Composition and structure of synaptic ectosomes exporting antigen receptor linked to functional CD40 ligand from helper T cells
- University of Oxford, United Kingdom
- University of Malta, Malta
Figures
Figure 1 with 1 supplement
CD40 dependent recruitment of CD40L to the IS and deposition in SE trail.
(A) Schematic of PSLB and mature IS. (B) Detection of CD40L with the anti-CD40L clone 24–31 as a function of CD40 in the PSLB. T cells were allowed to form IS for 10 min in the presence of Alexa …
Figure 1—figure supplement 1
Normalized maximum projections of Airyscan of CD40L (anti-CD40L Alexa Fluor 657, Red hot) within CD4+ T cell volume PSLB in the presence/absence of UCHT1-Fab and CD40, Scale bar: 5 µm.
https://doi.org/10.7554/eLife.47528.003
Figure 2 with 5 supplements
CD40L is incorporated in SE.
(A) Schematic of BSLB (B) Schematic of T cell (green) interacting with BSLB with UCHT1-Fab (magenta) and SE on BSLB after T cell removal. (C) % CD40L and TCR transfer to BSLB coated with incremental …
Figure 2—figure supplement 1
Gating Strategy for SE on BSLB and representative histograms following CRISPR/Cas9 knockdown.
(A) Gating Strategy for SE capture on BSLB. Forward/Side site scatter plot of beads and T cells following liberation of BSLB from with EDTA/PBS. Beads have a distinctive and narrow scatter …
Figure 2—figure supplement 2
ICOS significantly increases CD40L transfer to BSLB.
Per marker statistical analyses of heat maps shown in Figure 2D. P values < 0.05 were considering significant. Repeat Measures ANOVA with Geisser-Greenhouse correction was performed. Significance …
Figure 2—figure supplement 3
Efficient transfer of CD40L to BSLB at low UCHT1-Fab densities.
(A) Percent of protein transfer (%) from human T cells to BSLBs containing increasing densities of UCHT1-Fab (0–1800 molec./μm2) and two different BSLB compositions: UCHT1-Fab + ICAM-1 (left) or …
Figure 2—figure supplement 4
ICOSL increases T cell: BSLB conjugate formation and ICOS transfer.
(A) % T cell: BSLB conjugate formation in response to increasing densities of ICOSL. (B) % T cell: BSLB conjugate formation in response to increasing densities of UCHT1-Fab in the presence or …
Figure 2—figure supplement 5
BST2, CD63, CD81 and CD82 and CD40L localize to the synaptic cleft of UCHT-1 Fab stimulated cells.
(A) Representative TIRF and IRM images showing that proteins characterized by flow cytometry as synaptically transferred and deposited on PSLB containing ICAM-1, UCHT1-Fab, CD40 and ICOSL. T cells …
Figure 3
CD40L, BST2, and ICOSL localize to the synaptic cleft.
(A) Representative TIRFM images and IRM images showing staining of CD40L in the IS following incubation of HA specific clones on PSLB coated with ICAM-1 and either HLA-DR9HA or HLA-DR9CLIP monomers. …
Figure 4
Specificity of CD40L transfer- effects of bystander BSLB and general activation.
(A) Representative flow cytometry histograms and percentage marker transfer (CD40L, TCR and CD81) of ‘cognate’ (UCHT1-Fab +ve Atto488; green) and bystander (UCHT1-Fab –ve Atto565; red) BSLB and T …
Figure 5 with 3 supplements
SE contains ESCRTs and TCR signalosome.
(A) Proteins enriched by UCHT1-Fab on BSLB also containing ICAM-1, ICOSL and CD40. The network plot is based on known and predicted interactions from the STRING database (v11), with minimal …
Figure 5—figure supplement 1
Recruitment of EPN1 to the plasma membrane.
Airyscan confocal microscopy shows recruitment of EPN1 (green; white arrow) to the plasma membrane and at the synaptic cleft on PSLB in the presence of UCHT1-Fab (magenta) and CD40 (unlabeled).
Figure 5—figure supplement 2
Transfer to BSLB following CRISPR/Cas9 RNP electroporation.
(A) Percentage transfer of CD81, TCR, CD40L and CD4 to BSLB following electroporation of CD4+ T cells with either control CRISPR/Cas9-CD19gRNA RNP or CRISPR/Cas9-targetgRNA RNP, (B) WB showing …
Figure 5—figure supplement 3
Effect of ADAM10 inhibitor and CRISPR/Cas9 electroporation on transfer to BSLB.
