Lymphoid Progenitor Cell Line Research Articles

Transcription factor Zbtb1 interacts with bridging factor Lmo2 and maintains the T-lineage differentiation capacity of lymphoid progenitor cells

Hematopoietic stem and progenitor cells can differentiate into all types of blood cells. Regulatory mechanisms underlying pluripotency in progenitors, such as the ability of lymphoid progenitor cells to differentiate into T-lineage, remain unclear. We have previously reported that LIM domain only 2 (Lmo2), a bridging factor in large transcriptional complexes, is essential to retain the ability of lymphoid progenitors to differentiate into T-lineage. However, biochemical characterization of Lmo2 protein complexes in physiological hematopoietic progenitors remains obscure. Here, we identified approximately 600 Lmo2-interacting molecules in a lymphoid progenitor cell line by two-step affinity purification with LC-MS/MS analysis. Zinc finger and BTB domain containing 1 (Zbtb1) and CBFA2/RUNX1 partner transcriptional corepressor 3 (Cbfa2t3) were found to be the functionally important binding partners of Lmo2. We determined CRISPR/Cas9-mediated acute disruption of Zbtb1 or Cbfa2t3 in the lymphoid progenitor or bone marrow–derived primary hematopoietic progenitor cells causes significant defects in the initiation of T-cell development when Notch signaling is activated. Our transcriptome analysis of Zbtb1- or Cbfa2t3-deficient lymphoid progenitors revealed that Tcf7 was a common target for both factors. Additionally, ChIP-seq analysis showed that Lmo2, Zbtb1, and Cbfa2t3 cobind to the Tcf7 upstream enhancer region, which is occupied by the Notch intracellular domain/RBPJ transcriptional complex after Notch stimulation, in lymphoid progenitors. Moreover, transduction with Tcf7 restored the defect in the T-lineage potential of Zbtb1-deficient lymphoid progenitors. Thus, in lymphoid progenitors, the Lmo2/Zbtb1/Cbfa2t3 complex directly binds to the Tcf7 locus and maintains responsiveness to the Notch-mediated inductive signaling to facilitate T-lineage differentiation.

Hoxa9 Regulates Flt3 in Lymphohematopoietic Progenitors

Early B cell factor (EBF) is a transcription factor essential for specification and commitment to the B cell fate. In this study, we show downregulation of a developmentally regulated cluster of hoxa genes, notably hoxa9, coincides with induction of EBF at the Pro-B cell stage of B cell differentiation. Analysis of the hematopoietic progenitor compartment in Hoxa9(-/-) mice revealed significantly reduced frequencies and expression levels of Flt3, a cytokine receptor important for lymphoid priming and the generation of B cell precursors (BCPs). We show that Hoxa9 directly regulates the flt3 gene. Chromatin immunoprecipitation analysis revealed binding of Hoxa9 to the flt3 promoter in a lymphoid progenitor cell line. Knockdown of Hoxa9 significantly reduced Flt3 transcription and expression. Conversely, forced expression of Hoxa9 increased Flt3 transcription and expression in a Pro-B cell line that expressed low levels of Flt3. Hoxa9 inversely correlated with ebf1 in ex vivo-isolated bone marrow progenitors and BCPs, suggesting that EBF might function to silence a Hoxa9 transcriptional program. Restoration of EBF function in an EBF(-/-) cell line induced B lineage gene expression but did not directly suppress hoxa9 transcription, revealing alternate mechanisms of Hoxa9 regulation in BCPs. These data provide new insight into Hoxa9 function and regulation during lymphoid and B cell development. Furthermore, they suggest that failure to upregulate Flt3 provides a molecular basis for the lymphoid/early B cell deficiencies in Hoxa9(-/-) mice.

FTY720, a New and Alternative Strategy for Treating Blast Crisis CML and Ph1 ALL Patients.

Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome positive (Ph1) acute lymphoblastic leukemia (ALL) are two fatal BCR/ABL-driven leukemias against which the current therapy with Abl kinase inhibitors fails to induce a long-term response, as the majority of patients are either refractory or relapse after a few months of treatment. We recently reported that functional loss of the PP2A tumor suppressor occurs during CML disease progression and that restoration of PP2A activity impairs in vitro and in vivo BCR/ABL leukemogenesis. Here we assessed the therapeutic potential of the PP2A activator FTY720 in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL+ leukemias. FTY720 (500 nM-2.5 mM) induces caspase-dependent apoptosis (70–98% annexin V+) and impairs the clonogenic potential (70–95% inhibition) of imatinib/dasatinib-sensitive and -resistant (T315I) p210 and p190 BCR/ABL-expressing myeloid and lymphoid progenitor cell lines (Ph1 K562, 32D-p210BCR/ABL, 32D-p210(T315I)BCR/ABL and BaF3-p190BCR/ABL), respectively, and of primary bone marrow CML-BCCD34+ (n=11) and Ph1 ALLCD34+/CD19+ (n=12) patients cells. Interestingly the cytokine (IL-3 or IL-7)-dependent growth and differentiation of normal CD34+ myeloid and CD34+/CD19+ lymphoid progenitors (n=8) is not affected by FTY720 treatment. Furthermore, pharmacologic doses of FTY720 markedly suppress leukemogenesis in SCID mice (n=13 per group) transplanted with myeloid and lymphoid progenitor cells transformed with p210BCR/ABL and p190BCR/ABL, respectively. In fact, the median survival has not yet been reached in FTY720-treated (10 mg/kg/day) BCR/ABL+ cell-injected mice. Conversely, all of untreated 32D-p210BCR/ABL, 32D-p210BCR/ABL(T315I) and BaF3-p190BCR/ABL leukemic mice died of an overt acute leukemia-like process with a median survival of 4.3, 4.8 and 4.1 weeks, respectively (P<0.001). After 11 weeks of FTY720 treatment, 80% and 90% of p210 and p190 mice, respectively, were alive and in molecular remission. Moreover, long-term (189 days) FTY720 daily administration (10 mg/kg/day) did not induce any adverse effect, and achieved sustained absence of BCR/ABL+ cells (assessed by nested RT-PCR) in 50% of mice transplanted with myeloid progenitors expressing the imatinib/dasatinib-resistant T315I p210BCR/ABL mutant. Mechanistically, the anti-leukemic effects of FTY720 are sphingosine 1-phosphate receptor 1 (SIP1)-mediated and dependent on the ability of FTY720 to activate PP2A phosphatase. That, in turn, inhibits the activity and expression of wild type and mutant p210 and p190 BCR/ABL oncoproteins and important regulators (e.g. Akt) of malignant cell survival and proliferation. Altogether, these results not only reinforce the importance of the PP2A tumor suppressor in the biology of Ph1 leukemias but, because FTY720 has been shown to be feasible in Phase I-III clinical trials for multiple sclerosis or solid organ transplant patients, they strongly support the use of this PP2A activator as a novel therapeutic approach for CML-BC and Ph1 ALL.

E2F1 Promotes Cytokine Mediated Cell Survival via a Mechanism That Is Separable from Its Cell Cycle Regulatory Effects

Cytokines are important regulators of lymphocyte proliferation and survival during immune responses. The retinoblastoma pathway constitutes an important intracellular network that forms the basis of cell cycle regulation and cellular proliferation in all mammalian cells. Transcription factors of the E2F family form a central component of this pathway, and represent important targets for activation by mitogenic cytokines such as interleukin‐2 (IL‐2). We have previously described a model for study of the E2F1 transcription factor by stable overexpression in the cytokine‐dependent lymphoid progenitor cell line BaF‐B03. In this model of IL‐2 receptor signalling, E2F1 overexpressing BaF‐B03 cells exhibit cytokine‐independent cellular proliferation and survival, thereby supporting the concept that E2F activation is a critical step in the genesis of clonal expansion of antigen‐primed lymphocytes. Here, we provide evidence linking E2F1 to a serum‐dependent cell survival pathway that is separable from its cell cycle regulatory effects. Our data show that the serum glycerophospholipid lysophosphatidic acid is capable of mediating this survival effect via a mechanism that is sensitive to chemical inhibition of phosphatidylinositol 3‐kinase. IL‐2 mediated cell survival, but not cell cycle progression, is dependent upon this serum‐dependent cell survival pathway. The findings presented here provide an insight into how mitogenic cytokines such as IL‐2 regulate the apparently separate processes of lymphocyte proliferation and survival via recruitment of the retinoblastoma pathway.

Stromal Cells and Cytokines in the Induction of Recombination Activating Gene (RAG) Expression in a Human Lymphoid Progenitor Cell

The activation of recombination activating genes (RAGs) plays critical roles in the V(D)J gene recombination machinery and lymphocyte repertoire formation. However, the regulation of RAG gene expression in humans as well as animals is poorly understood. We show that RAG gene expression is activated in a human lymphoid progenitor cell line (FL8.2.4.4) by coculturing them on a bone marrow-derived stromal cell line (PA6) in the presence of cytokines. The RAG transcripts become detectable in 12 hours after initiation of culture, and the increased level is sustained at 24 hours. Among the cytokines, IL-3, IL-6, and IL-7, but not IL-2, IL-4, SCF, GM-CSF induces RAG activation. IL-3, IL-6, and IL-7 exert their effect synergistically on RAG activation. A cognate interaction between FL8.2.4.4 cells and PA6 stromal cells seems to be prerequisite for RAG activation. RAG transcripts are inducible in FL8.2.4.4 cells when cocultured on paraformaldehyde fixed-PA6 stromal cells in the presence of cytokines. These data indicate that two separate signals are both required for induction of RAG activation in lymphoid progenitors; one from the cell surface molecule(s) on stromal cells, and the other from recombinant cytokine(s). The expression of RAG mRNA in FL8.2.4.4 cells is concomitant with induction of recombinase activity. Thus, this system may provide a useful means for further understanding of the mechanisms controlling RAG activation and lymphocyte development in human system.

