pre-T Cell Receptor Research Articles

Synchronized development of thymic eosinophils and thymocytes.

The thymus is an organ required for T cell development and is also an eosinophil-rich organ; however, the nature and function of thymic eosinophils remain unclear. Here, we characterized the gene expression and differentiation mechanism of thymic eosinophils in mice. Thymic eosinophils showed a distinct gene expression profile compared with other organ-resident eosinophils. The number of thymic eosinophils was controlled by medullary thymic epithelial cells (mTECs). In Rag-deficient mice, the unique gene expression signature of thymic eosinophils was lost but restored by pre-T cell receptor signalling, which induces CD4+ CD8+ thymocyte differentiation, indicating that T cell differentiation beyond the CD4- CD8- stage is necessary and sufficient for the induction of thymic eosinophils. These results demonstrate that thymic eosinophils are quantitatively and qualitatively regulated by mTECs and developing thymocytes, respectively, suggesting that thymic eosinophils are a distinct, thymus-specific cell subset, induced by interactions with thymic cells.

Pre-T cell receptor localization and trafficking are independent of its signaling.

Expression of the pre-T cell receptor (preTCR) is an important checkpoint during the development of T cells, an essential cell type of our adaptive immune system. The preTCR complex is only transiently expressed and rapidly internalized in developing T cells and is thought to signal in a ligand-independent manner. However, identifying a mechanistic basis for these unique features of the preTCR compared with the final TCR complex has been confounded by the concomitant signaling that is normally present. Thus, we have reconstituted preTCR expression in non-immune cells to uncouple receptor trafficking dynamics from its associated signaling. We find that all the defining features of the preTCR are intrinsic properties of the receptor itself, driven by exposure of an extracellular hydrophobic region, and are not the consequence of receptor activation. Finally, we show that transitory preTCR cell surface expression can sustain tonic signaling in the absence of ligand binding, suggesting how the preTCR can nonetheless drive αβTCR lineage commitment.

Pre-T cell receptor self-MHC sampling restricts thymocyte dedifferentiation.

Programming T cells to distinguish self from non-self is a vital, multi-step process that occurs in the thymus1-4. Signalling through the pre-T cell receptor (preTCR), a CD3-associated heterodimer comprising an invariant pTα chain and a clone-specific β chain, is a critical early checkpoint in thymocyte development within the αβ T cell lineage5,6. PreTCRs arrayed on CD4-CD8- double-negative thymocytes ligate peptides bound to major histocompatibility complex molecules (pMHC) on thymic stroma, similar to αβ T cell receptors that appear on CD4+CD8+ double-positive thymocytes, but via a different molecular docking strategy7-10. Here we show the consequences of these distinct interactions for thymocyte progression using synchronized fetal thymic progenitor cultures that differ in the presence or absence of pMHC on support stroma, and single-cell transcriptomes at key thymocyte developmental transitions. Although major histocompatibility complex (MHC)-negative stroma fosters αβ T cell differentiation, the absence of preTCR-pMHC interactions leads to deviant thymocyte transcriptional programming associated with dedifferentiation. Highly proliferative double-negative and double-positive thymocyte subsets emerge, with antecedent characteristics of T cell lymphoblastic and myeloid malignancies. Compensatory upregulation of diverse MHC class Ib proteins in B2m/H2-Ab1 MHC-knockout mice partially safeguards in vivo thymocyte progression, although disseminated double-positive thymic tumours may develop with ageing. Thus, as well as promoting β chain repertoire broadening for subsequent αβ T cell receptor utilization, preTCR-pMHC interactions limit cellular plasticity to facilitate normal thymocyte differentiation and proliferation that, if absent, introduce developmental vulnerabilities.

Evolution of surrogate light chain in tetrapods and the relationship between lengths of CDR H3 and VpreB tails.

