pre-TCR Signaling Research Articles

IKAROS Family Transcription Factors in Lymphocyte Differentiation and Function.

The IKAROS family of transcription factors comprises four zinc-finger proteins (IKAROS, HELIOS, AIOLOS, and EOS), which over the last decades havebeen established to be critical regulators of the development and function of lymphoid cells. These factors act as homo- or heterodimers and are involved both in gene activation and repression. Their function often involves cross-talk with other regulatory circuits, such as the JAK/STAT, NF-κB, and NOTCH pathways. They control lymphocyte differentiation at multiple stages and are notably critical for lymphoid commitment in multipotent hematopoietic progenitors and for T and Bcell differentiation downstream of pre-TCR and pre-BCR signaling. They also control many aspects of effector functions in mature B and T cells. They are dysregulated or mutated in multiple pathologies affecting the lymphoid system, which range from leukemia to immunodeficiencies. In this chapter, we review the molecular and physiological function of these factors in lymphocytes and their implications in human pathologies.

Dual Targeting of Apoptotic and Signaling Pathways in T-Lineage Acute Lymphoblastic Leukemia.

Relapsed T-acute lymphoblastic leukemia (T-ALL) has limited treatment options. We investigated mechanisms of resistance to BH3 mimetics in T-ALL to develop rational combination strategies. We also looked at the preclinical efficacy of NWP-0476, a novel BCL-2/BCL-xL inhibitor, as single agent and combination therapy in T-ALL. We used BH3 profiling as a predictive tool for BH3 mimetic response in T-ALL. Using isogenic control, venetoclax-resistant (ven-R) and NWP-0476-resistant (NWP-R) cells, phosphokinase array was performed to identify differentially regulated signaling pathways. Typical T-ALL cells had increased dependence on BCL-xL, whereas early T-precursor (ETP)-ALL cells had higher BCL-2 dependence for survival. BCL-2/BCL-xL dual inhibitors were effective against both subtypes of T-lineage ALL. A 71-protein human phosphokinase array showed increased LCK activity in ven-R cells, and increased ACK1 activity in ven-R and NWP-R cells. We hypothesized that pre-TCR and ACK1 signaling pathways are drivers of resistance to BCL-2 and BCL-xL inhibition, respectively. First, we silenced LCK gene in T-ALL cell lines, which resulted in increased sensitivity to BCL-2 inhibition. Mechanistically, LCK activated NF-κB pathway and the expression of BCL-xL. Silencing ACK1 gene resulted in increased sensitivity to both BCL-2 and BCL-xL inhibitors. ACK1 signaling upregulated AKT pathway, which inhibited the pro-apoptotic function of BAD. In a T-ALL patient-derived xenograft model, combination of NWP-0476 and dasatinib demonstrated synergy without major organ toxicity. LCK and ACK1 signaling pathways are critical regulators of BH3 mimetic resistance in T-ALL. Combination of BH3 mimetics with tyrosine kinase inhibitors might be effective against relapsed T-ALL.

Stepwise progression of β-selection during T cell development involves histone deacetylation.

During T cell development, the first step in creating a unique T cell receptor (TCR) is genetic recombination of the TCRβ chain. The quality of the new TCRβ is assessed at the β-selection checkpoint. Most cells fail this checkpoint and die, but the coordination of fate at the β-selection checkpoint is not yet understood. We shed new light on fate determination during β-selection using a selective inhibitor of histone deacetylase 6, ACY1215. ACY1215 disrupted the β-selection checkpoint. Characterising the basis for this disruption revealed a new, pivotal stage in β-selection, bookended by up-regulation of TCR co-receptors, CD28 and CD2, respectively. Within this "DN3bPre" stage, CD5 and Lef1 are up-regulated to reflect pre-TCR signalling, and their expression correlates with proliferation. These findings suggest a refined model of β-selection in which a coordinated increase in expression of pre-TCR, CD28, CD5 and Lef1 allows for modulating TCR signalling strength and culminates in the expression of CD2 to enable exit from the β-selection checkpoint.