A Murine Monoclonal Antibody (928) Recognizing a New Epitope Formed with a Combination of [formula omitted] and [formula omitted] Gene Products

ABSTRACT: A murine monoclonal antibody (mAb), 928, that recognizes a cell surface antigen (928 Ag) on a human Epstein-Barr virus-transformed fetal liver-derived lymphoid progenitor cell line (FL4.4) was generated. The 928 mAb reacted with only FL4.4; it did not react with any other 57 cell lines tested. Two color flowcytometry analysis of peripheral blood mononuclear cells (PBMC) revealed that the 928 mAb reacted with B cell and monocyte fractions from only two individuals out of 63 unrelated donors. Biochemical analyses showed that the 928 Ag composes of two molecules (33 and 34 K d) and forms a SDS-resistant, noncovalently linked dimer conformation, the feature being similar to that of peptide-bound MHC class II molecules. Treatment of FL4.4 cells with the 928 mAb significantly facilitated homotypic cell aggregation. In addition, treatment of PBMC of the 928 Ag + donor with recombinant IL-4 augmented the expression of the 928 Ag on CD64 + monocytes. Typing of HLA-DRB1, DPA1 and DPB1 alleles of the 928 Ag expressing and nonexpressing cells revealed that the 928 Ag is expressed only on PBMC of HLA-DPA1∗0201 and DPB1∗0301 positive donors. Finally, anti-DP antibody precleared 928 Ag from the cell lysate. These results demonstrate that the 928 mAb recognizes a polymorphic determinant of HLA-DPA1∗0201 - DPB1∗0301 gene products. The possibility that amino acids in the groove of the peptide-binding site of HLA-DP molecules are critical for the 928 epitope is discussed.

Induction of Recombination Activating Gene Expression in a Human Lymphoid Progenitor Cell Line: Requirement of Two Separate Signals From Stromal Cells and Cytokines

The activation and expression of recombination activating genes (RAGs) plays critical roles in V(D)J gene recombination machinery and lymphocyte development. We showed that RAG gene expression was induced in freshly isolated human bone marrow cells and a human lymphoid progenitor cell line, FL8.2.4.4, by coculture on a monolayer of a murine bone marrow-derived stromal cell line, PA6, in the presence of a mixture of recombinant cytokines. The RAG transcripts were detected 12 hours after initiation of culture, and the increased level was sustained at 24 hours. Among recombinant cytokines, interleukin-3 (IL-3), IL-6, and IL-7, but not IL-2, IL-4, stem cell factor (SCF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) could induce RAG-1 activation in FL8.2.4.4 cells, and a significant synergistic effect between IL-3, IL-6, and IL-7 was observed. Using a double chamber culture technique, it was shown that a cognate interaction between FL8.2.4.4 cells and PA6 stromal cells was a prerequisite for RAG-1 activation. Furthermore, RAG-1 transcripts were induced in FL8.2.4.4 cells when they were cocultured on paraformaldehyde-fixed PA6 stromal cells in the presence of cytokines. These results suggest that two separate signals are both required for induction of RAG-1 activation in lymphoid progenitors; one from the cell surface molecule(s) on stromal cells, and the other from the recombinant cytokine(s). Finally, we showed that expression of RAG mRNA in FL8.2.4.4 cells was concomitant with induction of recombinase activity. This system may provide useful means for further understanding the mechanisms controlling RAG activation and lymphocyte development in the human system.

Expression of 18.6/CD23 antigen on human lymphoid progenitor cell lines and phorbol 12-myristate 13-acetate (PMA)-induced microglia-shaped cells.

