News and Publications

Regulation of T-Cell Development in the Thymus

J. Kaye, H. Shao, S. Bluvshteyn, L.-Y. Chen, E.M. Rubin, B. Lee, P. Han

Although many cell-surface markers have been described that delineate the stages of T-cell development in the thymus, little is known about the underlying mechanisms that regulate these events. This situation is particularly true for positive selection, a process in which developing thymocytes are driven to differentiate. In this process, antigen-specific T-cell receptors (TCRs) mediate recognition of MHC-encoded molecules expressed by thymic epithelial cells. Our laboratory is interested in the regulation of this differentiation, both at the level of the cellular interaction between thymocytes and epithelial cells and at the molecular level, where we are investigating signaling and changes in gene expression in responding thymocytes.

ROLE OF THE RAS--MAP KINASE SIGNALING PATHWAY IN POSITIVE SELECTION

Binding of the TCR leads to activation of the small GTPase Ras and the downstream MAP kinase cascade. Inhibition of this signaling pathway prevents differentiation of immature thymocytes into mature T cells. To investigate the specific function of this pathway in positive selection, we expressed a constitutively active form of Ras in an immature thymocyte cell line. This cell line maintains the ability to differentiate upon TCR activation. Cells that expressed active Ras underwent partial differentiation, including downregulation of CD8 genes. These results provide evidence for a direct link between Ras signaling pathways and coreceptor regulation during positive selection. Moreover, they suggest a link between Ras signaling and commitment to the CD4 or CD8 T cell lineages. We are exploiting this unique system to detect gene targets for Ras--MAP kinase signaling pathways and to understand the role of Ras in activation of cell adhesion.

Recent reports indicate that stimulation of thymocytes with suboptimal doses of phorbol ester and calcium ionophore can mimic positive selection. We are taking advantage of this system to understand the biochemical requirements of the differentiation of thymocytes. Our findings indicate that this differentiation requires sustained low-level activation of MAP kinase, consistent with the long-held view that positive selection is due to a low-affinity or low-avidity interaction.

The downstream consequences of "weak" signaling, however, are not clear. We found that the pattern of activation of MAP kinase plays a role in regulating the temporal pattern of gene expression during thymocyte differentiation. The particular temporal pattern may play a critical role in distinguishing the opposing consequences of "strong or weak" TCR-mediated activation of thymocytes, resulting in cell death or positive selection, respectively.

REGULATION OF GENE EXPRESSION DURING THYMIC SELECTION

Little is known about the regulation of gene expression during positive selection. We determined that the immediate-early gene Egr-1, which encodes a zinc finger transcription factor, may act as a mediator between signals on the cell surface and changes in gene expression during thymocyte selection. The Egr family of transcriptional regulators is coordinately upregulated in thymocytes as a result of TCR-mediated activation and differentiation, and expression of these proteins depends on Ras and calcineurin signaling pathways, as does positive selection. We are exploring the functional role of these transcription factors in T-cell development.

Other downstream targets for the Ras--MAP kinase signaling pathway in this context are largely unknown. One approach we are taking to address this issue is to ascertain changes in gene expression in an immature thymocyte cell line that expresses an active form of Ras. By screening high-density arrays of expressed sequence tags containing more than 14,000 unique murine cDNA clones, we can detect genes that are upregulated or downregulated as a consequence of Ras activation in this cell type. These studies should lead to a better understanding of positive selection at the molecular level.

PUBLICATIONS

DeKoning, J., Kaye, J. Requirements for differentiation of a CD4⁺8⁺ T cell line. Dev. Immunol. 5:91, 1997.

Ochoa-Garay, J., Kaye, J., Coligan, J.E. NF-B is required for peptide antigen-induced differentiation of a CD4⁺CD8⁺ thymocyte line. J. Immunol. 160:3835, 1998.

Pankhaniya, R., Jabrane-Ferrat, N., Gaufo, G.O., Sreedharan, S.P., Dazin, P., Kaye, J., Goetzl, E.J. Vasoactive intestinal peptide enhancement of antigen-induced differentiation of a cultured line of mouse thymocytes. FASEB J. 12:119, 1998.

Shao, H., Rubin, E., Chen, L.-Y., Kaye, J. A role for Ras signaling in coreceptor regulation during differentiation of a double positive thymocyte cell line. J. Immunol. 159:5773, 1997.