FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2044004846> ?p ?o ?g. }

• W2044004846 endingPage "5521" @default.
• W2044004846 startingPage "5511" @default.
• W2044004846 abstract "Article15 October 2003free access Determination of lymphoid cell fate is dependent on the expression status of the IL-7 receptor Sheetal J. Purohit Sheetal J. Purohit Department of Microbiology and Division of Developmental and Clinical Immunology, University of Alabama at Birmingham, Birmingham, AL, 35294-3300 USA Search for more papers by this author Robert P. Stephan Robert P. Stephan Department of Microbiology and Division of Developmental and Clinical Immunology, University of Alabama at Birmingham, Birmingham, AL, 35294-3300 USA Search for more papers by this author Hyung-Gyoon Kim Hyung-Gyoon Kim Department of Microbiology and Division of Developmental and Clinical Immunology, University of Alabama at Birmingham, Birmingham, AL, 35294-3300 USA Search for more papers by this author Brantley R. Herrin Brantley R. Herrin Department of Microbiology and Division of Developmental and Clinical Immunology, University of Alabama at Birmingham, Birmingham, AL, 35294-3300 USA Search for more papers by this author Larry Gartland Larry Gartland Department of Microbiology and Division of Developmental and Clinical Immunology, University of Alabama at Birmingham, Birmingham, AL, 35294-3300 USA Search for more papers by this author Christopher A. Klug Corresponding Author Christopher A. Klug Department of Microbiology and Division of Developmental and Clinical Immunology, University of Alabama at Birmingham, Birmingham, AL, 35294-3300 USA Search for more papers by this author Sheetal J. Purohit Sheetal J. Purohit Department of Microbiology and Division of Developmental and Clinical Immunology, University of Alabama at Birmingham, Birmingham, AL, 35294-3300 USA Search for more papers by this author Robert P. Stephan Robert P. Stephan Department of Microbiology and Division of Developmental and Clinical Immunology, University of Alabama at Birmingham, Birmingham, AL, 35294-3300 USA Search for more papers by this author Hyung-Gyoon Kim Hyung-Gyoon Kim Department of Microbiology and Division of Developmental and Clinical Immunology, University of Alabama at Birmingham, Birmingham, AL, 35294-3300 USA Search for more papers by this author Brantley R. Herrin Brantley R. Herrin Department of Microbiology and Division of Developmental and Clinical Immunology, University of Alabama at Birmingham, Birmingham, AL, 35294-3300 USA Search for more papers by this author Larry Gartland Larry Gartland Department of Microbiology and Division of Developmental and Clinical Immunology, University of Alabama at Birmingham, Birmingham, AL, 35294-3300 USA Search for more papers by this author Christopher A. Klug Corresponding Author Christopher A. Klug Department of Microbiology and Division of Developmental and Clinical Immunology, University of Alabama at Birmingham, Birmingham, AL, 35294-3300 USA Search for more papers by this author Author Information Sheetal J. Purohit1, Robert P. Stephan1, Hyung-Gyoon Kim1, Brantley R. Herrin1, Larry Gartland1 and Christopher A. Klug 1 1Department of Microbiology and Division of Developmental and Clinical Immunology, University of Alabama at Birmingham, Birmingham, AL, 35294-3300 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:5511-5521https://doi.org/10.1093/emboj/cdg522 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Signaling through the IL-7 receptor (IL-7R) is necessary for the development of the earliest B- and T-lineage cells. IL-7R is first expressed on common lymphoid progenitor cells and is not detected on primitive common myeloid progenitors. In this study, we show that enforced expression of IL-7R on multipotential stem cells does not influence lymphoid versus myeloid cell fate. T cell development was compatible with sustained IL-7R expression; however, we observed a near complete block in B cell development at the onset of B-lineage commitment. Unlike pre-proB cells from control animals, developmentally-arrested IL-7R+B220+cd19−NK1.1−Ly-6C− cells failed to express EBF and Pax5. These results suggest that transient downregulation of IL-7R signaling is a necessary event for induction of EBF and Pax5 expression and B-lymphocyte commitment. Introduction Growth factors and cytokines