Targeted knock-in mice expressing mutations of CD28 reveal an essential pathway for costimulation
Lindzy F Dodson1, Jonathan S Boomer, Christine M Deppong
1Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
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 C theta, and glycogen synthase kinase 3beta, 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.
Insights
The proximal tyrosine motif of CD28 does not require phosphatidylinositol 3-kinase (PI3-kinase) activation for normal T-cell function. The distal proline motif, however, initiates a critical pathway essential for T-cell signaling and immune responses.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- The precise role of phosphatidylinositol 3-kinase (PI3-kinase) activation in CD28 signaling remains debated.
- CD28 is a crucial co-stimulatory receptor for T-cell activation and function.
Purpose of the Study:
- To elucidate the specific roles of distinct CD28 cytoplasmic tail motifs in PI3-kinase activation and downstream signaling.
- To determine the necessity of PI3-kinase binding to CD28 for T-cell function in vivo.
Main Methods:
- Generation of knock-in mice with mutations in critical CD28 cytoplasmic tail domains.
- Analysis of T-cell signaling pathways, including phosphorylation events and cytokine secretion.
- Assessment of T-cell function in vitro and in vivo, including humoral responses and experimental allergic encephalomyelitis.
Main Results:
- Mutation of the proximal tyrosine motif disrupted PI3-kinase binding and PKB/Akt phosphorylation but did not affect IL-2 secretion or T-cell function.
- Signaling via the C-terminal proline motif was essential for phosphorylation of PDK1, PKCθ, and GSK3β, and contributed to PKB/Akt phosphorylation.
- T cells with mutated proline motifs showed impaired IL-2 secretion, humoral responses, and reduced disease severity in EAE.
Conclusions:
- Direct PI3-kinase activation by the proximal CD28 tyrosine motif is dispensable for normal T-cell function.
- The distal proline motif of CD28 initiates a critical, nonredundant signaling pathway essential for T-cell activation and immune responses.
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