Over the past 10 years, we have been studying two phenomena in cloned populations of CD4+ T lymphocytes referred to as costimulation and anergy. Both affect the production of the T cell growth and differentiation factor interleukin-2 (IL-2) produced following antigen activation of these cells. Costimulation entails a 30 to 100 fold enhancement of IL-2 production when signaling through the antigen-specific T cell receptor is supplemented with signaling through the CD28 receptor on the same cell. Clonal anergy is an anti-proliferative state that the T cell enters when it only receives a signal through the antigen-specific receptor. In this case, subsequent re-stimulation of IL-2 production, even in the presence of co-stimulation, is inhibited 10 to 50 fold. Our goals in this project have been to try and understand the molecular mechanisms behind these two phenomena.? The prominent affect that costimulation and anergy have on expression of the IL-2 gene has led us to explore its chromatin structure and transcriptional regulation. In FY-2008 we explored the differences in IL-2 gene expression between naive and previously activated T cells. T cell activation with anti-CD3 and anti-CD28 stimulates much more IL-2 mRNA (10 fold) at early time points (4-6 hours) in pre-activated or memory CD4+ T cells than in naive T cells; yet the total amount of IL-2 produced by 72 hours is comparable. To explain this paradox we have now uncovered a biphasic regulation in the transcription of IL-2 in naive T cells. During the first 10 hours after stimulation only 5-10% of naive T cells produce IL-2 in a capture assay. In contrast, greater than 65% of the T cells in a pre-activated population make IL-2 within 6 hours. The same low percentage of naive responding T cells was observed with cells from a GFP Knock-In mouse at the IL-2 locus, showing that there is a limitation in the number of cells transcribing the gene. Beyond 18 hours of stimulation, however, the naive population entered a second phase of IL-2 production which peaked around 48 to 60 hours with over 60% of the cells responding. This late phase expression required cRel, CD28 and TNFR signalling and was completely absent in cells from a double KO lacking both the p55 and the p75 TNF receptors. The late phase expression was correlated with a 10 fold increase in the cell surface expression of the p75 TNF receptor by 24 hours after stimulation. Once pre-activated, however, the remaining T cells no longer required TNFRs for IL-2 production, although they were still partially CD28 and cRel dependent. The quicker response (6 hours) of the pre-activated T cells was associated with a small increase in DNA accessibility at the IL-2 locus in the resting population and a faster and greater increase in accessibility following TCR stimulation. The latter was accompanied by nucleosome loss over the IL-2 promoter as was previously published by Shannons laboratory (Mol. Cell Biol. 25:3209, 2005). This loss was not observed in naive T cells even at the 48 hour time point. Finally, basal levels of cRel expression were higher in pre-activated T cells and greater levels were found sooner in the nucleus following TCR stimulation. Our current working model is that nucleosome disassembly facilitates the rapid initiation of IL-2 transcription in pre-activated T cells and that cRel signaling participates in this process. In naive CD4+ T cells the cRel component is rate-limiting and requires TNFR signalling to boost or sustain a high enough level to initiate IL-2 transcription.
| Choi, Heonsik; Cho, Sung-Yup; Schwartz, Ronald H et al. (2006) Dual effects of Sprouty1 on TCR signaling depending on the differentiation state of the T cell. J Immunol 176:6034-45 |
