Therapeutic Inducers of Natural Killer cell Killing (ThINKK): preclinical assessment of safety and efficacy in allogeneic hematopoietic stem cell transplant settings.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) remains the standard of care for chemotherapy-refractory leukemia patients, but cure rates are still dismal. To prevent leukemia relapse following HSCT, we aim to improve the early graft-versus-leukemia effect mediated by natural killer (NK) cells. Our approach is based on the adoptive transfer of Therapeutic Inducers of Natural Killer cell Killing (ThINKK). ThINKK are expanded and differentiated from HSC, and exhibit blood plasmacytoid dendritic cell (pDC) features. We previously demonstrated that ThINKK stimulate NK cells and control acute lymphoblastic leukemia (ALL) development in a preclinical mouse model of HSCT for ALL. Here, we assessed the cellular identity of ThINKK and investigated their potential to activate allogeneic T cells. We finally evaluated the effect of immunosuppressive drugs on ThINKK-NK cell interaction. METHODS: ThINKK cellular identity was explored using single-cell RNA sequencing and flow cytometry. Their T-cell activating potential was investigated by coculture of allogeneic T cells and antigen-presenting cells in the presence or the absence of ThINKK. A preclinical human-to-mouse xenograft model was used to evaluate the impact of ThINKK injections on graft-versus-host disease (GvHD). Finally, the effect of immunosuppressive drugs on ThINKK-induced NK cell cytotoxicity against ALL cells was tested. RESULTS: The large majority of ThINKK shared the key characteristics of canonical blood pDC, including potent type-I interferon (IFN) production following Toll-like receptor stimulation. A minor subset expressed some, although not all, markers of other dendritic cell populations. Importantly, while ThINKK were not killed by allogeneic T or NK cells, they did not increase T cell proliferation induced by antigen-presenting cells nor worsened GvHD in vivo. Finally, tacrolimus, sirolimus or mycophenolate did not decrease ThINKK-induced NK cell activation and cytotoxicity. CONCLUSION: Our results indicate that ThINKK are type I IFN producing cells with low T cell activation capacity. Therefore, ThINKK adoptive immunotherapy is not expected to increase the risk of GvHD after allogeneic HSCT. Furthermore, our data predict that the use of tacrolimus, sirolimus or mycophenolate as anti-GvHD prophylaxis regimen will not decrease ThINKK therapeutic efficacy. Collectively, these preclinical data support the testing of ThINKK immunotherapy in a phase I clinical trial.

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 ß-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 ß-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.

Patients' NK cell stimulation with activated plasmacytoid dendritic cells increases dinutuximab-induced neuroblastoma killing.

Targeted immunotherapy has improved the outcome of patients with high-risk neuroblastoma (NB). However, immune escape of tumor cells still occurs and about 40% of NB patients relapse and die from their disease. We previously showed that natural killer (NK) cell stimulation by Toll-like receptor (TLR)-activated plasmacytoid dendritic cells (pDC) increases the efficacy of dinutuximab-based immunotherapy against NB cell lines via the TRAIL death-receptor pathway. With the aim to translate our findings into a novel adoptive therapy of TLR-activated pDC, we investigated the pDC/NK cell axis in NB patients undergoing dinutuximab-based immunotherapy. We show that pDC counts were low at the beginning of immunotherapy but reached normal levels over time. Blood NK cell counts were normal in all patients, although a high proportion of CD56bright CD16low/- cells was observed. The stimulation of patient's blood cells with a TLR9 ligand led to IFN-α production by pDC, and TRAIL expression on NK cell surface. Patient's NK cells expressed high levels of CD69 and TRAIL after stimulation with activated pDC. Both CD56bright CD16low/- and CD56dim CD16+ NK cells degranulated against autologous target cells in the presence of dinutuximab. Importantly, pDC-induced NK cell activation increased the dinutuximab mediated autologous killing of patient-derived NB cells. Altogether, our study demonstrates that TLR-activated pDC strongly increase the cytotoxic functions of NK cells in high-risk NB patients undergoing immunotherapy. These results, therefore, support pDC adoptive immunotherapy as a novel approach to decrease the risk of relapse in patients with high-risk NB.

Activation of innate immunity in primary human cells using a plant virus derived nanoparticle TLR7/8 agonist.

