cells. Taken together, these data suggest that RhoH regulates TCR signaling via recruiting ZAP-70 and Lck to CD3f in the immunological synapse for activation of downstream signaling molecules. Materials and Methods Mice and T lymphocyte isolation 129Sv Rhoh-/- mice were generated by standard DNA homologous recombination and backcrossed in a C57BL/6J background as described previously[3]. p14tg/+; Rhoh-/- mice expressing p14 TCR (Va2/Vb8) transgene in a compound 129Sv and C57BL/6J background were generated as described previously[3]. Animals used were littermates derived from N20 backcross generation. 129S6/SvEvTac-Rag2-/- mice were pur- chased from Taconic Animal Models (Hudson, NY). All experiments involving animals were approved by the Institutional Animal Care and Use Committee of the Cincinnati Children’s Research Foundation. Single cell suspensions were generated from dissected thymus and lymph node. T cells were purified by negative selection using the Pan T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA). The purity of isolated T cells in all experiments was greater than 90%, as assessed by flow cytometry with anti-mouse CD3e Ab. For TCR activation, thymocytes or T cells were incubated with anti-mouse CD3e mAb (2c11, BD Pharmingen, San Diego, CA) and anti-mouse CD28 mAb (37.51, eBioscience, San Diego, CA) (both at 5 mg/107 cells). Cross-linking of mAbs was accomplished using anti-Hamster IgG Abs (5 mg/107 cells, BD Pharmingen). After incubation for 2 min at 37uC, cold phosphate-buffered saline (PBS, 10 vol) was added to stop the activation. Retroviral vector construction and retrovirus supernatant generation Myr-ZAP-70 was constructed by the addition of the avian src myristoylation sequence (MGSSKSKPK) at the N terminus of ZAP-70 cDNA[11] (kindly provided by Dr. Andrey Shaw, Washington University). Myr-ZAP-70 was amplified using a human full-length ZAP-70 cDNA with the following primers; (forward) 59-GACGAATTCATGGGCAGCAGCAAGAGCAA- GCCCAAGCCCGATCCCGCGGCGCAC-39 (MGSSKSKPK coding sequence was underlined), (reverse) 59-ACCGTCGACT- CAGCCACATGCAGCCTCGGCCACCTGT-39. Amplified PCR products were subcloned in pSF91 bicistronic retroviral vector expressing enhanced green fluorescence protein (EGFP) at EcoRI and SalI sites[12]. To produce the retroviral vector expressing wild type ZAP-70, the EcoRI/SalI fragment of full-length ZAP-70 in pGEM3Z was ligated into pSF91. The sequence and orientation of the DNA construct was verified by DNA sequencing. The MIEG3-HA-RhoH vector was described previously[6]. Recombi- nant retroviruses were produced using the ecotropic Phoenix packaging cell system[13]. Briefly, 8 mg retroviral vector plasmid DNA, 10 mg Moloney leukemia virus (MLV) gag-pol plasmid, and 3 mg ecotropic envelope plasmid were cotransfected into Phoenix cells using the CaPO4 coprecipitation procedure (Invitrogen, Carlsbad, CA). Retroviral supernatants were collected every 12 hours. Titer of recombinant retrovirus was determined by infecting NIH3T3 cells using serial dilution[14]. Mouse bone marrow transduction and reconstitution of RAG2-/- mice Low density bone marrow (LDBM) cells were harvested 4 days after 5-fluorouracil (5-FU, 150 mg/kg) injection. Retrovirus- mediated transduction of mouse LDBM cells was performed as RhoH as an Adaptor for ZAP-70 described previously[15]. Briefly, LDBM cells were cultured for 2 days in Iscove’s Modified Dulbecco’s Medium (Invitrogen) supplemented with 10% fetal calf serum (FCS, HyClone, Logan, UT), 2% penicillin and