T-cell development encompasses both the acquisition of appropriate recognition specificity and the acquisition of effector response capability. The long-term objective of this project has been to define the process of functional maturation undergone by developing thymocytes. We have focused on the acquisition of competence to express interleukin-2 (IL- 2) as a prototype effector function, for lL-2 expression is subject to exquisite induction requirements and to stringent developmental regulation at the level of cell-type specificity. Using in vivo and in vitro IL-2 induction as a sensitive probe of thymocyte physiology, two major results have emerged. One is that the potential to express IL-2 is acquired very early in intrathymic differentiation, prior to and independently of any expression of the T-cell receptor (TcR) for antigen. The other is that activation stimuli encountered in vivo by developing thymocytes actually result in the triggering of IL-2 expression in rare cells in several interesting developmental compartments. These results raise several questions which we propose to address. One is what relationship really exists between the initial acquisition of competence to express lL-2 and the commitment of a cell to the T-cell lineage. Another is what the developmental significance may be of each of the precisely limited episodes of intrathymic activation that results in lL-2 induction in vivo. Another, more broadly, is to what extent the different types of activation experiences that even post-thymic T cells undergo have directive or stochastic long-term effects on cell fate and function. To address these questions, we propose to take advantage of a newly- developed recombinase-dependent lineage tracing technique. With this technique, the site-specific Cre recombinase is introduced into mice as a transgene under the control of regulatory sequences that will activate it transiently at a developmental stage of interest. Site-specific recombination catalyzed by Cre in a second, marker transgene then heritably marks the progeny of the Cre-expressing cell, even after Cre itself is turned off. IL -2 regulatory sequences have been extensively studied by others and by ourselves, and it is possible to use known sequences to induce a transgene to he coexpressed with lL-2. By placing Cre under IL-2 control, we will be able to trace the progeny of cells that express lL-2 in vivo, in thymocyte subsets, in """"""""nonconventional"""""""" T-cell lineages, in different effector subsets of peripheral TcRalphabeta+ T cells, and even in non-T hematopoietic cells. The results should reveal how the subset of inductive contacts that lead to lL-2 expression may be linked with the determination of cell fate.
Specific aim l: To use the Cre-lox system under the control of lL-2 regulatory sequences to mark and trace the fate of cells derived from IL-2 producers.
Specific aim 2 : To test the relation between lL-2 inducing activation and cell fate in the T-cell lineage.
Specific aim 3 : To test the fidelity of T-cell lineage-restricted lL-2 expression by analysis of lL-2/Cre-dependent marking of non-T lineage cells in vivo.
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