play vital roles in lineage specification within mammalian developmental systems. For instance, within the mammalian nervous system, multipotent neural crest precursors (NCPs) exposed to glial growth factor differentiate into glial cells while transforming growth factor β stimulates NCP to develop into smooth muscle in a process that is independent of growth factor-mediated cell survival (Anderson, 1997). In the hematopoietic system, growth factor and cytokine signaling pathways have been shown to regulate both proliferation and survival of hematopoietic cells, while their function in lineage specification remains controversial (Enver et al., 1998; Metcalf, 1998). One recent example illustrating the potential instructive role of cytokine receptor signaling in lineage determination within the hematopoietic system was shown when the IL-2Rβ chain was expressed in common lymphoid progenitor (CLP) cells (Kondo et al., 2000). CLP cells are normally restricted in their developmental potential to the B, T and natural killer (NK) cell lineages. However, when IL-2Rβ was introduced into CLP cells using either an MHC class I promoter-driven transgene or from a retroviral vector, CLP were induced to differentiate along the myeloid cell pathway when stimulated at a clonal level with IL-2. Although these findings are informative in showing that lineage determination can be regulated by cytokine receptor signaling, it remains unclear whether IL-2Rβ has such a function in normal hematopoiesis. Signaling through the IL-7 receptor (IL-7R) is necessary for the development of the earliest B- and T-lineage cells (Peschon et al., 1994; Miller et al., 2002). IL-7R is comprised of two chains, the IL-7Rα ligand-binding chain and the IL-2Rγc signaling chain (Goodwin et al., 1990; Noguchi et al., 1993), which are both required for proper signaling to occur (Ziegler et al., 1995). The IL-7Rα chain also comprises part of the thymic stromal lymphopoietin (TSLP) receptor (Park et al., 2000; Leonard, 2002), while the IL-2Rγc chain is shared by the receptors for IL-2, IL-4, IL-9, IL-15 and IL-21 (Sugamura et al., 1996; Asao et al., 2001). During hematopoiesis, IL-7R expression is first detected at the CLP stage (Kondo et al., 1997). It is present on developing thymocytes at the CD4−cd8− stage, on CD4+ and CD8+ single positive T cells, and on immature B-lineage cells. It is not expressed on CD4+cd8+ double positive thymocytes or mature B cells (Sudo et al., 1993; Appasamy, 1999; Fry and Mackall, 2002). Based on findings from IL-7R knockout animals, IL-7R is also required for γδ T cell development but not for NK and dendritic cell development (He and Malek, 1996; Maki et al., 1996; Fry and Mackall, 2002). Blocking cell death by enforced expression of the anti-apoptotic gene, bcl-2, is sufficient to rescue αβ T cell development in IL-7R−/− animals (Akashi et al., 1997; Maraskovsky et al., 1998) but is not sufficient to rescue the block in early B cell development, suggesting that IL-7R signaling has unique functions within the earliest B- and T-lineage cells. Although myeloid-lineage cells, including the common myeloid progenitor (CMP), do not express IL-7R (Akashi et al., 2000), several experiments have shown that IL-7 can result in various effects on myeloid lineage cells both in vitro and in vivo. IL-7 synergistically increases myeloid colony formation of Lin−Sca-1+ progenitors when combined with various colony-stimulating factors, including GM-CSF and CSF-1 (Jacobsen et al., 1993; Appasamy, 1999). In addition, administration of recombinant IL-7 to mice has been shown to increase the numbers of immature myeloid lineage cells, which further supports a direct or indirect function of IL-7 in myelopoiesis (Faltynek et al., 1992). A possible role for IL-7R signaling in lymphoid lineage commitment is suggested by a number of observations. First, IL-7R is initially detected on CLP, but not on CMP or multipotent stem cells (Kondo et al., 1997; Akashi et al., 2000). Second, experiments involving the knockout of the B-lineage transcription factor Pax5 showed that Pax5−/− cells could be maintained at the pro-B stage in vitro in media containing IL-7 (Nutt et al., 1999). However, when IL-7 was removed or depleted from the cultures, the cells changed morphology and gave rise to various myeloid lineage cells both in vitro and in vivo (Nutt et