A set point in the selection of the αβTCR T cell repertoire imposed by pre-TCR signaling strength

Signaling via the T cell receptor (TCR) is critical during the development, maintenance, and activation of T cells. Quantitative aspects of TCR signaling have an important role during positive and negative selection, lineage choice, and ability to respond to small amounts of antigen. By using a mutant mouse line expressing a hypomorphic allele of the CD3ζ chain, we show here that the strength of pre-TCR–mediated signaling during T cell development determines the diversity of the TCRβ repertoire available for positive and negative selection, and hence of the final αβTCR repertoire. This finding uncovers an unexpected, pre-TCR signaling–dependent and repertoire–shaping role for β-selection beyond selection of in-frame rearranged TCRβ chains. Our data furthermore support a model of pre-TCR signaling in which the arrangement of this receptor in stable nanoclusters determines its quantitative signaling capacity.

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.

CTPS and IMPDH form cytoophidia in developmental thymocytes

The cytoophidium, a filamentous structure formed by metabolic enzymes, has emerged as a novel regulatory machinery for certain proteins. The rate-limiting enzymes of de novo CTP and GTP synthesis, cytidine triphosphate synthase (CTPS) and inosine monophosphate dehydrogenase (IMPDH), are the most characterized cytoophidium-forming enzymes in mammalian models. Although the assembly of CTPS cytoophidia has been demonstrated in various organisms including multiple human cancers, a systemic survey for the presence of CTPS cytoophidia in mammalian tissues in normal physiological conditions has not yet been reported. Herein, we examine major organs of adult mouse and observe that CTPS cytoophidia are displayed by a specific thymocyte population ranging between DN3 to early DP stages. Most of these cytoophidium-presenting cells have both CTPS and IMPDH cytoophidia and undergo rapid cell proliferation. In addition, we show that cytoophidium formation is associated with active glycolytic metabolism as the cytoophidium-presenting cells exhibit higher levels of c-Myc, phospho-Akt and PFK. Inhibition of glycolysis with 2DG, however, disrupts most of cytoophidium structures and impairs cell proliferation. Our findings not only indicate that the regulation of CTPS and IMPDH cytoophidia are correlated with the metabolic switch triggered by pre-TCR signaling, but also suggest physiological roles of the cytoophidium in thymocyte development.

Developing T cells form an immunological synapse for passage through the β-selection checkpoint.

The β-selection checkpoint of T cell development tests whether the cell has recombined its genomic DNA to produce a functional T cell receptor β (TCRβ). Passage through the β-selection checkpoint requires the nascent TCRβ protein to mediate signaling through a pre-TCR complex. In this study, we show that developing T cells at the β-selection checkpoint establish an immunological synapse in in vitro and in situ, resembling that of the mature T cell. The immunological synapse is dependent on two key signaling pathways known to be critical for the transition beyond the β-selection checkpoint, Notch and CXCR4 signaling. In vitro and in situ analyses indicate that the immunological synapse promotes passage through the β-selection checkpoint. Collectively, these data indicate that developing T cells regulate pre-TCR signaling through the formation of an immunological synapse. This signaling platform integrates cues from Notch, CXCR4, and MHC on the thymic stromal cell to allow transition beyond the β-selection checkpoint.

Critical role of WNK1 in MYC-dependent early mouse thymocyte development.

WNK1, a kinase that controls kidney salt homeostasis, also regulates adhesion and migration in CD4+ T cells. Wnk1 is highly expressed in thymocytes, and since migration is important for thymocyte maturation, we investigated a role for WNK1 in mouse thymocyte development. We find that WNK1 is required for the transition of double negative (DN) thymocytes through the β-selection checkpoint and subsequent proliferation and differentiation into double positive (DP) thymocytes. Furthermore, we show that WNK1 negatively regulates LFA1-mediated adhesion and positively regulates CXCL12-induced migration in DN thymocytes. Despite this, migration defects of WNK1-deficient thymocytes do not account for the developmental arrest. Instead, we show that in DN thymocytes WNK1 transduces pre-TCR signals via OXSR1 and STK39 kinases, and the SLC12A2 ion co-transporter that are required for post-transcriptional upregulation of MYC and subsequent proliferation and differentiation into DP thymocytes. Thus, a pathway regulating ion homeostasis is a critical regulator of thymocyte development.

