T CELL DEVELOPMENT AND SIGNAL TRANSDUCTION

DIETMAR J. KAPPES, Ph.D., Member

Our work aims to define the roles of specific signaling molecules in thymic development and to determine whether alternative developmental outcomes reflect quantitative or qualitative differences in signaling. During differentiation in the thymus, immature T cells undergo rearrangement of their T cell receptor (TCR) a and b genes to generate a diverse set of clonotypic TCRs. Immature thymocytes are selected for further maturation depending on the capacity of their TCRs to interact with self-peptides presented in the context of self-major histocompatibility complex (MHC) molecules. The resulting TCR-mediated signals may differ quantitatively and/or qualitatively. Different signals lead to the alternate developmental outcomes of maturation or apoptosis (positive versus negative selection). Positively selected thymocytes undergo alternate commitment to either the T killer or T helper lineages, which correlates precisely with a cell's TCR specificity towards MHC class I or II molecules, respectively. Lineage commitment is marked phenotypically by the loss of expression of one of the co-receptor molecules, CD8 or CD4. Immature thymocytes express both co-receptors (double positive [DP]), while T killer or T helper cells express only CD8 or CD4, respectively (single positive [SP] CD8+ or CD4+). It seems likely that co-engagement of class I or II MHC molecules by TCRs and the appropriate co-receptors on DP thymocytes initiates specific signals, which leads to commitment to the CD8+ killer and CD4+ helper lineages, respectively. The nature of these signals, and whether they are distinct or overlapping with those leading to positive selection, remains to be elucidated.

The TCR heterodimer is tightly associated with four independently encoded CD3 subunits (d, g, e, and z) which are required for efficient transport to the cell surface. CD3 subunits all possess long intracellular tails and are responsible for transducing signals upon TCR engagement. It remains unclear whether CD3 subunits have overlapping or specialized signaling functions. Arguing for functional overlap, a common motif, the immunoreceptor tyrosine-activating motif (ITAM), occurs within the cytoplasmic domains of all CD3 components. However, ITAMs also show significant sequence diversity and have different binding affinities to known intracellular ligands, leaving open the possibility that different CD3 subunits preferentially activate different signaling pathways. We and others have started to dissect the individual roles of CD3 components in vivo by generating mutant mice that either lack specific CD3 subunits entirely or express altered versions.

We have generated a CD3d deficient ( d-/-) mouse by deleting most of the protein-coding region from the CD3d locus. Absence of CD3d resulted in the failure of thymocytes to undergo positive selection, i.e. arrest at the DP stage. Developmentally controlled upregulation of surface TCR expression was also blocked. In addition, negative selection, as assayed by the elimination of thymocytes expressing self-reactive TCRs, failed to occur, indicating that CD3d is required for both types of selection. Cells of the minor T cell lineage expressing g/d rather than a/b TCRs develop normally in d-/- mice. It has recently been shown that gd T cells are not subject to MHC-restricted positive selection and usually omit the DP stage of maturation, consistent with the view that CD3d is specifically required for positive selection of DP thymocytes.

To determine whether the critical role of CD3d in thymic selection reflects a specific signaling function, we have now generated transgenics expressing a truncated form of CD3d lacking the cytoplasmic domain implicated in signaling (DCYT mice). This transgene was able to fully restore thymic selection in d-/- mice, indicating that the critical function of CD3d maps to its extracellular and/or transmembrane domains. This is consistent with a structural role for CD3d in achieving a critical surface density of the TCR complex, but does not exclude other roles such as providing a docking site for other cell surface molecules. We have exploited the fact that TCR surface density can be controlled in d-/- mice by the availability of DCYT protein to generate transgenic lines that express surface TCR levels 2, 4- or 8-fold below normal. These mice allow a precise definition of the role of TCR-mediated signal strength in regulating thymic development. Strikingly, progressive diminution of TCR density leads to a disproportionate decrease in the frequency of SP CD4+ cells such that at the lowest TCR levels, SP CD8+ cells become predominant in both the thymus and periphery (Figure 1). To test whether this reflects a specific blockade in the development of class II-restricted thymocytes or their alternate development to the CD8 lineage, we introduced the class II-restricted "AND" TCR transgene onto the DCYT background. "AND" transgenic mice showed a progressive diminution in the generation of SP CD4+ thymocytes with decreasing TCR density. However, there was no compensatory increase in the generation of SP CD8+ thymocytes at any level of TCR expression. These data indicate that reduced TCR-mediated signaling affected the efficiency of positive selection, but could not alter lineage commitment.

FIGURE 1. Reducing TCR surface levels reverses the normal CD4:CD8 T lymphocyte ratio. a. Flow cytometric measurement of TCR density on peripheral T lymphocytes from different transgenic lines showing stepwise reductions in TCR surface levels of 2, 4 and 8-fold below normal. b. Proportions of CD4+ and CD8+ peripheral T cells in the same transgenic lines. Note the relative decrease in SP CD4+ T cells and increase in SP CD8+ T cells from right to left.

SIGNALING PATHWAYS IN LINEAGE COMMITMENT. KAPPES, DAVÉ, KEEFE

It appears, as discussed above, that commitment to either T killer or helper lineages involves lineage-specific signals mediated by the TCR in conjunction with co-receptor molecules. Other signaling pathways, such as that mediated by the Notch receptor, may also be involved. The intracellular components of signaling pathways required for lineage specification remain essentially unknown. In our colony, we have recently discovered a spontaneous mouse mutant that almost completely lacks peripheral CD4+ helper T cells ("Helper deficient" [HD] mice). HD mice differ from other mutant mouse lines lacking CD4+ T cells in that the defect is intrinsic to developing thymocytes and appears to affect a critical pathway involved in lineage commitment. This novel mouse mutant affords a unique opportunity to explore the molecular mechanisms underlying lineage commitment, and to determine whether it is mechanistically distinct from positive selection. Consistent with such a mechanistic distinction, the HD defect does not impair TCR-mediated signaling, or the efficiency of positive selection. Most strikingly, we have demonstrated that the HD defect causes class II-restricted thymocytes to mature to the CD8 rather than the CD4 lineage, and that this redirection is independent of MHC class I expression (Figure 2). This has certain implications for the mechanism of lineage commitment and for the models that have been proposed to explain it. Current efforts are focussed on mapping the chromosomal position of the HD gene. Defining the molecular defect underlying this novel phenotype should lead to unique insights into the regulation of lineage commitment.

FIGURE 2. The HD mutation leads to redirection of MHC class II-restricted thymocytes to the CD8 lineage. Distribution of thymic and peripheral T cell subsets in HD-/-, b2m-/- and doubly deficient HD-/- b2m-/- mice. b2m-/- mice lack MHC class I expression, so that only class II-restricted thymocytes can mature in this background. Note the presence of SP CD8+ cells in doubly deficient HD-/- b2m-/- mice, but not in HD+ b2m-/- mice which generate only SP CD4+ T cells.

PUBLICATIONS

DAVÉ, V.P., ALLMAN, D., WIEST, D.L., KAPPES, D.J. Limiting TCR expression leads to quantitative but not qualitative changes in thymic selection. J. Immunol. (in press).

Paper in press at time of previous report:

DAVÉ, V.P., KEEFE, R., BERGER, M.A., DRBAL, K., PUNT, J.A., WIEST, D.L., ALARCON, B., KAPPES, D.J. Altered functional responsiveness of thymocyte subsets from CD3d-deficient mice to TCR/CD3 engagement. Int. Immunol. 10:1481-1490, 1998.

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998