The major goal of this project is to perform pilot studies for establishing the relationship between serum TSLP and IL-7 levels, and CRLF2 expression, ethnicity, and prognostic indicators in pediatric B-ALpatients and to use a TSLP and IL-7 cytokine-specific human B-ALL xenograft model to determine the interplay between TSLP and IL-7 in the progression of CRLF2d B-ALL and the survival of subclones of minimal residual disease.

Goal: Using a novel xenograft model transplanted with primary pediatric CRLF2 B-ALL cells we will study   the role of TSLP-CRLF2 interactions in leukemia cell survival/proliferation and gene expression in vivo.

IL-7 plays a central role in murine B cell production, however its role in human B lymphopoiesis is controversial. Early studies of mouse B cell development showed that IL-7 increases in vitro B cell production by ~50–fold while the loss of IL-7R signaling in knockout mice results in a block in B cell development.  In contrast, the addition of IL-7 to human cultures only increased B cell production by ~2-5 fold while patients with defects in IL-7 had normal numbers of peripheral blood B cells. This led to the conclusion that human B lymphopoiesis differs from that in mouse with respect to the requirement for IL-7. A more comprehensive analysis of IL-7 knockout mice showed that the production of B cells during the fetal/neonatal period was relatively normal, although these mice did indeed have a block in adult B cell production. More recent studies describe a “developmental switch” in murine B lymphopoiesis. In adult mice, signaling through the IL-7R is required for expression of the EBF transcription factor. EBF in turn upregulates a cascade of B lineage specific genes that are required for B cell development. During murine fetal and neonatal life, IL-7-independent expression of EBF allows for B lineage differentiation resulting in a developmental switch when this capacity is lost at ~two weeks of age.  The above findings are important because most of the data concerning the role of IL-7 in human B cell development was obtained from fetal or neonatal hematopoietic sources. In addition, human studies that examine adult B lymphopoiesis have relied on co-cultures that include murine stromal cell lines. We and others have shown that these stromal cell lines produce high levels of murine IL-7 that induces human IL-7R signaling, thus making it difficult to interpret the effects of exogenous human IL-7. Here we describe a novel, human-only model of in vitro B cell development based on co-culturing hematopoietic stem cells (HSCs) from umbilical cord blood (CB) or adult human bone marrow (BM) on primary human BM stroma. This culture model produces a substantial population of CD19+cμ– B cell precursors at 3 weeks with 11-16% of B lineage progeny maturing to the IgM+ stage by 5 weeks of culture. The percentage of in vitro-generated B cell precursors that express IL-7Rα (~35%) mirrors that observed for in vivo-generated B cell precursors in adult BM, thus providing evidence of the physiological relevance of our human-only culture model. Using the human-only culture model we examined the role of IL-7 in human B lymphopoiesis from lineage marker negative (Lin–) CD34+ HSCs in adult BM and from their CB counterparts that give rise to B cells during the neonatal period. IL-7 increased human B cell production by >60-fold from both CB and adult BM HSCs. IL-7-induced increases were dose-dependent and specific to CD19+ cells. STAT5 phosphorylation and expression of the Ki-67 proliferation antigen indicate that IL-7 acts directly on CD19+ cells to increase proliferation at the CD34+ and CD34– pro-B cell stages. These data provide evidence that IL-7 plays a critical role in human B lymphopoiesis as it does in the mouse. While IL-7-independent B cell production is essentially absent in human-only cultures with adult BM, HSCs in CB give rise to a small but consistent population of CD19LO B lineage cells that express EBF and PAX-5 and respond to subsequent IL-7 stimulation. Flt3 ligand, but not thymic stromal-derived lymphopoietin (TSLP), was required for the IL-7-independent B cell production observed in human-only cultures with CB HSCs. These data provide the first evidence of a developmental switch in human B lymphopoiesis. As compared to CB, adult BM shows a B lymphoid-specific reduction of in vitro generative capacity that is progressively more profound in developmentally sequential precursor populations, resulting in a ~50-fold reduction in IL-7-dependent B lineage generative capacity. Indeed, the IL-7-dependent B cell generative capacity of adult BM is similar to the IL-7-independent B cell generative capacity of CB. These data suggest that a mechanism in addition to the developmental switch observed in the mouse model may be responsible for the IL-7 dependency that we observe in adult human B lymphopoiesis.

