David L. Wiest

On the Dynamics of TCR:CD3 Complex Cell Surface Expression and Downmodulation

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.

Branching out to gain control: how the pre-TCR is linked to multiple functions

How is signaling specificity achieved by the pre-TCR during selection of T-cell fate? Like the TCR, this receptor controls many functions, and recent studies define which pathways couple the pre-TCR to the molecular events controlling survival, proliferation, allelic exclusion at the TCRbeta locus, and further differentiation.

Mechanistic basis of pre-T cell receptor-mediated autonomous signaling critical for thymocyte development.

The pre–T cell receptor (TCR) is crucial for early T cell development and is proposed to function in a ligand-independent way. However, the molecular mechanism underlying the autonomous signals remains elusive. Here we show that the pre-TCR complex spontaneously formed oligomers. Specific charged residues in the extracellular domain of the pre-TCR α-chain mediated formation of the oligomers in vitro. Alteration of these residues eliminated the ability of the pre-TCR α-chain to support pre-TCR signaling in vivo. Dimerization but not raft localization of CD3ε was sufficient to simulate pre-TCR function and promote β-selection. These results suggest that the pre-TCR complex can deliver its signal autonomously through oligomerization of the pre-TCR α-chain mediated by charged residues.

The molecular chaperone calnexin is expressed on the surface of immature thymocytes in association with clonotype-independent CD3 complexes.

Immature thymocytes express clonotype‐independent CD3 complexes that, when engaged by anti‐CD3 antibodies, can signal CD4‐CD8‐ thymocytes to differentiate into CD4+CD8+ cells. Clonotype‐independent CD3 complexes consist of CD3 components associated with an unknown 90 kDa surface protein. We now report the surprising finding that this 90 kDa surface protein is the molecular chaperone calnexin, an integral membrane protein previously thought to reside only in the endoplasmic reticulum (ER). We found that calnexin‐CD3 complexes escaping to the cell surface utilize interchain associations distinct from those utilized by calnexin‐CD3 complexes remaining within the ER. Specifically, we demonstrate that carbohydrate‐mediated luminal domain interactions that are necessary for formation of most internal calnexin‐CD3 complexes destined to be expressed on the cell surface, and we provide evidence that cytoplasmic domain interactions between calnexin and CD3 epsilon chains mask calnexins ER retention signal, permitting calnexin and associated proteins to escape ER retention. Thus, the present study demonstrates that partial T cell antigen receptor complexes can escape the ER of immature thymocytes in association with their molecular chaperone to be expressed at low levels on the cell surface where they may function as a signaling complex to regulate thymocyte maturation.

Marked Induction of the Helix-Loop-Helix Protein Id3 Promotes the γδ T Cell Fate and Renders Their Functional Maturation Notch Independent

alphabeta and gammadelta 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 gammadelta T cell receptor (gammadeltaTCR) play instructional roles in specifying the alphabeta and gammadelta T-lineage fates, respectively. Nevertheless, the signaling pathways differentially engaged to specify fate and promote the development of these lineages remain poorly understood. Here, we show that differential activation of the extracellular signal-related kinase (ERK)-early growth response gene (Egr)-inhibitor of DNA binding 3 (Id3) pathway plays a defining role in this process. In particular, Id3 expression served to regulate adoption of the gammadelta fate. Moreover, Id3 was both necessary and sufficient to enable gammadelta-lineage cells to differentiate independently of Notch signaling and become competent IFNgamma-producing effectors. Taken together, these findings identify Id3 as a central player that controls both adoption of the gammadelta fate and its maturation in the thymus.

Early Growth Response Transcription Factors Are Required for Development of CD4−CD8− Thymocytes to the CD4+CD8+ Stage

Progression of immature CD4−CD8− thymocytes beyond the β-selection checkpoint to the CD4+CD8+ stage requires activation of the pre-TCR complex; however, few of the DNA-binding proteins that serve as molecular effectors of those pre-TCR signals have been identified. We demonstrate in this study that members of the early growth response (Egr) family of transcription factors are critical effectors of the signals that promote this developmental transition. Specifically, the induction of three Egr family members (Egr1, 2, and 3) correlates with pre-TCR activation and development of CD4−CD8− thymocytes beyond the β-selection checkpoint. Enforced expression of each of these Egr factors is able to bypass the block in thymocyte development associated with defective pre-TCR function. However, Egr family members may play somewhat distinct roles in promoting thymocyte development, because there are differences in the genes modulated by enforced expression of particular Egr factors. Finally, interfering with Egr function using dominant-negative proteins disrupts thymocyte development from the CD4−CD8− to the CD4+CD8+ stage. Taken together, these data demonstrate that the Egr proteins play an essential role in executing the differentiation program initiated by pre-TCR signaling.