Differential survival requirements for Lck- and CD3e-dependent signaling defines two populations of ATMKO thymic T cell lymphomas

Abstract The product of theataxia-telangiectasia mutated gene (ATM) is a serine-threonine kinase thatcommunicates between molecules that detect dangerous DNA double strand breaksand effector mechanisms that maintain genomic integrity. ATM deficiency in bothhumans and mice is associated with genomic instability and increased malignancies. ATMKO mice exclusively develop thymic T cell lymphomas that resemble thymically-derivedT-ALL that occur in human ATM-deficient ataxia telangiectasia patients. Flowcytometry identified two subsets of ATMKO T cell lymphomas based on surfaceexpression of CD3ɛ, TCRβ and pre-Tα:Cells that express surface CD3ɛ, TCRβ and pre-Tα(surface receptor high; sRhi) and cells that do not express detectable surface CD3ɛ, TCRβ orpre-Tα(sRlo). When assayed for CD25 and CD44 expression sRhi lymphomas resemble DN3or DN4 cells while sRlo lymphomas resemble more immature DN1 or DN2 cells. Pre-TCRsignaling in normal DN3 and DN4 thymocytes provides an essential survivalsignal that is proposed to be constitutive and ligand-independent. Therefore, weasked if surface sRhi and sRlo cells are dependent on pre-TCR signaling byincubating cells with either Lck inhibitor, CAS 213743-31-8, or CD3ɛshRNA to inhibit pre-TCR signaling. Both treatments significantly inhibitedsurvival of sRhi lymphomas, while survival of sRlo cells was largely unaffected. These results suggest that ATMKO T celllymphomas arise from cells transformed at different stages of DN development andthat sRhigh cells, like normal DN3 and DN4 thymocytes, retain apre-TCR-dependent survival requirement. These results also suggest that pre-TCRsignaling may be a useful target for clinical intervention in treatment ofhuman T-ALL that express a pre-TCR.

The transcriptional repressor Bcl6 promotes pre-TCR-induced thymocyte differentiation and attenuates Notch1 activation.

Pre-T-cell receptor (TCR) signal transduction is required for developing thymocytes to differentiate from CD4-CD8- double-negative (DN) cell to CD4+CD8+ double-positive (DP) cell. Notch signalling is required for T-cell fate specification and must be maintained throughout β-selection, but inappropriate Notch activation in DN4 and DP cells is oncogenic. Here, we show that pre-TCR signalling leads to increased expression of the transcriptional repressor Bcl6 and that Bcl6 is required for differentiation to DP. Conditional deletion of Bcl6 from thymocytes reduced pre-TCR-induced differentiation to DP cells, disrupted expansion and enrichment of intracellular TCRβ+ cells within the DN population and increased DN4 cell death. Deletion also increased Notch1 activation and Notch-mediated transcription in the DP population. Thus, Bcl6 is required in thymocyte development for efficient differentiation from DN3 to DP and to attenuate Notch1 activation in DP cells. Given the importance of inappropriate NOTCH1 signalling in T-cell acute lymphoblastic leukaemia (T-ALL), and the involvement of BCL6 in other types of leukaemia, this study is important to our understanding of T-ALL.

