Evolution of differential 4‐1BB signaling in Human and Murine immune system

4‐1BB (CD137) is an inducible tumor necrosis factor receptor superfamily (TNFRSF) member and is currently being targeted in cancer immunotherapy and autoimmunity. 4‐1BB interacts with its cognate ligand 4‐1BBL to promote T lymphocyte activation and proliferation, but the dissimilarities in mouse and human molecules might result in differential engagement of this pathway. To elucidate the molecular mechanism underlying this distinct behavior, we have determined the crystal structures of human and mouse 4‐1BB/4‐1BBL complexes. Collectively, these structures provide insights into the structural differences that drive the species‐specific receptor‐ligand interactions. The most significant feature is that the TNF homology domain of human (h)4‐1BBL form non‐covalent homo‐trimeric arrangements, whereas the mouse (m)4‐1BBL formed a novel disulfide linked homo‐dimer. In the human 4‐1BB/4‐1BBL complex, each receptor binds at the crevice formed between two protomers of h4‐1BBL resulting in the formation of a “hetero‐hexameric” functional signaling complex (1), whereas each m4‐1BB receptor binds exclusively to a single m4‐1BBL protomer leading to a “tetrameric” signaling unit (2). As multimerization and clustering is a prerequisite for TNFR intracellular signaling, appearance of differential functional signaling units in human and mouse supports a unique mechanism of 4‐1BB signaling in both of these species. Of note, we have discovered that h4‐1BB forms a disulfide‐linked dimer and this covalent receptor dimerization amplifies h4‐1BB signaling by cross‐linking individual ligand‐receptor complexes to form a 2D network. Since the m4‐1BBL dimer can only recruit two monomeric 4‐1BB receptors, cross‐linking of two hetero‐tetrameric signaling units via Galectin‐9 binding to the N‐linked glycans on m‐41BB is required for receptor oligomerization and strong signaling (3). While all characterized TNFR/TNF members exhibit interspecies cross reactivity between human and mouse molecules, 4‐1BB and 4‐1BBL lacks this distinguishing feature. Our structural studies together with comparison of human and mouse 4‐1BB/4‐1BBL complexes highlighted several differences between the ligand and receptor binding interfaces and provide an explanation for the absence of inter species cross‐reactivity between them (2). Furthermore, our biochemical and mutational analysis discloses more variations between mouse and human molecules that could affect signaling and thus suggest the generation of better animal models to study 4‐1BB biology in humans. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .