Dysfunctional T cell response can lead to abnormal immune function and the progression of immune-related pathologies. CTLA-4 receptors function as a negative regulator of T cell response by effectively suppressing activation, and it has been shown that mutations in CTLA-4 led to deleterious auto-immune effects. Two ligands, CD80 and CD86, both bind CTLA-4, but it is not fully understood how these ligands differ in their effect on CTLA-4. Sansom and colleagues recently showed that binding properties of these ligands to CTLA-4 resulted in distinct differences in CTLA-4 fate during transendocytosis which affected their ability to regulate autoimmunity. Initial microscopy and flow cytometry studies were conducted to visualize and quantify transendocytosis of CD80 and CD86 ligands from donor cells to CTLA-4 positive recipient cells. During these studies, it was noted that CD86 appeared to be pH dependent, as a lysosomal acidification inhibitor helped with the detection of CD86, presumably due to its ability to stabilize the interaction between CD86 and CTLA-4. Indeed, confocal microscopy studies showed that CD80-CTLA-4 complexes resided in larger vesicles, while CD86 appeared to dissociate from CTLA-4 and were localized to smaller vesicles. CD80 binding to CTLA-4 resulted in measurable ubiquitination of the receptor in both heterologous cells and unmanipulated human T regulatory cells. Ubiquitination of CTLA-4 targeted the CD80-CTLA-4 complex for lysosomal degradation. Conversely, due to the weaker interaction of CTLA-4 and CD86, endosomal pH changes promoted the dissociation of the complex and recycling of CTLA-4 back to the plasma membrane. Recycling of CTLA-4 was validated in CRISPR models where key recycling compartmental proteins were knocked out. Interestingly, clinically relevant mutations in CTLA-4 were identified that affected binding to CD86 and not CD80, which affected CD86’s T cell response. Overall, these findings support the idea that ligand interaction promotes distinct effects on CTLA-4 which can have a significant impact on T cell function and immune regulation. Cytoskeleton Inc’s Ubiquitination Affinity Beads (Cat. #UBA01-Beads) were essential to measure endogenous ubiquitination of CTLA-4 in primary human T regulatory cells.
Above: Schematic showing high avidity binding of CD80 and CTLA-4, which results in ubiquitination of CTLA-4, transendocytosis, and lysosomal degradation.