 is recruited to the TCR signaling complex with streptavidin (8 mg/ml; Pierce) for the indicated time. A total of 50 mg using the same adaptor molecules employed by PLCg1. Thus, cell lysates was subjected to direct Western blotting analysis for detecting PLCg2 is expressed in T cells, is associated with LAT and SLP-76, phosphorylation at Y759 with a pY759 Ab (3874; Cell Signaling). The rest of the cell lysates were immunoprecipitated with anti-PLCg2 Abs, fol- and is activated upon TCR engagement. lowed by Western blotting analysis with an Ab that specifically recognizes PLCg2 plays a role in T cell development and activation SLP-76 (06-548; Millipore) or LAT (06-807; Millipore). For detecting Erk phosphorylation, FACS-sorted YFP+DP thymocytes or total thymocytes PLCg2 single deficiency has no detectable effect on T cell de- were coated with biotin anti-CD3, followed by cross-linking with strep- velopment or TCR-dependent activation, whereas PLCg1 single tavidin for the indicated time. The cells were lysed, and 50 mg of each cell deficiency markedly impairs TCR-mediated T cell development lysate was subjected to direct Western blot analysis using anti–p-Erk (sc- 7383; Santa Cruz Biotechnology) and anti-Erk (sc-93, sc-154; Santa Cruz and function (34, 35). However, it is possible that PLCg2 plays Biotechnology) Abs. a role in TCR-mediated T cell development and activation, and the The Journal of Immunology 3

FIGURE 1. Expression and activation of PLCg1 and PLCg2 in primary T cells. (A) Western blotting analysis of PLCg1 and PLCg2 expression in T cells. T cells at different developmental stages were purified. Total cell lysates (20 mg) of each sample were subjected to Western blotting analysis with the indicated Abs. The figure shown is a representative of three independent experiments. (B) Phosphorylation of both PLCg1 and PLCg2 following anti-CD3 Downloaded from stimulation. Purified splenic T cells were stimulated with plate-bound anti-CD3 (10 mg/ml). Cells were then subjected to IP-Western analysis using the indicated Abs. (C) Phosphorylation of PLCg2 on Y759 after TCR engagement. Purified splenic T cells were coated with biotin anti-CD3, followed by cross- link with streptavidin for the indicated time. Cell lysates were subjected to direct Western blotting analysis using an Ab that specifically recognizes phosphorylated tyrosine residue 759 of PLCg2. (D) Association of PLCg2 with LAT and SLP-76 following TCR engagement. Cells were stimulated, as described in (C), and cell lysates were then subjected to IP-Western blotting analysis using the indicated Abs. The figures in (B)–(D) are each a repre- sentative of two independent experiments. http://www.jimmunol.org/ role of PLCg2 might be revealed when the expression of PLCg1is compared with those in wild-type CD4Cre/YFP mice (Fig. 2A, absent or reduced. Thus, we compared T cell development in mice Table I). Importantly, the percentage of YFP+DP thymocytes was deficient for both PLCg1 and PLCg2 with that in mice lacking further increased, and the percentages and numbers of YFP+ only PLCg1. To this end, we generated CD4Cre/YFP/PLCg1fl/fl CD4SP and YFP+CD8SP thymocytes were further reduced in PLCg22/2, CD4Cre/YFP/PLCg1fl/fl, and CD4Cre/YFP mice. In CD4Cre/YFP/PLCg1fl/flPLCg22/2 relative to CD4Cre/YFP/ these mice, the CD4 enhancer/promoter/silencer initiates Cre ex- PLCg1fl/fl mice (Fig. 2A, Table I). Consistent with the defect in pression at the thymic DP stage (40), and Cre-mediated YFP thymus, splenic YFP+CD4+ and YFP+CD8+ T cell numbers were fl/fl expression can track floxed PLCg1 deletion (34, 37). Consistent significantly reduced in CD4Cre/YFP/PLCg1 mice compared by guest on September 25, 2021 with previous report (34), thymocytes from CD4Cre/YFP/ with wild-type CD4Cre/YFP mice (Table I), and were further PLCg1fl/fl mice displayed a severe block at DP to SP transition, reduced in CD4Cre/YFP/PLCg1fl/flPLCg22/2 mice compared as shown in the increase of the percentage of YFP+DP cells and with CD4Cre/YFP/PLCg1fl/fl mice (Table I). Percentage of YFP+ the reduction in the percentage and number of YFP+SP cells CD8+ cells was also substantially reduced in CD4Cre/YFP/

