Present address: Human Development and Health, University of Southampton, Southampton, SO16 6YD, UK.
The pathway of cross-presentation is influenced by the particle size of phagocytosed antigen
Version of Record online: 23 APR 2012
© 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd
Volume 136, Issue 2, pages 163–175, June 2012
How to Cite
Mant, A., Chinnery, F., Elliott, T. and Williams, A. P. (2012), The pathway of cross-presentation is influenced by the particle size of phagocytosed antigen. Immunology, 136: 163–175. doi: 10.1111/j.1365-2567.2012.03558.x
- Issue online: 23 APR 2012
- Version of Record online: 23 APR 2012
- Accepted manuscript online: 19 JAN 2012 09:05PM EST
- Received 19 September 2011; revised 11 January 2012; accepted 13 January 2012.
Figure S1. Control experiment to check the efficacy of MG132 in blocking direct presentation of cytosolic OVA. Both live and fixed cells were incubated with a hypertonic solution of OVA and subjected to a hypotonic shock.
Figure S2. Estimation of cross-presentation yield. (A) Calibration of the fluorescence intensity of an Alexa Fluor 647 F(ab′)2 fragment using QIFIKIT beads loaded with known numbers of mouse epitopes. Fluorescence was determined by flow cytometry. (B) Standard curve derived by plotting mean fluorescence intensity of the stained beads against the number of epitopes per bead. (C) Staining of SIINFEKL-MHC class I complexes on the surface of DC2.4 cells loaded with varying concentrations of exogenous SIINFEKL using the 25-D1.16 antibody. The number of complexes was calculated using the standard curve in (B). (D) Response of B3Z cells to DC2.4 cells loaded with varying concentrations of exogenous SIINFEKL. There is a degree of overlap between the upper range of the B3Z response curve and the lower range of sensitivity of the Kb-SIINFEKL-specific antibody. This overlap was used to translate B3Z response into Alexa Fluor 647 fluorescence intensity.
Figure S3. Distribution of actin around phagocytosed MS. DC2.4 cells were fed yellow-green fluorescent MS shown in green for 30 min, before fixing and staining cellular actin with a phalloidin-Alexa Fluor 647 conjugate (shown in red). Upper panels: cells were fed sonicated 3.0 μm MS. Lower panels: cells were fed aggregated 0.8 μm MS. Actin: single optical sections showing actin alone. Actin, MS: as before, but with the fluorescent MS overlaid. Actin z-stack: actin maximum projection of entire cells. Actin, MS z-stack: as before, but with the fluorescent MS overlaid. DIC: differential interference contrast image. Merge: Superimposition of fluorescence and DIC images. Arrows point to examples of phagosomes surrounded by actin. White bars represent 10 μm.
Figure S4. Translocation of c-Fos from cytoplasm to nucleus after phagocytosis of microspheres. 0.8 μm: DC2.4 cells were fed sonicated 0.8 μm MS according to Experimental Procedures. Cells were fixed and stained 1 hr (upper panels) or 4 hr (lower panels) after the start of the 30 min antigen pulse. C-Fos is shown in green and the nucleus in red. Areas of colocalisation appear yellow. DIC: differential interference contrast image. Merge: overlay of fluorescent and DIC images. 3.0 μm: cells were fed sonicated 3.0 μm MS. 0.8 μm Agg: cells were fed aggregated 0.8 μm MS. White bar represents 10 μm.
Video 1. Time-lapse animation of DC2.4 cells incubated for 24 hr in the absence of microspheres.
Videos 2–4. Time-lapse animations of DC2.4 cells incubated for 24 hr in the presence of OVA-coated 3.0 μm MS.
Data S1. Detailed Materials and methods.
|IMM_3558_sm_FigS1.PDF||21K||Supporting info item|
|IMM_3558_sm_FigS2.PDF||139K||Supporting info item|
|IMM_3558_sm_FigS3.PDF||1614K||Supporting info item|
|IMM_3558_sm_FigS4.PDF||2118K||Supporting info item|
|IMM_3558_sm_Supplemental-Methods.doc||34K||Supporting info item|
|IMM_3558_sm_Video-3.avi||5041K||Supporting info item|
|IMM_3558_sm_Video-4.avi||3106K||Supporting info item|
|IMM_3558_sm_Video-1.avi||3863K||Supporting info item|
|IMM_3558_sm_Video-2.avi||5037K||Supporting info item|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.