In the mammalian immune system, the surrogate light chain (SLC) shapes the antibody repertoire during B cell development by serving as a checkpoint for production of functional heavy chains (HC). Structural studies indicate that tail regions of VpreB contact and cover the third complementarity-determining region of the HC (CDR H3). However, some species, particularly bovines, have CDR H3 regions that may not be compatible with this HC-SLC interaction model. With immense structural and genetic diversity in antibody repertoires across species, we evaluated the genetic origins and sequence features of surrogate light chain components. We examined tetrapod genomes for evidence of conserved gene synteny to determine the evolutionary origin of VpreB1, VpreB2, and IGLL1, as well as VpreB3 and pre-T cell receptor alpha (PTCRA) genes. We found the genes for the SLC components (VpreB1, VpreB2, and IGLL1) only in eutherian mammals. However, genes for PTCRA occurred in all amniote groups and genes for VpreB3 occurred in all tetrapod groups, and these genes were highly conserved. Additionally, we found evidence of a new VpreB gene in non-mammalian tetrapods that is similar to the VpreB2 gene of eutherian mammals, suggesting VpreB2 may have appeared earlier in tetrapod evolution and may be a precursor to traditional VpreB2 genes in higher vertebrates. Among eutherian mammals, sequence conservation between VpreB1 and VpreB2 was low for all groups except rabbits and rodents, where VpreB2 was nearly identical to VpreB1 and did not share conserved synteny with VpreB2 of other species. VpreB2 of rabbits and rodents likely represents a duplicated variant of VpreB1 and is distinct from the VpreB2 of other mammals. Thus, rabbits and rodents have two variants of VpreB1 (VpreB1-1 and VpreB1-2) but no VpreB2. Sequence analysis of VpreB tail regions indicated differences in sequence content, charge, and length; where repertoire data was available, we observed a significant relationship between VpreB2 tail length and maximum DH length. We posit that SLC components co-evolved with immunoglobulin HC to accommodate the repertoire – particularly CDR H3 length and structure, and perhaps highly unusual HC (like ultralong HC of cattle) may bypass this developmental checkpoint altogether.

Monoallelic Heb/Tcf12 Deletion Reduces the Requirement for NOTCH1 Hyperactivation in T-Cell Acute Lymphoblastic Leukemia.

Early T-cell development is precisely controlled by E proteins, that indistinguishably include HEB/TCF12 and E2A/TCF3 transcription factors, together with NOTCH1 and pre-T cell receptor (TCR) signalling. Importantly, perturbations of early T-cell regulatory networks are implicated in leukemogenesis. NOTCH1 gain of function mutations invariably lead to T-cell acute lymphoblastic leukemia (T-ALL), whereas inhibition of E proteins accelerates leukemogenesis. Thus, NOTCH1, pre-TCR, E2A and HEB functions are intertwined, but how these pathways contribute individually or synergistically to leukemogenesis remain to be documented. To directly address these questions, we leveraged Cd3e-deficient mice in which pre-TCR signaling and progression through β-selection is abrogated to dissect and decouple the roles of pre-TCR, NOTCH1, E2A and HEB in SCL/TAL1-induced T-ALL, via the use of Notch1 gain of function transgenic (Notch1ICtg) and Tcf12+/- or Tcf3+/- heterozygote mice. As a result, we now provide evidence that both HEB and E2A restrain cell proliferation at the β-selection checkpoint while the clonal expansion of SCL-LMO1-induced pre-leukemic stem cells in T-ALL is uniquely dependent on Tcf12 gene dosage. At the molecular level, HEB protein levels are decreased via proteasomal degradation at the leukemic stage, pointing to a reversible loss of function mechanism. Moreover, in SCL-LMO1-induced T-ALL, loss of one Tcf12 allele is sufficient to bypass pre-TCR signaling which is required for Notch1 gain of function mutations and for progression to T-ALL. In contrast, Tcf12 monoallelic deletion does not accelerate Notch1IC-induced T-ALL, indicating that Tcf12 and Notch1 operate in the same pathway. Finally, we identify a tumor suppressor gene set downstream of HEB, exhibiting significantly lower expression levels in pediatric T-ALL compared to B-ALL and brain cancer samples, the three most frequent pediatric cancers. In summary, our results indicate a tumor suppressor function of HEB/TCF12 in T-ALL to mitigate cell proliferation controlled by NOTCH1 in pre-leukemic stem cells and prevent NOTCH1-driven progression to T-ALL.