FIGURE 2. T cell development in PLCg1 and PLCg2 double-deficient mice. Thymocytes (A) and splenocytes (B) derived from CD4Cre/YFP, CD4Cre/ YFP/PLCg1fl/fl, and CD4Cre/YFP/PLCg1fl/fl PLCg22/2 mice were examined for CD4 and CD8 expression profile. Percentages indicate cells gated in total or YFP+ lymphoid populations. Data shown are representative of at least three mice in each group. (C) Role of PLCg2 in TCR-mediated cell proliferation. Splenocytes were labeled with PKH Fluorescent Cell Linker Dye, according to the manufacturer’s recommendation, and stimulated as indicated for 72 h before analysis for cell proliferation. Data shown are a representative of two independent experiments. 4 PHOSPHOLIPASE Cg2 IN TCR SIGNALING

Table I. Analysis of YFP+ T cell populations in CD4Cre/YFP, CD4Cre/YFP/PLCg1fl/fl, and CD4Cre/YFP/PLCg1fl/flPLCg22/2 mice

Thymocytes Splenocytes

YFP+ YFP+DP YFP+CD4SP YFP+CD8SP YFP+ YFP+CD4+ YFP+CD8+ CD4Cre/YFP % 89 6 2.5 84 6 2.9 12 6 2.2 3.0 6 0.8 33 6 10 55 6 3.9 38 6 8.1 (n = 10) #(3106)916 30 77 6 26 10 6 3.3 2.7 6 1286 10 16 6 6.0 11 6 4.7 CD4Cre/YFP/PLCg1fl/fl %796 3.9** 96 6 0.6** 2.5 6 0.5** 0.7 6 0.3** 12 6 8.3** 34 6 17** 52 6 17* (n =8) #(3106) 108 6 27 105 6 26 2.7 6 0.6** 0.7 6 0.3** 9.1 6 3.4** 3.6 6 1.2** 4.2 6 2.3* CD4Cre/YFP/PLCg1fl/fl PLCg22/2 %846 3.9 98 6 0.9* 1.8 6 0.6* 0.2 6 0.06* 4.7 6 2.1 37 6 21 5.6 6 2** (n =3) #(3106)576 50 56 6 49 0.9 6 0.7* 0.1 6 0.04* 7.9 6 8.9 1.6 6 0.8* 0.6 6 0.7* The age of the mice analyzed was between 6 wk and 4 mo. Data presented are average percentage (%) or absolute number (#) of each T cell subset. Percentage of YFP+ cells is gated in total lymphocytes, and other populations are gated in YFP+ lymphocytes. The p value (two tailed) was calculated by comparing the percentage or absolute number of each T cell subset from CD4Cre/YFP mice with those from CD4Cre/YFP/PLCg1fl/fl mice (annotations added to the data derived from CD4Cre/YFP/PLCg1fl/fl mice), or by comparing the percentage or absolute number of each T cell subset from CD4Cre/YFP/PLCg1fl/fl mice with those from CD4Cre/YFP/PLCg1fl/flPLCg22/2 mice (annotations added to the data derived from CD4Cre/YFP/PLCg1fl/flPLCg22/2 mice). In this table, CD4Cre/YFP/PLCg1fl/fl included CD4Cre/YFP/PLCg1fl/fl and CD4Cre/YFP/PLCg1fl/2 genotypes. *p , 0.05, **p , 0.001.

PLCg1fl/flPLCg22/2 mice compared with CD4Cre/YFP/PLCg1fl/fl PLCg2 deficiency (35) and T cell-specific PLCg1 deficiency (data mice (Fig. 2B). T cells from CD4Cre/YFP/PLCg1fl/fl mice also not shown) caused certain level of lethality perinatally or several Downloaded from exhibited dramatic reduction of proliferation in response to weeks after birth, the frequency of obtaining CD4Cre/PLCg1fl/fl stimulation with anti-CD3, anti-CD3/anti-CD28, or anti-CD3/IL-2 PLCg22/2 mice that also carry an HY TCR transgene was ex- compared with those from CD4Cre/YFP mice, and this prolifer- tremely low. However, we observed that T cells in PLCg1+/2 mice ation was further reduced in T cells derived from CD4Cre/YFP/ displayed reduced expression of PLCg1 proteins (data not shown). PLCg1fl/flPLCg22/2 mice (Fig. 2C). These data demonstrate that Importantly, we observed that PLCg1+/2PLCg22/2 thymocytes