al., 1999; Mikkola et al., 2002). This suggests that both IL-7R signaling and Pax5 activity function to maintain B cell identity at the point of B-lineage commitment. Finally, further analysis of IL-7R−/− animals has indicated that both B and T lymphopoiesis are blocked prior to the earliest committed progenitor stage for both lineages (Peschon et al., 1994; Miller et al., 2002). Collectively, these observations raise the possibility that IL-7R signaling may play an important role in the specification of lymphoid cell fate from multipotent progenitor cells. In order to test whether IL-7R expression would restrict the developmental potential of multipotential cells to the lymphoid lineages, we transduced bone marrow progenitor cells with an IL-7R-expressing retrovirus and examined hematopoiesis derived from these cells in reconstituted mice. Analysis of transplant recipients showed that graded levels of IL-7R expression resulted in altered ratios of B versus myeloid cells in peripheral blood of all reconstituted animals. Mice transplanted with cells expressing a control green fluorescent protein (GFP) retrovirus showed no evidence of lineage skewing at high or low levels of GFP expression. Further analysis of B cell development in bone marrow of mice expressing IL-7R indicated a severe developmental block at the earliest B cell progenitor stage. Unlike their phenotypically equivalent counterparts that do not express IL-7R, developmentally arrested IL-7R+B220+cd19−NK1.1−Ly-6C− cells failed to express EBF and Pax5, which suggests that transient downregulation of IL-7R expression may be a necessary event for induction of EBF and Pax5 expression and B cell commitment. Sustained expression of IL-7R had no inhibitory effect on the development of T or myeloid- lineage cells. This indicates that IL-7R signaling does not influence lymphoid versus myeloid cell fate but rather promotes T versus B cell development in a manner dependent on its expression status. Results Retroviral expression of IL-7R rescues B and T cell development in IL-7R−/− cells In order to test whether enforced expression of IL-7R in multipotent stem cells would promote commitment to the lymphoid lineage, we used murine stem cell virus (MSCV) (Hawley et al., 1994) to coexpress the two components of IL-7R, IL-7Rα and IL-2Rγc, along with one of two spectrally distinct GFP variants. Expression of blue-excited GFP (Bex) and violet-excited GFP (Vex) can be detected in a single cell due to their differential excitation properties (Anderson et al., 1996). The two IL-7R chains were individually cloned upstream of an internal ribosome entry site (IRES) within MSCV (Figure 1A). Cells were transduced with both viruses in the event that the IL-2Rγc signaling chain would be limiting for IL-7R assembly or for the proper function of other IL-2Rγc-associated receptors including IL-2R, IL-4R, IL-9R, IL-15R and IL-21R. Stable virus-producing cell lines that produced both MSCV-IL-7Rα-Bex and MSCV-IL-2Rγc-Vex, or the control MSCV-Bex and MSCV-Vex viruses, were generated using the GP+E-86 cell line (see Materials and Methods). Figure 1.Characterization of IL-7Rα and IL-2Rγ retroviruses. (A) Schematic diagram of retroviral constructs generated in MSCV. MSCV consists of: LTR (long terminal repeat), IRES, Bex and Vex. (B) Rescue of T cell development in IL-7R−/− cells is shown in a representative FACS stain at four weeks post transplant. Thymocytes were gated on double-positive (Bex and Vex) cells and then analyzed by CD4 and CD8 staining. (C) γδ T cell development is rescued in IL-7R−/− cells by IL-7R expression. FACS analysis was done at 7 weeks post-transplantation. Download figure Download PowerPoint In order to confirm that a functionally competent IL-7R was being assembled on the cell surface in vivo, bone marrow was isolated from IL-7Rα knockout animals and then cocultured on double virus-producing GP+E-86 cells for 48 h before injection into Ly-5 congenic, lethally irradiated recipient mice. Four weeks post-transplant, thymi from reconstituted animals were analyzed for rescue of T-lineage development by staining for CD4 and CD8, since IL-7Rα knockout animals exhibit a severe developmental block prior to CD4 and CD8 expression (Peschon et al., 1994). T-cell development in animals reconstituted with cells expressing the control Bex/Vex vectors was not