Rod-shaped virus-like nanoparticles (VLNP) made of papaya mosaic virus (PapMV) coat proteins (CP) self-assembled around a single stranded RNA (ssRNA) were showed to be a TLR7 agonist. Their utilization as an immune modulator in cancer immunotherapy was shown to be promising. To establish a clinical relevance in human for PapMV VLNP, we showed that stimulation of human peripheral blood mononuclear cells (PBMC) with VLNP induces the secretion of interferon alpha (IFNα) and other pro-inflammatory cytokines and chemokines. Plasmacytoid dendritic cells (pDCs) were activated and secreted IFN-α upon VLNP exposure. Monocyte-derived dendritic cells upregulate maturation markers and produce IL-6 in response to PapMV VLNP stimulation, which suggests the activation of TLR8. Finally, when co-cultured with NK cells, PapMV induced pDCs promoted the NK cytolytic activity against cancer cells. These data obtained with primary human immune cells further strengthen the clinical relevance of PapMV VLNPs as a cancer immunotherapy agent.

Expression of Disialoganglioside (GD2) in Neuroblastic Tumors: A Prognostic Value for Patients Treated With Anti-GD2 Immunotherapy.

Neuroblastoma, a malignant neoplasm of the sympathetic nervous system, is one of the most aggressive pediatric cancers. Patients with stage IV high-risk neuroblastoma receive an intensive multimodal therapy ending with an immunotherapy based on a chimeric monoclonal antibody ch14.18. Although the use of ch14.18 monoclonal antibody has significantly increased the survival rate of high-risk neuroblastoma patients, about 33% of these patients still relapse and die from their disease. Ch14.18 targets the disialoganglioside, GD2, expressed on neuroblastic tumor (NT) cells. To better understand the causes of tumor relapse following ch14.18 immunotherapy, we have analyzed the expression of GD2 in 152 tumor samples from patients with NTs using immunohistochemical stainings. We observed GD2 expression in 146 of 152 samples (96%); however, the proportion of GD2-positive cells varied among samples. Interestingly, low percentage of GD2-positive cells before immunotherapy was associated with relapse in patients receiving ch14.18 immunotherapy. In addition, we demonstrated in vitro that the sensitivity of neuroblastoma cell lines to natural killer-mediated lysis was dependent on the proportion of GD2-positive cells, in the presence of ch14.18 antibody. In conclusion, our results indicate that the proportion of tumor cells expressing GD2 in NTs should be taken in consideration, as a prognostic marker, for high-risk neuroblastoma patients receiving anti-GD2 immunotherapy.

In vitro differentiated plasmacytoid dendritic cells as a tool to induce anti-leukemia activity of natural killer cells.

Acute lymphoblastic leukemia (ALL) is believed to be resistant to NK cell-mediated killing. To overcome this resistance, we developed an innovative approach based on NK cell stimulation with Toll-like receptor (TLR)-activated plasmacytoid dendritic cells (pDC). The translation of this approach into the clinic requires the production of high numbers of human pDC. Herein, we show that in vitro differentiation of cord blood CD34+ progenitors in the presence of aryl hydrocarbon receptor antagonists gives rise to clinically relevant numbers of pDC, as about 108 pDC can be produced from a typical cord blood unit. Blocking the aryl hydrocarbon receptor (AHR) pathway significantly increased the yield of pDC. When compared to pDC isolated from peripheral blood, in vitro differentiated pDC (ivD-pDC) exhibited an increased capacity to induce NK cell-mediated killing of ALL. Although ivD-pDC produced lower amounts of IFN-α than peripheral blood pDC upon TLR activation, they produced more IFN-λ2, known to play a critical role in the induction of anti-tumoral NK cell functions. Both TLR-9 and TLR-7 ligands triggered pDC-induced NK cell activation, offering the possibility to use any clinical-grade TLR-7 or TLR-9 ligands in future clinical trials. Finally, adoptive transfer of ivD-pDC cultured in the presence of an AHR antagonist cured humanized mice with minimal ALL disease. Collectively, our results pave the way to clinical-grade production of sufficient numbers of human pDC for innate immunotherapy against ALL and other refractory malignancies.

Efficient Killing of High Risk Neuroblastoma Using Natural Killer Cells Activated by Plasmacytoid Dendritic Cells.

