A mutational analysis of CD28 utilizing targeted knock-in mice (50.24)

Abstract

T cell activation requires signaling through the TcR and engagement of a costimulatory receptor. CD28 is the most important costimulatory receptor; however, the signaling pathways initiated by CD28 remain highly controversial. To examine this, we generated gene targeted knock-in mice that express mutations in either the 1) proximal YMNM (Y170F), 2) distal PYAP (AYAA), or 2) both the YMNM and PYAP (Y170F/AYAA) motifs of the cytoplasmic tail of CD28. Mutation of the PYAP motif resulted in impaired proliferation, cytokine secretion and B cell help. In contrast, the Y170F mutant had normal to slightly increased proliferation and IL-2 production in vitro, as well as a prolonged duration of inflammation in vivo. T cells from both mutant knock-in mice upregulated the activation markers CD25, CD69 and CD95. Interestingly, the CD28 knock-out and CD28-AYAA knock-in mice both fail to induce the expression of ICOS on T cells, a critical component for germinal center production. To determine if additional motifs mediate CD28-dependent functions, we tested the responses of the double mutant. The phenotype of the Y170F/AYAA double mutant was not substantially different than the single AYAA mutant. These data strongly suggest that the residual T cell responses present in the AYAA mouse are not due to signaling initiated by the YMNM motif, and that additional signaling pathways mediate the remaining CD28-dependent effects.