Supplemental Data
Supplemental Data for
Celli et al., Immunity , pp.
- Movie S1. CD4+ T Cells Establish Long-Lived Interaction with the First Ag-Bearing DC Encountered
MHC
class II-deficient recipients were injected with DCs isolated from female Ubi-GFP mice (green) and were adoptively transferred
with SNARF-labeled Marilyn CD4+ T cells (red). After 24 hr, mice were prepared from intravital imaging of the popliteal LN.
Time-lapse images were acquired with two-photon microscopy. After 11 min of imaging, 50 μg of Dby peptide was injected
intravenously through the jugular vein. Imaging was then continued for 1 hr.
- Movie S2. Zoomed-in
View of CD4+ T Cell-DC Interactions Early after Peptide Injection
CD4+ T cells and DCs are shown in red and green,
respectively.
- Movie S3. CD4+ T Cells Change Behavior after Peptide Injection
Comparison
of CD4+ T cell behavior before (left panel) and after (right panel) peptide injection. White circles indicate the positions
of long-lived interactions.
- Movie S4. Early CD4+ T Cell-DC Contacts Are Maintained at 2 hr
The
experiment was performed as described in the legend of Movie S1. Image acquisition was started 95 min after peptide injection.
- Movie S5. Early CD4+ T Cell-DC Contacts Are Maintained at 6 hr
The experiment was performed as
described in the legend of Movie S1. Image acquisition was started 6 hr after peptide injection.
- Movie
S6. Early CD4+ T Cell-DC Contacts Are Also Observed with OT-II T Cells
MHC class II-deficient recipients were
injected with DCs isolated from female Ubi-GFP mice (green) and were adoptively transferred with SNARF-labeled OT-II CD4+
T cells (red). After 24 hr, mice were prepared from intravital imaging of the popliteal LN. Time-lapse images were acquired
with two-photon microscopy. After 6 min of imaging, 50 μg of OVA323-339 peptide was injected intravenously through
the jugular vein.
- Movie S7. The Formation of Prolonged CD4+ T Cell-DC Contact Is Antigen Specific
MHC
class II-deficient recipients were injected with DCs isolated from female Ubi-GFP mice (green) and were adoptively transferred
with SNARF-labeled Marilyn T cells (red) and OT-II T cells (yellow) labeled with a mixture of CFSE and SNARF. After 24 hr,
mice were prepared from intravital imaging of the popliteal LN. Time-lapse images were acquired with two-photon microscopy.
During imaging, 50 μg of Dby peptide was injected intravenously through the jugular vein.
- Movie
S8. Comparison of Marilyn and OT-II CD4+ T Cell Behavior before and after Peptide Injection
White circles indicate
the positions of long-lived T cell-DC interactions.
- Movie S9. CD4+ T Cells Establish Long-Lived
Interactions with Male DCs in the Initial Stages of the Activation Process
B6 recipients were injected with DCs
isolated from male Ubi-GFP mice (green). After 24 hr, 20 × 106 SNARF-labeled Marilyn CD4+ T cells (red) were adoptively transferred.
Mice were immediately prepared for intravital two-photon imaging, and time-lapse imaging was started 1–2 hr after T cell transfer.
White circles indicate the positions of long-lived T cell-DC interactions.
- Movie S10. Visualizing
the Early Recruitment of CD4+ Marilyn T Cells by Male DCs
The movie shows an example of a crawling CD4+ T cell
encountering a male DC in the early phases of the activation process and subsequently establishing a long-lived interaction
with this DC. This experiment was performed with B6 recipients.
- Movie S11. Early Recruitment of
CD4+ Marilyn T Cells by Male DCs Also Occurs in MHC Class II-Deficient Recipients
The movie shows CD4+ T cells
establishing long-lived interactions with a male DC in the early phases of the activation. In addition, a crawling T cell
is seen encountering this DC and establishing a long-lived interaction. This experiment was performed with MHC class II-deficient
recipients.
- Movie S12. Disruption of Stable T Cell-DC Interactions In Vivo after the Injection
of MHC Class II Ab
MHC class II-deficient recipients were injected with DCs (green) isolated from female Ubi-GFP
mice and were adoptively transferred with SNARF-labeled Marilyn CD4+ T cells (red). After 24 hr, mice were injected intravenously
with 50 μg of Dby peptide. Mice were prepared for intravital two-photon imaging of the popliteal LN. Two hours after
peptide injection, 500 μg of Y3P MHC class II Ab (right panel) or a control Ab (left panel) was injected intravenously
through the jugular vein. Imaging was performed 2 hr after Ab injection. Circles indicate the presence of stable T cell-DC
interactions. White dots show motile T cells establishing only transient contacts with Ag-bearing DC.
