Human CTLA4 Stable Cell Line-CHO-K1

CD28 and CTLA-4 (CD152) play important roles in regulating T-cell immunity, balancing activation and inhibition of T-cell responses. Although both receptors share the same ligands, CD80 and CD86, the specific requirements for the two different ligands remain unclear. Here, researchers demonstrate that although CTLA-4 targets both CD80 and CD86 for destruction via transcytosis, this process results in different fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remains ligand-bound and is ubiquitinated and trafficked through late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 dissociates and recycles back to the cell surface in a pH-dependent manner, allowing further transcytosis. Researchers also identify clinically relevant mutations that cause autoimmune diseases and selectively impair CD86 transcytosis by affecting CTLA-4 recycling or CD86 binding. These observations provide a rationale for the two distinct ligands and suggest that defects in CTLA-4-mediated CD86 transcytosis are involved in autoimmunity.

To study CD80 and CD86 TEs, the researchers performed experiments using a variety of CTLA-4-expressing cells (Chinese hamster ovary (CHO) cells, Jurkat T cells, and primary human Treg cells) to capture fluorescent ligands (green fluorescent protein (GFP) or mCherry labeled) from contralateral cells during CTLA-4-ligand interaction. To ensure that the measured ligand transfer was caused only by TEs and not by cell doublets, the researchers labeled ligand donor cells with CellTrace Violet (CTV) and monitored the transfer of fluorescent ligands to CTLA-4+ cells by flow cytometry (Figure 1a-c). CTV+ donor cells clearly showed ligand loss (Figure 1b, c, upper quadrants), indicating that both CD80 and CD86 were effectively removed by TEs. Ligand acquisition by CTLA-4+ cells is shown as fluorescent protein uptake in the lower right quadrant, which is clearly visible when CTLA-4+ cells acquire CD80 (Figure 1b, lower right grey quadrant). In contrast, uptake of CD86 was consistently more difficult to detect (Figure 1b, lower right blue quadrant). Therefore, the researchers used the lysosomal acidification inhibitor NH4Cl to examine whether CD86 detection was pH sensitive, as this is important for endolysosomal fusion and lysosomal acidification. In the presence of NH4Cl, CD86 capture was now more easily observed (Figure 1b, right column, lower right quadrant). These data raise the possibility that CD80 and CD86 undergo different intracellular processing following TE and that CD86 detection within recipient cells is more pH sensitive.

Figure 1. TE of CD80 and CD86 reveals distinct ligand characteristics. (Kennedy A, et al., 2022)