rescued and displayed the IL-7R knockout phenotype of arrested αβ T cell development at the double negative CD4−cd8− stage (Figure 1B). In contrast, normal αβ T cell development and thymic cellularity was completely restored in animals reconstituted with IL-7R-expressing cells as indicated by CD4 and CD8 staining (Figure 1B). T cell development was rescued in the IL-7R knockout cells by IL-7Rα/IL-2Rγc or by IL-7Rα alone (Figure 1B; data not shown), which indicates that retrovirally-encoded IL-7R was functional. Constitutive IL-7R expression had no apparent detrimental effects on T cell development in that we continued to observe normal production of T-lineage cells for as long as 14 weeks post-transplantation, which was the longest time point analyzed. In addition, γδ T cell development was rescued in animals reconstituted with cells expressing the IL-7R, while it was absent in animals reconstituted with Bex/Vex-expressing IL-7R−/− cells (Figure 1C). Graded levels of IL-7R expression alter the ratio of B-lymphoid versus myeloid cells in the periphery To determine the effects of constitutive IL-7R expression on normal hematopoiesis, we analyzed peripheral blood from animals reconstituted with wild-type bone marrow cells transduced with either the control (Bex/Vex) or IL-7R-expressing retroviruses beginning at 3–4 weeks post-transplant. Cells that expressed a single fluorophore (Bex or Vex) or double-expressing cells were independently analyzed (Figure 2A). In all analyses, gates were drawn on Bex/Vex+ cells, which were all Ly5.1 (donor-marker) positive (data not shown). While the Bex/Vex control mice (henceforth referred to as control mice) showed a higher frequency of B220+ cells in peripheral blood (n = 15), mice reconstituted with IL-7Rα/IL-2Rγc-expressing cells (IL-7R mice) displayed a more Mac-1+ phenotype (n = 15). In IL-7R mice, both double positive IL-7Rα/IL-2Rγc (Bex/Vex) cells and single positive IL-7Rα (Bex) cells showed a large increase in the frequency of Mac-1+ cells, compared with single positive IL-2Rγc (Vex) cells (Figure 2A, d–f). The skewed ratio of B220+ versus Mac-1+ cells was only evident in IL-7Rα or in IL-7Rα/IL-2Rγc expressing cells, indicating that the Bex/Vex fluorophores do not inherently skew development between lymphoid and myeloid cells (Figure 2A, a–c). Further examination of the data revealed that increasing levels of IL-7Rα/IL-2Rγc expression within a given animal resulted in a statistically significant decrease in the frequency of B220+ cells along with a pronounced increase in the frequency of Mac-1+ cells (Figures 2B and C). Cells that expressed increasing levels of Bex (IL-7Rα) were gated along the diagonal (Figure 2B) to ensure that approximately stoichiometric levels of IL-7Rα and IL-2Rγc would be expressed within a given cell in the gated populations. It should be noted that the variability seen in the frequencies of individual lineages is due to the inherent variability associated with a reconstitution assay. The same phenotypic shift was observed in 15 mice per construct analyzed from three independent reconstitution experiments. Figure 2.Increased levels of IL-7Rα/IL-2Rγc expression results in a phenotypic skew to the myeloid lineages in peripheral blood. (A) Peripheral blood analysis at 4 weeks post transplant. Quadrants a, b, c and d, e, f, which gated the three different subpopulations of cells (Vex single positive, Bex/Vex double positive and Bex single positive, respectively), were analyzed for B220 (B cell) and Mac-1 (myeloid) expression. Dead cells were excluded by propidium iodide staining in all samples prior to gating. (B) IL-7Rα/IL-2Rγc-expressing peripheral blood cells were gated for increasing levels of Bex/Vex expression and then analyzed for B220 and Mac-1 expression. (C) A bar graph shows the percentage of B220+ and Mac-1+ cells for low and high levels of IL-7R expression from five IL-7R-reconstituted animals and five Bex/Vex reconstituted animals from a single experiment at 4 weeks post transplant. Similar observations were made in 15 total mice from three separate reconstitution experiments. Download figure Download PowerPoint Increased Bex/Vex levels correlate with increased surface expression of IL-7R To determine whether increased levels of Bex/Vex expression correlated with increased levels of IL-7Rα on the cell surface, we used an