streptomycin (P/S), and 100 ng/ml each of recombinant rat stem cell factor (SCF), megakaryocyte growth and development factor (MGDF) and granulocyte colony- stimulating factor (G-CSF) (all from Amgen, Thousand Oaks, CA). Pre-stimulated LDBM cells were infected twice with the high-titer retrovirus supernatant on fibronectin fragment CH296 (kindly provided by Takara Bio, Otsu, Japan). EGFP+ cells were sorted by a fluorescence-activated cell sorting (FACS) Vantage Sorter (BD Biotechnology, Franklin Lakes, NJ) and were injected intravenously into the sub-lethally irradiated (300 Rads using a 137Cs irradiator) Rag2-/- recipient mice. At 8 weeks post transplantation, single cell suspensions were prepared from thymus and subjected to flow cytometric analysis. Antibodies The following antibodies were used in immunoblot analyses at 1:1000 dilution: anti- ZAP-70 (1E7.2) and anti-phosphotyrosine (4G10) (Millipore, Billerica, MA); anti-HA (3F10) (Roche, Indianapolis, IN); anti-b-actin (AC15) (Sigma, St Louis, MO); anti-CD3f (3F67) and anti-Lck (3A5) (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-Src Tyr416 (2101), anti-phospho- ZAP-70 Tyr319 (2701), anti-phospho-ZAP-70 Tyr493 (2704), anti-Lck (2752), anti-ZAP-70 (99F2), anti-phospho-LAT Tyr191 (3584)anti-phospho-p44/p42 Thr202/Tyr204 (9101) and anti- p42/p44 (9102) (Cell Signaling Technology, Beverly, MA). Primary antibodies were detected with horseradish peroxidase- conjugated goat anti-mouse (Cell Signaling Technology), or goat anti-rabbit (Cell Signaling Technology), donkey anti-rat (Santa Cruz Biotechmology) or donkey anti-goat secondary (Santa Cruz Biotechmology) antibodies (1:3000 dilution) with enhanced chemiluminescence detection (Cell Signaling Technology) The following fluor-conjugated monoclonal anti-mouse antibodies (mAb) were used for flow cytometry: CD4 (RM4-5), CD8a (53- 6.7), and CD3e mAb (2c11) (all from BD Pharmingen). T cell–APC conjugation and immunofluorescence staining Lymph node cells were isolated from WT and Rhoh-/- p14 transgenic mice and positively selected for CD8+ T cells by a magnetic bead cell selection technique (Miltenyi Biotec, Auburn, CA). For positive selection of CD8+ T cells, lymph node T cells were labeled with biotinylated anti-CD8a mAb. These cells were then further incubated with anti-biotin magnetic beads (Miltenyi Biotec) and purified in accordance with the manufacturer’s recommendations. Antigen-presenting (APC) cells (CHB.2B B cell lymphoma cell line)[16] were pre-loaded with 1 mg/ml of gp33 peptide for 12 hours. The pre-loaded CHB.2B cells were mixed with CD8+ T cells from WT and Rhoh-/- p14 transgenic mice and then layered onto poly-L-lysine-coated coverslips. After 5 min, cells were fixed with BD Cytofix/Cytoperm (BD Biosciences, San Jose, CA)) and stained with Rhodamine (TRITC)-labeled phalloidin (Molecular Probes, Eugene, OR), anti-ZAP-70 (Cell Signaling) or anti-Lck (3A5, Santa Cruz Biotechnology) followed by anti–rabbit Alexa488 or anti-mouse Alexa555 (Molecular Probes). For the localization of Lck and ZAP-70 in Rhoh-/- T cells expressing Myr-ZAP-70, pan-T cells were isolated from LN by using Pan-T Cell Isolation Kit (Miltenyi Biotec). T cells were transduced with retroviruses co-expressing EGFP and Myr-ZAP- 70 or EGFP alone as described previously[3]. EGFP+ T cells were sorted with a FACSVantage and then were cultured without anti- CD3e Ab stimulation (IMDM with 10% FCS, 20 ng/ml IL2 PLoS ONE | www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e13970