High-risk neuroblastoma (NB) remains a major therapeutic challenge despite the recent advent of disialoganglioside (GD2)-antibody treatment combined with interleukin (IL)-2 and granulocyte monocyte-colony stimulating factor (GM-CSF). Indeed, more than one third of the patients still die from this disease. Here, we developed a novel approach to improve the current anti-GD2 immunotherapy based on NK cell stimulation using toll-like receptor (TLR)-activated plasmacytoid dendritic cells (pDCs). We demonstrated that this strategy led to the efficient killing of NB cells. When the expression of GD2 was heterogeneous on NB cells, the combination of pDC-mediated NK-cell activation and anti-GD2 treatment significantly increased the cytotoxicity of NK cells against NB cells. Activation by pDCs led to a unique NK-cell phenotype characterized by increased surface expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), with increased expression of CD69 on CD56dim cytotoxic cells, and strong interferon-γ production. Additionally, NB-cell killing was mediated by the TRAIL death-receptor pathway, as well as by the release of cytolytic granules via the DNAX accessory molecule 1 pathway. NK-cell activation and lytic activity against NB was independent of cell contact, depended upon type I IFN produced by TLR-9-activated pDCs, but was not reproduced by IFN-α stimulation alone. Collectively, these results highlighted the therapeutic potential of activated pDCs for patients with high-risk NB.

TRAIL-mediated killing of acute lymphoblastic leukemia by plasmacytoid dendritic cell-activated natural killer cells.

Acute lymphoblastic leukemia (ALL) still frequently recurs after hematopoietic stem cell transplantation (HSCT), underscoring the need to improve the graft-versus-leukemia (GvL) effect. Natural killer (NK) cells reconstitute in the first months following HSCT when leukemia burden is at its lowest, but ALL cells have been shown to be resistant to NK cell-mediated killing. We show here that this resistance is overcome by NK cell stimulation with TLR-9-activated plasmacytoid dendritic cells (pDCs). NK cell priming with activated pDCs resulted in TRAIL and CD69 up-regulation on NK cells and IFN-γ production. NK cell activation was dependent on IFN-α produced by pDCs, but was not reproduced by IFN-α alone. ALL killing was further enhanced by inhibition of KIR engagement. We showed that ALL lysis was mainly mediated by TRAIL engagement, while the release of cytolytic granules was involved when ALL expressed NK cell activating receptor ligands. Finally, adoptive transfers of activated-pDCs in ALL-bearing humanized mice delayed the leukemia onset and cure 30% of mice. Our data therefore demonstrate that TLR-9 activated pDCs are a powerful tool to overcome ALL resistance to NK cell-mediated killing and to reinforce the GvL effect of HSCT. These results open new therapeutic avenues to prevent relapse in children with ALL.

SCL, LMO1 and Notch1 reprogram thymocytes into self-renewing cells.

The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive NOTCH1 allele in all immature thymocytes is sufficient to sensitize them to SCL-LMO1, thereby increasing the pool of self-renewing cells. Surprisingly, hyperactive NOTCH1 cannot reprogram thymocytes on its own, despite the fact that NOTCH1 is activated by gain of function mutations in more than 55% of T-ALL cases. Rather, elevating NOTCH1 triggers a parallel pathway involving Hes1 and Myc that dramatically enhances the activity of SCL-LMO1 We conclude that the acquisition of self-renewal and the genesis of pre-LSCs from thymocytes with a finite lifespan represent a critical first event in T-ALL. Finally, LYL1 and LMO1 or LMO2 are co-expressed in most human T-ALL samples, except the cortical T subtype. We therefore anticipate that the self-renewal network described here may be relevant to a majority of human T-ALL.

Impaired interferon-alpha production by plasmacytoid dendritic cells after cord blood transplantation in children: implication for post-transplantation toll-like receptor ligand-based immunotherapy.

