Lymphoid Precursors Research Articles

Humoral and cellular immune responses to influenza vaccination in children with cancer receiving chemotherapy

The immune response to influenza vaccination in children with cancer is controversial. The objective of this study was to characterize the cellular and humoral immune responses to an influenza vaccine in children with cancer who were receiving chemotherapy. In this study, children with cancer, who were not previously immunized, received an influenza vaccine via intramuscular injection. Blood samples were obtained prior to and at 4 weeks after immunization. Antibodies were measured using a hemagglutination inhibition (HI) assay. Cell-mediated immunity was measured by specific lymphoproliferation with (3)H-thymidine incorporation and by measuring cell frequencies following staining with monoclonal antibodies (CD8, CD4, CD19, CD45RA and CD27) using flow cytometry following incubation with the influenza antigen for 5 days. Geometric mean titers (GMT), mean counts per minute (cpm), cell frequencies prior to and following vaccination and percentage patient responses were compared using the Mann-Whitney non-parametric U and Chi-square tests; where p<0.05 was considered to indicate a statistically significant result. A total of 56 children were included. Their mean age was 6.64±3.61 years. Acute lymphoblastic leukemia (ALL) was diagnosed in 75, solid tumors in 23 and lymphoma in 2% of the children. Subjects with titers ≥40 hemagglutination units (HU) increased from 43% prior to vaccination to 73% following vaccination (p=0.01), whereas the GMT increased from 31.35 [95% confidence interval (CI), 29-111] to 143.45 HU (95% CI, 284-640) following vaccination (p<0.001). An increase in CD45RA expression in CD8(+) T cells was observed following vaccination (p=0.01). An increase in CD27 expression was observed in the CD4/8-negative cell population stimulated with the influenza antigen following vaccination (p<0.05). No serious adverse effects were observed. An increase in the seropositivity rate and GMT values following influenza vaccination were also observed. Influenza immunization was well tolerated among these children with cancer and increased the humoral and cellular immune responses with the activation of probable lymphoid precursors.

Adipocytes inhibit B lymphopoiesis at the common lymphoid progenitor (CLP) to pre-proB cell stage (111.30)

Abstract Like in humans, B lymphopoiesis declines in rabbits with age, and we previously showed that adult rabbit BM stroma is unable to support generation of proB cells. We investigated changes in BM stroma of adult rabbits and found an increase in adipocytes similar to that in aged humans. We also found that both rabbit and human adipocytes secrete a soluble factor(s) that inhibits B lymphopoiesis. We investigated the stage at which the inhibitory molecule blocks B lymphopoiesis by co-culturing cord blood-derived human HSC and OP9 stromal cells with rabbit adipocyte-conditioned media (CM) and found that the inhibitory factor does not impair the generation of CLP but inhibits generation of pre-proB cells. We tested if the inhibitory molecule acts via stromal cells by treating OP9 stromal cells with adipocyte CM and then co-culturing them with rabbit BM mononuclear cells (MNC). The capacity of pre-treated OP9 stromal cells to support B lymphopoiesis was unchanged. In contrast, treatment of BM MNC of adult rabbit with the adipocyte CM and subsequent co-culture with OP9 stromal cells without adipocyte CM, showed that pre-treated cells were unable to differentiate into proB cells. We conclude that the adipocyte-derived soluble factor acts on early lymphoid precursors to inhibit B lymphopoiesis from the CLP to pre-proB cell stage. Such inhibition likely contributes to the decline of B lymphopoiesis in aged rabbits and humans and can be exploited to enhance immunity in elderly.