PLCg2 plays a role in TCR-mediated thymic DP to SP transition showed reduced TCR-mediated Erk activation (Fig. 4A). Therefore, http://www.jimmunol.org/ and TCR-mediated proliferation. we examined thymic selection in PLCg1+/2PLCg22/2HY mice, in which PLCg2 was absent and PLCg1 expression is reduced. PLCg2 plays a role in TCR signal transduction in thymocytes The breeding was set up in a way to generate wild-type HY, Thymic DP to SP transition is driven by signals emanating from the PLCg22/2/HY, PLCg1+/2/HY, and PLCg1+/2PLCg22/2/HY TCR. Our observation that PLCg2 played a role in thymic DP to SP mice and ensure each experimental mouse to carry one copy of HY transition suggested contribution of PLCg2 to TCR signal trans- transgene. The total thymocyte cell numbers were comparable in duction in thymocytes. To determine the role of PLCg2 in TCR- the four types of female HY transgenic mice (Table II). The per- mediated signaling, we examined TCR-regulated signaling events centages of CD8SP thymocytes and positively selected T3-70high in DP thymocytes from CD4Cre/YFP, CD4Cre/YFP/PLCg1fl/fl,and CD8SP cells were not significantly different in female wild-type by guest on September 25, 2021 CD4Cre/YFP/PLCg1fl/flPLCg22/2 mice. YFP+DP thymocytes from HY, PLCg22/2/HY, and PLCg1+/2/HY mice (Fig. 4B, Table II), CD4Cre/YFP/PLCg1fl/fl mice displayed markedly reduced, but not indicating that absence of PLCg2 alone or reduction of PLCg1 abolished, TCR-induced Ca2+ flux compared with those derived from wild-type CD4Cre/YFP mice (Fig. 3A). In contrast, YFP+DP thy- mocytes derived from CD4Cre/YFP/PLCg1fl/flPLCg22/2 mice were unable to elicit TCR-induced Ca2+ flux (Fig. 3A). Similarly, TCR- mediated Erk activation was substantially reduced in CD4Cre/YFP/ PLCg1fl/fl YFP+DP thymocytes, and was abolished in CD4Cre/YFP/ PLCg1fl/flPLCg22/2 YFP+DP thymocytes (Fig. 3B). Collectively, these data demonstrate that, in addition to PLCg1, PLCg2plays a role in TCR-mediated signaling, and their functions are additive in TCR signal transduction in DP thymocytes. PLCg2 plays a role in T cell selection TCR-mediated Erk activation in DP thymocytes is important for thymic positive selection (27–29, 41–43). Our observation that PLCg2 played a role in thymic DP to SP transition (Fig. 2A, Table I) and TCR-mediated Erk activation in DP thymocytes (Fig. 3B) FIGURE 3. Role of PLCg2 in TCR-mediated signal transduction. prompted us to examine the role of PLCg2 in thymic positive se- Thymocytes were purified from CD4Cre/YFP, CD4Cre/YFP/PLCg1fl/fl, lection. Previously, we reported that PLCg1 plays a role in T cell and CD4Cre/YFP/PLCg1fl/flPLCg22/2 mice. (A) TCR-induced Ca2+ flux positive and negative selection using the HY TCR transgenic model in PLCg1/PLCg2 double-deficient DP thymocytes. Thymocytes were (34). HY TCR transgenic mice express a transgenic TCR that is loaded with indo-1 and were stained with Abs to CD4 and CD8. Biotin- conjugated anti-CD3 (first arrow) and subsequently streptavidin (second specific for a peptide derived from the HY male Ag in the context of 2+ b arrow) were added to the cells to cross-link TCR. Induction of Ca flux MHC class I D molecule (44). T cells with the HY transgenic TCR 2+ + b wasdeterminedbyLSRII.Ca analysis was gated on YFP DP thymo- are negatively selected in H-2 male mice, resulting in deletion of B + cytes. ( ) TCR-induced Erk activation in PLCg1/PLCg2 double-deficient CD8 T cells expressing the transgenic TCR. However, HY + b DP thymocytes. FACS-sorted YFP DP thymocytes were stimulated with transgenic T cells are positively selected in female H-2 mice, biotin-conjugated anti-CD3 plus streptavidin. Cell lysates were subjected + leading to skewed development of thymocytes toward the CD8 to SDS-PAGE, followed by Western blotting analysis with the indicated lineage. Selection of T cells bearing the transgenic TCR can be Ab. Data shown in (A) and (B) are a representative of two independent followed by the clonotypic Ab, T3-70 (45, 46). Because both experiments. The Journal of Immunology 5