Plasmacytoid dendritic cells (pDCs) initiate both innate and adaptive immune responses, making them attractive targets for post-transplantation immunotherapy, particularly after cord blood transplantation (CBT). Toll-like receptor (TLR) agonists are currently studied for pDC stimulation in various clinical settings. Their efficacy depends on pDC number and functionality, which are unknown after CBT. We performed a longitudinal study of pDC reconstitution in children who underwent bone marrow transplantation (BMT) and single-unit CBT. Both CBT and unrelated BMT patients received antithymocyte globulin as part of their graft-versus-host disease prophylaxis regimen. pDC blood counts were higher in CBT patients than in healthy volunteers from 2 to 9 months after transplantation, whereas they remained lower in BMT patients. We showed that cord blood progenitors gave rise in vitro to a 500-fold increase in functional pDCs over bone marrow counterparts. Upon stimulation with a TLR agonist, pDCs from both CBT and BMT recipients upregulated T cell costimulatory molecules, whereas interferon-alpha (IFN-α) production was impaired for 9 months after CBT. TLR agonist treatment is thus not expected to induce IFN-α production by pDCs after CBT, limiting its immunotherapeutic potential. Fortunately, in vitro production of large amounts of functional pDCs from cord blood progenitors paves the way for the post-transplantation adoptive transfer of pDCs.

Post-transcriptional down-regulation of Toll-like receptor signaling pathway in umbilical cord blood plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (PDCs) from human umbilical cord blood (UCB) produce lower amounts of IFN-α upon TLR stimulation compared with adult counterparts. This difference may play a role in the low graft-versus-host disease rate after UCB transplantation and in the impaired immune response of the neonate to pathogens. Comparing UCB PDC to their adults counterparts, we found that they exhibited a mature surface phenotype and a normal antigen uptake. They upregulated costimulatory molecules upon activation, although with delayed kinetics. Protein, but not ARN, levels of TLR-9, MyD88, IRAK1 and IRF-7, involved in the TLR-9 signaling pathway were reduced. The expression levels of miR-146a and miR-155, known to be involved in the post-transcriptional down-regulation of immune responses, were higher. These data point out a post-transcriptional down-regulation of the TLR-9/IRF-7 signaling pathway in UCB PDC.

Defects in CD54 and CD86 up-regulation by plasmacytoid dendritic cells during pregnancy.

Physiological modulation of the immune system is required for foetal tolerance during pregnancy. However, this immune regulation might lead to impaired self-defence against pathogens. Indeed, pregnant women are more susceptible to newly encountered viruses comparing to non-pregnant women, as exemplified by the prevalence of severe complications in pregnant women infected with the pandemic influenza virus in 2009. Plasmacytoid dendritic cells (pDCs) are specialized dendritic cells that recognise viral antigens and initiate both innate and adaptive immune responses. We therefore sought to determine whether the number and/or the functions of peripheral blood pDCs are regulated during pregnancy. pDC maturation and interferon (IFN)-α production were analysed in response to Toll-like receptor (TLR) stimulation of peripheral blood mononuclear cells from pregnant and non-pregnant women. Our results reveal that pDC frequency is slightly decreased, while the IFN-α production in response to TLR stimulation increases during pregnancy. Interestingly, the up-regulation of the co-stimulatory receptors CD54 (ICAM1) and CD86 is significantly decreased in pDCs from pregnant women as compared to controls, suggesting a possible impact on T-cell responses. In conclusion, we propose that the modulation of CD54 and CD86 expression on peripheral blood pDCs during pregnancy might decrease the initiation of adaptive antiviral immune responses.

Modeling T-cell acute lymphoblastic leukemia induced by the SCL and LMO1 oncogenes.

Deciphering molecular events required for full transformation of normal cells into cancer cells remains a challenge. In T-cell acute lymphoblastic leukemia (T-ALL), the genes encoding the TAL1/SCL and LMO1/2 transcription factors are recurring targets of chromosomal translocations, whereas NOTCH1 is activated in >50% of samples. Here we show that the SCL and LMO1 oncogenes collaborate to expand primitive thymocyte progenitors and inhibit later stages of differentiation. Together with pre-T-cell antigen receptor (pre-TCR) signaling, these oncogenes provide a favorable context for the acquisition of activating Notch1 mutations and the emergence of self-renewing leukemia-initiating cells in T-ALL. All tumor cells harness identical and specific Notch1 mutations and Tcrbeta clonal signature, indicative of clonal dominance and concurring with the observation that Notch1 gain of function confers a selective advantage to SCL-LMO1 transgenic thymocytes. Accordingly, a hyperactive Notch1 allele accelerates leukemia onset induced by SCL-LMO1 and bypasses the requirement for pre-TCR signaling. Finally, the time to leukemia induced by the three transgenes corresponds to the time required for clonal expansion from a single leukemic stem cell, suggesting that SCL, LMO1, and Notch1 gain of function, together with an active pre-TCR, might represent the minimum set of complementing events for the transformation of susceptible thymocytes.