Aberrant SYK Expression As a Potential Therapeutic Target in T/NK Cell Neoplasms

Background: During T cell development spleen tyrosine kinase (SYK) is highly expressed at pre-TCR signaling stages, and gradually becomes undetectable at the mature stage. We have previously reported aberrant expression of SYK in peripheral T-cell lymphoma (PTCL). Additionally, ITK-SYK fusion protein detected in a subset of PTCL, mimics a constitutively active TCR signal and drives oncogenesis in mouse models of PTCL. Multiple SYK inhibitors are currently under active investigation in clinical trials for patients with B cell lymphoma and acute myeloid leukemia, and show promising results. Multiparameter flow cytometry can quantitate protein levels at single cell resolution in immunophenotypically defined neoplastic populations. In this study, we employ multiparameter flow cytometry to quantitate the SYK expression in T and NK cell malignancies and to guide the clinical trial design and as potential tool to evaluate the therapeutic responses. Methods: Patients with T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), T-cell large granular lymphocyte leukemia (T-LGL), HTLV-1+ adult T-cell leukemia/lymphoma (ATLL), T-cell prolymphocytic leukemia (T-PLL), angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), mixed phenotype acute leukemia (MPAL) and natural killer cell large granular lymphocyte leukemia (NK-LGL) were included. Abnormal samples included the peripheral blood (46 cases), bone marrow aspirates (10 cases) or tissue (9 cases) from 65 patients diagnosed with T or NK cell malignancies, including AITL (8 cases), ATLL (5 cases), ALCL (2 cases), CTCL (25 cases), PTCL-NOS (5 cases), T-PLL (7 cases), T-LGL (7 cases), NK-LGL (1 case), MEITL (1 case), MAPL (3 cases) and T-ALL (1 case). Normal controls included peripheral blood from healthy donors (15 cases), or patient derived- peripheral blood (6 cases), bone marrow aspirates (13 cases) and lymph node (16 cases) with diagnostic normal immunophenotype. Results: SYK was highly expressed in all the B and most NK cells from peripheral blood, bone marrow and lymph node of normal controls and there was no difference in the percentage of positive cells or intensity of staining in different specimen types. In normal T cell subsets SYK was expressed in a small proportion of T cells and at very low level. Only 3.9% (95% CI: 3.3% - 6.3%) of the CD4+ T cells from peripheral blood, 2.6% (1.5% - 6.6%) from bone marrow, and 1.9% (1.4% - 5.8%) from lymph node were positive for SYK. Similar to CD4+ T cells, SYK was expressed in 5.4% (95% CI: 4.3% - 7.8%) of the CD8+ T cells from peripheral blood. These data suggest SYK expression is nearly undetectable in the normal mature T cells which are part of adaptive immunity. In contrast, lymphoid cells of innate immunity such as NK cells express high levels of SYK. Similar to normal T-cell subsets, the neoplastic T-cells of most PTCL believed to originate from cells of adaptive immunity (AITL, ATLL, ALCL, CTCL, PTCL-NOS and T-PLL) showed low levels of SYK expression (median: 5.8%; 95% CI: 5.8% -10.4%) with rare exceptions (Figure 1). In contrast, the T and NK cell neoplasms of innate immunity origin (NK-LGL, MEITL) and immature precursor T-cell origin (MPAL and T-ALL) showed high levels of SYK expression (median 91.1%, 95% CI as 97.1% - 99.6%). SYK expression in T-LGL was more modest and variable (median 43.6%, 95% CI as 43.6% - 78.0% (Figure 1). Conclusion: SYK is highly expressed in the neoplastic lymphoid cells of innate immunity such as NK-LGL, MEITL and T-LGL cases, and immature hematopoietic neoplasms with T-cell differentiation. The expression of SYK in neoplastic cells from patients with mature T cell lymphoma of adaptive immunity remains low. Given the significant physiological role of SYK in innate immunity, SYK expression in NK/T-cell neoplasms is likely to have an oncogenic role, suggesting that SYK might be a good therapy target for these tumors. Disclosures Roshal: Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services; Physicians' Education Resource: Other: Provision of services. Dogan:Roche: Consultancy, Research Funding; Novartis: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Seattle Genetics: Consultancy; Corvus Pharmaceuticals: Consultancy.

Notch and the pre-TCR coordinate thymocyte proliferation by induction of the SCF subunits Fbxl1 and Fbxl12.

Proliferation is tightly regulated during T cell development and is limited to immature CD4−CD8− thymocytes. The major proliferative event is initiated at the ‘β-selection’ stage following successful rearrangement of Tcrβ and is triggered by and dependent on concurrent signaling by Notch and the pre-TCR; however, it is unclear how these signals cooperate to promote cell proliferation. Here we found that β-selection-associated proliferation required the combined activity of two SCF ubiquitin ligase complexes that included as substrate recognition subunits the F-box proteins Fbxl1 or Fbxl12. Both SCF complexes targeted the cyclin-dependent kinase inhibitor Cdkn1b for ubiquitinylaton and degradation. We found that Notch signals induced the transcription of Fbxl1 whereas pre-TCR signals induced the transcription of Fbxl12. Thus, concurrent Notch and pre-TCR signaling induced the expression of two genes, Fbxl1 and Fbxl12, whose products functioned identically but additively to promote degradation of Cdkn1b, cell cycle progression, and proliferation of β-selected thymocytes.