Notch-induced endoplasmic reticulum-associated degradation governs mouse thymocyte β-selection.

Signals from the pre-T cell receptor and Notch coordinately instruct β-selection of CD4-CD8-double negative (DN) thymocytes to generate αβ T cells in the thymus. However, how these signals ensure a high-fidelity proteome and safeguard the clonal diversification of the pre-selection TCR repertoire given the considerable translational activity imposed by β-selection is largely unknown. Here, we identify the endoplasmic reticulum (ER)-associated degradation (ERAD) machinery as a critical proteostasis checkpoint during β-selection. Expression of the SEL1L-HRD1 complex, the most conserved branch of ERAD, is directly regulated by the transcriptional activity of the Notch intracellular domain. Deletion of Sel1l impaired DN3 to DN4 thymocyte transition and severely impaired mouse αβ T cell development. Mechanistically, Sel1l deficiency induced unresolved ER stress that triggered thymocyte apoptosis through the PERK pathway. Accordingly, genetically inactivating PERK rescued T cell development from Sel1l-deficient thymocytes. In contrast, IRE1α/XBP1 pathway was induced as a compensatory adaptation to alleviate Sel1l-deficiency-induced ER stress. Dual loss of Sel1l and Xbp1 markedly exacerbated the thymic defect. Our study reveals a critical developmental signal controlled proteostasis mechanism that enforces T cell development to ensure a healthy adaptive immunity.

A general chemical crosslinking strategy for structural analyses of weakly interacting proteins applied to preTCR-pMHC complexes.

T lymphocytes discriminate between healthy versus infected or cancerous cells via T-cell receptor mediated recognition of peptides bound and presented by cell-surface-expressed major histocompatibility complex molecules (MHCs). Pre-T cell receptors (preTCRs) on thymocytes foster development of αβT lymphocytes through their β chain interaction with MHC displaying self-peptides on thymic epithelia. The specific binding of a preTCR with a peptide-MHC complex (pMHC) has been identified previously as forming a weak affinity complex with a distinct interface from that of mature αβTCR. However, a lack of appropriate tools has limited prior efforts to investigate this unique interface. Here we designed a small-scale linkage screening protocol using bismaleimide linkers for determining residue-specific distance constraints between transiently interacting protein pairs in solution. Employing linkage distance restraint-guided molecular modeling, we report the oriented solution docking geometry of a preTCRβ-pMHC interaction. The linkage model of preTCRβ-pMHC complex was independently verified with paramagnetic pseudocontact chemical shift (PCS) NMR of the unlinked protein mixtures. Using linkage screens, we show that the preTCR binds with differing affinities to peptides presented by MHC in solution. Moreover, the C-terminal peptide segment is a key determinant in preTCR-pMHC recognition. We also describe the process for future large-scale production and purification of the linked constructs for NMR, X-ray crystallography and single molecule electron microscopy studies.

Pre-T cell receptors topologically sample self-ligands during thymocyte β-selection.

Self-discrimination, a critical but ill-defined molecular process programmed during thymocyte development, requires myriad pre-T cell receptors (preTCRs) and αβTCRs. Using x-ray crystallography, we show how a preTCR applies the concave β-sheet surface of its single variable domain (Vβ) to "horizontally" grab the protruding MHC α2-helix. By contrast, αβTCRs purpose all six complementarity-determining region (CDR) loops of their paired VαVβ module to recognize peptides bound to major histocompatibility complex molecules (pMHCs) in "vertical" head-to-head binding. The preTCR topological fit ensures that CDR3β reaches the peptide's featured C-terminal segment for pMHC sampling, establishing the subsequent αβTCR canonical docking mode. "Horizontal" docking precludes germline CDR1β- and CDR2β-MHC binding to broaden β-chain repertoire diversification before αβTCR-mediated selection refinement. Thus, one subunit successively attunes the recognition logic of related multicomponent receptors.