IL-7 enhances survival of human CD56bright NK cells.

Lymphoid differentiation and activation critically depend on cytokine stimulation and the interleukin-7 (IL-7) signaling in particular. Although it has been demonstrated that IL-7 may play a role in natural killer (NK) cell maturation, the effect of IL-7 stimulation on mature human NK cells has not been studied. We, therefore, investigated the expression and functional activity of IL-7Ralpha on mature NK populations from adult blood. In this article, we demonstrate that IL-7Ralpha is specifically expressed in the CD56bright noncytotoxic cytokine-producing NK subset. Importantly, this expression is thymus independent, contrary to what is observed in mice. In addition, we show that IL-7Ralpha is expressed at higher levels on NKG2A+CD56bright NK cells. In contrast to IL-15 stimulation, IL-7 does not increase NK cell cytotoxicity, interferon-gamma production, or the expression of activation markers, indicating that these cytokines play different functions in NK homeostasis and activation. However, IL-7 promotes the survival of the CD56bright NK subset and inhibits apoptosis by increasing BCL2 expression. These data should be taken into account when considering the clinical use of IL-7, particularly after stem cell transplantation.

Scl regulates the quiescence and the long-term competence of hematopoietic stem cells.

The majority of long-term reconstituting hematopoietic stem cells (LT-HSCs) in the adult is in G(0), whereas a large proportion of progenitors are more cycling. We show here that the SCL/TAL1 transcription factor is highly expressed in LT-HSCs compared with short-term reconstituting HSCs and progenitors and that SCL negatively regulates the G(0)-G(1) transit of LT-HSCs. Furthermore, when SCL protein levels are decreased by gene targeting or by RNA interference, the reconstitution potential of HSCs is impaired in several transplantation assays. First, the mean stem cell activity of HSCs transplanted at approximately 1 competitive repopulating unit was 2-fold decreased when Scl gene dosage was decreased. Second, Scl(+/-) HSCs were at a marked competitive disadvantage with Scl(+/+) cells when transplanted at 4 competitive repopulating units equivalent. Third, reconstitution of the stem cell pool by adult HSCs expressing Scl-directed shRNAs was decreased compared with controls. At the molecular level, we found that SCL occupies the Cdkn1a and Id1 loci in primary hematopoietic cells and that the expression levels of these 2 regulators of HSC cell cycle and long-term functions are sensitive to Scl gene dosage. Together, our observations suggest that SCL impedes G(0)-G(1) transition in HSCs and regulates their long-term competence.

ETO2 coordinates cellular proliferation and differentiation during erythropoiesis.

The passage from proliferation to terminal differentiation is critical for normal development and is often perturbed in malignancies. To define the molecular mechanisms that govern this process during erythropoiesis, we have used tagging/proteomics approaches and characterized protein complexes nucleated by TAL-1/SCL, a basic helix-loop-helix transcription factor that specifies the erythrocytic lineage. In addition to known TAL-1 partners, GATA-1, E2A, HEB, LMO2 and Ldb1, we identify the ETO2 repressor as a novel component recruited to TAL-1 complexes through interaction with E2A/HEB. Ectopic expression and siRNA knockdown experiments in hematopoietic progenitor cells show that ETO2 actively represses erythroid TAL-1 target genes and governs the expansion of erythroid progenitors. At the onset of erythroid differentiation, a change in the stoichiometry of ETO2 within the TAL-1 complex activates the expression of known erythroid-specific TAL-1 target genes and of Gfi-1b and p21(Cip), encoding two essential regulators of erythroid cell proliferation. These results suggest that the dynamics of ETO2 recruitment within nuclear complexes couple cell proliferation to cell differentiation and determine the onset of terminal erythroid maturation.

SCL assembles a multifactorial complex that determines glycophorin A expression.