IL-7 is critical for B cell production in adult mice, however its role in human B lymphopoiesis is controversial. Using a novel, human-only culture model we show that IL-7 increases human B cell production by >60-fold from hematopoietic stem cells (HSCs) in both cord blood (CB) and adult bone marrow (BM). IL-7-induced increases are dose-dependent and specific to CD19+ cells. STAT5 phosphorylation and expression of the Ki-67 proliferation antigen indicate that IL-7 acts directly on CD19+ cells to increase proliferation at the CD34+ and CD34– pro-B cell stages, but not among CD19– B lineage precursors. As compared to CB, adult BM shows a reduction of in vitro generative capacity that is progressively more profound in developmentally sequential B cell precursor populations, resulting in a ~50-fold reduction in IL-7-dependent B lineage generative capacity. Without IL-7, HSCs in CB, but not BM, give rise to a small but consistent population of CD19LO B lineage cells that express EBF and PAX-5 and respond to subsequent IL-7 stimulation. Flt3 ligand, but not TSLP, was required for the IL-7-independent production of human B lineage cells. These data provide evidence that human B cell production from adult BM is dependent on IL-7 and suggest that a mechanism in addition to the “developmental switch” observed in the mouse model may be responsible for IL-7 dependency in adult human B lymphopoiesis. Supported by NIH K01 DK066163 (KJP).

Ikaros encodes a zinc finger protein essential for lymphoid development. Ikaros localizes to pericentromeric heterochromatin (PC-HC) where it recruits target genes, resulting in their activation or repression via chromatin remodeling. Ikaros'''''''' function is controlled by post-translational modification. Our previous studies show that phosphorylation of Ikaros by CK2 kinase controls its PC-HC localization, and its ability to bind the upstream regulatory region (URE) and regulate expression of TdT during thymocyte differentiation. Here, we demonstrate, by co-immunoprecipitation, that Ikaros interacts in vivo with a specific phosphatase for which it is a substrate. Mutational analysis identified specific residues essential for Ikaros-phosphatase interaction. Mutant Ikaros protein with disrupted phosphatase interaction undergoes CK-2-mediated hyperphosphorylation that inhibits its ability to bind the TdT URE, and its PC-HC localization. Alanine mutations at the CK2-phosphorylated residues restore both Ikaros'''''''' DNA-binding ability and PC-HC localization, regardless of its ability to interact with the phosphatase. We propose a model whereby the regulation of TdT expression by Ikaros is controlled by CK2 kinase and interaction with a specific phosphatase and that the balance of these two signal transduction pathways is essential for normal T cell differentiation. Supported by NIH K22CA111392 (SD)

Murine studies show that immature transitional B cells travel from the bone marrow to the spleen where they become mature follicular and marginal zone (MZ) B cells. Based on phenotypic and functional studies performed in the mouse, multiple models have been developed to define developmentally sequential subsets of transitional B cells in the spleen. However, studies of transitional B cells in human spleen have not been reported, although data from peripheral blood (PB) suggests that surface markers used to define transitional B cell subsets in the mouse can be used to identify their human counterparts in the circulation. Thus, studies of murine splenic B cell development are controversial and it is not known whether the developmental pathway(s) present in the mouse spleen exist in humans. Here we utilized transitional B cell markers identified in murine spleen and human peripheral blood to examine B cell development in human spleen. Human fetal and adult spleens were stained for four-color flow cytometry to assess co-expression of IgD, IgM, CD21, CD23, CD24, and CD38. Surface phenotypes observed in human spleen are consistent with those reported for mature and transitional 1 (T1) and transitional 2 (T2) B cells in mouse spleen and in human PB.  CD38, CD21 and CD24 appear to be the most powerful markers for discriminating phenotypically distinct populations. Almost all B cells present in human fetal spleen showed a phenotype characteristic of T1 and T2 B cells, including high levels of CD38 expression. Phenotypic analysis of B cell subsets in the adult spleen showed the presence of T2 and mature follicular and MZ B cells, but few T1 cells. The emergence of B cells that exhibit a transitional phenotype early in the fetal period suggests that these cells are likely to be the developmental counterparts of transitional B cells identified in mice.  Current functional studies are aimed at establishing a developmental sequence of human transitional B cell subsets. During the transitional stages of B cell development selection events prevent the production of autuoreactive B cells. These studies will provide a foundation for understanding and treating B cell-mediated autoimmunities. This work was supported by NIH K01-DK066163 (to KJP)