FIGURE 4. Thymic selection in PLCg1 heterozy- gous- and PLCg2 homozygous-deficient mice. (A) TCR- induced Erk activation in PLCg1+/2PLCg22/2 thymo- cytes. Total thymocytes from wild-type and PLCg1+/2 PLCg22/2 mice were stimulated with biotin-conjugated anti-CD3 plus streptavidin. Cell lysates were subjected to direct Western blotting analysis with the indicated Abs. Data are a representative of two independent experiments. (B) Positive selection in PLCg1+/2PLCg22/2 HY mice is impaired. CD4 and CD8 expression profile of thymocytes from 4- to 10-wk-old wild-type HY, PLCg22/2/HY, PLCg1+/2/HY, and PLCg1+/2PLCg22/2/HY female mice. (C) Negative selection is reduced in PLCg1+/2 PLCg22/2 HY mice. CD4 and CD8 expression profile of thymocytes from 4- to 10-wk-old wild-type HY, PLCg22/2/HY,PLCg1+/2/HY, and PLCg1+/2PLCg22/2/ HY male mice. (D) Impaired negative selection of PLCg1+/2PLCg22/2 thymocytes in an in vitro negative selection assay. DP thymocytes from individual wild- Downloaded from type (n = 3) and PLCg1+/2PLCg22/2 (n = 2) thymo- cytes were cultured in a 24-well plate with medium alone (med) or coated overnight with anti-CD3 and anti- CD28. The cells were harvested 20 h later, stained with PI, and analyzed by FACS. *p , 0.05. http://www.jimmunol.org/ protein was not able to significantly affect positive selection. PLCg22/2/HY mice displayed marked increase in the percentages However, compared with female wild-type HY mice, female of CD8SP and T3-70high CD8SP thymocytes (Fig. 4C, Table II), PLCg1+/2PLCg22/2/HY mice displayed a marked reduction in the indicating impaired negative selection in PLCg1+/2PLCg22/2/HY percentages of both CD8SP thymocytes and T3-70high CD8SP mice. The increase in the percentage of CD8SP thymocytes was thymocytes (Fig. 4B, Table II). Compared with female PLCg22/2/HY also significant in male PLCg1+/2PLCg22/2/HY mice, when and PLCg1+/2/HY mice, female PLCg1+/2PLCg22/2/HY mice compared with male PLCg22/2/HY and PLCg1+/2/HY mice. also showed a significant reduction of T3-70high CD8SP thymocyte Thus, PLCg2 deficiency impairs thymic negative selection when percentage (Table II). Thus, PLCg2 deficiency impairs thymic PLCg1 expression is reduced in the HY transgenic model. We also by guest on September 25, 2021 positive selection when PLCg1 expression is reduced. examined the role of PLCg2 in negative selection using an in vitro Next, we examined the role of PLCg2 in negative selection thymic negative selection model, in which DP thymocytes were using the HY transgenic mice. There was no significant differ- induced to undergo apoptosis with plate-bound anti-CD3 and anti- ence in thymic cellularity and percentages of CD8SP and T3- CD28 (47). Less PLCg1+/2PLCg22/2 thymocytes underwent ap- 70high CD8SP thymocytes within male wild-type HY, PLCg22/2/ optosis than wild-type thymocytes (Fig. 4D). The reduced apo- HY, and PLCg1+/2/HY mice, suggesting a similar level of negative ptosis was not due to heterozygous PLCg1 deficiency, because selection in these mice. Compared with male wild-type HY, there was no significant difference in the levels of cell apoptosis PLCg22/2/HY, and PLCg1+/2/HY mice, PLCg1+/2PLCg22/2/HY between wild-type and PLCg1+/2 thymocytes (data not shown). mice showed some level of increase in thymocyte cellularity (Table Taken together, these data demonstrate that PLCg2 plays a role in II). Compared with male wild-type HY mice, male PLCg1+/2 both thymic positive and negative selection.

Table II. PLCg2 is important for thymic selection in HY TCR transgenic mice

HY Females

Genotype Thymocyte No. (3106) CD8SP% in Thymocytes T3-70highCD8SP% in Thymocytes HY (n = 9) 112 6 56 21 6 11 18 6 10 PLCg22/2/HY (n = 7) 100 6 27 16 6 5.0 13 6 3.8 PLCg1+/2/HY (n = 7) 101 6 77 17 6 7.9 14 6 7.5 PLCg1+/2PLCg22/2/HY (n = 10) 109 6 53 11 6 3.3* 8.5 6 3.2** HY Males

