The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement. Phosphorylated LAT binds many critical signaling molecules. The central role of this molecule in TCR-mediated signaling has been demonstrated by experiments in a LAT-deficient cell line. To probe the role of LAT in T cell development, the LAT gene was disrupted by targeting. LAT-deficient mice appeared healthy. Flow cytometric analysis revealed normal B cell populations but the absence of any mature peripheral T cells. Intrathymic development was blocked within the CD4- CD8- stage. No gross abnormality of NK or platelet function was observed. LAT is thus critical to both T cell activation and development.

Current data indicate that CD5 functions as an inhibitor of TCR signal transduction. Consistent with this role, thymocyte selection in TCR transgenic/CD5−/− mice is altered in a manner suggestive of enhanced TCR signaling. However, the impact of CD5 deletion on thymocyte selection varies depending on the transgenic TCR analyzed, ranging from a slight to a marked shift from positive toward negative selection. An explanation for the variable effect of CD5 on selection is suggested by the observation that CD5 surface expression is regulated by TCR signal intensity during development and CD5 surface levels on mature thymocytes and T cells parallel the avidity of the positively selecting TCR/MHC/ligand interaction. In this study, we generated mice that overexpress CD5 during thymocyte development (CD5-tg), and then examined the effect of CD5 overexpression or CD5 deletion (CD5−/−) on selection of thymocytes that express the same TCR transgenes. The results demonstrate that the effect on thymocyte selection of altering CD5 expression depends on the avidity of the selecting interaction and, consequently, the level of basal (endogenous) CD5 surface expression. Substitution of endogenous CD5 with a transgene encoding a truncated form of the protein failed to rescue the CD5−/− phenotype, demonstrating that the cytoplasmic domain of CD5 is required for its inhibitory function. Together, these results indicate that inducible regulation of CD5 surface expression during thymocyte selection functions to fine tune the TCR signaling response.

T cell antigen receptor (TCR) and pre-TCR complexes are composed of clonotypic heterodimers in association with dimers of signal transducing invariant subunits (CD3␥, -␦, -, and ). The role of individual invariant subunits in T cell development has been investigated by generating gene-specific mutations in mice. Mutation of CD3␥, -␦, or results in an incomplete block in development, characterized by reduced numbers of mature T cells that express low levels of TCR. In contrast, mature T cells are absent from CD3 ؊͞؊ mice, and thymocyte development is arrested at the early CD4 ؊ CD8 ؊ stage. Although these results suggest that CD3 is essential for pre-TCR and TCR expression͞function, their interpretation is complicated by the fact that expression of the CD3␥ and CD3␦ genes also is reduced in CD3 ؊͞؊ mice. Thus, it is unclear whether the phenotype of CD3 ؊͞؊ mice ref lects the collective effects of CD3␥, -␦, and -deficiency. By removing the selectable marker (PGK-NEO) from the targeted CD3 gene via Cre͞loxP-mediated recombination, we generated mice that lack CD3 yet retain normal expression of the closely linked CD3␥ and CD3␦ genes. These (CD3 ⌬͞⌬ ) mice exhibited an early arrest in T cell development, similar to that of CD3 ؊͞؊ mice. Moreover, the developmental defect could be rescued by expression of a CD3 transgene. These results identify an essential role for CD3 in T cell development not shared by the CD3␥, CD3␦, or -family proteins and provide further evidence that PGK-NEO can inf luence the expression of genes in its proximity.Differentiation of thymocytes into mature, functional T cells requires the input of signals delivered through the T cell antigen receptor (TCR) and a precursor form of the TCR, the pre-TCR (1, 2). The TCR complexes expressed on mature T cells are composed of subunits originating from six different genes: TCR␣ and TCR␤ (or TCR␥ and TCR␦), CD3␥, CD3␦, CD3 , and (or a related family member) (3). The clonotypic (TCR␣͞␤ or TCR␥͞␦) chains, which specify distinct lineages of T cells, are responsible for ligand recognition and are generated during development by programmed rearrangement of germline [V-(D)-J] gene segments. TCR␣͞␤ and TCR␥͞␦ heterodimers lack inherent signaling activity but associate noncovalently with dimers composed of the invariant signal transducing CD3 and -family subunits. The generally accepted stoichiometry for the TCR complex is TCR␣␤ or TCR␥␦͞CD3␥ ͞CD3␦ ͞ .Rearrangement of genes encoding the lineage-specific ␣␤-and ␥␦-TCR chains is initiated in immature CD4 Ϫ CD8 Ϫ [or double negative (DN)] thymocytes that constitutively express the CD3␥, CD3␦, CD3 , and subunits (4). Productive rearrangement of both the TCR␥ and TCR␦ genes results in surface expression of ␥␦TCR complexes and commitment of DN thymocytes to the ␥␦-T cell lineage (5). On the other hand, productive rearrangement of the TCR␤ locus results in surface expression of a ''pre-TCR'' complex composed of TCR␤ chain paired with an invariant, pre-T␣ chain in association with CD3 and -chain dimers (2). Signa...

The T cell antigen receptor (TCR) and pre-TCR complexes are composed of multiple signal-transducing subunits (CD3γ, CD3δ, CD3ε, and ζ) that each contain one or more copies of a semiconserved functional motif, the immunoreceptor tyrosine-based activation motif (ITAM). Although biochemical studies indicate that individual TCR-ITAMs may bind selectively or with different affinity to various effector molecules, data from other experiments suggest that at least some ITAMs are functionally equivalent. In this study, we examined the role of CD3ε ITAM-mediated signals in T cell development by genetically reconstituting CD3ε-deficient mice with transgenes encoding either wild-type or ITAM-mutant (signaling defective) forms of the protein. The results demonstrate that signals transduced by CD3ε are not specifically required for T cell maturation but instead contribute quantitatively to TCR signaling in a manner similar to that previously observed for ζ chain. Unexpectedly, analysis of TCR-transgenic/CD3ε-mutant mice reveals a potential role for CD3ε signals in T cell survival.

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