Ikaros encodes a zinc finger protein that is essential for lymphoid development. The absence of Ikaros results in a loss of B cells and a severe arrest in T cell differentiation. In hematopoietic cells, Ikaros localizes to pericentromeric heterochromatin (PC-HC) where it recruits its target genes, resulting in their activation or repression via chromatin remodeling. The function of Ikaros is controlled by post-translational modifications. Using in vivo labeling of murine thymocytes combined with phosphopeptide mapping, we identified four novel Ikaros phosphorylation sites. In vivo inhibition of CK2 kinase activity with a CK2-specific inhibitor abolished Ikaros’ phosphorylation at these residues, while in vitro kinase assays showed that CK2 kinase directly phosphorylates these sites. In vivo experiments that examined the function of phosphomimetic Ikaros mutants demonstrated that CK2-mediated phosphorylation regulates Ikaros’ localization to PC-HC. Additionally, the ability of Ikaros to bind the upstream regulatory elements of its known target gene–terminal deoxynucleotidetransferase (TdT)–was decreased by phosphorylation of two amino acids. In thymocytes, Ikaros acts as a repressor of the TdT gene.  Induction of differentiation of thymocytes with PMA plus ionomycin results in transcriptional repression of TdT expression. This process has been associated with increased binding of Ikaros to the upstream regulatory element of TdT. Phosphopeptide analysis of in vivo-labeled thymocytes revealed that Ikaros undergoes dephosphorylation during induction of thymocyte differentiation and that dephosphorylation is responsible for the increased DNA-binding affinity of Ikaros toward the TdT promoter. We propose a model whereby reversible phosphorylation of Ikaros at specific amino acids by CK2 kinase, controls its subcellular localization and its ability to regulate TdT expression during thymocyte differentiation.

Human B lymphopoiesis is thought to differ from that in mouse with respect to the requirement for IL-7. Here we investigated the role of IL-7 in human B cell development using an in vitro model based on co-culturing human hematopoietic stem cells (HSCs) on primary stroma from adult human bone marrow (BM). Addition of IL-7 to this co-culture model increased B cell production by ~40-fold. IL-7-induced increases were dose-dependent and specific to CD19+ cells. Flow cytometry analysis of STAT5 phosphorylation, IL-7Rα, and the proliferation antigen, ki-67, indicate that IL-7 acts directly on B cell precursors where it increases proliferation, but not cell survival. The effects of IL-7 were most profound in cultures with adult BM HSCs where few, if any, human B lineage cells are generated in the absence of IL-7 activity. When co-cultures initiated with cord blood (CB) and BM HSCs were compared, IL-7-induced increases were similar in magnitude, and B cell precursors responded similarly to IL-7. However, a comparison of the generative capacity of CB and BM HSCs showed that the ability of BM HSCs to give rise to CD19+, but not CD19– cells, was 40 times less than that of CB HSCs. Our results provide evidence that IL-7 is critical for B cell production from HSCs in adult BM due to their low B lymphoid generative capacity as compared to HSCs in CB. Supported by NIH K01 DK066163 and the Dept. of Pathology and Human Anatomy and the Center for Health Disparities and Molecular Medicine, Loma Linda University School of Medicine

Ikaros encodes a zinc finger protein that is involved in heritable gene silencing. Biological experiments showed that Ikaros is essential for normal hematopoiesis and that it acts as a tumor suppressor. In hematopoietic cells, Ikaros localizes to pericentromeric heterochromatin (PC-HC) where it recruits its target genes, resulting in their activation or repression via chromatin remodeling. The function of Ikaros is controlled by post-translational modifications. CK2 kinase has been shown previously to phosphorylate Ikaros at its C-terminus, affecting cell cycle progression. We used in vivo labeling of murine thymocytes and the VL3-3M2 leukemia cell line, followed by phosphopeptide mapping, to identify four novel Ikaros phosphorylation sites. Functional analysis of phosphomimetic mutants showed that the phosphorylation of two individual amino acids determines the affinity of Ikaros toward probes derived from PC-HC. In vivo experiments demonstrated that the targeting of Ikaros to PC-HC is regulated by phosphorylation. The ability of Ikaros to bind the upstream regulatory elements of its known target gene–terminal deoxynucleotidetransferase (TdT) – was decreased by the phosphomimetic mutation of two amino acids. In vivo treatment of VL3-3M2 cells with a specific inhibitor of CK2 kinase - 5,6-Dichloro-1-b-D-ribofuranosyl benzimidazole (DRB) abolished phosphorylation of all four phosphorylation sites. This suggests that the phosphorylation of these amino acids is dependent on the CK2 kinase signal transduction pathway.  CK2 kinase has pro-oncogenic activity and its overexpression contributes to development of T cell leukemia in mice. We propose a model whereby signaling through the CK2 kinase transduction pathway induces reversible phosphorylation of Ikaros at specific amino acids. This mechanism regulates the subcellular localization of Ikaros, as well as its ability to control TdT expression during thymocyte differentiation.