Genotype Thymocyte No. (3106) CD8SP% in Thymocytes T3-70highCD8SP% in Thymocytes HY (n = 6) 8.4 6 6.0 11 6 6.2 10 6 6.0 PLCg22/2/HY (n = 5) 6.4 6 1.8 13 6 4.1 11 6 4.7 PLCg1+/2/HY (n = 5) 7.9 6 3.4 12 6 3.9 11 6 3.5 PLCg1+/2PLCg22/2/HY (n =9) 146 8.9 19 6 5.7* 17 6 5.6* The age of the mice analyzed was between 4 and 10 wk. Data presented are average thymocyte number (No.) or percentage (%) of thymocyte subsets. The p values (two tailed) were calculated by comparing the total thymocyte cell number or percentage of each thymocyte subset from PLCg22/2/HY, PLCg1+/2/HY, or PLCg1+/2PLCg22/2/HY mice with those from HY mice. *p , 0.05, **p , 0.01. 6 PHOSPHOLIPASE Cg2 IN TCR SIGNALING

Discussion Disclosures Immunodepletion of PLCg1, the predominate isoform in T cells, The authors have no financial conflicts of interest. effectively depletes TCR-induced PLCg activity from Jurkat T cell extracts (16, 33). In addition, PLCg1 deficiency in T cells References markedly impairs TCR signaling and TCR-mediated T cell de- 1. Godfrey, D. I., J. Kennedy, T. Suda, and A. Zlotnik. 1993. A developmental velopment (34). However, PLCg2 deficiency seems not to affect pathway involving four phenotypically and functionally distinct subsets of CD32 TCR signaling and TCR-mediated T cell development (35). Thus, CD42CD82 triple-negative adult mouse thymocytes defined by CD44 and CD25 PLCg1 is generally believed to be responsible for TCR-induced expression. J. Immunol. 150: 4244–4252. 2. Petrie, H. T., and J. C. Zu´n˜iga-Pflu¨cker. 2007. Zoned out: functional mapping of PLCg activity. Nonetheless, the presence of a residual TCR- stromal signaling microenvironments in the thymus. Annu. Rev. Immunol. 25: induced Ca2+ influx in the absence of PLCg1 suggests a poten- 649–679. 3. Petrie, H. T., F. Livak, D. Burtrum, and S. Mazel. 1995. T cell receptor tial involvement of PLCg2 in TCR signaling. In the current study, recombination patterns and mechanisms: cell death, rescue, and T cell produc- we found that PLCg2 was expressed in T cells and played a role in tion. J. Exp. Med. 182: 121–127. TCR-mediated Ca2+ mobilization, Erk activation, thymic DP to SP 4. Herna´ndez-Munain, C., B. P. Sleckman, and M. S. Krangel. 1999. A develop- mental switch from TCR delta enhancer to TCR alpha enhancer function during transition, and positive and negative selection. The functions of thymocyte maturation. Immunity 10: 723–733. PLCg1 and PLCg2 appear to be redundant to some degree in 5. Starr, T. K., S. C. Jameson, and K. A. Hogquist. 2003. Positive and negative T cells. The observation that the absence of PLCg2 alone has no selection of T cells. Annu. Rev. Immunol. 21: 139–176. 6. Anderson, G., P. J. Lane, and E. J. Jenkinson. 2007. Generating intrathymic effect on TCR-mediated function implicates that PLCg2 defi- microenvironments to establish T-cell tolerance. Nat. Rev. Immunol. 7: 954–963. ciency can be largely masked by compensatory effects of the full 7. Page, D. M., L. P. Kane, T. M. Onami, and S. M. Hedrick. 1996. Cellular and

