Supporting Online Material


Abstract
Full Text
Impaired B and T Cell Antigen Receptor Signaling in p110Greek Letter Delta PI 3-Kinase Mutant Mice
Klaus Okkenhaug, Antonio Bilancio, Géraldine Farjot, Helen Priddle, Sara Sancho, Emma Peskett, Wayne Pearce, Stephen E. Meek, Ashreena Salpekar, Michael D. Waterfield, Andrew J. H. Smith, Bart Vanhaesebroeck

Supporting Online Material

Methods

Reagents

Antibodies against the PI3K subunits have been described previously (1, 2). Antibodies against the following cell surface markers were from BD Pharmingen: CD3Greek Letter Epsilon (145-2C11), CD4 (L3T4), CD8Greek Letter Alpha (53-6.6), CD21 (7G6), CD23 (B3B4), CD28 (37.51), CD44 (IM7), CD45/B220 (RA6-6B2), CD40L (MR1), IgM (AF6-78). Anti-Akt/PKB (pSer473 and total) were from Cell Signaling Technologies, anti-Erk (pTyr204, (E-4)) from Santa Cruz Technologies, anti-Erk (total) from Promega, anti-IgM F(ab�)2 anti-hamster IgG were from Jackson ImmunoResearch, anti-actin (AC-15) and rabbit anti-mouse Ig from Sigma. rIL-2 and rIL-4 were from R&D Systems. LPS, PdBu, ionomycin and anti-actin were from Sigma. Polystyrene beads were from Polyscience Inc. Indo-1 and BCECF-AM were from Molecular Probes. IL-2 concentrations were measured using an ELISA kit from BD Pharmingen. Ig isotype titers were determined by ELISA (Southern Biotech). TNP-KLH, TNP-Ficoll, and TNP-Ova were from Biosearch Technologies, and Alum from Serva.

Mice

Mice were kept in individually ventilated cages and cared for according to Home Office regulations. The mice used in these experiments were backcrossed to the C57BL/6 background for 3-5 generations, and WT littermates were used as controls. The DO11-10 mice (3) were provided to us by Claire Lloyd, Imperial College London. The p110Greek Letter DeltaD910A/D910A DO11-10 mice had been backcrossed on the Balb/C background for 5 generations.

Cell purification

B and T cells were purified from spleen and/or lymph nodes, as indicated, by positive or negative selection, using the following antibody-coated magnetic beads from Miltenyi Biotec: anti-MHC class II, anti-CD8, anti-CD43, anti-CD90.

PI3K assay

Lipid kinase assays were performed essentially as described (1). Briefly, p110Greek Letter Alpha, p110Greek Letter Beta or p110Greek Letter Delta were immunoprecipitated using specific antibodies or non-immune control antibodies and subjected to an in vitro kinase reaction using PtdIns(4,5)P2 as a substrate. Alternatively, tyrosine-phosphorylated PDGF-R peptide coupled beads were used to precipitate total class IA PI3K activity as described previously (4). The lipids were resolved by thin-layer chromatography and quantified on a Molecular Imager FX (Bio-Rad). The relative radioactivity of the PtdIns(3,4,5)P3 spots, after subtracting background values from immunoprecipitates made using control antibodies (rabbit anti-mouse Ig), are expressed as arbitrary PhosphorImager units (a.u.).

Flow cytometry and Ca++ analysis

Cell surface expression patterns were analyzed in 3 or 4 colors on a FACSCalibur instrument (Becton Dickinson). For Ca++ analysis, spleen or lymph node cells were incubated with 1 Greek Letter Mug/ml Indo-1 at room temperature for 45 min. The cells were surface-stained for B220, CD4 and CD8 and six color analysis was performed using a BD LSR instrument to determine the ratio of bound to free dye in the lymphocytes as a function of the amount of free Ca++ in the cytosol (expressed as the ratio of Greek Letter Lambda401 (high calcium) to Greek Letter Lambda475 (zero calcium)).

Proliferation assays

Purified B or CD4+ T cells were incubated at 2x105 cells per well in 96 well plates with the indicated stimuli in 200 Greek Letter Mul media (RPMI, 10% FCS, 5 x 10-5 M Greek Letter Beta-mercaptoethanol, antibiotics). [3H]-thymidine was added for the last 6 h of a 48 h culture, and the cells were harvested and incorporated [3H]-thymidine incorporation quantified by scintillation counting. DO11-10 T cells were incubated with 2x105mitomycin C (50 Greek Letter Mug/ml, 30 min) treated purified Balb/C B cells.

Adhesion assay

Purified spleen and lymph nodes T cells were labeled with BCECF-AM. After pre-incubation with 1 Greek Letter Mug/ml of soluble anti-CD3 for 30 min at 37°C, cells were transferred to 96-well microtiter plates coated with human fibronectin (Gibco) or mouse ICAM-1 (gift from Andrew Smith and Nancy Hogg, CR UK) and stimulated by crosslinking with anti-hamster antibodies at 15 Greek Letter Mug/ml for 30 min at 37°C. The non-adherent cells were then removed by filling the wells with culture medium and inverting the plates for 15 min at 37°C. Adherent cells remaining in the wells were quantified by measuring BCECF fluorescence.

