Modulation of TCRß surface expression during TCR revision.

TCR revision is a tolerance mechanism by which self-reactive TCRs expressed by mature CD4(+) peripheral T cells are replaced by receptors encoded by genes generated by post-thymic DNA rearrangement. The downmodulation of surface TCR expression initiates TCR revision, and serves as a likely trigger for the induction of the recombinase machinery. We show here in a Vß5 transgenic mouse model system that downregulation of the self-reactive transgene-encoded TCR is not maintained by transgene loss or diminished transcription or translation. The downregulation of surface TCR expression likely occurs in two stages, only one of which requires tolerogen expression.

Cutting Edge: Rag deletion in peripheral T cells blocks TCR revision.

Mature CD4(+)Vbeta5(+) T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or TCR revision. In Vbeta5 transgenic mice, this latter tolerance pathway results in the appearance of CD4(+)Vbeta5(-)TCRbeta(+) T cells, coinciding with Rag1, Rag2, and TdT expression and the accumulation of V(beta)-DJ(beta) recombination intermediates in peripheral CD4(+) T cells. Because postthymic RAG-dependent TCR rearrangement has remained controversial, we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We show in this study that Rag deletion in post-positive selection T cells in Vbeta5 transgenic mice blocks TCR revision in vivo and that mature peripheral T cells sorted to remove cells bearing endogenous TCRbeta-chains can express newly generated TCRbeta molecules in adoptive hosts. These findings unambiguously demonstrate postthymic, RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4(+) T cells.

Cutting edge: two distinct motifs within the Fas ligand tail regulate Fas ligand-mediated costimulation.

The cytoplasmic domain of Fas ligand is sufficient to costimulate CD8(+) T cells by driving Fas ligand recruitment into lipid rafts and association with select Src homology 3-containing proteins, activating PI3K and MAPK pathways, mediating nuclear translocation of the transcription factors NFAT and AP-1, and enhancing IFN-gamma production and Ag-specific CD8(+) T cell proliferation. We now show that Fas ligand molecules lacking amino acids 45-54 in the proline-rich region of the cytoplasmic domain fail to costimulate but serve as effective death inducers. Death induction and costimulation by Fas ligand are therefore clearly separable functions. Further, upon Fas ligand-mediated costimulation, casein kinase I phosphorylates Fas ligand, in which two conserved casein kinase I binding sites regulate NFAT activation and costimulation. These results help resolve how one molecule can serve as a double-edged immunomodulator by directing discrete biological consequences.

The cytoplasmic domain of Fas ligand costimulates TCR signals.

Productive T cell activation generally requires costimulation in addition to a signal delivered through the TCR. Although FasL is well-characterized for its capacity to deliver a death signal through Fas, this TNF family member can also transmit a reverse signal to enhance Ag-driven T cell proliferation. In this study, we define this reverse signal through FasL as costimulation by showing it requires TCR coengagement and is CD28 independent. We demonstrate that FasL-mediated costimulation drives FasL recruitment into lipid rafts and association with select Src homology 3 (SH3)-containing proteins. We further show that the proline-rich intracellular domain of FasL is sufficient to costimulate by enhancing the phosphorylation of Akt, ERK1/2, JNK, and FasL itself, by activating the transcription factors NFAT and AP-1, and by enhancing IFN-gamma production. These results elucidate the pathway of costimulation through the death inducer FasL, and comprise the first mechanistic analysis of a newly emerging group of costimulators, the TNF family.