biochemical requirements for thymocyte negative selection. Semin. Immunol. 8: Downloaded from expression of PLCg1. The impairment of TCR signaling by 69–82. PLCg1 deficiency cannot be compensated by mere existence of 8. Marrack, P., and J. Kappler. 1997. Positive selection of thymocytes bearing alpha PLCg2, but can be further deteriorated by lack of PLCg2. Thus, beta T cell receptors. Curr. Opin. Immunol. 9: 250–255. 9. Samelson, L. E. 2002. Signal transduction mediated by the T cell antigen re- an unappreciated role of PLCg2 in TCR signaling can only be ceptor: the role of adapter proteins. Annu. Rev. Immunol. 20: 371–394. revealed when PLCg1 expression is reduced or absent. 10. Zhang, W., J. Sloan-Lancaster, J. Kitchen, R. P. Trible, and L. E. Samelson. PLCg1 and PLCg2 contribute to TCR signal transduction; 1998. LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 92: 83–92. however, it is not known whether the contributions made by the 11. Finco, T. S., T. Kadlecek, W. Zhang, L. E. Samelson, and A. Weiss. 1998. LAT is http://www.jimmunol.org/ two PLCg are quantitatively or qualitatively different. Our study required for TCR-mediated activation of PLCgamma1 and the Ras pathway. Immunity 9: 617–626. suggests that PLCg2 is recruited to the TCR signaling complex 12. Yablonski, D., M. R. Kuhne, T. Kadlecek, and A. Weiss. 1998. Uncoupling of through its interaction with SLP-76 and LAT, which is a mecha- nonreceptor tyrosine kinases from PLC-gamma1 in an SLP-76-deficient T cell. nism employed by PLCg1. Similar receptor coupling mechanism Science 281: 413–416. 13. Zhang, W., B. J. Irvin, R. P. Trible, R. T. Abraham, and L. E. Samelson. 1999. is also employed by PLCg2 in human platelets following collagen Functional analysis of LAT in TCR-mediated signaling pathways using a LAT- receptor activation (48). Both activated PLCg hydrolyze phos- deficient Jurkat cell line. Int. Immunol. 11: 943–950. 14. Granja, C., L. L. Lin, E. J. Yunis, V. Relias, and J. D. Dasgupta. 1991. PLC phatidylinositol 4,5-bisphosphate to generate DAG and IP3, which 2+ gamma 1, a possible mediator of T cell receptor function. J. Biol. Chem. 266: lead to PKC and Ras/ERK activation as well as Ca influx (19– 16277–16280. 24). Both the magnitude and duration of PKC and Ras/ERK ac- 15. Park, D. J., H. W. Rho, and S. G. Rhee. 1991. CD3 stimulation causes phos- by guest on September 25, 2021 2+ phorylation of -gamma 1 on serine and tyrosine residues in tivation and Ca flux are therefore dependent upon the amount of a human T-cell line. Proc. Natl. Acad. Sci. USA 88: 5453–5456. PLCg activity and can differentially affect activation of a number 16. Weiss, A., G. Koretzky, R. C. Schatzman, and T. Kadlecek. 1991. Functional of transcription factors, including AP-1, NF-kB, and NFAT (22– activation of the T-cell antigen receptor induces tyrosine phosphorylation of phospholipase C-gamma 1. Proc. Natl. Acad. Sci. USA 88: 5484–5488. 26). The degree of both PLCg1 and PLCg2 activation, and thus 17. Secrist, J. P., L. Karnitz, and R. T. Abraham. 1991. T-cell antigen receptor li- the total amount of PLCg activity, reflects the strength of TCR gation induces tyrosine phosphorylation of phospholipase C-gamma 1. J. Biol. signaling and ultimately determines the nature of the T cell re- Chem. 266: 12135–12139. 