antibody coupled with fluorochrome-filled liposomes as a secondary reagent to detect staining with the IL-7Rα primary antibody. In our hands, the commercially available IL-7R antibody generally gave a weak fluorescence signal that did not allow for clear separations of IL-7R+ and IL-7R− cell populations. The use of the liposome-conjugated reagent resulted in a 10- to 100-fold increase in fluorescence intensity without a significant increase in non-specific background staining (Scheffold et al., 2000). This approach allowed us to detect a significant range of IL-7Rα expression on transduced cells, which was critical for establishing a correlation between IL-7R cell-surface expression as a function of increasing Bex/Vex fluorescence intensity. IL-7Rα liposome staining of Bex/Vex control bone marrow showed a low percentage of IL-7Rα-expressing cells, which was expected since early B cell progenitors express endogenous IL-7Rα (Figure 3A, left panel) (Sudo et al., 1993). The liposome staining of bone marrow from animals reconstituted with IL-7R-expressing cells showed a substantial increase in the number of Mac-1+ cells that expressed IL-7Rα (Figure 3A, middle panel). To assess the specificity of the staining, cells were blocked with unconjugated anti-IL-7Rα antibody prior to addition of the conjugated anti-IL-7Rα liposome secondary antibody (Figure 3A, right panel). This showed that IL-7Rα was specifically detected with the liposome method. Notably, IL-7R expression from the retroviral vectors (Figure 3A, middle panel) was not higher than levels of endogenous IL-7R detected on control bone marrow cells (Figure 3A, compare left and middle panels). Figure 3.Increased levels of Bex/Vex expression correlate in a non-linear manner with increased IL-7R expression on the cell surface. (A) Bone marrow analysis at 9 weeks post-transplant using IL-7R liposome staining. Representative control Bex/Vex (n = 2) and IL-7Rα/IL-2Rγc (n = 2) mice were analyzed by staining with IL-7R liposome (see Materials and methods) and Mac-1 (myeloid) antibodies. Non-specific background staining using the liposome approach was assessed by first blocking with 100–1000-fold excess of unlabeled, primary IL-7R antibody prior to staining with the labeled IL-7Rα antibody (right panel). (B) IL-7Rα/IL-2Rγc-expressing bone marrow cells were gated at varying levels of Bex/Vex expression and then analyzed for IL-7R expression using IL-7R liposome staining. The mean fluorescence intensity of IL-7R expression represented by each histogram is shown on the right. Download figure Download PowerPoint Using the IL-7R liposome stain, we found that IL-7R surface expression increased as levels of Bex/Vex expression increased (Figure 3B), although there was not a direct linear correlation between Bex/Vex fluorescence levels and those observed for IL-7R (as measured by the mean fluorescence intensities of the gated populations and histograms). The highest levels of Bex/Vex expression only represented a 2.5-fold increase in IL-7R surface expression when compared with IL-7R expression in non-transduced cells (Figure 3B). Similar results were obtained when we analyzed expression of cells expressing only the IL-7Rα chain (Bex+ cells; data not shown). In addition, surface IL-7R expression was not detected above background staining at low levels of Bex/Vex expression even using the highly sensitive liposome staining approach. Collectively, these results indicate that increased surface expression of IL-7R correlates with the phenotypic shift from B220+ to Mac-1+ cells in the periphery and that levels of IL-7R expression from the retroviral vectors was similar to endogenous IL-7R levels on the cell surface. Constitutive expression of IL-7R results in an early B cell developmental block The correlation between IL-7R levels and increased myelopoiesis in peripheral blood led us to consider two possible scenarios to explain our results. First, constitutive IL-7R expression in bone marrow could lead to a block in B cell development, which would result in a perceived increase of mature myeloid cells in the periphery. Second, IL-7R signaling in multipotential cells might stimulate commitment to the myeloid versus lymphoid cell pathway or promote myelopoiesis post-commitment. To investigate these possibilities, we analyzed B cell development and early myelopoiesis