Raft recruitment

Following stimulation, the cells were fixed and stained with FITC-cholera toxin B subunit (Sigma), which labels the GM1 ganglioside enriched in lipid rafts, as described (5, 6). The efficiency of raft recruitment was evaluated by visual analysis of about 50 to 100 cell/bead complexes for each experiment, using a fluorescent microscope. In unstimulated cells, a similar amount of GM1 was distributed equally over the cell surface in WT and p110Greek Letter DeltaD910A/D910A T cells, as assessed by confocal microscopy and FACS analysis (not shown).

Immunization

Four WT and 4 p110Greek Letter DeltaD910A/D910A mice were immunized with 100 Greek Letter Mug TNP-KLH adsorbed to alum or with 100 Greek Letter Mug TNP-Ficoll in PBS. Serum obtained from tail bleeds on day 12 was serially diluted in 96-well plates coated with Ova-TNP, followed by detection of TNP-specific antibodies by ELISA. The EC50 values were determined by non-linear regression analysis of the dilution curves using Prism 3.0 software (GraphPad).

Histological analysis

Tissue sections from more than 50 organs were examined. Details can be found at http://www.frimorfo.com/Morphomeprogrmas/MorphomeGoldHistomorphology.htm


Supplemental Figure 1. Gene targeting. Two genomic clones covering the coding region of the p110Greek Letter Delta gene, Pik3cd, were isolated from a 129/Ola genomic library. DNA sequencing revealed that the p110Greek Letter Delta gene is encoded by 22 coding exons (sequence deposited to GenBank; accession number AF532989). A knock-in vector containing the last 4 exons of the Pik3cd locus was constructed and designed to introduce a point mutation, converting the conserved DFG motif in the C-terminal p110Greek Letter Delta kinase domain to AFG. Two additional features were incorporated into the targeting vector: a sequence encoding a Myc-epitope tag introduced immediately 5� of the stop codon (to allow for recognition of the mutated protein with epitope-specific antibodies) and a reporter/selection cassette flanked by loxP sites inserted into the 3� UTR sequence. This cassette contained an IRES sequence followed by the Greek Letter Beta-galactosidase (lacZ) coding sequence and a neomycin resistance gene expressed from its own promoter (MC1neopA). Note also that a vector-derived HindIII site was introduced just 5� of the cassette insertion site.


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The targeting construct was transfected into E14Tg2a embryonic stem (ES) cells, and the transfected cells subjected to G418 selection. Targeted clones were identified by Southern blot analysis of AseI and HindIII-digested DNA using probes (D77-78 and D43-39) flanking the 5� and 3� sides of the vector integration positions respectively, at a frequency of around 25%. Sequencing was used to confirm the presence of the AFG-encoding mutation. Male chimeras generated from 3 independently derived clones with the AFG-encoding mutation were bred with C57BL/6 females and germ line transmission confirmed by Southern blot analysis of tail DNA. These lines were backcrossed onto a C57BL/6 background for 3-5 generations. Deletion of the selection cassette was achieved by intercrossing p110Greek Letter Delta+/- mice with C57BL/6 mice carrying a Cre-recombinase transgene (7), and verified by Southern blot analysis, as shown, and PCR (not shown).

(A) Organization of the p110Greek Letter Delta gene. DFG represents the conserved amino acid motif in the C-terminal p110Greek Letter Delta kinase domain. (B) Linearized p110Greek Letter Delta targeting vector showing the AFG-encoding mutation. (C) Targeted p110Greek Letter Delta allele. (D) Targeted p110Greek Letter Delta allele after Cre recombinase-mediated deletion of the reporter/selection cassette. (E) Southern blot analysis of HindIII-digested genomic DNA from tails of pups derived from p110Greek Letter Delta+/-matings of mice with and without the reporter/selection cassette using 3� flanking probe D43-39. Analysis revealed a HindIII restriction site polymorphism between 129/Ola and C57BL/6 mice. Southern blot analysis detects a 7 kb HindIII WT fragment in C57BL/6 mice.

Key: Exon sequences are represented by filled black rectangles, intron sequences by a thick black line and plasmid sequence by a thin black line. Restriction sites are A (AseI), H (HindIII), M (MscI) and S (ScaI) and the relevant restriction fragments highlighted by a dotted line with arrowheads. Probe fragments are represented by gray lines. The Myc tag is shown as a filled red rectangle, the floxed IRES/lacZ/MC1neopA cassette as gray, blue and green filled rectangles respectively and the loxP sites as pink triangles with the pointed end indicating orientation.


Supplemental Table 1. Peripheral blood cell counts.
WT/WT mean ± SD (n = 7) D910A/D910A mean ± SD (n = 8)
Erythrocytes (x106/Greek Letter Mul) 9.45 ± 0.459.53 ± 0.63 (n.s.)
Total leukocytes (x103/Greek Letter Mul) 5.39 ± 1.38 7.73 ± 1.87 (P < 0.05)
Segmented neutrophils/Greek Letter Mul 719 ± 1991447± 406 (P < 0.01)
Eosinophils/Greek Letter Mul 94± 93 60± 79 (n.s.)
Basophils/Greek Letter Mul 30 ± 31 43 ± 48 (n.s.)
Monocytes/Greek Letter Mul 100 ± 61 216 ± 126 (n.s.)
Lymphocytes/Greek Letter Mul 4439 ± 1245 5955 ± 1551 (n.s.)


P values were calculated using the non-parametric Mann-Whitney U test (comparisons with WT mice). P values Less than or Equal to Sign 0.05 were considered to be statistically significant. The number of animals in each group is indicated by n. n.s. = not significant.

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