CD28 mediates acquisition of APC-derived membrane fragments upon B7 binding. (A) Left: Confocal microscopy of a conjugate between a CD80-GFP+ Raji B cell and a CD80-GFP+ Jurkat T cell. Image shows CD80-GFP accumulation in CTLA4-associated vesicles and CTLA4-independent acquisition of membrane fragments enriched in CD80-GFP (white arrowhead). Right: Correlated light electron micrograph (CLEM) of confocal inset in A. Red arrowhead indicates discrete membrane fragments acquired by Jurkat from Raji APC. (B) Confocal micrographs of CD28−/− Jurkat cells co-expressing CD28-mCherry and CTLA4-HaloTag (JFX-646) in conjugation with CD80−/−CD86−/− Raji cells expressing CD80-GFP. Insets show CD28 and CTLA4 colocalization with Raji-derived membrane projections. (C) Coomassie brilliant blue stained SDS-PAGE of purified MBP-28.3scFv. (D) Representative confocal micrographs of a Raji (CD80−/−CD86−/−CD80-GFP+):Jurkat (CD28−/−CD28-mCherry+) conjugate (left) and a Raji (CD80−/−CD86−/−CD86-GFP+):Jurkat (CD28−/−CD28-mCherry+) conjugate (right), with or without the presence of 28.3scFv. Scatter plots on the immediate right show the synaptic enrichment indices of CD28. In differential interference contrast (DIC) images, R denotes Raji, J denotes Jurkat. Scale bar: 5 µm. n = 20 conjugates. (E) Cartoon depicting EM labeling strategy of human regulatory T cells following conjugation with Raji B cells expressing CD80-HaloTag (JFX-549). Laser excitation at 561 nm induces oxidative polymerization of electron-dense DAB proximal to CD80. (F) Representative electron micrographs of human Treg cells upon contact with Raji APC expressing CD80-Halo in the presence or absence of 28.3scFv, with CD80-Halo stained by DAB. The rightmost two images under the −28.3scFv condition show zoomed in view of another Treg cell after Raji contact, highlighting possible fusion of CD80-Halo–associated membrane fragments. Error bars in D are SD from 20 Raji:Jurkat conjugates under each indicated condition. ***, P < 0.001; Student’s t test. Source data are available for this figure: SourceData F1.
Figure 1.

CD28 mediates acquisition of APC-derived membrane fragments upon B7 binding. (A) Left: Confocal microscopy of a conjugate between a CD80-GFP+ Raji B cell and a CD80-GFP+ Jurkat T cell. Image shows CD80-GFP accumulation in CTLA4-associated vesicles and CTLA4-independent acquisition of membrane fragments enriched in CD80-GFP (white arrowhead). Right: Correlated light electron micrograph (CLEM) of confocal inset in A. Red arrowhead indicates discrete membrane fragments acquired by Jurkat from Raji APC. (B) Confocal micrographs of CD28−/− Jurkat cells co-expressing CD28-mCherry and CTLA4-HaloTag (JFX-646) in conjugation with CD80−/−CD86−/− Raji cells expressing CD80-GFP. Insets show CD28 and CTLA4 colocalization with Raji-derived membrane projections. (C) Coomassie brilliant blue stained SDS-PAGE of purified MBP-28.3scFv. (D) Representative confocal micrographs of a Raji (CD80−/−CD86−/−CD80-GFP+):Jurkat (CD28−/−CD28-mCherry+) conjugate (left) and a Raji (CD80−/−CD86−/−CD86-GFP+):Jurkat (CD28−/−CD28-mCherry+) conjugate (right), with or without the presence of 28.3scFv. Scatter plots on the immediate right show the synaptic enrichment indices of CD28. In differential interference contrast (DIC) images, R denotes Raji, J denotes Jurkat. Scale bar: 5 µm. n = 20 conjugates. (E) Cartoon depicting EM labeling strategy of human regulatory T cells following conjugation with Raji B cells expressing CD80-HaloTag (JFX-549). Laser excitation at 561 nm induces oxidative polymerization of electron-dense DAB proximal to CD80. (F) Representative electron micrographs of human Treg cells upon contact with Raji APC expressing CD80-Halo in the presence or absence of 28.3scFv, with CD80-Halo stained by DAB. The rightmost two images under the −28.3scFv condition show zoomed in view of another Treg cell after Raji contact, highlighting possible fusion of CD80-Halo–associated membrane fragments. Error bars in D are SD from 20 Raji:Jurkat conjugates under each indicated condition. ***, P < 0.001; Student’s t test. Source data are available for this figure: SourceData F1.

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