Structures of mouse and human GITR–GITRL complexes reveal unique TNF superfamily interactions

Abstract

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and GITR ligand (GITRL) are members of the tumor necrosis superfamily that play a role in immune cell signaling, activation, and survival. GITR is a therapeutic target for directly activating effector CD4 and CD8 T cells, or depleting GITR-expressing regulatory T cells (Tregs), thereby promoting anti-tumor immune responses. GITR activation through its native ligand is important for understanding immune signaling, but GITR structure has not been reported. Here we present structures of human and mouse GITR receptors bound to their cognate ligands. Both species share a receptor–ligand interface and receptor–receptor interface; the unique C-terminal receptor–receptor enables higher order structures on the membrane. Human GITR–GITRL has potential to form a hexameric network of membrane complexes, while murine GITR–GITRL complex forms a linear chain due to dimeric interactions. Mutations at the receptor–receptor interface in human GITR reduce cell signaling with in vitro ligand binding assays and minimize higher order membrane structures when bound by fluorescently labeled ligand in cell imaging experiments.

Atypical TNF-TNFR superfamily binding interface in the GITR-GITRL complex for T cell activation

“…Therefore, the findings in the present study indi-cate that the unique interface of GITR/GITRL is conserved in human and mouse, which is distinct from the typical TNFSF/ TNFRSF interface. Wang et al (2021) reported the mGITR/ mGITRL and hGITR/hGITRL complexes and showed that the overall structures of the GITR/GITRL complexes in human and mouse are similar to one another, which confirms our speculation and fills gaps in the knowledge of the overall binding mode and specific molecular mechanisms in our studies.…”

“…The DIV in mGITR and KFS in hGITR determined the binding specificity of these two receptors to their ligands. The recently reported hGITR/hGITRL complex structure also supports that KFS in hGITR plays critical roles in interaction with its ligand (Wang et al, 2021). Substitution of DIV with KFS in mGITR attenuated binding of mGITR to mGITRL but enabled cross-recognition of mGITR with hGITRL and vice versa.…”

Structural delineation and phase-dependent activation of the costimulatory CD27:CD70 complex

“…Group 3 TNF ligands are the most structurally divergent members and include CD70, CD30L, GITRL, 4-1BBL and OX40L (34). Co-crystal structures of human GITRL, 4-1BBL and OX40L with their respective receptors were published previously, but this report is the first publication of the co-crystal structure of CD70 (34,(44)(45)(46)(47). The overall trimeric CD70 assembly adopts a "blooming flower" structural arrangement that most resembles OX40L (Fig.…”

Therapeutic antibody activation of the glucocorticoid-induced TNF receptor by a clustering mechanism

“…Sequence conservation in the TNF receptor family is low to moderate, but the general receptor structure features an N-terminal ectodomain that harbors cysteine-rich domains (CRDs) and one transmembrane helix that connects to an unstructured intracellular C-terminal domain (1,3). Many structures of TNF receptor ectodomains have been reported alone or in complex with ectodomains for their ligands (1,11,(18)(19)(20). However, no structures of full-length TNF receptors have been solved.…”

“…Structural work on GITR, GITRL, and the GITR/GITRL complex has focused exclusively on soluble ectodomains from mouse and human orthologs (11,(21)(22)(23)(24). Structures of mouse GITR (mGITR) and human GITR (hGITR) have shown that, despite low sequence identity (59%), the two ectodomains have nearly identical structures [root mean square deviation (RMSD) < 1 Å] and both ectodomains form monomers in solution (fig.…”