Human bone marrow stromal cells simultaneously support B and T/NK lineage development from human haematopoietic progenitors: a principal role for flt3 ligand in lymphopoiesis

The regulation of human early lymphopoiesis remains unclear. B- and T-lineage cells cannot develop simultaneously with conventional stromal cultures. Here we show that telomerized human bone marrow stromal cells supported simultaneous generation of CD19(+) CD34(lo/-) CD10(+) cyCD79a(+) CD20(+/-) VpreB(-) pro-B cells and CD7(+) CD34(+) CD45RA(+) CD56(-) cyCD3(-) early T/Natural Killer (NK) cell precursors from human haematopoietic progenitors, and the generation of both lymphoid precursors was promoted by flt3 ligand (flt3L). On the other hand, stem cell factor or thrombopoietin had little or no effect when used alone. However, both acted synergistically with flt3L to augment the generation of both lymphoid precursors. Characteristics of these lymphoid precursors were evaluated by gene expression profiles, rearrangements of IgH genes, or replating assays. Similar findings were observed with primary human bone marrow stromal cells. Notably, these two lymphoid-lineage precursors were generated without direct contact with stromal cells, indicating that early B and T/NK development can occur, at least in part, by stromal cell-derived humoral factors. In serum-free cultures, flt3L elicited similar effects and appeared particularly important for B cell development. The findings of this study identified the potential of human bone marrow stromal cells to support human early B and T lymphopoiesis and a principal role for flt3L during early lymphopoiesis.

Bone Marrow B cell Precursor Number after Allogeneic Stem Cell Transplantation and GVHD Development

Patients without chronic graft-versus-host disease (cGVHD) have robust B cell reconstitution and are able to maintain B cell homeostasis after allogeneic hematopoietic stem cell transplantation (HSCT). To determine whether B lymphopoiesis differs before cGVHD develops, we examined bone marrow (BM) biopsies for terminal deoxynucleotidyl transferase (TdT) and PAX5 immunostaining early post-HSCT at day 30 when all patients have been shown to have high B cell activating factor (BAFF) levels. We found significantly greater numbers of BM B cell precursors in patients who did not develop cGVHD compared with those who developed cGVHD (median = 44 vs 2 cells/high powered field [hpf]; respectively; P < .001). Importantly, a significant increase in precursor B cells was maintained when patients receiving high-dose steroid therapy were excluded (median = 49 vs 20 cells/hpf; P = .017). Thus, we demonstrate the association of BM B cell production capacity in human GVHD development. Increased BM precursor B cell number may serve to predict good clinical outcome after HSCT.

Synergistic, Context-Dependent, and Hierarchical Functions of Epithelial Components in Thymic Microenvironments

Specialized niche environments specify and maintain stem and progenitor cells, but little is known about the identities and functional interactions of niche components in vivo. Here, we describe a modular system for the generation of artificial thymopoietic environments in the mouse embryo. Thymic epithelium that lacks hematopoietic function but is physiologically accessible for hematopoietic progenitor cells is functionalized by individual and combinatorial expression of four factors, the chemokines Ccl25 and Cxcl12, the cytokine Scf, and the Notch ligand DLL4. The distinct phenotypes and variable numbers of hematopoietic cells in the resulting epithelial environments reveal synergistic, context-dependent, and hierarchical interactions among effector molecules. The surprisingly simple rules determining hematopoietic properties enable the in vivo engineering of artificial environments conducive to the presence of distinct myeloid or T or B lymphoid lineage precursors; moreover, synthetic environments facilitate the procurement of physiological progenitor cell types for analytical purposes and future therapeutic applications.

Differentiation of stem and progenitor cells in activated immune cells with anti-malignant properties

Studies on the biology of immune cells are mainly focused on their role as immune activators and modulators. In their appropriate cultivation and/or modifications, they have shown abilities for an enhanced expression of specific effective molecules. These properties have characterized them as promising candidates for construction of novel safe vaccines and gene-engineering products on their basis. In this aspect, in the last years, attention is directed towards the development of new safe therapeutic methods and techniques with immune cells. In this connection, according to results obtained, in vitro-co-cultivation derived by myeloid and lymphoid precursors with positive and negative on the additionally-inserted copy of the oncogene Dcn1 transfected cells, no immune reaction is observed against both genetically-manipulated cell types, which could be accepted as a proof for their safety. On the other hand, signs of increased degree for both myeloid and lymphoid differentiation in the presence of transfected cells, positive on additionally-inserted copy of the oncogen are indicated. Key words: Progenitor cells, immune cell precursors, gene constructs, oncogenes, tumor-suppresser genes.