in bone marrow. The ordered development of B-lineage cells in bone marrow has been defined by the differential expression of cell-surface markers and by the rearrangement status of immunoglobulin genes (Hardy et al., 1991; Li et al., 1996; Hardy and Hayakawa, 2001). The pre-pro-B cell stage, also termed Fraction (Fr.) A, is B220+cd43+cd19−IgM− (Li et al., 1996; Rolink et al., 1996). Fraction B/C cells are B220+cd43+CD19+IgM− and represent committed B-lineage cells that have undergone rearrangement of D–J gene segments at the immunoglobulin heavy chain locus. Bone marrow analysis of IL-7R mice stained with B220 and Mac-1 showed a similar reduction in B-lineage cells with increasing levels of IL-7R expression (Figure 4A). Further analysis of IL-7R mice revealed a near complete block in B lymphopoiesis at the transition between Fr. A and Fr. B cells as indicated by the absence of B220+cd19+ IL-7R-expressing cells (Figure 4B). The mean frequency of B220+cd19+ cells in bone marrow was 0.57% for IL-7R mice (n = 6). These cells were also missing among cells that only expressed high levels of the IL-7Rα chain (single Bex+ cells; data not shown). The frequency of B220+cd19+ cells was not affected in control mice that expressed high levels of Bex/Vex (Figure 4B, mean frequency of 13.6%, n = 6). Analysis of the thymus by CD4 and CD8 staining showed no difference in T cell development between control and IL-7R animals that expressed high levels of Bex/Vex (Figure 4C, n = 4 for each construct). These data show that T cell development, in contrast to B cell development, is compatible with sustained IL-7R expression. Figure 4.Constitutive IL-7R expression results in a B cell developmental block at the Hardy Fr. A to B transition. (A) Bone marrow analysis at four weeks post-transplant of IL-7Rα/IL-2Rγc mice gated for increasing Bex/Vex levels. Data are representative of Bex/Vex and for IL-7Rα/IL-2Rγc mice. (B) Bone marrow analysis of Bex/Vex control mice and IL-7Rα/IL-2Rγc mice to assess early B cell development. Both low and high Bex/Vex levels were gated and then analyzed for B220 and CD19 expression. Fr. A cells are B220+cd19− while B220+CD19+ cells represent bone marrow Fractions B–F (Hardy et al., 1991). (C) Analysis of control and IL-7R thymi by staining with CD4 and CD8. Cells are gated through high Bex/Vex or IL-7Rα/IL-2Rγc levels and then stained with CD4 and CD8 (Bex/Vex, n = 4; IL-7Rα/IL-2Rγc, n = 4). Download figure Download PowerPoint Lymphoid-lineage commitment is not affected by enforced IL-7R expression We originally questioned whether IL-7R expression in stem cells might promote lymphoid lineage commitment over myeloid development. If there were enhanced commitment to the B cell pathway due to IL-7R expression in stem cells, we might expect to see an increase in the frequency and absolute number of IL-7R-expressing Fr. A cells and a decrease in the frequency of myeloid progenitor cells in bone marrow of IL-7R animals. Bone marrow cells from both control and IL-7R mice were gated for both high and low IL-7R levels and 4000 cells from each gate were then sorted and plated in triplicate into methylcellulose M3434 media supplemented with or without IL-7. Control bone marrow was also plated using the same parameters. Colonies of >50 cells were typed and counted 10 days later. The predominant colony types in all the samples, from both the control and IL-7R animals, were granulocyte–macrophage colony-forming units (CFU-GM). We observed no significant difference in colony type or number between cells from Bex/Vex control animals or IL-7R animals at any level of IL-7R expression, whether in the presence or absence of IL-7 (Table I). Based on this observation, it is likely that IL-7R expression does not promote lymphopoiesis at the expense of myelopoiesis. In addition, the reduced number of Fr. A cells observed among cells expressing higher levels of IL-7R (Figure 4B) is not due to IL-7-mediated promotion of early myelopoiesis, otherwise we would have expected higher numbers of CFU-GM progenitors in platings of IL-7R high cells, which was not the case (Table I). Table 1. Myeloid colony-forming cell assay initiated with 4000 Bex/Vex-positive bone marrow cells FACS-purified from IL-7R and control animals Mouse Bex/Vex level CFU-GM (with IL-7) CFU-GM (without IL-7) Bex/Vex Low 26 13a Bex/Vex High 11 17a IL-7R