Unequal Contribution of Akt Isoforms in the Double-Negative to Double-Positive Thymocyte Transition

Pre-TCR signals regulate the transition of the double-negative (DN) 3 thymocytes to the DN4, and subsequently to the double-positive (DP) stage. In this study, we show that pre-TCR signals activate Akt and that pharmacological inhibition of the PI3K/Akt pathway, or combined ablation of Akt1 and Akt2, and to a lesser extent Akt1 and Akt3, interfere with the differentiation of DN3 and the accumulation of DP thymocytes. Combined ablation of Akt1 and Akt2 inhibits the proliferation of DN4 cells, while combined ablation of all Akt isoforms also inhibits the survival of all the DN thymocytes. Finally, the combined ablation of Akt1 and Akt2 inhibits the survival of DP thymocytes. Constitutively active Lck-Akt1 transgenes had the opposite effects. We conclude that, following their activation by pre-TCR signals, Akt1, Akt2, and, to a lesser extent, Akt3 promote the transition of DN thymocytes to the DP stage, in part by enhancing the proliferation and survival of cells undergoing β-selection. Akt1 and Akt2 also contribute to the differentiation process by promoting the survival of the DP thymocytes.

Developmental regulation of alpha beta T cell antigen receptor expression results from differential stability of nascent TCR alpha proteins within the endoplasmic reticulum of immature and mature T cells.

The alpha beta T cell antigen receptor (TCR) that is expressed on most T lymphocytes is a multisubunit transmembrane complex composed of at least six different proteins (alpha, beta, gamma, delta, epsilon and zeta) that are assembled in the endoplasmic reticulum (ER) and then transported to the plasma membrane. Expression of the TCR complex is quantitatively regulated during T cell development, with immature CD4+CD8+ thymocytes expressing only 10% of the number of surface alpha beta TCR complexes that are expressed on mature T cells. However, the molecular basis for low TCR expression in developing alpha beta T cells is unknown. In the present study we report the unexpected finding that assembly of nascent component chains into complete TCR alpha beta complexes is severely impaired in immature CD4+CD8+ thymocytes relative to their mature T cell progeny. In particular, the initial association of TCR alpha with TCR beta proteins, which occurs relatively efficiently in mature T cells, is markedly inefficient in immature CD4+CD8+ thymocytes, even for a matched pair of transgenic TCR alpha and TCR beta proteins. Inefficient formation of TCR alpha beta heterodimers in immature CD4+CD8+ thymocytes was found to result from the unique instability of nascent TCR alpha proteins within the ER of immature CD4+CD8+ thymocytes, with nascent TCR alpha proteins having a median survival time of only 15 min in CD4+CD8+ thymocytes, but > 75 min in mature T cells. Thus, these data demonstrate that stability of TCR alpha proteins within the ER is developmentally regulated and provide a molecular basis for quantitative differences in alpha beta TCR expression on immature and mature T cells. In addition, these results provide the first example of a receptor complex whose expression is quantitatively regulated during development by post‐translational limitations on receptor assembly.

Inactivation of ribosomal protein L22 promotes transformation by induction of the stemness factor, Lin28B

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 ∼ 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.

A Spontaneously Arising Mutation in the DLAARN Motif of Murine ZAP-70 Abrogates Kinase Activity and Arrests Thymocyte Development

Development of immature CD4+ CD8+ thymocytes into functionally mature CD4+ and CD8+ T cells is driven by selection events that require signals transduced through the T cell antigen receptor (TCR). Transduction of TCR signals in the thymus involves tyrosine phosphorylation of the protein tyrosine kinase ZAP-70 by p56(lck) and results in induction of ZAP-70 enzymatic activity. We have identified a novel, spontaneously arising point mutation within a highly conserved motif (DLAARN) in the kinase domain of murine ZAP-70 that uncouples tyrosine phosphorylation of ZAP-70 from induction of ZAP-70 kinase activity. Mice homozygous for this mutation are devoid of mature T cells because thymocyte development is arrested at the CD4+ CD8+ stage of differentiation. The developmental arrest is due to the inability of CD4+ CD8+ thymocytes to propagate TCR signals in the absence of ZAP-70 kinase activity despite tyrosine phosphorylation of TCR-associated ZAP-70 molecules.