The bullseye synapse formed between CD4+ T
cells and staphylococcal enterotoxin B -pulsed
DCs plays a suppressive role in the CD4+
T-cell activation
Wei Lin1,3*, Zhichao Fan2*, Yuanzhen Suo2, Yuting Deng1,3,Min Zhang1,3, Jiyang Wang1,3,
Xunbin Wei2**, Yiwei Chu1,3**
Supplementary Figure 1. The effect of H57-597 mAb Alexa Fluor 647 antibody on T-cell
activation. (A) The expression of CD25 was measured in CD4+ T cells labelled or unlabelled with
H57-597 mAb after T cells contacted SEB-pulsed DCs for 8 h and compared to T cells without
DC contact (left panel). Percentage of CD25+ T cells among the CD4+ T cells labelled or
unlabelled with H57-597, or control cells, was quantitatively analysed (right column). (B) The
expression of CD69 was measured in CD4+ T cells labelled or unlabelled with H57-597 mAb after
CD4+ T cells contacted SEB-pulsed DCs for 8 h and compared to CD4+ T cells without DC
contact (left panel). Percentage of CD69+ T cells among the CD4+ T cells labelled or unlabelled
with H57-597, or control cells, was quantitatively analysed (right column). (C) The expression of
Ki67 was measured in CD4+ T cells labelled or unlabelled with H57-597 mAb after CD4+ T cells
contacted SEB-pulsed DCs for 24 h and compared to CD4+ T cells without DC contact (left panel).
Percentage of Ki67+ T cells among the CD4+ T cells labelled or unlabelled with H57-597, or
control cells, was quantitatively analysed (right column). (D) The expression of PI was measured
in CD4+ T cells labelled or unlabelled with H57-597 mAb after CD4+ T cells contacted
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SEB-pulsed DCs for 24 h and compared to T cells without DC contact (left panel). Percentage of
PI+ T cells among the CD4+ T cells labelled or unlabelled with H57-597, or control cells, was
quantitatively analysed (right column). SEB = 100 ng/ml. The means ± s.e.m. of three independent
experiments are shown. ***p<0.001 (two-tailed Student’s t-test).
Supplementary Figure 2. The accumulation of CD28 in a bullseye IS, a multifocal IS or a
non-specific synapse. Bullseye IS and multifocal IS were formed at the interface of SEB-specific
CD4+ T cells and DCs. Non-specific synapse was formed at the interface of non-SEB-specific
CD4+ T cells and DCs. BF image shows the contact between a CD4+ T cell and DC. Dotted white
line depicts the contact boundary of the CD4+ T cell and DC. Images are a cross-section of a 3D
plane rotated in an en face view of a synapse. TCR is shown red, ICAM-1 is green, CD28 is bule.
Scale bar is 2 μm.
Supplementary Figure 3. Calcium responses in T cells forming a bullseye IS or multifocal IS
were measured and presented by △F/F.
(A-C) Representative Ca2+ responses were measured in
the individual T cells stimulated with DCs pulsed with 10 ng/ml, 100 ng/ml or 1μg /ml SEB. Data
showed the Ca2+ signalling response during the period of 5 min after T cells contacted DCs.
Images were obtained every 4-s. (D) The average calcium response was measured in T cells
forming a bullseye IS or multifocal IS. Data are shown as means ± s.e.m., n=25, from 3
independent experiments. **P<0.01 (two-tailed Student’s t-test).
Supplementary Figure 4. Calcium responses in T cells were measured and presented by △F/F.
(A) The relationship between the motility (pink) and calcium signal (blue) of a T cell was
analyzed in the process of bullseye IS formation. (B) The relationship between the motility (pink)
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and calcium response (blue) of a T cell was analyzed in the conversion process from a multifocal
IS to
a bullseye IS. (C) The calcium responses were measured in CD4+CD25- T cells (pink line)
or CD4+CD25+ T cells (blue line), n=25. (D) Calcium responses occured in CD4+CD25+ T cells
forming a bullseye IS (blue line) or multifocal IS (pink line). (E) Calcium responses occur in
CD4+CD25- T cells forming a bullseye IS (blue line) or multifocal IS (pink line). (F) The mean
peak calcium response in CD4+CD25+ T cells or CD4+CD25- T cells forming different type of
synapse. Cell contacts are measured from three independent experiments (n=25). (G) Calcium
responses were measured in CD4+ T cells treated with anti-CTLA-4 antibody (n=50). Data are
shown as means ± s.e.m., *P<0.05, **P<0.01, ***P<0.001 (two-tailed Student’s t-test, one-way
ANOVA). (C-E) and (G) show the Ca2+ signalling response during the period of 10 min after T
cells contacted DCs. Images were obtained every 10-s.
Supplementary Figure 5. (A)The expression of Foxp3 in CD4+CD25+ T cells. The expression of
Foxp3 was measured in a CD4+CD25+ T cell forming a bullseye IS. TCR (red) and ICAM-1(green)
are labeled to show the synapse structure. Nuclei are stained with HOECHST (blue). Foxp3
(yellow) expressed in the nucleus of CD4+CD25+ T cell forming a bullseye IS. (B) Bullseye IS
formation in CD4+CD25+Foxp3-GFP T cells. TCR (red) and LFA-1(blue) are labelled to show the
synapse structure. Foxp3-GFP (green) was expressed in a CD4+CD25+ T cell forming a bullseye
IS. The last three panels are the interface of T-DC to show the structure of synapse. Dotted white
line depicts the contact boundary of the CD4+ T cell and DC. Scale bar is 2 μm.
Supplementary Figure 6. The construction of ICAM-1-EGFP/DC2.4 cell line. DC2.4 cell line
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is a C57BL/6 mouse bone marrow derived DC cell line. (A) The plasmid of pEGFP-ICAM-1 was
identified by digested reaction; (B) Expression of ICAM-1 fused with EGFP (110 KD) was
identified by western blotting. (C) Expression of ICAM-1-EGFP in DC2.4 was measured by
immunofluorescence microscopy. (D) The expression of EGFP fluorescence and ICAM-1 in
DC2.4 cells was analyzed by flow cytometry. Anti-ICAM-1-PE was used to stain ICAM-1.
Percentages were shown in each quadrant. (E) Phenotype and maturation of DC2.4 cell line (upper
row) or ICAM-1-EGFP/DC2.4 cell line (bottom row) were analyzed by flow cytometry. Dotted
line shows the cells stained with isotype antibody as a control. The solid line showed the cells
after LPS and INF-γ induction, the grey line showed the cells before LPS and INF-γ induction.
Supplementary Movie 1. The 3D structure of bullseye IS. Imaris was used to generate 3D
volumes and rotate between en face and side views. cSMAC is marked by TCR (red), while
pSMAC is marked by ICAM-1 (green).
Supplementary Movie 2. The 3D structure of multifocal IS structure. Imaris was used to generate
3D volumes and rotate between en face and side views. A number of TCR clusters (red) are
crossed with ICAM-1(green).
Supplementary Movie 3. The bullseye IS was formed directly when the CD4+ T cell contacted
the DC. The last frame is the bullseye IS.
Supplementary Movie 4. The bullseye IS was converted from multifocal IS. The last two frames
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are the moltifocal and the bullseye IS.
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