The utility of multiparametric flow cytometry for the detection of minimal residual disease in acute lymphoblastic leukemia

The quantification of the amount of minimal residual disease (MRD) in the bone marrow (BM) of patients with acute lymphoblastic leukemia (ALL) at several points of the treatment of the disease has proven to be an important independent predictor of treatment outcome(1-5). More recently, treatment protocols have been designed to adjust chemotherapy according to the presence or absence of MRD on day 8 or 15 of the induction therapy and at the end of induction (day 29) in order to deliver sufficient chemotherapy to cure the patient and minimize acute and long-term side effects. However, this approach is only justified in controlled clinical studies. Flow cytometry, an essential component of the diagnostic work-up in ALL, is used to determine the cell lineage of leukemic blasts, to quantify antigen expression and to detect aberrant antigen expressions. These data are essential for the detection of abnormal cell populations in the BM in the study of MRD. It has been shown that leukemic blasts may be distinguished from normal lymphoid precursors in up to 87% of ALL cases. Moreover, if the technique is well standardized, it is able to detect one abnormal cell among 10,000 to 100,000 normal BM cells(3). Several different panels of antibody combinations have been described with some also considering the cost-effectiveness and simplicity(1,5). However, combinations of at least four colors are essential to give reliable results. The following figure presents the analysis of a case of B-cell ALL. The abnormal blasts may be distinguished from the normal ones by their deficient and variable expression of CD45 (Figure 1). Figure 1 Minimal residual disease study of a B-cell acute lymphoblastic leukemia case. Phenotype at diagnosis: CD45dim variable, CD19+, CD10+ and aberrant expression of CD13/ CD33. Residual blasts in yellow (dim and variable expression of CD45, positive for CD19, CD10 ...

SUMO-Specific Protease 1 Is Critical for Early Lymphoid Development through Regulation of STAT5 Activation

Small ubiquitin-like modifier (SUMO) modification has emerged as an important regulatory mechanism during embryonic development. However, it is not known whether SUMOylation plays a role in the development of the immune system. Here, we show that SUMO-specific protease 1 (SENP1) is essential for the development of early T and B cells. STAT5, a key regulator of lymphoid development, is modified by SUMO-2 and is specifically regulated by SENP1. In the absence of SENP1, SUMO-2 modified STAT5 accumulates in early lymphoid precursors, resulting in a block in its acetylation and subsequent signaling. These results demonstrate a crucial role of SENP1 inthe regulation of STAT5 activation during early lymphoid development.

Linfoma no Hodgkin centrofacial relacionado a VIH. Reporte de un caso y revisión de la literatura

Non-Hodgkin lymphoma (NHL) is the second most common AIDS-related malignancy. The maxillary sinuses, nasal cavity and ethmoid sinus are the most common sites of NHL (33%). In about 3% of cases defined as AIDS, NHL is present at the start of the course of HIV infection. NHL seems to result from uncontrolled proliferation of immature lymphoid precursors that have lost the ability to differentiate and thus accumulate progressively in the host. Most patients have a relatively low CD4 cell count (100-200 cells/mm3). HIV often infects T lymphocytes and the loss of T-cell regulatory function leads to hypergammaglobulinemia and polyclonal B-cell hyperplasia The treatment of head and neck NHL usually involves radiation, chemotherapy and surgery or a combination thereof.Primary NHL lesions in the oral soft tissues are usually asymptomatic, relatively soft in consistency, and appear as a diffuse increase in volume that affects mainly the buccal mucosa, gums and posterior portion of the hard palate.

