Ineavely Baez

IL-7 Dependence in human B lymphopoiesis increases during progression of ontogeny from cord blood to bone marrow.

IL-7 is critical for B cell production in adult mice; however, its role in human B lymphopoiesis is controversial. One challenge was the inability to differentiate human cord blood (CB) or adult bone marrow (BM) hematopoietic stem cells (HSCs) without murine stroma. Here, we examine the role of IL-7 in human B cell development using a novel, human-only model based on coculturing human HSCs on primary human BM stroma. In this model, IL-7 increases human B cell production by >60-fold from both CB and adult BM HSCs. IL-7-induced increases are dose-dependent and specific to CD19+ cells. STAT5 phosphorylation and expression of the Ki-67 proliferation Ag indicate that IL-7 acts directly on CD19+ cells to increase proliferation at the CD34+ and CD34− pro-B cell stages. 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 (early B cell factor) and PAX-5 and respond to subsequent IL-7 stimulation. Flt3 ligand, but not thymic stromal-derived lymhopoietin (TSLP), was required for the IL-7-independent production of human B lineage cells. As compared with CB, adult BM shows a reduction of in vitro generative capacity that is progressively more profound in developmentally sequential populations, resulting in an ∼50-fold reduction in IL-7-dependent B lineage generative capacity. These data provide evidence that IL-7 is essential for human B cell production from adult BM and that IL-7-induced expansion of the pro-B compartment is increasingly critical for human B cell production during the progression of ontogeny.

Suppression of dendritic cell activation by diabetes autoantigens linked to the cholera toxin B subunit

Antigen presenting cells, specifically dendritic cells (DCs) are a focal point in the delicate balance between T cell tolerance and immune responses contributing to the onset of type I diabetes (T1D). Weak adjuvant proteins like the cholera toxin B subunit when linked to autoantigens may sufficiently alter the balance of this initial immune response to suppress the development of autoimmunity. To assess adjuvant enhancement of autoantigen mediated immune suppression of Type 1 diabetes, we examined the cholera toxin B subunit (CTB)-proinsulin fusion protein (CTB-INS) activation of immature dendritic cells (iDC) at the earliest detectable stage of the human immune response. In this study, Incubation of human umbilical cord blood monocyte-derived immature DCs with CTB-INS autoantigen fusion protein increased the surface membrane expression of DC Toll-like receptor (TLR-2) while no significant upregulation in TLR-4 expression was detected. Inoculation of iDCs with CTB stimulated the biosynthesis of both CD86 and CD83 co-stimulatory factors demonstrating an immunostimulatory role for CTB in both DC activation and maturation. In contrast, incubation of iDCs with proinsulin partially suppressed CD86 co-stimulatory factor mediated DC activation, while incubation of iDCs with CTB-INS fusion protein completely suppressed iDC biosynthesis of both CD86 and CD83 costimulatory factors. The incubation of iDCs with increasing amounts of insulin did not increase the level of immune suppression but rather activated DC maturation by stimulating increased biosynthesis of both CD86 and CD83 costimulatory factors. Inoculation of iDCs with CTB-INS fusion protein dramatically increased secretion of the immunosuppressive cytokine IL-10 and suppressed synthesis of the pro-inflammatory cytokine IL12/23 p40 subunit protein suggesting that linkage of CTB to insulin (INS) may play an important role in mediating DC guidance of cognate naïve Th0 cell development into immunosuppressive T lymphocytes. Taken together, the experimental data suggests Toll like receptor 2 (TLR-2) plays a dominant role in CTB mediated INS inhibition of DC induced type 1 diabetes onset in human Type 1 diabetes autoimmunity. Further, fusion of CTB to the autoantigen was found to be essential for enhancement of immune suppression as co-delivery of CTB and insulin did not significantly inhibit DC costimulatory factor biosynthesis. The experimental data presented supports the hypotheses that adjuvant enhancement of autoantigen mediated suppression of islet beta cell inflammation is dependent on CTB stimulation of dendritic cell TLR2 receptor activation and co-processing of both CTB and the autoantigen in the same dendritic cell.