SCL/TAL1 is a hematopoietic-specific transcription factor of the basic helix-loop-helix (bHLH) family that is essential for erythropoiesis. Here we identify the erythroid cell-specific glycophorin A gene (GPA) as a target of SCL in primary hematopoietic cells and show that SCL occupies the GPA locus in vivo. GPA promoter activation is dependent on the assembly of a multifactorial complex containing SCL as well as ubiquitous (E47, Sp1, and Ldb1) and tissue-specific (LMO2 and GATA-1) transcription factors. In addition, our observations suggest functional specialization within this complex, as SCL provides its HLH protein interaction motif, GATA-1 exerts a DNA-tethering function through its binding to a critical GATA element in the GPA promoter, and E47 requires its N-terminal moiety (most likely entailing a transactivation function). Finally, endogenous GPA expression is disrupted in hematopoietic cells through the dominant-inhibitory effect of a truncated form of E47 (E47-bHLH) on E-protein activity or of FOG (Friend of GATA) on GATA activity or when LMO2 or Ldb-1 protein levels are decreased. Together, these observations reveal the functional complementarities of transcription factors within the SCL complex and the essential role of SCL as a nucleation factor within a higher-order complex required to activate gene GPA expression.

Biallelic transcriptional activation of oncogenic transcription factors in T-cell acute lymphoblastic leukemia.

Aberrant expression of transcription factor oncogenes such as HOX11, HOX11L2, TAL1/SCL, LYL1, LMO1, and LMO2 can be detected in lymphoblasts from up to 80% of patients with acute T-cell lymphoblastic leukemia (T-ALL). Transcriptional activation of these oncogenes in leukemic cells typically results from chromosomal rearrangements that place them next to highly active cis-acting transcriptional regulatory elements. However, biallelic activation of TAL1 in some T-ALL cases has been previously proposed. We have used allele-specific mRNA analysis to show that trans-acting mechanisms leading to biallelic overexpression of TAL1 are involved in 10 (42%) of 24 TAL1+ informative T-ALL cases, 2 (17%) of 12 HOX11+ informative cases, and 7 (64%) of 11 LMO2+ informative cases. We propose that aberrant expression of oncogenic transcription factors in a significant fraction of T-ALLs may result from loss of the upstream transcriptional mechanisms that normally down-regulate the expression of these oncogenes during T-cell development.

Growth factor independence-1B expression leads to defects in T cell activation, IL-7 receptor alpha expression, and T cell lineage commitment.

T cell differentiation in the thymus is dependent upon signaling through the TCR and is characterized by the resulting changes in expression patterns of CD4 and CD8 surface coreceptor molecules. Although recent studies have characterized the effects of proximal TCR signaling on T cell differentiation, the downstream integration of these signals remains largely unknown. The growth factor independence-1 (GFI1) and GFI1B transcriptional repressors may regulate cytokine signaling pathways to affect lymphocyte growth and survival. In this study, we show that Gfi1 expression is induced upon induction of the T cell program. Gfi1B expression is low and dynamic during T cell development, but is terminated in mature thymocytes. Transgenic expression of GFI1 and GFI1B in T cells allowed us to determine the functional consequences of constitutive expression. GFI1 potentiates response to TCR stimulation and IL-2, whereas GFI1B-transgenic T cells are defective in T cell activation. Moreover, GFI1B-transgenic thymocytes display reduced expression of the late-activation marker IL-7R alpha, and a decrease in CD4(-)8(+) single-positive T cells that can be mitigated by transgenic expression of BCL2 or GFI1. These data show that GFI1 and GFI1B are functionally unique, and implicate a role for GFI1 in the integration of activation and survival signals.

Regulation of pT alpha gene expression by a dosage of E2A, HEB, and SCL.

The expression of the pT alpha gene is required for effective selection, proliferation, and survival of beta T-cell receptor (beta TCR)-expressing immature thymocytes. Here, we have identified two phylogenetically conserved E-boxes within the pT alpha enhancer sequence that are required for optimal enhancer activity and for its stage-specific activity in immature T cells. We have shown that the transcription factors E2A and HEB associate with high affinity to these E-boxes. Moreover, we have identified pT alpha as a direct target of E2A-HEB heterodimers in immature thymocytes because they specifically occupy the enhancer in vivo. In these cells, pT alpha mRNA levels are determined by the presence of one or two functional E2A or HEB alleles. Furthermore, E2A/HEB transcriptional activity is repressed by heterodimerization with SCL, a transcription factor that is turned off in differentiating thymocytes exactly at a stage when pT alpha is up-regulated. Taken together, our observations suggest that the dosage of E2A, HEB, and SCL determines pT alpha gene expression in immature T cells.