Targeted Knock-In Mice Expressing Mutations of CD28 Reveal an Essential Pathway for Costimulation

Abstract

Despite extensive study, the role of phosphatidylinositol 3-kinase (PI3-kinase) activation in CD28 function has been highly contentious. To definitively address this question, we generated knock-in mice expressing mutations in two critical domains of the cytoplasmic tail of CD28. Mutation of the proximal tyrosine motif interrupted PI3-kinase binding and prevented CD28-dependent phosphorylation of protein kinase B (PKB)/Akt; however, there was no detectable effect on interleukin-2 (IL-2) secretion, expression of Bcl-X_L, or on T-cell function in vivo. Furthermore, we demonstrate that signaling initiated by the C-terminal proline motif is directly responsible for tyrosine phosphorylation of phosphoinosotide-dependent kinase 1, protein kinase Cθ, and glycogen synthase kinase 3β, as well as contributing to threonine phosphorylation of PKB. T cells mutated in this domain were profoundly impaired in IL-2 secretion, and the mice had marked impairment of humoral responses as well as less severe disease manifestations in experimental allergic encephalomyelitis. These data demonstrate that the distal proline motif initiates a critical nonredundant signaling pathway, whereas direct activation of PI3-kinase by the proximal tyrosine motif of CD28 is not required for normal T-cell function.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

This work was supported by a grant to J.M.G. from the NIH (HL062683). L.F.D. and C.M.D. are supported by NIH training grant T32HL07317. We thank the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St. Louis, MO, for the use of the Embryonic Stem Cell Core, which provided embryonic stem cell transfection services. The Siteman Cancer Center is supported in part by an NCI Cancer Center Support grant (P30 CA91842).

We thank A. Shaw, W. Swat, and A. Gelman for helpful discussion and critical review of the manuscript. We thank Ron McCarthy for expert microinjections.

We have no conflicting financial interests.

L.D.F. contributed to the data presented in Fig. 1, 2, 3, 4, 7, and 8. J.S.B. contributed to the data presented in Fig. 5, 6, and 9. C.D. contributed to data presented in Fig. 4, 7, and 8. D.D.S. designed and generated the knock-in construct and performed all Southern analysis and verifications and contributed to the data presented in Fig. S1 in the supplemental material. T.B. maintained the breedings and contributed to the data presented in Fig. S1 of the supplemental material. J.S. and J.H.R. contributed to the data presented in Fig. 8. J.M.G. oversaw and directed all phases of this project. All authors have reviewed the final version of the manuscript and concur with the submission.