The nature of lymphoid progenitors and factor(s) determining commitment to either the T- or B-lymphocyte pathway are poorly understood in the human system. In this study, we generated a monoclonal antibody (MoAb), 18.6, that recognizes a cell surface antigen on a human lymphoid progenitor cell line (FL4.4). MoAb 18.6 reacted with lymphoid progenitor lines, B lymphoid cell lines, and myelomonocytic cell lines. It did not react with any T cell or erythroid leukemic cell lines. Two color FACS analyses of normal lymphoid tissues showed that MoAb 18.6 reacted with a majority of CD20+ mature B cells and a minority of CD64+ monocytes. Molecules of 3 different sizes with MW of 34, 45, and 68 Kd were precipitated with MoAb 18.6 from the lymphoid progenitor cell line. The 18.6 antigen was not expressed on a fetal liver-derived lymphoid progenitor-like cell line, FL1.4, which has the capacity to differentiate into microglia-shaped cells upon PMA-stimulation. Stimulation of FL1.4 cells with PMA induced expression of the 18.6 antigen within 24 hr and the microglia-shaped cells stained positively with MoAb 18.6. Finally, cloning of a cDNA that encoded the 18.6 antigen revealed that the 18.6 antigen is identical to the CD23 antigen. Taken together, these data suggest that the 18.6/CD23 antigen is expressed on lymphoid precursors at a very early stage of differentiation.

Signal transmission through MHC class II molecules in a human B lymphoid progenitor cell line: different signaling pathways depending on the maturational stages of B cells.

The function of MHC class II HLA-DR molecules expressed on a human B lymphoid progenitor cell line FL8.2.4.4 (abbreviated as FL4.4) was examined. FL4.4 cells expressed HLA-DR molecules and stimulation of the DR molecules by anti-DR mAb or by superantigen TSST-1 induced strong augmentation of homocytic aggregation and protein tyrosine phosphorylation in FL4.4 cells. Induced homocytic aggregation in FL4.4 consists both of LFA-1/ICAM-1-dependent and -independent pathways as revealed by mAb blocking experiments. Metabolic inhibitors, NaN3 and cytochalasin B, blocked the induced homocytic aggregation of FL4.4. Early mature Daudi B cell lines also showed a similar type of homocytic aggregation by stimulation with anti-DR mAb. Daudi cells are more sensitive to protein kinase inhibitors herbimycin A and H7 than FL4.4 cells in their blocking of induced homocytic aggregation, while W7 showed stronger inhibitory effects on FL4.4 cells than on Daudi cells. Western blotting analysis revealed that the stimulation of DR molecules induced protein tyrosine phosphorylation of 100-kDa, 90-kDa, 60-kDa and 55-kDa proteins in FL4.4 cells, while, in Daudi cells 110-kDa, 100-kDa and 80-kDa proteins were phosphorylated. These results suggest that different signaling pathways through class II molecules are employed depending on the maturational stage of B-cell differentiation.

Clonal lymphoid progenitor cell lines expressing the BCR/ABL oncogene retain full differentiative function.

The early stages of hematopoiesis have been difficult to study due to problems in obtaining homogeneous populations of progenitor cells that retain both self-renewal and differentiative capacities. We have developed an in vitro system in which transformation of murine bone-marrow cells with the BCR/ABL oncogene, a gene associated with stem-cell leukemias, leads to the outgrowth of clonal lines that have an early lymphoid progenitor cell phenotype. The progenitor cells retain immunoglobulin heavy and light chain genes in a germ-line configuration. These cells give rise in vitro to pre-B cells that have diverse diversity-joining (D-J) region rearrangements, and on transfer to mice with severe combined immune deficiency, differentiate to surface IgM+, immunoglobulin-secreting B cells that respond to T-cell help and function in an antigen-specific fashion. Although their growth is stimulated by BCR/ABL, the progenitor cells depend for continued growth on a stromal cell-derived soluble factor distinct from the pre-B-cell growth factor, interleukin 7. These findings show that BCR/ABL can promote proliferation of an early hematopoietic progenitor cell without preventing its differentiation. This system provides a means of studying the complete B-cell developmental process from clonal progenitor cell to end-stage plasma cell.

Continuing rearrangement of immunoglobulin and T-cell receptor genes in a Ha-ras-transformed lymphoid progenitor cell line.

The arrangement of immunoglobulin genes has been examined in a series of lymphoid cell lines transformed with the Harvey murine sarcoma virus. One cell line, HAFTL-1, expresses antigenic markers characteristic of B-lymphoid cells and undergoes frequent rearrangement at the JH locus (where J = joining and H = heavy chain) during propagation in culture. By molecular cloning and nucleotide sequence determination, these rearrangements were found to represent the earliest postulated step in heavy chain gene assembly: the joining of a diversity (D) segment to a JH segment. The HAFTL-1 cell line also undergoes infrequent D beta-to-J beta joining at the T-cell receptor beta locus in culture. The observations presented here suggest that the HAFTL-1 cell line represents the early stage of B-cell differentiation at which immunoglobulin gene rearrangement is initiated.