The CD28 Transmembrane Domain Contains an Essential Dimerization Motif

Abstract

CD28 plays a critical role in regulating immune responses both by enhancing effector T cell activation and differentiation and controlling the development and function of regulatory T cells. CD28 is expressed at the cell surface as a disulfide linked homodimer that is thought to bind ligand monovalently. How ligand binding triggers CD28 to induce intracellular signaling as well as the proximal signaling pathways that are induced are not well-understood. In addition, recent data suggest inside-out signaling initiated by the T cell antigen receptor can enhance CD28 ligand binding, possibly by inducing a rearrangement of the CD28 dimer interface to allow for bivalent binding. To understand how possible conformational changes during ligand-induced receptor triggering and inside-out signaling are mediated, we examined the CD28 transmembrane domain. We identified an evolutionarily conserved YxxxxT motif that is shared with CTLA-4 and resembles the transmembrane dimerization motif within CD3ζ. We show that the CD28 transmembrane domain can drive protein dimerization in a bacterial expression system at levels equivalent to the well-known glycophorin A transmembrane dimerization motif. In addition, ectopic expression of the CD28 transmembrane domain into monomeric human CD25 can drive dimerization in murine T cells as detected by an increase in FRET by flow cytometry. Mutation of the polar YxxxxT motif to hydrophobic leucine residues (Y145L/T150L) attenuated CD28 transmembrane mediated dimerization in both the bacterial and mammalian assays. Introduction of the Y145L/T150L mutation of the CD28 transmembrane dimerization motif into the endogenous CD28 locus by CRISPR resulted in a dramatic loss in CD28 cell surface expression. These data suggest that under physiological conditions the YxxxxT dimerization motif within the CD28 transmembrane domain plays a critical role in the assembly and/or expression of stable CD28 dimers at the cell surface.

De novodesigned transmembrane domains tune engineered receptor functions

“…Mechanistically, our data show that the high cytokine secretion driven by the HER2-CD8-CD28-ζ CAR is directly linked to the 23-amino-acid CD28 TMD itself. This effect could be related to a highly conserved self-association motif recently identified in the CD28 TMD (Leddon et al, 2020) that is very similar to motifs that stabilize ζζ TMD homodimers and the TCRαβ TMD heterodimer (Call et al, 2006;Dong et al, 2019;Krshnan et al, 2016) and may therefore drive unintended interactions between CARs and endogenous CD28 to enhance cytokine production.…”

“…For convenience, most CARs incorporate the TMD sequence of the same protein from which the adjacent hinge or signalling domains were derived; that is, most commonly from endogenous T cell proteins such as CD4, CD8α, CD28 or the T cell receptor (TCR)-associated ζ chain. At least some of these TMD sequences can engage in molecular interactions that drive self-association and/or assembly with the essential T cell proteins from which they were derived (Bridgeman et al, 2010(Bridgeman et al, , 2014Call et al, 2002Call et al, , 2006Hennecke and Cosson, 1993;Leddon et al, 2020) and thereby impact CAR surface expression and functional properties in ways that reduce control over signalling outcomes. The ζ TMD, for example, drives both self-association (to form homodimers) and incorporation into endogenous TCR-CD3 complexes in the T-cell membrane (Call et al, 2002(Call et al, , 2006Cosson et al, 1991;Dong et al, 2019;Rutledge et al, 1992) .…”

“…The sequence determinants of these homotypic and heterotypic interactions are overlapping (Call et al, 2006) and therefore difficult to disentangle, and yet both appear to exert significant control over CAR signalling (Bridgeman et al, 2010(Bridgeman et al, , 2014 . The hinge and TMD sequences used in current FDA-approved CAR-T cell therapies derive from either CD8α or CD28; although experimentally less well characterised, these sequences carry similar risks of confounding interactions with endogenous T cell signalling proteins (Fujiwara et al, 2020;Hennecke and Cosson, 1993;Leddon et al, 2020) , thus hampering the rational design of CARs with predictable properties.…”

T Cell Activation Machinery: Form and Function in Natural and Engineered Immune Receptors

“…There is also evidence that TCR-mediated signals enhance CD28 affinity for its ligands [ 103 , 104 ] in an example of inside-out signaling reminiscent of integrin activation [ 105 ]. Structural changes involved in bi-directional signaling may be transmitted through the CD28 TM domain, which self-associates through a highly conserved polar motif Yx 4 T, where Y is tyrosine, x is any amino acid and T is threonine [ 106 ]. Notably, very similar Yx 4 T motifs also contribute to dimerization in CTLA-4 [ 106 ], ζ chain [ 52 ], FcRIγ [ 107 ] and TCRαβ [ 55 ].…”

“…Structural changes involved in bi-directional signaling may be transmitted through the CD28 TM domain, which self-associates through a highly conserved polar motif Yx 4 T, where Y is tyrosine, x is any amino acid and T is threonine [ 106 ]. Notably, very similar Yx 4 T motifs also contribute to dimerization in CTLA-4 [ 106 ], ζ chain [ 52 ], FcRIγ [ 107 ] and TCRαβ [ 55 ]. Whether this motif serves a homologous functional purpose in each of these receptors remains to be determined.…”