BMS-936564/MDX-1338: A Fully Human Anti-CXCR4 Antibody Induces Apoptosis In Vitro and Shows Anti Tumor Activity In Vivo

Abstract 1543BMS-936564/MDX-1338 is a fully human monoclonal antibody that specifically recognizes human CXCR4 and is currently in phase 1 studies in patients with relapsed/refractory acute myeloid leukemia (AML) and multiple myeloma (MM). CXCR4 has been identified as a prognostic indicator for AML and other malignancies, in which greater expression of CXCR4 correlates with disease severity. CXCR4 is a seven-transmembrane, G-protein-coupled receptor in the CXC chemokine receptor family. In response to stimulation by its ligand, the chemokine CXCL12, CXCR4 activates calcium flux, chemotaxis and mediates directional migration of hematopoietic cells. In healthy adults, the receptor is predominantly expressed on B and T cells, monocytes, macrophages, NK and dendritic cells, as well as lymphoid and myeloid precursor cells. Expression of CXCR4 is elevated in a variety of cancers and the interaction of CXCR4 on tumor cells with CXCL12 in the bone marrow promotes tumor cell survival and growth. An antagonist of this pathway is predicted to be efficacious in a variety of hematologic malignancies. In vitro studies demonstrate that BMS-936564/MDX-1338 binds to CXCR4expressing cells with low nanomolar affinity. The antibody blocks CXCL12 binding to CXCR4 expressing cells and inhibits CXCL12 induced migration and calcium flux with low nanomolar EC50 values. When given as monotherapy on established tumors, the antibody exhibits anti-tumor activity in multiple AML, NHL and MM xenograft models. BMS-936564/MDX-1338 is an IgG4 and thus does not elicit complement dependent cytotoxicity (CDC) or antibody dependent cell mediated cytotoxicity (ADCC). In vitro and in vivo studies suggest that BMS-936564/MDX-1338 induces apoptosis as one mechanism of tumor growth inhibition. Here we describe the in vitro and in vivo characterization and activities of BMS-936564/MDX-1338. Disclosures:Kuhne:Bristol-Myers Squibb: Employment. Mulvey:Bristol-Myers Squibb: Employment. Chen:Bristol-Myers Squibb: Employment. Pan:Bristol-Myers Squibb: Employment. Chong:Bristol-Myers Squibb: Employment. Niekro:Bristol-Myers Squibb: Employment. Kempe:Bristol-Myers Squibb: Employment. Henning:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Korman:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment.

Acute Lymphocytic Leukemia and Down Syndrome

Abstract SCI-8 Children with Down syndrome are at a markedly increased risk for acute lymphoblastic leukemia (DS-ALL). These leukemias are exclusively of the B lymphoid precursor phenotype and occur in a similar age to “common” sporadic ALLs with the striking absence of infant ALL. Recent studies reveal that DS-ALLs are heterogeneous and differ from sporadic ALLs. Only about a fifth of DS-ALLs carry the common cytogenetic aberrations typical to childhood ALL. Genomic rearrangements leading to the expression of a cytokine receptor, CRLF2, are detected in 60% of DS-ALL in comparison with up to 10% of sporadic ALLs. CRLF2 heterodimerizes with Interleukin 7 receptor-α (IL7R) to form the receptor to thymic stromal lymphopoietin (TSLP). This receptor is usually present in macrophages, dendritic cells, and some T lymphocytes and participates in allergic and inflammatory processes. The aberrant expression of the TSLP receptor is DS-ALL (and sporadic ALL) is often associated with additional mutations that cause constitutive activation of the downstream JAK-STAT and mTOR growth signaling pathways. These are either lymphoid specific activating mutations of JAK2 or JAK1 or mutations in CRLF2 or IL7R that cause ligand-independent receptor dimerization. The role of the trisomy in selecting these somatic abnormalities is presently unknown. Clinically, the prognosis of DS-ALL is inferior to sporadic ALL mainly because of increased treatment toxicity. However, recent data suggest that the inferior outcome may also be related to the genetic properties of the leukemic cells and that excessive chemotherapy dose reduction may not be appropriate for these patients. Therefore increased vigilance for infectious complications and optimal supportive care are required during periods of intensive chemotherapy. The common activation of the TSLP signaling pathway in DS-ALLs suggests a future for targeted therapy with JAK and/or mTOR inhibitors. Importantly, research of DS-ALL has proven relevant for the general patient population with ALL, as somatic mutations in the TSLP pathway have been discovered in children and adults with sporadic ALL. A major research challenge is the elucidation of the roles of constitutional and somatic trisomy 21 in leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