Spatial proteomics reveal that the protein phosphatase PTP1B interacts with and may modify tyrosine phosphorylation of the rhomboid protease RHBDL4

Rhomboid-like proteins are evolutionarily conserved, ubiquitous polytopic membrane proteins, including the canonical rhomboid intramembrane serine proteases and also others that have lost protease activity during evolution. We still have much to learn about their cellular roles, and evidence suggests that some may have more than one function. For example, RHBDL4 (rhomboid-like protein 4) is an endoplasmic reticulum (ER)–resident protease that forms a ternary complex with ubiquitinated substrates and p97/VCP (valosin-containing protein), a major driver of ER-associated degradation (ERAD). RHBDL4 is required for ERAD of some substrates, such as the pre–T-cell receptor α chain (pTα) and has also been shown to cleave amyloid precursor protein to trigger its secretion. In another case, RHBDL4 enables the release of full-length transforming growth factor α in exosomes. Using the proximity proteomic method BioID, here we screened for proteins that interact with or are in close proximity to RHBDL4. Bioinformatics analyses revealed that BioID hits of RHBDL4 overlap with factors related to protein stress at the ER, including proteins that interact with p97/VCP. PTP1B (protein-tyrosine phosphatase nonreceptor type 1, also called PTPN1) was also identified as a potential proximity factor and interactor of RHBDL4. Analysis of RHBDL4 peptides highlighted the presence of tyrosine phosphorylation at the cytoplasmic RHBDL4 C terminus. Site-directed mutagenesis targeting these tyrosine residues revealed that their phosphorylation modifies binding of RHBDL4 to p97/VCP and Lys63-linked ubiquitinated proteins. Our work lays a critical foundation for future mechanistic studies of the roles of RHBDL4 in ERAD and other important cellular pathways.

PRE‐T CELL RECEPTOR SIGNALING DRIVES LEUKEMOGENESIS AND IS A THERAPEUTIC TARGET IN T CELL ACUTE LYMPHOBLASTIC LEUKEMIA

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive lymphoid neoplasm that results from the malignant transformation of T-cell progenitors. The biology of T-ALL is strongly influenced by its developmental origins. For example, mutations in NOTCH1, a critical regulator of thymocyte development, are found in approximately 60% of patient tumors. Less is known, however, about the extent to which other determinants of thymocyte development participate in the leukemogenesis of T-ALL. Using a thymus transplantation-based, spontaneous mouse model of T-ALL, we found that multiple β-selection checkpoint factors were upregulated in leukemic T cells, including Ptcra, a subunit of the pre-T cell receptor (pre-TCR) complex. Genetic ablation of Ptcra in the mouse model dramatically reduced the occurrence of T-ALL. In human T-ALL cell lines, CRISPR/Cas9 knockout of PTCRA reduced in vitro proliferative capacity and the ability to form tumors in vivo. Analysis of clinical T-ALL datasets and patient samples demonstrated that PTCRA is highly and specifically expressed in leukemic T cells but not in normal, mature T cells, suggesting an appropriate therapeutic window for targeted therapy in T-ALL. Cumulatively, our findings highlight an important role for pre-TCR signaling in driving and sustaining T-ALL and support the further evaluation of PTCRA-directed therapies in T-ALL. Keywords: Notch pathway; T-cells. Disclosures: Kuhnert, F: Employment Leadership Position: Regeneron Pharmaceuticals Inc.

NMR: an essential structural tool for integrative studies of T cell development, pMHC ligand recognition and TCR mechanobiology.