18. Nishibe, S., M. I. Wahl, S. M. Herna´ndez-Sotomayor, N. K. Tonks, S. G. Rhee, sponse. However, our previous study has found that over- and G. Carpenter. 1990. Increase of the catalytic activity of phospholipase C- expression of PLCg1inPLCg2-deficient B cells fails to restore gamma 1 by tyrosine phosphorylation. Science 250: 1253–1256. 19. Rhee, S. G., and K. D. Choi. 1992. Regulation of inositol phospholipid-specific BCR-mediated B cell proliferation or maturation, indicating that phospholipase C isozymes. J. Biol. Chem. 267: 12393–12396. PLCg1 and PLCg2 have qualitatively different contributions 20. Rhee, S. G., and Y. S. Bae. 1997. Regulation of phosphoinositide-specific to signal transduction (49). PLCg1 and PLCg2 may couple the phospholipase C isozymes. J. Biol. Chem. 272: 15045–15048. 21. Rhee, S. G. 2001. Regulation of phosphoinositide-specific phospholipase C. TCR signaling pathway to different downstream signaling path- Annu. Rev. Biochem. 70: 281–312. ways. This possibility assumes that PLCg1andPLCg2have 22. Tognon, C. E., H. E. Kirk, L. A. Passmore, I. P. Whitehead, C. J. Der, and phospholipase-independent signaling functions. This assumption R. J. Kay. 1998. Regulation of RasGRP via a phorbol ester-responsive C1 do- main. Mol. Cell. Biol. 18: 6995–7008. is supported by the finding that a catalytically inactive mutant of 23. Dower, N. A., S. L. Stang, D. A. Bottorff, J. O. Ebinu, P. Dickie, PLCg1 can initiate a full mitogenic response in fibroblasts (50, H. L. Ostergaard, and J. C. Stone. 2000. RasGRP is essential for mouse thy- mocyte differentiation and TCR signaling. Nat. Immunol. 1: 317–321. 51). Consistently, the Src homology 3 other than the catalytic 24. Ebinu, J. O., D. A. Bottorff, E. Y. Chan, S. L. Stang, R. J. Dunn, and J. C. Stone. domain of PLCg1 is able to promote PC12 and NIH 3T3 cell 1998. RasGRP, a Ras guanyl nucleotide-releasing protein with calcium- and growth and possesses guanine nucleotide exchange factor activity diacylglycerol-binding motifs. Science 280: 1082–1086. 25. Isakov, N., and A. Altman. 2002. Protein kinase C(theta) in T cell activation. for the mitogenic GTPase PIKE (50–53). It is possible that either Annu. Rev. Immunol. 20: 761–794. in addition to or instead of the phospholipase activity, other ac- 26. Rao, A., C. Luo, and P. G. Hogan. 1997. Transcription factors of the NFAT tivities of PLCg1 and/or PLCg2 may make qualitatively different family: regulation and function. Annu. Rev. Immunol. 15: 707–747. 27. Alberola-Ila, J., K. A. Forbush, R. Seger, E. G. Krebs, and R. M. Perlmutter. contributions to TCR-mediated function. 1995. Selective requirement for MAP kinase activation in thymocyte differen- tiation. Nature 373: 620–623. 28. Alberola-Ila, J., K. A. Hogquist, K. A. Swan, M. J. Bevan, and R. M. Perlmutter. Acknowledgments 1996. Positive and negative selection invoke distinct signaling pathways. J. Exp. We thank Dr. Kristin A. Hogquist and Dr. Debra Newman for valuable Med. 184: 9–18. 29. Page`s, G., S. Gue´rin, D. Grall, F. Bonino, A. Smith, F. Anjuere, P. Auberger, and discussion of the data and critical reading of the manuscript. We thank J. Pouysse´gur. 1999. Defective thymocyte maturation in p44 MAP kinase (Erk 1) Dr. B.J. Folks for the kind gift of T3-70 hybridoma. knockout mice. Science 286: 1374–1377. The Journal of Immunology 7