Evaluation of Role for flt3L in Human Early B and T/NK Lymphopoiesis in a Novel Stromal Cell Culture System

Abstract 2385The regulatory mechanism of human early lymphopoiesis remains less defined. A major limitation of conventional in vitro assay is that B and T lymphopoiesis cannot be assessed simultaneously. We exploded a novel culture assay and found that the hTERT-transduced telomerized human stromal cells support the generation of CD19+CD34lo/-CD10+cyCD79a+CD20+/−VpreB− pro B- and CD7+CD34+CD45RA+CD56−cyCD3− early T-cell precursors from human hematopoietic progenitors without cytokines, which was enhanced by flt3L (2010, ASH). We further characterized the generated lymphoid precursors, and verified that lineage-specific transcription factors for B (Pax-5 and EBF) and T/NK-cell precursors (GATA-3, HEB, Id2) were expressed by the CD19+ and CD7+CD56− cells, respectively. The CD7+CD56− cells showed the differentiation potential to T- and NK-lineage cells, by replating with OP9-Delta1 in the presence of flt3L+IL-7, and with the telomerized-stromal cells in the presence of flt3L+IL-7+IL-15, respectively. In serum-free culture condition, B-cell differentiation was minimally supported by the stromal cells, while low number of CD7+ cells was observed. Nonetheless, a significant number of CD7+, CD19-cyCD79a+, and CD19+ cells developed in the presence of flt3L, suggesting an important role for flt3L in the generation of early T/NK- and, particularly, B-lineage cell precursors on the stromal cells. The cellular interaction between Notch and Notch ligand Delta-1 or -4 in the presence of appropriate cytokines is considered to be crucial in T-lineage cell development in mice. We therefore examined whether Notch pathway is involved in the T/NK-cell development under this culture condition. While the gene expression of Delta-1 or -4 was detected in the telomerized stromal cells, the protein expression of these ligands was not detected with Western-blotting analysis. After co-culture of hematopoietic progenitor cells with the telomerized stromal cells, the gene expression of Notch target gene, HES1, was not increased. These data suggest that Notch signaling is not involved in the generation of early T/NK cell precursors on the stromal cells. This novel in vitro culture system suggests that the development of early B- and T/NK- cell precursors from hematopoietic precursors take places on the stromal cells in a Notch-independent manner and that flt3L plays a principal role in the stimulation of the early B and T/NK lymphopoiesis. Disclosures:No relevant conflicts of interest to declare.

