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Blood, Vol. 113, Issue 14, 3198-3208, April 2, 2009
p85β phosphoinositide 3-kinase regulates CD28 coreceptor function
Blood Alcázar et al.
113: 3198
Supplemental materials for: Alcazar et al
T-cell activation protocols
For in vivo studies of the immune response (IR) in p85β−∕− mice, Candida albicans (strain 3153; National Collection of Pathogenic Fungi, Bristol, UK) was used as a pathogen. Briefly, yeast cells were cultured in YPD medium (yeast extract/peptone/dextrose; 24°C, 24 h), harvested by centrifugation, washed twice with phosphate-buffered saline (PBS) and suspended at 5 × 108/ml in PBS. Yeast were heat-inactivated (68°C, 2 h) with continuous movement. To induce primary responses, yeast cells (108/0.2 ml) were injected intraperitoneally (i.p.) into mice. To induce secondary responses, an identical i.p. injection was given 21 days after the first injection. For in vivo studies in p85β−∕− × F5 TCR Tg mice, the NP366–374 influenza peptide (74 nmol/0.2 ml) was injected i.p. once (day 0) or twice (days 0 and 10) for primary and secondary IR, respectively. For in vitro TCR-specific T-cell activation, T-cell–depleted APC (antigen-presenting cell) suspensions were pulsed (37°C, overnight) with influenza peptide NP366–374, (2 µg/ml, except where indicated), then treated with mitomycin C (25 µg/ml; Sigma; 37°C, 1 h) with occasional shaking, and washed three times with PBS. APC were mixed with T cells (1:1) for assays. For biochemical analysis, we incubated purified mouse T cells, Jurkat T cells, or Jurkat transfectants for 2 h in DMEM with 0.1% BSA (bovine serum albumin Fraction V low endotoxin; Sigma); cells were then washed and resuspended in serum-free medium. For short-term activation (≤1 h), 5–10 × 106 cells were incubated in 1.5-ml Eppendorf tubes with soluble anti-CD3 (1 µg/ml) or anti-CD3+anti-CD28 (1 µg/ml each; 15 min, on ice), then crosslinked with secondary antibody (1 µg/ml; 10 min, on ice), followed by incubation for various time periods at 37°C. Cells were collected and processed for analysis. For biochemical and proliferation assays, plastic wells were coated with anti-CD3 (5 µg/ml) or anti-CD3 plus anti-CD28 (2.25 µg/ml each). For parallel comparison of CD3, CD28 and CD3+CD28, cells 10 × 106 cells were treated with anti-CD3 (0.5 µg/ml) or anti-CD28 (2 µg/ml), or both. Coupling of peptides to Actigel resin
Peptides were synthesized by Alta Bioscience (University of Birmingham, UK), and were at least 90% pure as determined by HPLC. We used the following peptides: CD28-Y191* = SRLLHSDYMNMTPRR (M1); CD28-Y191-PO3* = SRLLHSDY(-PO3)MNMTPRR (M1p); CD28-Y218* = KHYQPYAPPRDFAAYRS (M2); CD28-Y218-PO3* = KHYQPYAPPR-DFAAY( PO3)RS (M2p) and PDGFR-Y751(-PO3** = Y(-PO3)VPMLG (PDGFR peptide) (*NP_006130; ** NP_002600). To couple peptides to resin, 500 µl (packed volume) Actigel-ADL superflow resin (Sterogene Bioseparations, Arcadia, CA) was washed 5 times in 100 mM PBS (pH 8; coupling buffer). Peptides (1 mg/ml) were dissolved in coupling buffer and added to the resin, followed by NaCNBH3 (coupling solution; 100 mM final concentration), and mixed (4°C, 16 h). The column was washed with 10 volumes of 500 mM NaCl in100 mM Tris-HCl (pH 8) and uncoupled sites were blocked with Tris-HCl (100 mM; pH 8) for 2 h. In vitro transcription translation, PI3K and PKB kinase assays
Human and mouse p85α and p85β were transcribed and translated in vitro in the presence of 35Smethionine using the TNT T7-coupled reticulocyte lysate system (Promega). PI3K and PKB kinase assays were as described.42 In vitro binding of proteins to peptide columns
Reticulocytes (5 µl) were diluted in binding buffer (50 mM phosphate buffer, pH 7.2; 150 mM NaCl; 0.02% Triton X-100; 2 mM EDTA and 200 mM sodium orthovanadate), and mixed with the appropriate peptide resin (4°C, 2 h, with rotation). The column was washed with 50 volumes of binding buffer. Bound proteins were removed from the resin by boiling in SDS/PAGE sample buffer and analyzed by autoradiography. Immunoprecipitation and Western blot assays
