Over the past several years, we have been studying two phenomena in cloned populations of CD4+ T lymphocytes referred to as costimulation and anergy. The former entails a 30-100-fold enhancement of interleukin-2 (IL-2) production when signaling through the antigen-specific T cell receptor is supplemented with signaling through the CD28 receptor on the same cell. Anergy is an anti-proliferative state that the T cell enters if it only receives a signal through the antigen-specific receptor. Our recent work has focused on the molecular mechanisms behind these two phenomena. Using IL-2 RNA accumulation and transcription reporter assays, CD28 costimulation was shown to increase the stability of IL-2 messenger(m) RNA and not to enhance the initiation of transcription. An early component of the CD28 effect was nuclear, however, as a six-fold enhancement with anti-CD28 stimulation was observed for unspliced mRNA. During this past year, we have confirmed and extended these results by using a luciferase reporter construct containing an internal ribosomal entry sequence (IRES). This sequence, which permits translation of the reporter no matter where it is located in the message, allowed us to map the CD28 regulatory element(s) to the 3' end of the message. Interestingly, control sequences were found in both the third and fourth exons suggesting a more complex regulation than expected. Our previous work on anergy provided evidence that the transactivation of the IL-2 gene by the AP-1 transcription factor was impaired in anergized T cell clones. We have now extended these findings with a human IL-2 reporter construct consisting of an enhancer composed of response elements (NF-AT and Oct) that are not directly negatively regulated by anergy. This reporter could be affected if we added back a large region of the IL-2 enhancer containing several other known response elements. Mutations in this segment identified two critical regions at -150 and -180 that were important for the anergic effect. The former site binds AP-1, but the latter site bound no detectable protein in electrophoretic mobility shift assays. The importance of this -180 site was confirmed, however, by mutating it in the normal mouse IL-2 enhancer and showing that this blocked the readout of an anergic effect without affecting the expression of the reporter. These results suggest that anergy is a cis-dominant negative form of regulation that is mediated by several transcription factors.
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