Mice
Mice were housed and experiments performed according to the institutional guidelines for animal experimentation of the Peter MacCallum Cancer Centre. PTPRCA-Ly5.1+ mice and severe combined immunodeficiency mice were purchased from the Walter and Eliza Hall Institute of Medical Research (Melbourne, VIC, Australia). Dlg1+/gt12, Lgl1+/- (13), FVBN/C57Bl/6-Scribble+/null-deficient or C57Bl6 Scribble+/null11 and HEL Rag SHH/+ SHL/− Rag−/− mice (SWHel)38 were bred and maintained at the Peter MacCallum Cancer Centre.
Generation of chimeric mice
Timed matings were established between heterozygote polarity-deficient animals. Embryos were harvested at embryonic day 14.5 and genotyped by PCR on single-cell suspensions from fetal livers. PTPRCA-Ly5.1+ or severe combined immunodeficiency recipients received two doses of 5.5 Gy administered greater than 3 h apart. 1 × 106 fetal liver cells were injected intravenous (i.v.). Reconstituted mice were maintained on neomycin sulphate-supplemented drinking water for 6 weeks post irradiation. Six to ten weeks post reconstitution peripheral blood was isolated from mice and stained with combinations of Ly5.2, CD3, B220, CD19, IgM and IgD to determine reconstitution efficiency.
NP-KLH Immunization and enzyme-linked immunosorbent assay
In vivo immunization and detection of NP-specific antibody via enzyme-linked immunosorbent assay was performed as described in Hagn et al.41
Cell culture of mouse B cells
Mouse B cells were prepared, CFSE labelled and cultured as described in Hawkins et al.37 Cell numbers were determined by reference to fluorescence-activated cell sorting calibration beads as described in Hawkins et al.20 B cells were typically >95% B220+, CD19+, IgM+, IgD+ as determined by flow cytometry. Stimuli were added as follows: lipopolysaccharide derived from Salmonella typhosa (LPS, Sigma-Aldrich, St Louis, MO, USA) and anti-CD40 antibody (clone FGK4.5, WEHI) were at used at 20 μg ml−1. IL-4 (R&D Systems, Minneapolis, USA) was added at 20 ng ml−1. In SWHEL-transgenic B cell cultures, HEL (Sigma-Aldrich) and FGK4.5 was bound to 4.5 μM epoxy beads as described previously42. For in vitro differentiation studies, B cells were fixed in 2% PFA for 30 min at 4 °C and permeabilized in 0.01% Tween-20 overnight then, stained for expression of CD138 (clone 281-2, BD Pharmingen), IgM or IgG1.
B cell-bead immuno-fluorescence
B cell-bead conjugates were fixed at 5, 24 or 48 h post incubation and prepared as described in Oliaro et al.15 Proteins of interest were detected with rabbit anti-Scribble (Santa Cruz, Cat. no. sc-28737), rabbit anti-tubulin (Rockland, Cat. no. 600-401-880), together with rat anti-mouse IgM (BioLegend, Cat. no. 406508) followed by the secondary antibodies (anti-rabbit-Alexa-488 and anti-rat Alexa-546, Molecular Probes). Cells were imaged using a Fluoview FV1000 confocal microscope (Olympus, Japan) with a 12.9 mW 488 nm multi-ion Argon laser, 1 mW 543 nm multi-ion green HeNe laser and 11 mW 633 nm red HeNe laser. All images were captured using a PlanApoN × 60 oil immersion objective (NA=1.42). IgM polarization to the bead was used to select conjugates for scoring of the localization of Scribble.
Live cell differentiation assay
B cells were isolated and cultured in LPS for 24 h using the conditions described above. After 24 h, B cells were serially diluted directly into eight-well glass chamber slides (Ibidi, Munich, Germany) with 125 × 125 × 60 μM micro-paddocks (Daniel Day, Swinburne University, Hawthorn, VIC 3122, Australia). B cells were cultured as described above with 20 μg ml−1 LPS and 80 ng ml−1 anti-CD18-PE antibody (BD Pharmingen) and imaged for 4 days in an environment-controlled microscopy chamber maintained at 37 °C and 10% CO2. Bright-field images were acquired at 5 min intervals and PE images were acquired at 15 min time intervals. Post acquisition, proliferation (cell division), death (morphology) and differentiation (acquisition of PE+ staining) was quantified by manually.
Influenza infection
Viral infections and assays were performed as previously described on mice maintained on a C57Bl/6 background30. Briefly, mice were inoculated intranasally with 104.5 p.f.u. of the HKx31 (H3N2) influenza virus28,43. To measure recall responses, mice were first inoculated i.p. with 107 p.f.u. of the heterologous A/PR/8/34 (H1N1, PR8, Mt Sinai strain) influenza virus and then challenged intranasally with 104.5 p.f.u. of HKx31 6–8 weeks later28,43. Antiviral T-cell responses were enumerated by staining with PE-labelled MHC class-I tetrameric complexes specific for the nucleoprotein (DbNP366-374) (28,43). To measure antigen-specific cytokine expression, splenocytes enriched for CD8+ T cells were stimulated in the presence of 1 μM DbNP366-374 (ref. 44) peptide and 1 μg ml−1 Brefeldin A (Epicenter Biotechnologies) for 5 h. Cells were then stained for relevant surface molecules, fixed and permeabilized with the Cytofix/Cytoperm reagent (BD Biosciences) and costained for interferon-γ.