Low 21 21 IL-7R High 9 6 Numbers represent an average of the granulocyte-macrophage colonies from two independent methylcellulose assay experiments. a Data from one experiment plated in triplicate. Constitutive IL-7R expression blocks EBF and Pax5 induction in B220+cd19−NK1.1−Ly-6C− Pre-proB cells The Fr. A subset has been further characterized for cells that have B-lineage potential by staining for NK1.1 and Ly-6C expression (Tudor et. al., 2000). The B220+cd19−NK1.1−Ly-6C− subset of Fr. A contains the only cells that have B-lineage potential. To characterize the developmental block in IL-7R-expressing pre-proB cells, Pax5, EBF and E47 mRNA expression was analyzed by RT–PCR in sorted populations from control or IL-7R mice. Pax5 is required for the maintenance of the B-cell committed state (Nutt et al., 1999; Mikkola et al., 2002) and is sufficient to completely block the earliest stage of T cell development when ectopically expressed in hematopoietic stem cells (Souabni et al., 2002; Cotta et al., 2003). EBF and E47 are also necessary for the earliest differentiation events within the B cell pathway. Targeted disruption of either factor in mice results in a complete block in B cell developmental prior to the onset of immunoglobulin heavy chain D–J rearrangement and CD19 expression (Bain et al., 1994; Zhuang et al., 1994; Lin and Grosschedl, 1995). Five B220+cd19−NK1.1−Ly-6C− cells gated for Bex/Vex expression from control or IL-7R mice (Figure 5A) were directly sorted into wells of a 96-well plate for reverse transcription and subsequent PCR using primers specific for EBF, E47 or Pax5. A β2 microglobulin primer was included in all reactions as an internal control (Figure 5B). All cells were sorted once and then re-sorted before deposition into the plate to ensure >99% purity of the sorted populations. At least two independent sorting experiments showed that 16/18 samples from control Bex/Vex mice were positive for Pax5 transcript while only 2/28 samples were positive for Pax5 among B220+cd19−NK1.1−Ly-6C− cells that expressed IL-7R (Figure 5B). EBF was expressed in 11/22 pre-pro-B cell samples sorted from control Bex/Vex animals and in only 4/27 samples from IL-7R mice. E47 expression was detected at approximately equivalent frequencies among control or IL-7R pre-pro-B cell samples (9/22 and 15/27, respectively). These results suggest that downregulation of IL-7R expression may be a necessary event for EBF and Pax5 induction at the earliest defined stage of B cell development. Figure 5.Pax5 and EBF are not expressed B220+cd19−NK1.1−Ly-6C− cells that constitutively express IL-7R. (A) High IL-7Rα/IL-2Rγc (or control Bex/Vex) cells were gated as B220+cd19−NK1.1−Ly-6C− and then five cells were sorted directly into a 96-well plate (see Materials and methods). Dead cells were excluded by propidium iodide staining. (B) Representative RT–PCR analysis of Pax5, EBF, E47 and β2 microglobulin. Each lane represents RT–PCR of five cells of the gated phenotype sorted into an individual well from control mice (lanes 1–6) or IL-7R mice (8–14). Lane 7 contained a control reaction that received five cells from control mice without the addition of reverse transcriptase. (C) IL-7R liposome stain of B220+cd19−NK1.1−Ly-6C− cells from the bone marrow of wild-type, C57B/6 mice. (D) IL-7R+ or IL-7R− B220+cd19−NK1.1−Ly-6C− cells gated as shown in Figure 5C were sorted once and then resorted before culture on the T220–29 ce" @default.
• W2044004846 created "2016-06-24" @default.
• W2044004846 creator A5014915463 @default.
• W2044004846 creator A5048761937 @default.
• W2044004846 creator A5061407555 @default.
• W2044004846 creator A5071302695 @default.
• W2044004846 creator A5083203391 @default.
• W2044004846 creator A5089561180 @default.
• W2044004846 date "2003-10-15" @default.
• W2044004846 modified "2023-10-18" @default.
• W2044004846 title "Determination of lymphoid cell fate is dependent on the expression status of the IL-7 receptor" @default.
• W2044004846 cites W1484466186 @default.
• W2044004846 cites W1569343698 @default.
• W2044004846 cites W1617267715 @default.
• W2044004846 cites W1649547160 @default.
• W2044004846 cites W1809670150 @default.
• W2044004846 cites W1939229836 @default.
• W2044004846 cites W1944598762 @default.
• W2044004846 cites W1966630605 @default.
• W2044004846 cites W1968561263 @default.
