Defining the Role of CD3 Zeta ITAMs in T Cell Signaling and Development

Alpha beta T cells express a T cell receptor (TCR) composed of an alpha beta heterodimer that is responsible for ligand recognition, which is associated with six invariant signaling chains, termed the CD3 subunits. These are organized into two independent signaling modules, the CD3 gamma epsilon / delta epsilon heterodimer ?and the CD3 zeta zeta?homodimer. In their cytoplasmic tail, the CD3 chains contain multiple copies of a motif termed an ITAM. The TCR contains 10 ITAMs, one in each CD3 gamma, delta and epsilon chain, and three in each CD3 zeta chain. The TCR complex, when isolated from thymocytes and peripheral T cells, contains a constitutively tyrosine-phosphorylated CD3 zeta molecule, p21, which results from ongoing interactions between the TCR and peptide/MHC molecules. The magnitude, extent, and duration of the CD3 ITAM phosphorylations are critical for the proper development of several subsets of T cells, including conventional T cells and invariant natural killer T (iNKT) cells. The mechanism by which these phosphorylations are regulated is not fully resolved. Herein, we report that p21 results from TCR interactions with MHC molecules prior to selection, supporting a model that the TCR has an inherent avidity for MHC molecules. Biochemical analyses of the TCR complex before and after TCR stimulation suggested that p21, which exists complexed to ZAP-70, is excluded from new TCR-driven signals. Conventional T cell development proceeds with as few as 4/10 functional CD3 ITAMs; however, iNKT cell development was significantly reduced when one CD3 zeta ITAM was rendered non-functional. This was due to an early block in development, at least in part attributable to an increase in cell death. iNKT cells are thought to be critical for the prevention of inflammatory arthritis following infection with the spirochete, Borrelia burgdorferi. Despite significantly reduced iNKT cells in the CD3 zeta ITAM mutant mice, there was no difference in the severity of Lyme arthritis in these mice compared to wild type controls following B. burgdorferi infection. These data suggest that CD3 ITAMs provide important signals during the selection and development of conventional and iNKT cells. Furthermore, these data suggest that iNKT cells are not the primary cell type responsible for preventing Lyme arthritis.