Purpose of Study: Extensive studies of mouse B cell development have used expression of surface markers to define transitional stages of immature B cell development in the mouse spleen. Studies of human B cell development are much more limited and the parallels between B cell production in mouse and human are unclear. To our knowledge there are no reports that examine transitional stages of immature B cell development in human spleen. The aim of this study is to identify human transitional B cell populations in the fetal spleen based on patterns of surface marker expression that have been used to characterize transitional stages of immature B cell development in mouse spleen and in human peripheral blood (PB). Methods Used: Human fetal spleen cells were stained for 4-color flow cytometry to detect expression of surface markers that have previously been used to identify transitional B cell populations.  Co-expression of CD24, CD38, IgD, CD21, and CD23 was examined in gated CD19+ cells. Staining patterns in human fetal spleen were compared to those reported for transitional B cell subsets in mouse spleen and/or human PB. Summary of Results: Based on patterns of surface marker expression reported for transitional 1 (T1) and transitional 2 (T2) B cell populations in mouse spleen and human PB, we identified phenotypic T1 and T2 B cell populations in  human fetal spleen. Size differences in human T1 and T2 cells in fetal spleen are consistent with differences in frequency of cell division that have been reported for T1 and T2 cells in human PB. Conclusions: Populations of transitional B cells, identified by surface immunophenotypes in the mouse model and in studies of human PB, are present in human spleen at a very early point in human development. Future studies are aimed at comparing and characterizing human B cell development and function in fetal and adult spleen. Further knowledge of B cell development in the spleen is important, given the potential of emerging therapies for B cell driven autoimmune diseases. These include B cell directed therapies such as humanized anti-CD20 and antibodies against BLyS and TACI.

The importance in understanding differences between B1 and B2 cells is critical for understanding immune responses against autoreactive antibodies or foreign antigens.  Therefore, demarcating differences in differentiation and development between these subsets is crucial to understand potential clinical effects following the loss of a subset or aberrant function by a subset.  Current understanding of these differences indicate that B1 cells are fetally derived and their renewal capacity is limited while B2 cells derive from the bone marrow and have a high renewal capacity.  These subset characteristics gain importance in the clinical setting particularly in irradiation treatment and subsequent bone marrow transplantation.  However, the majority of B cell differentiation and development data comes from mouse models.  Utilizing this data to understand human B cell development is helpful but it should not be taken as generic for all species including humans.  To date, no single model for human development has been characterized as well as the mouse model.  Therefore, understanding if B1 and B2 cells correlate with functional subsets in humans is a step toward characterizing a human model.  For mice, B1 and B2 cells are isolated based on several markers but particularly CD5.  B1 cells are CD5+ while B2 cells are CD5 negative.  B1 cells display T-independent immune responses and B2 cells have T-dependent responses.  Also, B1 cells tend to display selective incorporation of immunoglobulin (Ig) heavy chain segments with limited somatic mutation due to absence of TdT expression.  On the other hand, B2 cells exhibit random incorporation of heavy chain segments and high levels of nucleotides that are randomly  inserted by TdT at the junctions of V, D, and J segments during Ig heavy chain gene rearrangement.  Therefore, do these characteristics hold true for humans?  To answer this question we looked at immunoglobulin heavy chain rearrangement of human cord blood CD5+ B cells using single-cell RT-PCR.  Our preliminary data indicates a preferential usage of V gene family 3, but not of V gene segments proximal to the D segment as observed in B1 cells from mice.  Further studies, will examine the role of IL-7, Ikaros, and TdT in human B1 and B2 cell differentiation and development focusing on analysis of V gene usage.