Cutting Edge: Hematopoietic Stem Cell Expansion and Common Lymphoid Progenitor Depletion Require Hematopoietic-Derived, Cell-Autonomous TLR4 in a Model of Chronic Endotoxin

Hematopoietic stem and progenitors cells (HSPCs) are activated through TLR4 in vitro. However, it remains unclear whether in vivo TLR4 sensing by HSPCs occurs directly or via other cell intermediates. In this study, we examined the cellular mechanisms underlying murine hematopoietic stem cell (HSC) expansion and common lymphoid progenitor (CLP) depletion in a model of chronic low-dose LPS. Using adoptive-transfer approaches, we show that HSC and CLP sensitivity to chronic LPS depends on hematopoietic-derived, cell subset–autonomous TLR4. Like murine progenitors, human HSPCs are activated by TLR4 in vitro. Using humanized mice, a preclinical model relevant to human physiology, we show that persistent endotoxin increases the frequency of Ki-67+ HSCs and severely depletes CLPs and B precursors. Together, our findings show that murine HSPCs directly respond to endotoxin in vivo and that persistent LPS, a feature of several diseases of global health significance, impairs human lymphopoiesis.

Differences in Mouse and Human Nonmemory B Cell Pools

Identifying cross-species similarities and differences in immune development and function is critical for maximizing the translational potential of animal models. Coexpression of CD21 and CD24 distinguishes transitional and mature B cell subsets in mice. In this study, we validate these markers for identifying analogous subsets in humans and use them to compare the nonmemory B cell pools in mice and humans, across tissues, and during fetal/neonatal and adult life. Among human CD19+IgM+ B cells, the CD21/CD24 schema identifies distinct populations that correspond to transitional 1 (T1), transitional 2 (T2), follicular mature, and marginal zone subsets identified in mice. Markers specific to human B cell development validate the identity of marginal zone cells and the maturation status of human CD21/CD24 nonmemory B cell subsets. A comparison of the nonmemory B cell pools in bone marrow, blood, and spleen in mice and humans shows that transitional B cells comprise a much smaller fraction in adult humans than mice. T1 cells are a major contributor to the nonmemory B cell pool in mouse bone marrow, in which their frequency is more than twice that in humans. Conversely, in spleen, the T1:T2 ratio shows that T2 cells are proportionally ∼8-fold higher in humans than in mice. Despite the relatively small contribution of transitional B cells to the human nonmemory pool, the number of naive follicular mature cells produced per transitional B cell is 3- to 6-fold higher across tissues than in mice. These data suggest differing dynamics or mechanisms produce the nonmemory B cell compartments in mice and humans.

A novel xenograft model to study the role of TSLP-induced CRLF2 signals in normal and malignant human B lymphopoiesis

Thymic stromal lymphopoietin (TSLP) stimulates in vitro proliferation of human fetal B-cell precursors. However, its in vivo role during normal human B lymphopoiesis is unknown. Genetic alterations that cause overexpression of its receptor component, cytokine receptor-like factor 2 (CRLF2), lead to high-risk B-cell acute lymphoblastic leukemia implicating this signaling pathway in leukemogenesis. We show that mouse thymic stromal lymphopoietin does not stimulate the downstream pathways (JAK/STAT5 and PI3K/AKT/mTOR) activated by the human cytokine in primary high-risk leukemia with overexpression of the receptor component. Thus, the utility of classic patient-derived xenografts for in vivo studies of this pathway is limited. We engineered xenograft mice to produce human thymic stromal lymphopoietin (+T mice) by injection with stromal cells transduced to express the cytokine. Control (−T) mice were produced using stroma transduced with control vector. Normal levels of human thymic stromal lymphopoietin were achieved in sera of +T mice, but were undetectable in −T mice. Patient-derived xenografts generated from +T as compared to −T mice showed a 3–6-fold increase in normal human B-cell precursors that was maintained through later stages of B-cell development. Gene expression profiles in high-risk B-cell acute lymphoblastic leukemia expanded in +T mice indicate increased mTOR pathway activation and are more similar to the original patient sample than those from −T mice. +T/−T xenografts provide a novel pre-clinical model for understanding this pathway in B lymphopoiesis and identifying treatments for high-risk B-cell acute lymphoblastic leukemia with overexpression of cytokine-like factor receptor 2.

TSLP or IL‐7 provide an IL‐7Rα signal that is critical for human B lymphopoiesis

Thymic stromal lymphopoietin (TSLP) and IL‐7 are cytokines that signal via the IL‐7 receptor alpha (IL‐7Rα) to exert both overlapping and unique functions during early stages of mouse B‐cell development. In human B lymphopoiesis, the requirement for IL‐7Rα signaling is controversial and the roles of IL‐7 and TSLP are less clear. Here, we evaluated human B‐cell production using novel in vitro and xenograft models of human B‐cell development that provide selective IL‐7 and human TSLP (hTSLP) stimulation. We show that in vitro human B‐cell production is almost completely blocked in the absence of IL‐7Rα stimulation, and that either TSLP or IL‐7 can provide a signal critical for the production and proliferation of human CD19+ PAX5+ pro‐B cells. Analysis of primary human bone marrow stromal cells shows that they express both IL‐7 and TSLP, providing an in vivo source of these cytokines. We further show that the in vivo production of human pro‐B cells under the influence of mouse IL‐7 in a xenograft scenario is reduced by anti‐IL‐7 neutralizing antibodies, and that this loss can be restored by hTSLP at physiological levels. These data establish the importance of IL‐7Rα mediated signals for normal human B‐cell production.

