The bullseye synapse formed between CD4+ T cells and staphylococcalenterotoxin B -pulsed DCsplays 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 ofCD25 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 ofCD69 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 ofKi67 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 ofPI 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 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 Figure2.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 imageshows the contact between a CD4+ T cell and DC. Dotted whiteline 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 Figure3. Calcium responses in T cells forming a bullseye IS or multifocal ISwere measured andpresented 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 theCa2+signalling responseduring 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 asmeans±s.e.m.,n=25, from 3independent experiments. **P<0.01 (two-tailed Student’s t-test).

Supplementary Figure4. Calcium responsesin T cells were measured andpresented by△F/F.(A)The relationship between the motility (pink) and calcium signal (blue) of a T cellwas analyzed in the process of bullseye IS formation. (B)The relationship between the motility (pink) 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 forminga bullseye IS (blue line) or multifocal IS (pink line). (E) Calcium responsesoccur in CD4+CD25-T cells forminga 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 contactsare 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 asmeans±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 responseduring the period of 10 min after T cells contacted DCs. Images were obtained every10-s.

Supplementary Figure5.(A)The expression of Foxp3 inCD4+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-GFPT cells.TCR (red) and LFA-1(blue) are labelled to show the synapse structure.Foxp3-GFP (green)was expressedin 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 whiteline depicts the contact boundary of the CD4+ T cell and DC. Scale bar is 2 μm.

Supplementary Figure6. The construction of ICAM-1-EGFP/DC2.4 cell line. DC2.4 cell line 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 bywestern blotting. (C) Expression of ICAM-1-EGFP in DC2.4 was measured by immunofluorescence microscopy. (D) The expression ofEGFP fluorescence and ICAM-1 in DC2.4 cellswas analyzed byflow 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 byflow 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 framesarethe moltifocal and the bullseye IS.

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