SD4 : Supplementary Informations on Methods
FACS analysis of phagosomes
Phagosomes were analyzed by FACS according to the method described by
Ramachandra et al9. Briefly, cells were pulsed/chased for indicated times with 3 m latex
beads (Polysciences) and washed 4 times in cold PBS. After homogeneization in HB (3 mM
imidazole, 250 mM sucrose, 1mM DTT, protease inhibitor coktail), post nuclear supernatant
containing phagosomes was fixed with 1% paraformaldehyde-PBS for 10 mn on ice. Fixation
was stopped by addition of PBS-glycine 0,2 M. Then, the post nuclear supernatant containing
phagosomes was resuspended in cold PBS supplemented with 1% FCS, 0.1 % BSA for
staining with antibodies raised against the cytosolic portion of Lamp2, calnexin and TAP2.
Preparations were analysed after gating on a particular FSC/SSC region (corresponding to
single beads in a solution, not shown), which allows the distinction from other organelles. The
presence of membranes surrounding the beads of phagosomes was checked using the DiOc6
lipidic probe in the absence FCS and BSA (over 95% of the beads were stained, not shown).
The proportion of beads with no phagosomes was also estimated using Lamp2 staining (see
figure 1). Over 85% of the phagosomes were stained with Lamp2 antibody, as compared to
the pharmingen isotype matched control, in all the phagosome preparation used (not shown).
For labeling of phagosomes with biotinylated 25-D1.16, D1 cells were pulsed for 30
min and chased for 2 hrs with 3 m latex beads. After disruption of the cells, phagosomes
were fixed with 1% paraformaldehyde as before, and permeabilized with 0.5% TX-100 in
PBS, for 10 min at 4°C. Staining was revealed using a mixture of streptavidin Alexa 633 and
anti-mouse Alexa 647. After indirect staining with 25-D1.16 antibody, or an isotype matched
control, immunofluorescence on 25000 phagosomes containing single beads was analyzed by
FACS as before. Early OVA-phagosomes or OVA-beads (without phagosomes) were
indirectly stained with 25-D1.16 antibody, or an isotype matched control.
Immunofluorescence on phagosomes containing single beads (see the gate in FSC-SSC,
defined with beads alone) was analyzed by FACS.
For the experiment shown in figure 3F, D1 cells were incubated with a mixture of
FITC-BSA-beads and OVA-beads at a 1:1 ratio or with FITC-BSA-beads alone, for 30 min.
Under the mixture conditions used, under 20% of the BSA-beads are derived from cells that
have not phagocytosed an OVA-bead. After gating on single-bead phagosomes, gates of
FITC-positive BSA-phagosomes, or on FITC-negative OVA-phagosomes were performed
independently on the FL1-FSC blots (on 25000 phagosomes). The FL1 intermediate
phagosome population was excluded from the analysis, since it is unclear if this population
corresponds to FITC-BSA phagosomes that have lost part of their labeling (either due to
degradation 9, acidification or transport to the cytosol) or to some OVA-phagosomes that had
acquired FITC-BSA by vesicular exchange with BSA-phagosomes.
The Kolmogorov-Smirnov statistical analysis was performed using Cellquest software
(Becton Dickinson). The difference between the stainings for 25-D1.16 and isotype controls
were calculated, on the OVA- and the BSA-phagosomes from cells that took up both OVAand BSA-beads (mixture), as well as BSA-phagosomes from the cells that took up BSA-beads
alone (BSA alone).
In vitro TAP and loading assays
A TAP transport assay was performed as in Daniel et al.19. Phagosomes purified after
a 30 min pulse of D1 cells with 0.8 m latex beads were incubated in cytosol buffer (0.1%
BSA, MgCl2 2mM, KCl 130mM, 10mM NaCl, 1mM CaCl2, 2mM EGTA, 1mM DTT, 5mM
Hepes, pH7.3) with the iodinated R-10-F* synthetic peptide (RYWANATRSF, 40pmol)
encompassing a N-glycosylation sequence in the presence or not of ATP 2mM. After the
indicated times (figures 4A and C) or after 15 min (figures 4 B and D), phagosomes were
transferred to 4°C, and the excess of unincorporated peptide was eliminated by phagosome
centrifugation at 20 000g. Then, phagosomes were lysed (Tris 50mM, NaCl 150mM, MgCl2
5mM, NP40 1%, protease inhibitors coktails). Glycosylated R-10-F* was immunoprecipitated
from supernatants on ConA-sepharose and quantified after 4 washes in lysis buffer by gamma
counting. The same procedure was used for HLA-A2 loading assay, except that HLA-A2
bound peptide was immunoprecipitated on anti-HLA-A2- or control sepharose. In figure 4G,
loading of S-9-L* was competed by a 100 fold exess of the indicated peptides.