Molecular cloning of pTAC12 an alternative splicing product of the CD3gamma chain as a component of the pre-T cell antigen-receptor complex.

We have reported that a 12-kDa molecule (pTAC12 as a pre-T cell receptor (TCR)-associated chain) was associated as a dimer with the pre-TCR complex as well as the clonotype-independent CD3 complex on the cell surface of immature thymocytes. We now report by protein sequencing and molecular cloning that pTAC12 is an alternatively spliced product of the CD3gamma chain lacking exon 4 containing the transmembrane region. The transcript of pTAC12 is expressed in most T cell lineages and parallels the expression of CD3gamma. However, the pTAC12 protein is expressed on the cell surface of immature thymocytes but not mature T cells, despite the fact that mature T cells express a low level of pTAC12 in association with the TCR complex within the cells. These results indicate that pTAC12 may play a special role for the transport/expression and assembly of the pre-TCR.CD3 complex as well as the clonotype-independent CD3 complex in immature thymocytes.

A novel 90-kDa tyrosine-phosphorylated protein associated with TCR complex in thymocytes.

Ligation of the TCR-CD3 complex initiates a cascade of tyrosine phosphorylation that results in T cell activation. Initial activation of tyrosine kinases depends on the phosphorylation of activation motifs on CD3 chains. We previously found that a 90-kDa protein was tyrosine phosphorylated upon TCR cross-linking and the induction of the phosphorylation was dependent on the structure of the CD3 complex. In this study, we further characterized p90 phosphorylation. Phosphorylation of p90 was induced only by stimulation through the TCR-CD3 complex but not by other kinds of stimulation including CD28- or hydrogen peroxide-mediated activation and was dynamically regulated. Phosphorylated p90 was associated with the TCR-CD3 complex upon T cell activation. In a normal T cell population, thymocytes but not splenic T cells induced the tyrosine phosphorylation of p90 upon TCR cross-linking. These results suggest that p90 is a novel phosphoprotein associated with the TCR-CD3 complex and may play a role in TCR signaling during thymocyte differentiation.

A new 12-kilodalton dimer associated with pre-TCR complex and clonotype-independent CD3 complex on immature thymocytes.

We have characterized the pre-TCR/CD3 complex and the clonotype-independent CD3 (CIC) complex expressed on the cell surface of the immature cell line KKF which we previously identified as the cell line expressing only TCR beta-chain associated with the CD3 complex on the cell surface. We now show that KKF expresses the pT alpha-beta heterodimer-CD3 complex and also the CIC complex on the cell surface by surface biotinylation. In addition, not only the CIC complex but also the pT alpha-beta complex was found to be associated with the molecular chaperon calnexin. Whereas the CD3 zeta-chain was very weakly associated with the pre-TCR/CD3 complex, a 12-kDa molecule (designated pTAC12 as pre-TCR-associated chain) was more stably associated as a dimer with the pre-TCR complex as well as the CIC complex on KKF cells. The significance of pTAC12 dimer in the CD3 complex of immature thymocytes in vivo was demonstrated by coprecipitation of pTAC12 with the CD3 complex in thymocytes from SCID mice. These results show that pTAC12 is a component of the pT alpha-beta complex as well as the CIC complex, and they suggest that pTAC12 may play a role in signaling through and/or in the expression of the pre-TCR/CD3 complex.

Developmental arrest of NK1.1+ T cell antigen receptor (TCR)-alpha/beta+ T cells and expansion of NK1.1+ TCR-gamma/delta+ T cell development in CD3 zeta-deficient mice.

The relationship between the structure of the T cell antigen receptor (TCR)-CD3 complex and development of NK1.1+ T cells was investigated. The TCR complex of freshly isolated NK1.1+ TCR-alpha/beta+ thymocytes contained CD3 zeta homodimers and CD zeta-FcR gamma heterodimers, whereas that of the majority of NK1.1- T cells did not contain FcR gamma. The function of CD3 zeta and FcR gamma in the development of NK1.1+ T cells was determined by analyzing CD3 zeta- and FcR gamma-deficient mice. The NK1.1+ T cells from wild-type and CD3 zeta-deficient mice had equal levels of CD3 expression. However, the development of NK1.1+ TCR-alpha/beta+ T cells was almost completely disrupted in thymus and spleen in CD3 zeta-deficient mice, whereas no alteration was observed in FcR gamma-deficient mice. In contrast, the number of novel NK1.1+ TCR-gamma/delta+ thymocytes expressing a surface phenotype similar to NK1.1+ TCR-alpha/beta+ thymocytes increased approximately six times in CD3 zeta-deficient mice. These findings establish the distinct roles of the CD3 zeta chain in the development of the following different thymic T cell compartments: NK1.1- TCR+, NK1.1+ TCR-alpha/beta+, and NK1.1+ TCR-gamma/delta+ thymocytes, which cannot be replaced by CD3 eta or FcR gamma chains.

Differential contribution of the FcR gamma chain to the surface expression of the T cell receptor among T cells localized in epithelia: analysis of FcR gamma-deficient mice.

The function of the Fc receptors gamma chain (FcR gamma) for the expression of the T cell receptor (TCR) complex and for T cell development, especially for T cells localized in epithelia, was investigated by analyzing FcR gamma-deficient mice. In wild-type mice, CD8 alpha alpha + beta -TCR alpha beta + T cells of intestinal intraepithelial lymphocytes (i-IEL) utilized CD3 zeta homodimers and zeta-FcR gamma heterodimers, whereas CD8 alpha alpha + beta -TCR gamma delta + i-IEL used zeta-FcR gamma and FcR gamma homodimers in the TCR complex. On the other hand, these T cells in FcR gamma-deficient mice contained only zeta homodimers. The surface expression of the TCR complex was reduced in CD8 alpha alpha + beta -i-IEL and dendritic epidermal T cells (DETC) in these mice, whereas the development of these T cells was normal. The degree of reduction appeared to depend on the expression level of FcR gamma. In contrast to these populations, TCR gamma delta + intraepithelial T cells in reproductive organs (r-IEL) were dramatically decreased, suggesting that the development of r-IEL is FcR gamma-dependent, probably due to the predominant usage of FcR gamma homodimers in the TCR complex. These results indicate that the FcR gamma chain contributes differently to the TCR expression and to the development of T cells localized in epithelia.