Early studies of T cell structural biology using X-ray crystallography, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) focused on a picture of the αβT cell receptor (αβTCR) component domains and their cognate ligands (peptides bound to MHC molecules, i.e. pMHCs) as static interaction partners. Moving forward requires integrating this corpus of data with dynamic technologies such as NMR, molecular dynamics (MD) simulations and real-time single molecule (SM) studies exemplified by optical tweezers (OT). NMR bridges relevant timescales and provides the potential for an all-atom dynamic description of αβTCR components prior to and during interactions with binding partners. SM techniques have opened up vistas in understanding the non-equilibrium nature of T cell signaling through the introduction of force-mediated binding measurements into the paradigm for T cell function. In this regard, bioforces consequent to T-lineage cell motility are now perceived as placing piconewton (pN)-level loads on single receptor-pMHC bonds to impact structural change and αβT-lineage biology, including peptide discrimination, cellular activation, and developmental progression. We discuss herein essential NMR technologies in illuminating the role of ligand binding in the preT cell receptor (preTCR), the αβTCR developmental precursor, and convergence of NMR, SM and MD data in advancing our comprehension of T cell development. More broadly we review the central hypothesis that the αβTCR is a mechanosensor, fostered by breakthrough NMR-based structural insights. Collectively, elucidating dynamic aspects through the integrative use of NMR, SM, and MD shall advance fundamental appreciation of the mechanism of T cell signaling as well as inform translational efforts in αβTCR and chimeric T cell (CAR-T) immunotherapies and T cell vaccinology.

NMR-directed design of pre-TCRβ and pMHC molecules implies a distinct geometry for pre-TCR relative to αβTCR recognition of pMHC

The pre-T cell receptor (pre-TCR) guides early thymocytes through maturation processes within the thymus via interaction with self-ligands displayed on thymic epithelial cells. The pre-TCR is a disulfide-linked heterodimer composed of an invariant pre-TCR α (pTα) subunit and a variable β subunit, the latter of which is incorporated into the mature TCR in subsequent developmental progression. This interaction of pre-TCR with peptide-major histocompatibility complex (pMHC) molecules has recently been shown to drive robust pre-TCR signaling and thymocyte maturation. Although the native sequences of β are properly folded and suitable for NMR studies in isolation, a tendency to self-associate rendered binding studies with physiological ligands difficult to interpret. Consequently, to structurally define this critical interaction, we have re-engineered the extracellular regions of β, designated as β-c1, for prokaryotic production to be used in NMR spectroscopy. Given the large size of the full extracellular domain of class I MHC molecules such as H-Kb, we produced a truncated form termed Kb-t harboring properties favorable for NMR measurements. This system has enabled robust measurement of a pre-TCR-pMHC interaction directly analogous to that of TCRαβ-pMHC. Binding surface analysis identified a contact surface comparable in size to that of the TCRαβ-pMHC but potentially with a rather distinct binding orientation. A tilting of the pre-TCRβ when bound to the pMHC ligand recognition surface versus the upright orientation of TCRαβ would alter the direction of force application between pre-TCR and TCR mechanosensors, impacting signal initiation.

Pre-T Cell Receptors (Pre-TCRs) Leverage Vβ Complementarity Determining Regions (CDRs) and Hydrophobic Patch in Mechanosensing Thymic Self-ligands

The pre-T cell receptor (pre-TCR) is a pTα-β heterodimer functioning in early αβ T cell development. Although once thought to be ligand-autonomous, recent studies show that pre-TCRs participate in thymic repertoire formation through recognition of peptides bound to major histocompatibility molecules (pMHC). Using optical tweezers, we probe pre-TCR bonding with pMHC at the single molecule level. Like the αβTCR, the pre-TCR is a mechanosensor undergoing force-based structural transitions that dynamically enhance bond lifetimes and exploiting allosteric control regulated via the Cβ FG loop region. The pre-TCR structural transitions exhibit greater reversibility than TCRαβ and ordered force-bond lifetime curves. Higher piconewton force requires binding through both complementarity determining region loops and hydrophobic Vβ patch apposition. This patch functions in the pre-TCR as a surrogate Vα domain, fostering ligand promiscuity to favor development of β chains with self-reactivity but is occluded by α subunit replacement of pTα upon αβTCR formation. At the double negative 3 thymocyte stage where the pre-TCR is first expressed, pre-TCR interaction with self-pMHC ligands imparts growth and survival advantages as revealed in thymic stromal cultures, imprinting fundamental self-reactivity in the T cell repertoire. Collectively, our data imply the existence of sequential mechanosensor αβTCR repertoire tuning via the pre-TCR.