Human B lymphopoiesis is thought to differ from that in mouse with respect to the requirement for IL-7. Here we investigated the role of IL-7 in human B cell development using an in vitro model based on co-culturing human hematopoietic stem cells (HSCs) on primary stroma from adult human bone marrow (BM). Addition of IL-7 to this co-culture model increased B cell production by ~40-fold. IL-7-induced increases were dose-dependent and specific to CD19+ cells. Flow cytometry analysis of STAT5 phosphorylation, IL-7Rα, and the proliferation antigen, ki-67, indicate that IL-7 acts directly on B cell precursors where it increases proliferation, but not cell survival. The effects of IL-7 were most profound in cultures with adult BM HSCs where few, if any, human B lineage cells are generated in the absence of IL-7 activity. When co-cultures initiated with cord blood (CB) and BM HSCs were compared, IL-7-induced increases were similar in magnitude, and B cell precursors responded similarly to IL-7. However, a comparison of the generative capacity of CB and BM HSCs showed that the ability of BM HSCs to give rise to CD19+, but not CD19– cells, was 40 times less than that of CB HSCs. Our results provide evidence that IL-7 is critical for B cell production from HSCs in adult BM due to their low B lymphoid generative capacity as compared to HSCs in CB.

The importance in understanding differences between B1 and B2 cells is critical for understanding immune responses against autoreactive antibodies or foreign antigens.  Therefore, demarcating differences in differentiation and development between these subsets is crucial to understand potential clinical effects following the loss of a subset or aberrant function by a subset.  Current understanding of these differences indicate that B1 cells are fetally derived and their renewal capacity is limited while B2 cells derive from the bone marrow and have a high renewal capacity.  These subset characteristics gain importance in the clinical setting particularly in irradiation treatment and subsequent bone marrow transplantation.  However, the majority of B cell differentiation and development data comes from mouse models.  Utilizing this data to understand human B cell development is helpful but it should not be taken as generic for all species including humans.  To date, no single model for human development has been characterized as well as the mouse model.  Therefore, understanding if B1 and B2 cells correlate with functional subsets in humans is a step toward characterizing a human model.  For mice, B1 and B2 cells are isolated based on several markers but particularly CD5.  B1 cells are CD5+ while B2 cells are CD5 negative.  B1 cells display T-independent immune responses and B2 cells have T-dependent responses.  Also, B1 cells tend to display selective incorporation of immunoglobulin (Ig) heavy chain segments with limited somatic mutation due to absence of TdT expression.  On the other hand, B2 cells exhibit random incorporation of heavy chain segments and high levels of nucleotides that are randomly  inserted by TdT at the junctions of V, D, and J segments during Ig heavy chain gene rearrangement.  Therefore, do these characteristics hold true for humans?  To answer this question we looked at immunoglobulin heavy chain rearrangement of human cord blood CD5+ B cells using single-cell RT-PCR.  Our preliminary data indicates a preferential usage of V gene family 3, but not of V gene segments proximal to the D segment as observed in B1 cells from mice.  Further studies, will examine the role of IL-7, Ikaros, and TdT in human B1 and B2 cell differentiation and development focusing on analysis of V gene usage.

B cells are lymphocytes that produce antibodies. B cells develop from hematopoietic stem cells (HSC’s) in the bone marrow (BM) with the aid of stromal cells in the BM, that provide cell-to-cell interactions and the cytokine, IL-7, to support the developing B cell precursors. At sites of early B cell development in human BM, the stromal cells express CD10. Our lab has developed an in vitro model of B cell development based on co-culturing human HSCs and primary BM stromal cells. The stromal cells in our culture model express IL-7 and CD10. This culture model can also be used to support the survival of human pre-B Acute Lymphocytic leukemia (pre-B-ALL) cells. However, there are several disadvantages of using primary human BM stroma: 1) human BM from which we derive stroma is very expensive, 2) donor to donor variation in stroma may affect experiments, and 3) growth of stroma from BM is slow.  Here we are studying two human BM stromal cell lines, HS-27 and HS-5, to see if they can replace primary human BM stroma in supporting the growth of human B cell precursors.  RT-PCR indicate that the HS-27, but not HS-5, stromal cell line expressed IL-7.  Flow cytometry showed that HS-27 and HS-5 stromal cells both expressed CD10, but a higher percentage of HS-27 cells were CD10+. When human pre-B-All cells were placed in co-culture with HS-27 and HS-5 stromal cell lines, HS-27 supported better long-term survival of pre-B ALL cells. In addition, HS27, but not HS5 stromal cells were able to support the differentiation of CD19+ B lineage cells from human hematopoietic stem cells. Our data suggest that the HS-27 cell line is better able to support human B cell precursors and may be able to replace primary human BM stroma in our in vitro model of human B cell development.