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.

Notch Signaling Controls Transcription via the Recruitment of RUNX1 and MYB to Enhancers during T Cell Development.

Tcrd and Tcrg display identical developmental programs that depend on the activity of the enhancers Eδ and Eγ being "on" in pre-β-selection thymocytes to activate transcription and V(D)J recombination of the unrearranged genes and "off" in post-β-selection CD4+CD8+ double-positive thymocytes to inhibit transcription of the rearranged genes and avoid the expression of TCR δ- and TCR γ-chains in αβ T lymphocytes. Eδ and Eγ activity depends on transcription factor binding to essential Runx and Myb sites and parallels that of Notch signaling. We performed Notch gain- and loss-of-function experiments and found that Notch signaling activates Tcrd and Tcrg transcription by favoring the recruitment of RUNX1 and MYB to the enhancers. Our results suggest that the dissociation of RUNX1 and MYB from Eδ and Eγ chromatin in double-positive thymocytes, which results in enhancer inactivation, is caused by decreased Notch signaling triggered by pre-TCR signaling, thereby deciphering the molecular mechanism of Tcrd and Tcrg silencing during β-selection. These findings reveal a novel molecular mechanism for gene regulation via Notch signaling through the recruitment of RUNX1 and MYB to enhancer chromatin during thymocyte development.

A clonotype-independent pre-TCR complex and its downstream signals can support transformation of thymic T cells.

Abstract The kinase ataxia telangiectasia mutated (ATM) communicates between molecules that sense DNA damage in the form of double stranded breaks (DSB) and effector mechanisms that maintain genomic integrity. Persistent DSBs can lead to transformation and ATM-deficiency is associated with increased lymphoid malignancies in both humans and mice. ATM-deficient mice exclusively develop thymic T cell lymphomas with high penetrance. We explored the requirement for specific components of the TCR and its signaling pathway for the induction of these thymic lymphomas. We generated multiple strains of ATM-deficient mice in which mature T cell are absent by deletion of CD3ɛ, an invariant component of the pre-TCR complex; by deletion of RAG or TCRβ, in which recombining TCR variable components are deleted; or by deletion of LAT, an important component of pre-TCR and TCR signaling. These strains all showed a similar and severe developmental arrest at thymic DN3. Despite this arrest, mice deficient in RAG or TCRβ, exclusively developed thymic T cell lymphomas. In contrast, mice deficient in CD3ɛ or LAT, developed no or few T cell tumors, respectively, but developed B cell lymphomas. These findings suggest the importance of a clonotype-independent TCR complex and its downstream signals in transformation of T cells. Experiments are in progress to ask if survival of ATMKO T cell lymphomas after transformation is similarly dependent on CD3ɛ and LAT signaling.

Id Proteins Suppress E2A-Driven Invariant Natural Killer T Cell Development prior to TCR Selection.

A family of transcription factors known as E proteins, and their antagonists, Id proteins, regulate T cell differentiation at critical developmental checkpoints. Id proteins promote the differentiation of conventional αβ T cells and suppress the expansion of innate-like αβ T cells known as invariant natural killer T (iNKT) cells. However, it remains to be determined whether Id proteins differentially regulate these distinct lineage choices in early stages of T cell development. In this manuscript, we report that in Id-deficient mice, uninhibited activity of the E protein family member E2A mediates activation of genes that support iNKT cell development and function. There is also biased rearrangement in Id-deficient DP cells that promotes selection into the iNKT lineage in these mice. The observed expansion of iNKT cells is not abrogated by blocking pre-TCR signaling, which is required for conventional αβ T cell development. Finally, E2A is found to be a key transcriptional regulator of both iNKT and γδNKT lineages, which appear to have shared lineage history. Therefore, our study reveals a previously unappreciated role of E2A in coordinating the development of the iNKT lineage at an early stage, prior to their TCR-mediated selection alongside conventional αβ T cells.

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.

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.

The RNA-Binding Proteins Zfp36l1 and Zfp36l2 Enforce the Thymic β-Selection Checkpoint by Limiting DNA Damage Response Signaling and Cell Cycle Progression.

