Figure 4. Functional consequences of chimeric antigen receptor (CAR)-CD28 association in CAR T cells.

(a) Model of the CD28TM interface generated by mutagenesis of the CD3ζ TMD (PDB: 2HAC). Polar residues of the CD28 dimerization motif (orange) with predicted hydrogen bonds depicted (dotted lines). (b) Surface expression and (c) SDS-PAGE and immunoblot analysis of HER2 CARs possessing WT CD28TM or CD28TM mutations depicted in (a) expressed in the BW5147 cell line. (d) Quantitation of target cell killing measured by chromium release assay and cytokine production by primary mouse CD8⁺ CAR T cells in response to the MC57-HER2 target cell line (n = 4). Experiments performed as in Figure 3. p-Values determined by paired t-tests. (e) Representative immunofluorescent confocal images of CAR-CD28 co-clustering in primary mouse CAR T cells. CAR clustering was induced with anti-Myc primary followed by crosslinking with fluorescent secondary antibody (magenta). Cells were then labeled for CD28 (cyan). Images are Z-projections over 12 m, scale bar represents 3 m. (f) Quantitation of CAR-CD28 co-clustering, each dot representing the percentage of CAR clusters in one cell that co-localized with a CD28 cluster. Lines show mean CAR-CD28 co-clustering percentage/per cell ± SEM, n ≥ 30 cells. p-Values determined by unpaired t-tests.