• W2044004846 cites W1975870825 @default.
• W2044004846 cites W1979026253 @default.
• W2044004846 cites W1981444875 @default.
• W2044004846 cites W1983359974 @default.
• W2044004846 cites W1996418577 @default.
• W2044004846 cites W199729741 @default.
• W2044004846 cites W1998913815 @default.
• W2044004846 cites W2002573132 @default.
• W2044004846 cites W2010639752 @default.
• W2044004846 cites W2018338402 @default.
• W2044004846 cites W2023010212 @default.
• W2044004846 cites W2025533436 @default.
• W2044004846 cites W2026708020 @default.
• W2044004846 cites W2036340292 @default.
• W2044004846 cites W2036388406 @default.
• W2044004846 cites W2061307900 @default.
• W2044004846 cites W2065388713 @default.
• W2044004846 cites W2068778527 @default.
• W2044004846 cites W2069501059 @default.
• W2044004846 cites W2074521753 @default.
• W2044004846 cites W2077311610 @default.
• W2044004846 cites W2082746204 @default.
• W2044004846 cites W2082797461 @default.
• W2044004846 cites W2084228983 @default.
• W2044004846 cites W2087832707 @default.
• W2044004846 cites W2090786449 @default.
• W2044004846 cites W2096632716 @default.
• W2044004846 cites W2117455815 @default.
• W2044004846 cites W2125131118 @default.
• W2044004846 cites W2136706405 @default.
• W2044004846 cites W2139410381 @default.
• W2044004846 cites W2146499290 @default.
• W2044004846 cites W2152414109 @default.
• W2044004846 cites W2153011776 @default.
• W2044004846 cites W2153713615 @default.
• W2044004846 cites W2163584800 @default.
• W2044004846 cites W2271511280 @default.
• W2044004846 cites W2399252479 @default.
• W2044004846 cites W2406980108 @default.
• W2044004846 cites W26593026 @default.
• W2044004846 doi "https://doi.org/10.1093/emboj/cdg522" @default.
• W2044004846 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/213776" @default.
• W2044004846 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14532123" @default.
• W2044004846 hasPublicationYear "2003" @default.
• W2044004846 type Work @default.
• W2044004846 sameAs 2044004846 @default.
• W2044004846 citedByCount "39" @default.
• W2044004846 countsByYear W20440048462012 @default.
• W2044004846 countsByYear W20440048462013 @default.
• W2044004846 countsByYear W20440048462015 @default.
• W2044004846 countsByYear W20440048462019 @default.
• W2044004846 countsByYear W20440048462020 @default.
• W2044004846 countsByYear W20440048462021 @default.
• W2044004846 countsByYear W20440048462022 @default.
• W2044004846 countsByYear W20440048462023 @default.
• W2044004846 crossrefType "journal-article" @default.
• W2044004846 hasAuthorship W2044004846A5014915463 @default.
• W2044004846 hasAuthorship W2044004846A5048761937 @default.
• W2044004846 hasAuthorship W2044004846A5061407555 @default.
• W2044004846 hasAuthorship W2044004846A5071302695 @default.
• W2044004846 hasAuthorship W2044004846A5083203391 @default.
• W2044004846 hasAuthorship W2044004846A5089561180 @default.
• W2044004846 hasBestOaLocation W20440048461 @default.
• W2044004846 hasConcept C153911025 @default.
• W2044004846 hasConcept C170493617 @default.
• W2044004846 hasConcept C199360897 @default.
• W2044004846 hasConcept C41008148 @default.
• W2044004846 hasConcept C54355233 @default.
• W2044004846 hasConcept C86803240 @default.
• W2044004846 hasConcept C90559484 @default.
• W2044004846 hasConcept C95444343 @default.
• W2044004846 hasConceptScore W2044004846C153911025 @default.
• W2044004846 hasConceptScore W2044004846C170493617 @default.
• W2044004846 hasConceptScore W2044004846C199360897 @default.
• W2044004846 hasConceptScore W2044004846C41008148 @default.
• W2044004846 hasConceptScore W2044004846C54355233 @default.
• W2044004846 hasConceptScore W2044004846C86803240 @default.
• W2044004846 hasConceptScore W2044004846C90559484 @default.

• next