Graft-Versus-Host Disease Impairs Early B Lymphopoiesis in the Bone Marrow

Abstract 2976Graft-versus-host disease (GVHD) and infectious complications are main causes of non-relapse mortality after allogeneic stem cell transplantation (SCT). Impaired immune function after SCT is usually attributed to the immunosuppressive medication applied for GVHD prophylaxis or therapy. Using a major histocompatibility complex (MHC)–mismatched murine model of GVHD (C57BL/6→BALB/c), we now examined the influence of GVHD on B cell immunity after SCT in the absence of pharmacologic immunosuppression. Lethally irradiated BALB/c (H-2d) recipients were transplanted with T cell-depleted bone marrow (TCD BM; 2.5×106) from C57BL/6 (H-2b) donors and parallel groups received CD4+CD25− conventional donor T cells (Tconv; 0.25 × 106) 2d later. Mice that received TCD BM alone (n =10) did not develop GVHD and showed a rapid and complete reconstitution of B cells in peripheral blood (PB) (25 ± 7% CD19+ B cells at d21; 55 ± 5% at d100). Mice that received additional donor Tconv cells (n =12) developed severe GVHD and completely lacked donor and host B cells in PB until their early death or throughout the observation period of 100d (p<0.001). Animals that were protected from severe GVHD by the co-infusion of donor CD4+CD25+Foxp3+ regulatory T cells (Treg; 0.25×106 Treg transplanted together with TCD BM; 2d later transfusion of 0.25 × 106 Tconv) showed a delayed, but finally full reconstitution of their B cell compartment in PB (9 ± 12% CD19+ B cells at d21; 42 ± 17% at d100). Similarly, animals without GVHD after TCD BMT and animals protected from GVHD by co-transplanted Treg cells showed a complete reconstitution of their B cell compartment in spleen and BM at d100 (spleen: 26±4, 7×106 and 31 ± 9.3×106 CD19+ B cells, respectively; BM: 2, 2 ± 0, 3×106 and 2.9 ± 0.9×106 B cells, respectively). In contrast, B cells were not only undetectable in peripheral lymphoid organs in animals with severe GVHD but also in the BM, suggesting that B cell precursors were affected. To examine whether GVHD solely impedes B cell regeneration or actively contributes to B cell eradication, GVHD was induced after B cell reconstitution at d21 after TCD BMT by donor lymphocyte infusions (DLI). Within 1wk after the transfer of 8×106 or 12×106 donor CD4+ lymphocytes, a significant reduction of B cells in PB was detected (from 30.3 ± 5.2% to 10 ± 6.9% and 36.3 ± 9.2% to 5.9 ± 1.3%, respectively; n =4). Thus, GVHD not only affected B cell reconstitution, but even eradicated stem cell-derived B cells that were syngeneic to the GVHD-inducing T cells, suggesting that GVHD-induced inflammation contributed to B cell depletion. To examine the influence of GVHD on precursor cells, serial transplants were performed. Yet, TCD BM from both, animals with and without GVHD, reconstituted their B cell compartment upon secondary transplantation (n =18; 33.1 ± 14.8% vs. 32.4 ± 17% at d100), thereby proving that the stem cell compartment was not affected. Next, we examined the effect of GVHD on precursor cells. Multipotent BM precursors (lin−, Sca-1+, c-kit+ [LSK]) were not significantly different in GVHD animals (TCD BM plus Tconv; n =12) as compared to controls (TCD BM only; n =10; 3.5×103 ± 2.8×103 vs. 5.8×103 ± 2.5×103, respectively). However, common lymphoid precursors (CLP; Lin−, FLt3+, CD127+) in the BM were significantly reduced in animals with GVHD (0.3×103 ± 0.17×103) as compared to transplant recipients without GVHD (4.4×103 ± 2.2×103, p<0.001). These results suggest that the dysregulated production of pro-inflammatory cytokines during GVHD is toxic for early B cell precursors and/or that the alloresponse destroys the BM niche for developing B cells. As IFN- γ and TNF are known to be elevated in GVHD and to impair B lymphopoiesis even in a non-transplant setting, we generated mixed chimeras using BM from wt and cytokine receptor deficient animals. Yet, a selective B cell reconstitution from receptor deficient BM was not observed in GVHD, suggesting that neither of these cytokines is exclusively responsible for its toxic effects on B cell precursors. Taken together, our results show that GVHD not solely affects immune reconstitution by the well known destruction of secondary lymphoid organs, but it disturbs early lymphoid progenitors in the BM through inflammatory, but not necessarily allo-specific immune responses. Disclosures:No relevant conflicts of interest to declare.