The RNA-binding proteins Zfp36l1 and Zfp36l2 act redundantly to enforce the β-selection checkpoint during thymopoiesis, yet their molecular targets remain largely unknown. In this study, we identify these targets on a genome-wide scale in primary mouse thymocytes and show that Zfp36l1/l2 regulate DNA damage response and cell cycle transcripts to ensure proper β-selection. Double-negative 3 thymocytes lacking Zfp36l1/l2 share a gene expression profile with postselected double-negative 3b cells despite the absence of intracellular TCRβ and reduced IL-7 signaling. Our findings show that in addition to controlling the timing of proliferation at β-selection, posttranscriptional control by Zfp36l1/l2 limits DNA damage responses, which are known to promote thymocyte differentiation. Zfp36l1/l2 therefore act as posttranscriptional safeguards against chromosomal instability and replication stress by integrating pre-TCR and IL-7 signaling with DNA damage and cell cycle control.

236. Role of P53 on T Lymphopoiesis from Human Embryonic Stem Cells

Although pluripotent stem cells are well recognized as a potential source of cell therapy, it is still needed to improve efficiency to differentiate into target cell lineages. Tumor suppressor P53 regulates multiple signaling pathways triggered by diverse cellular stresses including DNA damages, oncogenic stimulations, and hypoxic stress, resulting in cell-cycle arrest, apoptosis, and senescence. P53 signaling is also important for double-stranded DNA breaks (DSBs) induced during physiologic events, i.e., rearrangement of antigen-specific receptors. It has been reported that P53-mediated DSB checkpoint contribute to normal murine T lymphopoiesis, especially at the double-negative (DN) stage which is defined as CD4-CD8- fraction in thymus and requires rearrangements of the T cell receptor (TCR) β locus and successful pre-TCR signaling. Here we defined the role of P53 on lymphopoiesis from human embryonic stem cells (ESCs).Firstly we modified P53 gene of human ESC H1 by utilizing of zinc finger nuclease targeting the P53 gene, kindly provided by Sangamo BioSciences. Sequencing analysis of the P53 knockout (KO) ES cells showed the successful deletion which induced the frame shift of the downstream sequence in both of its alleles. Western blot analysis of P53 phosphorylation status in P53 KO ESCs showed undetectable levels of phosphorylated or non-phosphorylated P53 proteins when cultured in the presence or absence of apoptotic signal triggered by mitomycin C (MMC). In consistent with this, P53 KO ESCs showed significant resistance to MMC-induced cell death. In addition, P53 KO ESCs lacked apoptotic stimulation-induced upregulation of P53 downstream target genes including P53 up-regulated modulator of apoptosis (PUMA).We then induced hematopoietic differentiation of P53 KO ESCs through embryoid body formation. Erythroid lineage cells developed from human ESCs were significantly suppressed in the absence of P53 signaling during embryoid body maturation. Pharmacological inhibition of P53 had the same effect as genetic disruption of P53 gene. CD34+ hematopoietic precursors were isolated from embryoid bodies originated from H1 and P53 KO ECSs, plated on OP9-DL1 stromal cells, and cultured in the presence of stem cell factor (SCF), FLT3 ligand, and interleukin (IL)-7. After 3-4 weeks of culture, CD45+CD3+ T lineage cells were induced from both H1 and P53 KO ECSs-derived CD34+ cells. Among these cells, most of the cells were in CD4+CD8+ double-positive (DP) stage, with increase in the yield of DP cells in the absence of P53 signaling (H1: 343 cells/1 × 10^6 input CD34+ cells; P53 KO: 2476 cells /1 × 10^6 input CD34+ cells; FigureFigure). Whether pharmacological inhibition of P53 had the similar effect on T lymphopoiesis as genetic disruption of P53 gene needs to be investigated furthermore.Our data indicate that P53 mediated signaling regulate in vitro early T lymphopoiesis from human pluripotent stem cells, especially at the transition from double negative into DP stage. These observations promoted us to perform high throughput transcriptome analysis including cDNA microarray analysis between early T lineage cells derived from H1 and P53 KO ESCs. Genes associated with the early T lymphopoiesis from human ESCs were identified and currently under further characterization.View Large Image | Download PowerPoint Slide