Genetic studies of Shc function in T cells

Shc is an adaptor protein containing an SH2 and a PTB domain that has been implicated in signaling events leading to mitogenesis and differentiation in several receptor systems. The importance of Shc in vivo was underscored by the embryonic lethality in mice with a disruption of the shc gene. However, the embryonic lethality of the shc null mutation severely limits further study. Previous work has demonstrated that the p52 and p46 isoforms of Shc become phosphorylated upon T cell receptor crossinglinking in T cell hybridomas and primary mature T cells, and that Shc can associate with the phosphorylated TCR-ζ chain. However, whether Shc plays a role during T cell development and function in vivo was not known.; To determine if Shc plays a role during T cell development, mice were generated that either can conditionally express a dominant negative form of Shc (ShcFFF) or were targeted at the shc locus with loxP sites for cinditonal deletion. These mice were then crossed with transgenic mice expressing Cre under the transcriptional control of the lck proximal promoter. In both the transgenic (lck-Cre-ShcFFF) and knockout (lck-Cre-shcfl/fl) mice, we observed that total thymic cellularity was profoundly decreased, with a developmental block in the double-negative (CD4−CD8 −) subset, resulting from an apparent block in transition from the DN3 CD44−CD25+ to the DN4 CD44 −CD25− stage. Recombination at the TCRβ locus and β chain expression appeared to be unaffected.; By crossing lck-Cre-ShcFFF mice with DO11.10 TCR transgenic mice, we found that the introduced T cell receptor cannot overcome the developmental block due to ShcFFF, these findings strongly suggest a role for Shc in pre-TCR signaling. We also found that pre-TCR mediated cell differentiation and cell proliferation is dependent on Shc, while Shc seems to be dispensable for allelic exclusion at TCR β locus and DN3 cell survival. To explore the mechanism of Shc mediated regulation of pre-TCR signaling, we crossed the lck -Cre-ShcFFF mice with mice that carry LckF505 (constitutively active Lck) or Raf-CAAX espression cassette, which are driven by the lck -proximal promoter. Shc was found to be phosphorylated by LckF505, but LckF505 could not rescue the DN3 developmental block by ShcFFF, which places Shc downstream of Lck in pre-TCR signaling. We also demonstrated that Shc mediates pre-TCR signaling through MAPK dependent and independent pathway, since Raf-CAAX alone was not able to rescue the phenotype of ShcFFF expressing mice.; We also demonstrated that Shc is required for mature T cell function, T cell proliferation and IL-2 production after TCR crosslinking by anti-CD3 is greatly decrased by loss of Shc. In summary, we have used two genetic tools to investigate the role of Shc during thymic T cell development and in primary mature T cell function. Our data suggest a nonredundant role of Shc in both thymic T cell development and mature T cell function.