30. Fischer, A. M., C. D. Katayama, G. Page`s, J. Pouysse´gur, and S. M. Hedrick. 42. O’Shea, C. C., T. Crompton, I. R. Rosewell, A. C. Hayday, and M. J. Owen. 2005. The role of erk1 and erk2 in multiple stages of T cell development. Im- 1996. Raf regulates positive selection. Eur. J. Immunol. 26: 2350–2355. munity 23: 431–443. 43. Werlen, G., B. Hausmann, and E. Palmer. 2000. A motif in the alphabeta T-cell 31. Falasca, M., S. K. Logan, V. P. Lehto, G. Baccante, M. A. Lemmon, and receptor controls positive selection by modulating ERK activity. Nature 406: J. Schlessinger. 1998. Activation of phospholipase C gamma by PI 3-kinase- 422–426. induced PH domain-mediated membrane targeting. EMBO J. 17: 414–422. 44. von Boehmer, H. 1990. Developmental biology of T cells in T cell-receptor 32. Schlessinger, J. 1997. Phospholipase Cgamma activation and phosphoinositide transgenic mice. Annu. Rev. Immunol. 8: 531–556. hydrolysis are essential for embryonal development. Proc. Natl. Acad. Sci. USA 45. Teh, H. S., P. Kisielow, B. Scott, H. Kishi, Y. Uematsu, H. Blu¨thmann, and 94: 2798–2799. H. von Boehmer. 1988. Thymic major histocompatibility complex antigens and 33. Coggeshall, K. M., J. C. McHugh, and A. Altman. 1992. Predominant expression the alpha beta T-cell receptor determine the CD4/CD8 phenotype of T cells. and activation-induced tyrosine phosphorylation of phospholipase C-gamma 2 in Nature 335: 229–233. B lymphocytes. Proc. Natl. Acad. Sci. USA 89: 5660–5664. 46. Teh, H. S., H. Kishi, B. Scott, and H. Von Boehmer. 1989. Deletion of auto- 34. Fu, G., Y. Chen, M. Yu, A. Podd, J. Schuman, Y. He, L. Di, M. Yassai, specific T cells in T cell receptor (TCR) transgenic mice spares cells with normal D. Haribhai, P. E. North, et al. 2010. Phospholipase Cgamma1 is essential for TCR levels and low levels of CD8 molecules. J. Exp. Med. 169: 795–806. T cell development, activation, and tolerance. J. Exp. Med. 207: 309–318. 47. Punt, J. A., B. A. Osborne, Y. Takahama, S. O. Sharrow, and A. Singer. 1994. 35. Wang, D., J. Feng, R. Wen, J. C. Marine, M. Y. Sangster, E. Parganas, Negative selection of CD4+CD8+ thymocytes by T cell receptor-induced apo- A. Hoffmeyer, C. W. Jackson, J. L. Cleveland, P. J. Murray, and J. N. Ihle. 2000. ptosis requires a costimulatory signal that can be provided by CD28. J. Exp. Phospholipase Cgamma2 is essential in the functions of B cell and several Fc Med. 179: 709–713. receptors. Immunity 13: 25–35. 48. Gross, B. S., S. K. Melford, and S. P. Watson. 1999. Evidence that phospholipase 36. Ji, Q. S., G. E. Winnier, K. D. Niswender, D. Horstman, R. Wisdom, C-gamma2 interacts with SLP-76, Syk, Lyn,LATandtheFcreceptorgamma- M. A. Magnuson, and G. Carpenter. 1997. Essential role of the tyrosine kinase chain after stimulation of the collagen receptor glycoprotein VI in human substrate phospholipase C-gamma1 in mammalian growth and development. platelets. Eur. J. Biochem. 263: 612–623. Proc. Natl. Acad. Sci. USA 94: 2999–3003. 49. Wen, R., Y. Chen, J. Schuman, G. Fu, S. Yang, W. Zhang, D. K. Newman, and 37. Srinivas, S., T. Watanabe, C. S. Lin, C. M. William, Y. Tanabe, T. M. Jessell, and D. Wang. 2004. An important role of phospholipase Cgamma1 in pre-B-cell F. Costantini. 2001. Cre reporter strains produced by targeted insertion of EYFP development and allelic exclusion. EMBO J. 23: 4007–4017. and ECFP into the ROSA26 locus. BMC Dev. Biol. 1: 4. 50. Smith, M. R., Y. L. Liu, N. T. Matthews, S. G. Rhee, W. K. Sung, and Downloaded from 38. Watanabe, D., S. Hashimoto, M. Ishiai, M. Matsushita, Y. Baba, T. Kishimoto, H. F. Kung. 1994. Phospholipase C-gamma 1 can induce DNA synthesis by T. Kurosaki, and S. Tsukada. 2001. Four tyrosine residues in phospholipase C- a mechanism independent of its activity. Proc. Natl. Acad. Sci. USA 91: gamma 2, identified as Btk-dependent phosphorylation sites, are required for B cell 6554–6558. antigen receptor-coupled calcium signaling. J. Biol. Chem. 276: 38595–38601. 51. Huang, P. S., L. Davis, H. Huber, P. J. Goodhart, R. E. Wegrzyn, A. Oliff, and 39. Kim, Y. J., F. Sekiya, B. Poulin, Y. S. Bae, and S. G. Rhee. 2004. Mechanism of D. C. Heimbrook. 1995. An SH3 domain is required for the mitogenic activity of B-cell receptor-induced phosphorylation and activation of phospholipase C- microinjected phospholipase C-gamma 1. FEBS Lett. 358: 287–292. gamma2. Mol. Cell. Biol. 24: 9986–9999. 52. Bae, S. S., Y. H. Lee, J. S. Chang, S. H. Galadari, Y. S. Kim, S. H. Ryu, and

40. Lee, P. P., D. R. Fitzpatrick, C. Beard, H. K. Jessup, S. Lehar, K. W. Makar, P. G. Suh. 1998. Src homology domains of phospholipase C gamma1 inhibit http://www.jimmunol.org/ M. Pe´rez-Melgosa, M. T. Sweetser, M. S. Schlissel, S. Nguyen, et al. 2001. A nerve growth factor-induced differentiation of PC12 cells. J. Neurochem. 71: critical role for Dnmt1 and DNA methylation in T cell development, function, 178–185. and survival. Immunity 15: 763–774. 53. Ye, K., B. Aghdasi, H. R. Luo, J. L. Moriarity, F. Y. Wu, J. J. Hong, K. J. Hurt, 41. Swan, K. A., J. Alberola-Ila, J. A. Gross, M. W. Appleby, K. A. Forbush, S. S. Bae, P. G. Suh, and S. H. Snyder. 2002. Phospholipase C gamma 1 is J. F. Thomas, and R. M. Perlmutter. 1995. Involvement of p21ras distinguishes a physiological guanine nucleotide exchange factor for the nuclear GTPase positive and negative selection in thymocytes. EMBO J. 14: 276–285. PIKE. Nature 415: 541–544. by guest on September 25, 2021