Depletion and recovery of lymphoid subsets following morphine administration

Opioid use and abuse has been linked to significant immunosuppression, which has been attributed, in part, to drug-induced depletion of lymphocytes. We sought to define the mechanisms by which lymphocyte populations are depleted and recover following morphine treatment in mice. Mice were implanted with morphine pellets and B- and T-cell subsets in the bone marrow, thymus, spleen and lymph nodes were analysed at various time points. We also examined the effects of morphine on T-cell development using an ex vivo assay. The lymphocyte populations most susceptible to morphine-induced depletion were the precursor cells undergoing selection. As the lymphocytes recovered, more lymphocyte precursors proliferated than in control mice. In addition, peripheral T-cells displayed evidence that they had undergone homeostatic proliferation during the recovery phase of the experiments. The recovery of lymphocytes following morphine-induced depletion occurred in the presence of morphine and via increased proliferation of lymphoid precursors and homeostatic proliferation of T-cells.

Canonical Wnt Signaling Regulates Hematopoiesis in a Dosage-Dependent Fashion

Canonical Wnt signaling has been implicated in the regulation of hematopoiesis. By employing a Wnt-reporter mouse, we observed that Wnt signaling is differentially activated during hematopoiesis, suggesting an important regulatory role for specific Wnt signaling levels. To investigate whether canonical Wnt signaling regulates hematopoiesis ina dosage-dependent fashion, we analyzed the effect of different mutations in the Adenomatous polyposis coli gene (Apc), a negative modulator of the canonical Wnt pathway. By combining different targeted hypomorphic alleles and a conditional deletion allele of Apc, a gradient of five different Wnt signaling levels was obtained invivo. We here show that different, lineage-specific Wnt dosages regulate hematopoietic stem cells (HSCs), myeloid precursors, and T lymphoid precursors during hematopoiesis. Differential, lineage-specific optimal Wnt dosages provide a unifying concept that explains the differences reported among inducible gain-of-function approaches, leading to either HSC expansion or depletion of the HSC pool.

T cells of patients with myelodysplastic syndrome are frequently derived from the malignant clone.

T cells of patients with myelodysplastic syndrome are frequently derived from the malignant clone.

Miz-1 Is Required To Coordinate the Expression of TCRβ and p53 Effector Genes at the Pre-TCR “β-Selection” Checkpoint

Miz-1 is a Broad-complex, Tramtrack and Bric-à-brac/pox virus zinc finger domain (BTB/POZ)-containing protein expressed in lymphoid precursors that can activate or repress transcription. We report in this article that mice expressing a nonfunctional Miz-1 protein lacking the BTB/POZ domain (Miz-1(ΔPOZ)) have a severe differentiation block at the pre-T cell "β-selection" checkpoint, evident by a drastic reduction of CD4(-)CD8(-) double-negative-3 (DN3) and DN4 cell numbers. T cell-specific genes including Rag-1, Rag-2, CD3ε, pTα, and TCRβ are expressed in Miz-1-deficient cells and V(D)J recombination is intact, but few DN3/DN4 cells express a surface pre-TCR. Miz-1-deficient DN3 cells are highly apoptotic and do not divide, which is consistent with enhanced expression of p53 target genes such as Cdkn1a, PUMA, and Noxa. However, neither coexpression of the antiapoptotic protein Bcl2 nor the deletion of p21(CIP1) nor the combination of both relieved Miz-1-deficient DN3/DN4 cells from their differentiation block. Only the coexpression of rearranged TCRαβ and Bcl2 fully rescued Miz-1-deficient DN3/DN4 cell numbers and enabled them to differentiate into DN4TCRβ(+) and double-positive cells. We propose that Miz-1 is a critical factor for the β-selection checkpoint and is required for both the regulation of p53 target genes and proper expression of the pre-TCR to support the proliferative burst of DN3 cells during T cell development.

