Phospholipase 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 genes 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 phosphatase, 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.