TCR downmodulation following ligation by MHC:peptide complexes is considered to be a pivotal event in T cell activation. Here, we analyzed the dynamics of TCR:CD3 cell surface expression on resting and antigen-activated T cells. We show that the TCR:CD3 complex is very stable and is rapidly internalized and recycled in resting T cells. Surprisingly, the internalization rate is not increased following TCR ligation by MHC:peptide complexes, despite significant TCR downmodulation, suggesting that constitutive internalization rather than ligation-induced downmodulation serves as the force that drives serial ligation. Furthermore, TCR downmodulation is mediated by the intracellular retention of ligated complexes and degradation by lysosomes and proteasomes. Thus, our data demonstrate that ligation induces TCR downmodulation by preventing recycling rather than inducing internalization.

The role of the T cell antigen receptor complex (TCR) in alphabeta/gammadelta lineage commitment remains controversial, in particular whether different TCR isoforms intrinsically favor adoption of a certain lineage. Here, we demonstrate that impairing the signaling capacity of a gammadeltaTCR complex enables it to efficiently direct thymocytes to the alphabeta lineage. In the presence of a ligand, a transgenic gammadeltaTCR mediates almost exclusive adoption of the gammadelta lineage, while in the absence of ligand, the same gammadeltaTCR promotes alphabeta lineage development with efficiency comparable to the pre-TCR. Importantly, attenuating gammadeltaTCR signaling through Lck deficiency causes reduced ERK1/2 activation and Egr expression and diverts thymocytes to the alphabeta lineage even in the presence of ligand. Conversely, ectopic Egr overexpression favors gammadelta T cell development. Our data support a model whereby gammadelta versus alphabeta lineage commitment is controlled by TCR signal strength, which depends critically on the ERK MAPK-Egr pathway.

SUMMARY αβ and γδ T-cells arise from a common thymocyte progenitor during development in the thymus. Emerging evidence suggests that the pre-T cell receptor (pre-TCR) and γδ T-cell receptor (γδTCR) play instructional roles in specifying the αβ and γδ T-lineage fates, respectively. Nevertheless, the signaling pathways differentially engaged to specify the fate and promote the development of these lineages remain poorly understood. Here we show that differential activation of the ERK - early growth response gene (Egr) - inhibitor of DNA binding 3 (Id3) pathway plays a defining role in this process. In particular, Id3 expression serves to regulate adoption of the γδ fate. Moreover, Id3 is both necessary and sufficient to enable γδ-lineage cells to differentiate independently of Notch signaling and become competent IFNγ-producing effectors. Taken together, these findings identify Id3 as a central player that controls both adoption of the γδ fate and their maturation in the thymus.

Ribosomal protein (RP) mutations in diseases such as 5q؊ syndrome both disrupt hematopoiesis and increase the risk of developing hematologic malignancy. However, the mechanism by which RP mutations increase cancer risk has remained an important unanswered question. We show here that monoallelic, germline inactivation of the ribosomal protein L22 (Rpl22) predisposes T-lineage progenitors to transformation. Indeed, RPL22 was found to be inactivated in ϳ 10% of human T-acute lymphoblastic leukemias. Moreover, monoallelic loss of Rpl22 accelerates development of thymic lymphoma in both a mouse model of T-cell malignancy and in acute transformation assays in vitro. We show that Rpl22 inactivation enhances transformation potential through induction of the stemness factor, Lin28B. Our finding that Rpl22 inactivation promotes transformation by inducing expression of Lin28B provides the first insight into the mechanistic basis by which mutations in Rpl22, and perhaps some other RP genes, increases cancer risk. (Blood. 2012;120(18):3764-3773) IntroductionIn addition to their role as structural components of ribosomes, ribosomal proteins (RPs) are increasingly understood to play critical roles in development and disease, in some cases from outside of the ribosome. These include roles in regulation of cell-cycle progression, apoptosis, 1 and translation, through direct interactions with mRNA. 2 Mutations in RPs cause diseases collectively termed ribosomopathies, which include myelodysplastic syndromes (MDS) and diamond blackfan anemia (DBA). DBA is caused by mutations in a variety of RPs, with approximately one-half of all cases resulting from mutations in RPS19, RPS26, RPL5, and RPL11, whereas a type of myelodysplastic syndrome known as 5qϪ syndrome has been attributed to the monoallelic loss of RPS14. 3,4 RPS14 haploinsufficiency in 5qϪ syndrome, as well as the ribosome dysfunction observed in other bone marrow failure syndromes, is associated with increased risk in patients for the development of hematologic malignancies. 5 Observations in animal models have similarly linked RP gene mutations with alterations in cancer risk because loss of one copy of numerous, essential RP genes increased susceptibility to tumor formation in zebrafish, 6 suggesting that some RPs may serve as haploinsufficient tumor suppressors. Nevertheless, neither the basis by which RP function as tumor suppressors nor the way RP mutations predispose to malignancy has been explained.The ribosomal protein L22 (Rpl22) is an RNA-binding component of the 60S ribosomal subunit that is not thought to be required for global cap-dependent translation, but its normal physiologic role is poorly understood. We have determined that despite the ubiquitous expression of Rpl22, its germline ablation in mouse is not lethal, unlike ablation of most RP genes. 7,8 Instead, mice in which the Rpl22 gene is biallelically inactivated in the germline are viable, fertile, and grossly normal, with the only striking defect being an exquisitely specific block in the development ...

Summary It remains controversial whether the highly-homologous ribosomal protein (RP) paralogs found in lower eukaryotes have distinct functions and this has not been explored in vertebrates. Here we demonstrate that despite ubiquitous expression, the RP paralogs, Rpl22 and Rpl22-like1 (Rpl22l1) play essential, distinct, and antagonistic roles in hematopoietic development. Knockdown of rpl22 in zebrafish embryos selectively blocks the development of T lineage progenitors after they have seeded the thymus. In contrast, knockdown of the rpl22 paralog, rpl22l1, impairs the emergence of hematopoietic stem cells (HSC) in the aorta-gonad-mesonephros by abrogating Smad1 expression and the consequent induction of essential transcriptional regulator, Runx1. Indeed, despite the ability of both paralogs to bind Smad1 RNA, Rpl22 and Rpl22l1 have opposing effects on Smad1 expression. Accordingly, circumstances that tip the balance of these paralogs in favor of Rpl22 (e.g., Rpl22l1 knockdown or Rpl22 overexpression) result in repression of Smad1 and blockade of HSC emergence.