Cells were suspended in lysis buffer (1% Triton-X 100, 50 mM HEPES, 150 mM NaCl, 5 mM NaF, 1 mM sodium orthovanadate, 1 mM PMSF (phenylmethanesulphonylfluoride), 1 mM EDTA (ethylenediaminetetraacetic acid), 1 µg/ml each aprotinin and leupeptin, and 2 nM okadaic acid), and incubated (15–30 min, on ice). After centrifugation (15,000 × g, 15 min, 4°C), supernatants were assayed for total protein (DC protein assay, Bio-Rad) and equivalent amounts of protein for each sample were used for immunoprecipitation analysis or SDS-PAGE. To examine CBL levels, lysates were performed with RIPA buffer (20 mM Tris-HCl pH 8.0, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing phosphatse and protease inhibitors, as above. For immunoprecipitation, protein A Sepharose beads and the appropriate antibodies were used. TCR downregulation analyses
To determine ligand-dependent TCR downregulation, Jurkat cells or suspensions of pooled mouse spleen and lymph node cells were incubated (37°C) without antibody, or with plate-bound anti-CD3 (5 µg/ml) or anti-CD3+anti-CD28 (2.25 µg/ml) for the time specified. For surface staining, mouse cells were incubated with FITC–anti-TCRβ (H57–597), RPE–anti-CD4 (GK1.5) and SPRD–anti-CD8 (53–6.7, all from Pharmingen); human cells were stained with FITC–anti-human–pan-TCRαβ (BMA031, Endogen, Rockford, IL), RPE–anti-human-CD4 (T4) and SPRD–anti-human-CD8 (T8; both from Beckman Coulter). TCR downregulation in CD4+ or CD8+ T cells was determined by FACS analysis. The percentage of TCR downregultion was determined based on the mean fluorescence intensity (MFI) of TCR expression on stimulated vs. unstimulated T cells, as follows: %TCR downregulation (time t) = 100× ((MFI-unstimulated T cells (time t) − MFI-stimulated T cells (time t))/MFI unstimulated T cells (time 0). Flow cytometry
Spleen and lymph node cell suspensions were prepared; erythrocytes were lysed, and cells counted. Cells were surface-stained with saturating concentrations of fluorescein isothiocyanate (FITC), phycoerythrin (PE) or biotin-conjugated antibodies (4°C, 30 min). Biotinylated antibodies were developed with streptavidin SPRD (spectral red; Southern Biotechnology, Birmingham, AL). Antibodies used were CD3 (145-2C11, Beckman Coulter, Fullerton, CA), CD4 (GK1.5, BD Pharmingen, San Diego, CA), L3T4, Southern Biotechnology), CD8α (53–6.7, BD Pharmingen), Lyt-2, Southern Biotechnology), as well as CD44 (IM7), CD62L (MEL-14), CD25 (7D4), and CD69 (H1.2F3; all from BD Pharmingen). Cells were analyzed on an EPICS XL cytometer with System II software (Beckman Coulter). Proliferation assays
Peptide-pulsed APC and T cells were incubated in 96-well U-bottom plates; IL-2 (interleukin 2, 10 U/ml) was added to some wells. After incubation (48 h), 3Hthymidine (1 µCi) was added; cells were harvested 16 h later to measure proliferation. Quantitative RT-PCR
The reverse transcription reaction was performed using 1µg total RNA per reaction with the Archive cDNA synthesis kit for RT-PCR (PE Applied Biosystem). The cDNA synthesis reaction mixture was diluted 50-fold and 500-fold in distilled water before use in quantitative PCR. Primers for PCR reactions were designed using the on-line tool Universal ProbeLibrary Assay Design Center (Roche) to have melting temperatures of 57–62°C, generate PCR products of approximately 100bp and span two different exons. Primer sequences were as follows: human PIK3R1, 5′-CCAGCAGCCGTTTACAGTG-3′, 5′-CGACCTAATAAGCTTCTTCAATAGC-3′; human PIK3R2, 5′-CGAGACCAGTACCTCG-TGTG-3′, 5′-TAATCCCCAGCCACTCGTT-3′; murine PIK3R1, 5′-GAATGTTCG-ACTCTATACAGAACACAA-3′, 5′-CATCTCCAAGTCCACTGACG-3′; murine PIK3R2, 5′-CCGGGACATACGTGGAGTT-3′, 5′-CCAGGTCTGGGAGTATGTGG-3′. Each PCR reaction contained 4µL Fluocycle SYBR Green mix for real-time PCR (Euroclone), 3µL cDNA and 0.3µM of each primer, in a final volume of 8µL. PCR reactions were run in triplicate in an ABI Prism 7900HT Sequence Detection System (PE Applied Biosystems). Initial denaturing time was 10min at 95°C, followed by 40 PCR cycles of 95°C, 15s and 60°C, 90s. Identity of the PCR products was confirmed by melting temperature analysis after the PCR run. Quantitation of mRNA transcripts in the different cell populations was done by the standard curve method. Briefly, known amounts (30, 300, 3000, 30000 and 300000 copies) of the given amplicons were subjected to real time PCR. Threshold cycles (Ct) were plotted against the logarithm of the starting quantities. Amplicon amounts present in the unkown samples were then interpolated from the Ct of the different reactions and expressed as copies per ng of RNA.
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