(A) Representative histograms showing synaptic transfer of TCR, CD40L and CD81 to BSLB following treatment of CD4+ T cells with either vehicle control (green) or ADAM10/17 inhibitor (magenta). (B) …
Figure 6
Size distribution SE captured from immunological synapses on PSLB.
(A) Schematic of SE deposition on PSLB. CD4+ T cell blasts were incubated for 90 min on supported lipid bilayers coated with ICAM-1 (200 molec./µm2), UCHT1-Fab (30 molec./µm2), CD40 (500 molec./µm2) …
Figure 7 with 3 supplements
Nanoscale structure of SE.
CD4+ T cell blasts were allowed to form IS for 90 min on PSLB with ICAM-1 (200 molec./µm2), ICSOL (200 molec./µm2), CD40 (500 molec./µm2) and UCHT1-Fab (30 molec./µm2) and then released with cold …
Figure 7—figure supplement 1
TCR co-localization with BST2 and ICOS and segregation from CD40L.
(A) Representative dSTORM images showing TCR (green) and CD40L (magenta) on WGA labeled SE (gray). Insets show examples of SEs containing only TCR, only CD40L or both proteins. (B) Multiple examples …
Figure 7—figure supplement 2
CD81 co-localization with TCR and CD40L.
Multiple examples of SEs released by CD4+ T cells incubated for 90 min on PSLB coated with ICAM-1, UCHT1-Fab, CD40 and ICOSL. The SEs were stained with anti-TCRαβ-AF488, anti-CD81-AF568 to visualize …
Figure 7—figure supplement 3
Distinct cross-correlation distances for TCR with BST2 and ICOS versus CD40L.
Examples of cross-correlation analysis for positive control data (A), between TCR and CD40L (B), TCR and ICOS (C), or between TCR and BST2 (D) from multiple cells.
Figure 8 with 1 supplement
CD40L-positive SE left by T cells help DC and high-density vesicular CD40L is sufficient for DC maturation.
(A, B) HLA-DR (A) or CD83 (B) expression on the surface of DCs stimulated for 24 hr on PSLB prepared as indicated to present SE. Each symbol represents the mean fluorescent intensity of a cell (n ≥ 2…
Figure 8—figure supplement 1
SE captured on PSLB efficiently activate moDCs.
(A) Overlaid histograms illustrating the expression of ICAM-1, CD80, CD83 and CD86 maturation markers on DCs following 24 hr of stimulation on PSLBs loaded with SEs derived from CD4+ T cells. …
Videos
Video 1
Live TIRFM imaging of CD40L at the IS.
CD4+ T cells were incubated in the presence of anti-CD40L antibody with PSLB coated with ICAM-1, 30 molec./µm^2 of UCHT1-Fab in the presence or absence of CD40 at 37C and imaged for the first 15 …
Video 2
Sequence of horizontal slices of CD4+ T cells.
https://doi.org/10.7554/eLife.47528.005
Video 3
Live TIRFM of CD4+ T cells showing CD40 clustering at the IS.
https://doi.org/10.7554/eLife.47528.006
Video 4
Live TIRFM of CD4+ Tcells showing kinapse formation on PSLB.
https://doi.org/10.7554/eLife.47528.007
Video 5
Airyscan of CD4+ T cell (green) interacting with BSLB coated with UCHT1-Fab (magenta) and ICAM-1.
https://doi.org/10.7554/eLife.47528.014Additional files
-
Supplementary file 1
Legend.
Proteins Identified by MS/MS analysis and represented in Figure 5. Fold and Log Fold two represent fold change of BSLB coated with ICAM-1, UCHT1-Fab, CD40, ICOSL over BSLB coated with ICAM-1, CD40 and ICOSL. Colors represent proteins are found for particular pathways than is predicted by chance as determined by reactome pathway
- https://doi.org/10.7554/eLife.47528.028
-
Supplementary file 2
Supplementary tables.
(A) Summary of antibodies used for determination of relative and absolute enrichment of bead-transferred proteins. (B) Surface CD40L levels and densities on T cells exposed to different BSLB substrates (either No UCHT1-Fab ± CD40 or UCHT1-Fab ± CD40). (C) Estimated number of CD40L molecules per SE and estimated CD40L densities on SE using parameters obtained by dSTORM and quantitative FCM. (D) Size distribution of SUVs as determined by Nanoparticle Tracking Analyses using light scattering and Brownian motion.
- https://doi.org/10.7554/eLife.47528.029
-
Transparent reporting form
- https://doi.org/10.7554/eLife.47528.030