Phospholipase Cγ1 is required for pre-TCR signal transduction and pre-T cell development.

Pre-T cell receptor (TCR) signaling is required for pre-T cell survival, proliferation, and differentiation from the CD4 and CD8 double negative (DN) to the double positive (DP) stage. However, the pre-TCR signal transduction pathway is not fully understood and the signaling molecules involved have not been completely identified. Phospholipase Cγ (PLCγ) 1 is an important signaling molecule that generates two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, that are important to mediate PKC activation and intracellular Ca2+ flux in many signaling pathways. Previously, we have shown that PLCγ1 is important for TCR-mediated signaling, development and T-cell activation, but the role of PLCγ1 in pre-TCR signal transduction and pre-T cell development is not known. In this study, we demonstrated that PLCγ1 expression level in pre-T cells was comparable to that in mature T cells. Deletion of PLCγ1 prior to the pre-TCR signaling stage partially blocked the DN3 to DN4 transition and reduced thymic cellularity. We also demonstrated that deletion of PLCγ1 impaired pre-T cell proliferation without affecting cell survival. Further study showed that deficiency of PLCγ1 impaired pre-TCR mediated Ca2+ flux and Erk activation. Thus our studies demonstrate that PLCγ1 is important for pre-TCR mediated signal transduction and pre-T cell development.

Non-canonical antagonism of PI3K by the kinase Itpkb delays thymocyte β-selection and renders it Notch-dependent.

β-selection is the most pivotal event determining αβ T cell fate. Here, surface-expression of a pre-T cell receptor (pre-TCR) induces thymocyte metabolic activation, proliferation, survival and differentiation. Besides the pre-TCR, β-selection also requires co-stimulatory signals from Notch receptors - key cell fate determinants in eukaryotes. Here, we show that this Notch-dependence is established through antagonistic signaling by the pre-TCR/Notch effector, phosphoinositide 3-kinase (PI3K), and by inositol-trisphosphate 3-kinase B (Itpkb). Canonically, PI3K is counteracted by the lipid-phosphatases Pten and Inpp5d/SHIP-1. In contrast, Itpkb dampens pre-TCR induced PI3K/Akt signaling by producing IP4, a soluble antagonist of the Akt-activating PI3K-product PIP3. Itpkb(-/-) thymocytes are pre-TCR hyperresponsive, hyperactivate Akt, downstream mTOR and metabolism, undergo an accelerated β-selection and can develop to CD4(+)CD8(+) cells without Notch. This is reversed by inhibition of Akt, mTOR or glucose metabolism. Thus, non-canonical PI3K-antagonism by Itpkb restricts pre-TCR induced metabolic activation to enforce coincidence-detection of pre-TCR expression and Notch-engagement.

Metabolic inhibition by inositol-tetrakisphosphate delays thymocyte β-selection and renders it Notch-dependent (HEM2P.241)

Abstract In β-selection, surface-expression of a pre-T cell receptor (pre-TCR) induces thymocyte metabolic activation, proliferation, survival and differentiation. This pivotal αβ T cell fate-determining event requires co-stimulatory Notch signals. Here, we show that this Notch dependence is enforced through antagonistic signaling by inositol-trisphosphate 3-kinase B (Itpkb) and phosphoinositide 3-kinase (PI3K). Itpkb produces soluble inositol-tetrakisphosphate which competes with the PI3K lipid-product PIP3 for binding to the effector-kinase Akt. Itpkb-/- thymocytes are pre-TCR hyperresponsive, hyperactivate Akt, downstream mTOR and metabolism, undergo an accelerated β-selection and can develop to CD4+CD8+ cells without Notch. This is reversed by inhibition of Akt, mTOR or glucose-metabolism. Thus, Itpkb restricts pre-TCR induced metabolic activation to enforce coincidence-detection of pre-TCR expression and Notch-engagement, and to prevent premature thymocyte maturation.