Abstract 2912: Protein pathway activation mapping of leukemia-associated JAK1 mutants

Abstract The Janus kinase 1 (JAK1) is a cytoplasmic tyrosine kinase that noncovalently associates with a variety of cytokine receptors and plays a nonredundant role in lymphoid cell precursor proliferation, survival and differentiation. We documented that somatic mutations in JAK1 occur in individuals with acute lymphoblastic leukemia (ALL) (Flex et al. 2008, J Exp Med 205:751-8). JAK1 mutations were more prevalent among adult subjects with T-cell precursor ALL, where they accounted for approximately 20% of cases, and were associated with poor response to therapy and overall prognosis. In order to understand the molecular and cellular mechanisms by which aberrant JAK1 function contribute to leukemogenesis we analysed the signaling architecture of BaF3 cell lines stably expressing a series of leukemia-associated JAK1 gene mutations by Reverse Phase Protein Microarray (RPMA). We utilized BaF3 cell lines, stably expressing human wild type JAK1 and five ALL-associated mutants bearing missense mutations in different domains of the kinase. Cells were serum starved for 16 hours and lysed before and after induction with IL-3 for 20 minutes. RPMA analysis was performed to determine the phosphorylation/activation state of 102 different key signalling proteins involved in the major intracellular signal transduction pathways. Results showed that intacellular signaling downstream JAK/STAT is differentially modulated between cell lines expressing the different ALL-associated JAK1 mutants. Statistical analysis revealed that mutations affecting the pseudo-kinase and in the kinase domains resulted in a different phosphorylation pathway network compared to mutations involving the FERM and SH2 domains. Our results support a model in which mutations affecting distinct domains of JAK1 have a differential perturbing effect on JAK1 function and signalling. Characterization of the consequences of leukemia-associated JAK1 mutants on the protein signalling network may help to identify new therapeutic targets for ALL. The present findings further document that functional protein pathway activation mapping using RPMA represents a powerful approach to uncovering mutation-driven events, and elucidating the specific events associated with different mutations within a single protein. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2912. doi:10.1158/1538-7445.AM2011-2912

E47 regulates hematopoietic stem cell proliferation and energetics but not myeloid lineage restriction (153.1)

Abstract The immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins, the widely expressed basic helix-loop-helix (bHLH) transcription factors, contribute to HSC and MPP activity but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches, we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. E47 deficient progenitors show competent myeloid production in short-term assays in vitro and in vivo. However, long-term myeloid and lymphoid differentiation is compromised due to a progressive loss of HSC self-renewal that is associated with diminished p21 expression and hyper-proliferation. The activity of E47 is shown to be cell-intrinsic. Moreover, E47 deficient HSCs and MPPs have altered expression of genes associated with cellular energy metabolism, and the size of the MPP pool but not downstream lymphoid precursors in bone marrow or thymus is partially rescued in vivo by anti-oxidant. Together, these observations suggest a role for E47 in the tight control of HSC proliferation and energy metabolism, and demonstrate that E47 is not required for short-term myeloid differentiation.

E47 regulates hematopoietic stem cell proliferation and energetics but not myeloid lineage restriction

AbstractThe immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins, the widely expressed basic helix-loop-helix transcription factors, contribute to HSC and MPP activity, but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches, we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. E47-deficient progenitors show competent myeloid production in short-term assays in vitro and in vivo. However, long-term myeloid and lymphoid differentiation is compromised because of a progressive loss of HSC self-renewal that is associated with diminished p21 expression and hyperproliferation. The activity of E47 is shown to be cell-intrinsic. Moreover, E47-deficient HSCs and MPPs have altered expression of genes associated with cellular energy metabolism, and the size of the MPP pool but not downstream lymphoid precursors in bone marrow or thymus is rescued in vivo by antioxidant. Together, these observations suggest a role for E47 in the tight control of HSC proliferation and energy metabolism, and demonstrate that E47 is not required for short-term myeloid differentiation.