CD28 Regulation of Interleukin-2 Production

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2010.CD28 is an important costimulatory molecule and critical for T cell activation survival, proliferation and differentiation. Previous studies have shown that CD28 regulates IL-2 production by upregulation of IL-2 transcription and stabilization of IL-2 mRNA. CD28 regulation of IL-2 transcription has been suggested to be mediated by an YMNM motif within the cytosolic tail. Upon tyrosine phosphorylation, the YMNM can recruit the SH2 domain containing protein, phosphoinositide-3-kinase, and lead to the activation of transcription factors necessary for IL-2 transcription. However, little is known about the mechanism of CD28-mediated stabilization of IL-2 mRNA. Our first observation is that the CD28 truncation, Δ179, lost its ability to stabilize IL-2 mRNA, indicating that the deleted 20 amino acid residues from the C-terminus of the cytosolic tail are necessary for CD28-mediated stabilization of IL-2 mRNA. Thus, we generated several CD28 mutations to identify the motif within the CD28 cytosolic tail that is necessary for CD28-mediated stabilization of IL-2 mRNA. We found that the prolines at positions 187 and 190 within the C-terminal proline-rich motif, PYAPAR, are necessary for CD28 to stabilize IL-2 mRNA. In addition, an 8 amino acid segment containing this C-terminal proline-rich motif is both necessary and sufficient to stabilize IL-2 mRNA. These results suggest that CD28 regulation of IL-2 mRNA stability is mediated by the recruitment of signaling molecules that contain SH3 domains. However, single amino acid mutations at the key residues of the proline-rich motif (P187, P190 and R192) that are necessary for interaction of SH3 domains do not affect the ability of CD28 to stabilize IL-2 mRNA. It is possible that this is due to some redundant motifs within the CD28 cytosolic tail that can stabilize IL-2 mRNA. It is also possible that the interaction of the downstream signaling molecule with C-terminal proline-rich motif is not mediated by a conventional SH3 domain. The motif we identified that mediates CD28 stabilization of IL-2 mRNA is different from the motif mediating CD28 regulation of IL-2 transcription, indicating that CD28 regulation of IL-2 transcription and IL-2 mRNA are two independent pathways. To confirm this, we generated two reciprocal truncation mutations, Δ166-186 and Δ187-199, and examined their effects on IL-2 transcription and IL-2 mRNA stability. CD28 mutation Δ166-186 contains the YMNM motif, but not the C-terminal poly-proline motif, while its reciprocal mutation Δ187-199 contains the C-termianl poly-proline motif, but not YMNM motif. We found that the mutation Δ166-186 can enhance IL-2 transcription, but lost its ability to stabilize IL-2 mRNA, while its reciprocal mutation Δ187-199 can stabilize IL-2 mRNA, but cannot upregulate IL-2 transcription. In addition, the mutation P187,190A, which cannot stabilize IL-2 mRNA, can promote NF-κB translocation to the nucleus that has been shown to correlate with IL-2 transcription level. These results demonstrate that the CD28-downstream pathways of regulation of IL-2 transcription and IL-2 mRNA stability are independent. We further found that the C-terminal proline-rich motif within the CD28 cytosolic tail is necessary for CD28 regulation of IL-2 protein produced by T cells. By quantifying the amount of IL-2 mRNA and protein, we found that CD28 mediated regulation of IL-2 mRNA stability that is mediated by the C-terminal proline-rich motif contributes to the total IL-2 mRNA level. However, this effect does not completely account for the enhancement of CD28 on IL-2 production, which is also mediated by C-terminal proline-rich motif. In collaborative studies we have found that CD28 signaling through the PYAPAR motif can enhance exocytosis of IL-2. Thus, CD28 can regulate cytokine synthesis at the transcriptional, posttranscriptional and post-translational levels