IL-7 coordinates proliferation, differentiation and Tcra recombination during thymocyte β-selection.

pre-T cell receptor (TCR) and Notch signaling induce transient self-renewal or “β-selection” of TCRβ+ CD4 CD8 double-negative-3 (DN3) and DN4 progenitors that differentiate into CD4 CD8 double positive (DP) thymocytes which then rearrange Tcra. Interleukin-7 (IL-7) promotes Bcl2-dependent survival of TCRβ− DN thymocytes, but IL-7 functions during β-selection remain unclear. Here, we show that IL-7 signals TCRβ+ DN3 and DN4 thymocytes to upregulate genes involved in cell growth and represses Bcl6. Accordingly, IL-7-deficient DN4 cells lacked trophic receptors and did not proliferate, but rearranged Tcra prematurely and differentiated rapidly. Bcl6 deletion, but not BCL2 over-expression, partially restored DN4 self-renewal in the absence of IL-7. Thus, IL-7 critically collaborates with pre-TCR and Notch signaling to coordinate proliferation, differentiation and Tcra recombination during β-selection.

Metabolic inhibition by inositol-tetrakisphosphate delays thymocyte β-selection and renders it notch-dependent (HEM4P.227)

Abstract In β-selection, surface-expression of a pre-T cell receptor (pre-TCR) induces thymocyte metabolic activation, proliferation, survival and differentiation. This pivotal αβ T cell fate-determining event requires co-stimulatory Notch signals. Here, we show that this Notch dependence is enforced through antagonistic signaling by inositol-trisphosphate 3-kinase B (Itpkb) and phosphoinositide 3-kinase (PI3K). Itpkb produces soluble inositol-tetrakisphosphate which competes with the PI3K lipid-product PIP3 for binding to the effector-kinase Akt. Itpkb-/- thymocytes are pre-TCR hyperresponsive, hyperactivate Akt, downstream mTOR and metabolism, undergo an accelerated β-selection and can develop to CD4+CD8+ cells without Notch. This is reversed by inhibition of Akt, mTOR or glucose-metabolism. Thus, Itpkb restricts pre-TCR induced metabolic activation to enforce coincidence-detection of pre-TCR expression and Notch-engagement, and to prevent premature thymocyte maturation.

Enforcement of γδ-lineage commitment by the pre–T-cell receptor in precursors with weak γδ-TCR signals

Developing thymocytes bifurcate from a bipotent precursor into αβ- or γδ-lineage T cells. Considering this common origin and the fact that the T-cell receptor (TCR) β-, γ-, and δ-chains simultaneously rearrange at the double negative (DN) stage of development, the possibility exists that a given DN cell can express and transmit signals through both the pre-TCR and γδ-TCR. Here, we tested this scenario by defining the differentiation outcomes and criteria for lineage choice when both TCR-β and γδ-TCR are simultaneously expressed in Rag2(-/-) DN cells via retroviral transduction. Our results showed that Rag2(-/-) DN cells expressing both TCRs developed along the γδ-lineage, down-regulated CD24 expression, and up-regulated CD73 expression, showed a γδ-biased gene-expression profile, and produced IFN-γ in response to stimulation. However, in the absence of Inhibitor of DNA-binding 3 expression and strong γδ-TCR ligand, γδ-expressing cells showed a lower propensity to differentiate along the γδ-lineage. Importantly, differentiation along the γδ-lineage was restored by pre-TCR coexpression, which induced greater down-regulation of CD24, higher levels of CD73, Nr4a2, and Rgs1, and recovery of functional competence to produce IFN-γ. These results confirm a requirement for a strong γδ-TCR ligand engagement to promote maturation along the γδ T-cell lineage, whereas additional signals from the pre-TCR can serve to enforce a γδ-lineage choice in the case of weaker γδ-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells serves to dictate its lineage choice.

Pre-T cell receptor for improved expansion of TCR alpha disrupted T cells

Meeting abstracts Recent data have emerged from adoptive T-cell therapies where exogenous expression of a chimeric antigen receptor (CAR) has been shown to confer cancer recognition on autologous T cells. However, the ability to apply this technology in an allogeneic setting would permit the