Western blotting
Cell lysates were prepared as follows. For polarity proteins, cells were washed twice in PBS and lysed in 100 mM NaCl, 20 mM Tris-Cl (pH 8.0), 0.5 mM EDTA and 0.5% (v/v) NP-40 in the presence of complete-Mini protease inhibitors (Roche, Mannheim, Germany). For phospho-ERK detection, cell pellets were lysed in 10% SDS, 30% Glycerol, 250 mM Tris-HCL pH 6.8 in the presence of Mini protease inhibitors and PhosStop, complete phosphatase inhibitors (Roche, Mannheim, Germany). Protein content was determined using a Bradford protein assay kit (Bio-Rad, Hercules, Ca). Lysates were boiled in either 2:1 ratio of 14 M βHYPERLINK \l "M (Sigma) to 2% bromophenol blue stock or 250 mM Tris-HCL pH 6.8, 50% glycerol W/V, 10% SDS W/V, 2.5% β-mercaptoethanol with bromophenol blue for phospho-ERK or polarity protein detection, respectively, then size-fractionated on polyacrylamide gels, and transferred to nitrocellulose membranes by electroblotting. Non-specific binding of antibodies was blocked by incubation in 5% skimmed milk and 0.05% Tween-20. For polarity proteins, the following antibodies were used: anti-Par3 (Rabbit—Upstate Biotechnology, NY, USA), Dlg-family—PSD-95 family (Upstate Biotechnology, NY, USA), PINS/GPSM2/LGN (Lifespan Biosciences, Seattle, WA, USA) and Scribble mouse monoclonal C6-D10 (ref. 44). Loading was determined by probing the same membrane for anti-tubulin (Cell Signalling, Danvers, MA, USA). For phospho-ERK detection, membranes were first probed with mouse anti-mouse phospho-ERK (Cell Signalling, Danvers, MA, USA Cat. no. 9106). For loading, membranes were stripped with 50 mM Tris-HCL pH 6.8, 2% SDS, 0.1 mM β-mercaptoethanol at 50 °C for 30 min, washed and membranes blocked overnight in 5% skimmed milk and 0.05% Tween-20, then re-probed with rabbit anti-mouse Total ERK (Cell Signalling, Danvers, MA, USA Cat. no. 9102). Bound antibodies were visualized by enhanced chemiluminescence (Amersham Pharmacia).
Immunohistochemistry
Immunohistochemistry was performed as described in Smith et al.40 Antibodies used were unlabelled rat anti-GL-7 (ref. 40) and biotinylated anti-IgD (Southern Biotech, Birmingham, Alabama, USA). GL-7 was detected with anti-rat HRP (Invitrogen) and biotinylated anti-IgD with streptavidin-Alkaline-phosphatase in subsequent steps.
In vitro activation of F5 CD8 T cells for analysis of ACD
F5 CD8+ T cells were prepared from lymph node preps of F5.Rag1KO mice and activated with endogenous antigen-presenting cells where specified. T cells were labelled with 2.5 μM of CellTrace Violet (CTV, Invitrogen) as described previously32. Cells were seeded into culture at 1 million cells 1 ml−1 per well in 24-well plate then activated with different combinations of NP68 (10 nM, Mimotopes), anti-CD3 (145-2C11, eBioscience, either plate-bound at 10 μg ml−1 or in solution at 0.5 μg ml−1), anti-CD28 (37.51, eBioscience, plate-bound at 10 μg/ml) and recombinant ICAM1-Fc fusion (R&D Systems, plate-bound at 10 μg ml−1). When indicated, cytochalasin B (10 nM, SIGMA) was added for the final 4 h of the culture period as described by others14,16. Cells were cultured for up to 72 h at 37 degrees and 5% CO2. At point of harvest, cells were first stained for CD8a (53-6.7, FITC conjugated, eBioscience) and the LIVE/DEAD fixable near infrared dye (Invitrogen) followed by fixation for 30 min in IC fixation buffer (eBioscience). Cells were then permeabilised and stained with goat anti PKC-zeta (Santa Cruz, sc-216-G) and an AF647 labelled donkey anti-goat secondary (Invitrogen).
Flow cytometry
For CFC analysis, cells were stained with 7-Aminoactinomycin D and run on a 3 laser, 8 detector CANTO-II (BD). Analysis was performed in flowJo (Tree Star, USA) by gating on singlet lymphocytes (FSC/SSC gate then FSC-A/FSC-W gate) and then live cells based on LIVE/DEAD fixable near infrared dye signal exclusion. For IFC analysis the optimum harvest window was established by measuring commitment to cycling and first division using CTV dilution, 7-AAD and Ki-67 (Supplementary Fig. S5). Cells were stained as for CFC but then labelled with propidium iodide (1 μg ml−1) and acquired on a fully calibrated ImageStream X (ISx, Amnis, USA) system as described previously using × 40 magnification32,46). IFC analysis was performed using IDEAS software (Amnis) and Image J (v1.43, NIH, USA) as described previously25,32. Briefly, IDEAS was used to identify live mitotic cells with a known divisional history based on CTV signal dilution, bright-field and nuclear imagery, and up to nine objective pixel intensity-based features32. Multispectral images of CD8, PKC-zeta and CTV were the exported for each fully validated mitotic image and analysed in Image J for signal distribution (% of total) across the plane of cytokinesis25,32 (Supplementary Fig. S6). Polarity index was calculated using the following equation: (Square root ((%-50) squared)))/50. The threshold for asymmetry was determined using the internal control of the total protein signal as defined by CTV binding to amine groups to account for the limits of inherent protein asymmetry33,34, as well as any potential issues with imaging cells in flow32.