An inter-species amino acid variation modulates signaling strength and function of CD28 in vitro and in vivo

Abstract

Abstract Animal models do not always reflect the human condition. One such example was a phase one clinical trial of a CD28 super agonist antibody (CD28SA). In mice, CD28SA treatment expanded Tregs and reduced autoimmune disease; yet when human volunteers received CD28SA, they experienced life-threatening cytokine storm and multi-organ failure. CD28 is constitutively expressed on T cells and provides the canonical co-stimulatory “signal 2” needed for T cell activation. The proline residues in the CD28 PYAP domain are required for downstream signaling and this motif is conserved across species. However, the human PYAP domain is adjacent to an additional proline, while murine PYAP is followed by an inactive alanine. Previous in vitro studies have suggested this single amino acid variation modulates downstream signaling. We have developed a “humanized” mouse with a CD28 A to P substitution at residue 210 adjacent to the PYAP domain (CD28 A210P). In vitro, CD28 A210PT cells had increased p-Akt, JunB abundance, and produced more IL-2 compared to WT T cells when stimulated in vitro. In vivo, immunization with antigen/CFA emulsion, CD28 A210Pmice display increased antibody production, suggestive of enhanced CD28-mediated Tfh activity. In addition, we assessed the response of the “humanized” CD28 mice to the CD28 super agonist antibody. After injection with CD28 super agonist, CD28 A210Pmice had significantly increased weight loss, elevated and sustained pro-inflammatory cytokine production, and less Treg expansion compared to WT mice. This study suggests interspecies variation in CD28 signaling due to differences outside of the well-studied signaling domains can alter outcomes of CD28 ligation and promote divergent outcomes when targeted in vivo.