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying the mechanisms of normal and malignant hematopoiesis and are the gold standard for identifying effective chemotherapies for many malignancies. PDX models are possible because many of the mouse cytokines also act on human cells. However, this is not the case for all cytokines, including many that are critical for studying normal and malignant hematopoiesis in human cells. Techniques that engineer mice to produce human cytokines (transgenic and knock-in models) require significant expense before the usefulness of the model has been demonstrated. Other techniques are labor intensive (injection of recombinant cytokine or lentivirus) and in some cases require high levels of technical expertise (hydrodynamic injection of DNA). This report describes a simple method for generating PDX mice that have exogenous human cytokine (TSLP, thymic stromal lymphopoietin) via weekly intraperitoneal injection of stroma that have been transduced to overexpress this cytokine. Use of this method provides an in vivo source of continuous cytokine production that achieves physiological levels of circulating human cytokine in the mouse. Plasma levels of human cytokine can be varied based on the number of stromal cells injected, and cytokine production can be initiated at any point in the experiment. This method also includes cytokine-negative control mice that are similarly produced, but through intraperitoneal injection of stroma transduced with a control vector. We have previously demonstrated that leukemia cells harvested from TSLP-expressing PDX, as compared to control PDX, exhibit a gene expression pattern more like the original patient sample. Together the cytokine-producing and cytokine-negative PDX mice produced by this method provide a model system that we have used successfully to study the role of TSLP in normal and malignant hematopoiesis.

Abstract B46: A novel patient-derived xenograft model for evaluating therapies that target the CRLF2 signaling pathway to reduce health disparities for Hispanic children with leukemia

The purpose of the studies described here was to identify drug targets and develop a preclinical model for testing therapies that can reduce health disparities for Hispanic children with high-risk acute lymphoblastic leukemia (ALL). Hispanic children are 1.24 times more likely to develop ALL than non-Hispanic whites and that number rises to 2.09 by adolescence and early adulthood. A major contributor to this health disparity is a type high-risk B-cell ALL called CRLF2 B-ALL. CRLF2 B-ALL occurs 5 times more often in Hispanic children than others, is prevalent in adolescents and young adults, and is associated with a high relapse rate and poor prognosis. CRLF2 B-ALL is caused by genetic alterations that result in over expression of the cytokine receptor, CRLF2. The CRLF2 receptor is activated by the cytokine, TSLP, causing downstream activation of the JAK/STAT5 and PI3/AKT/MTOR pathways. A gene target of activated STAT5 in B cell precursors is Mcl-1, a Bcl2 family pro-survival molecule. In addition, Mcl-1 protein levels are known to be increased through post-transcriptional mechanisms by activation of the mTOR pathway. We hypothesized that the normal level of circulating TSLP cytokine could induce CRLF2 activation leading to increased Mcl-1 expression in CRLF2 B-ALL cells. Our data show that TSLP increases phosphorylation of STAT5, as well as AKT and S6 (downstream of mTOR) in primary CRLF2 B-ALL cells from Hispanic pediatric patients, even when activating JAK mutations are present. When CRLF2 B-ALL cells from Hispanic pediatric patients were cultured for 3 days with and without physiological levels of TSLP, flow cytometry showed that expression of the Mcl-1 protein was significantly increased in cultures with TSLP as compared to cultures without TSLP. CRLF2 B-ALL cells treated in vitro with Mcl-1 inhibitor showed dose-dependent increases in caspase 3 activation and apoptosis as indicated by flow cytometry. These data provide evidence that TSLP can contribute to leukemia cell survival and identify Mcl-1 inhibitor as a candidate therapy for CRLF2 B-ALL. Our next step was to develop a preclinical model for testing therapies that target genes, such as Mcl-1, that are regulated by TSLP-induced CRLF2 signals in this disease. Patient-derived xenograft (PDX) models produced by transplanting leukemia cells from patients into immune deficient mice provide an in vivo model of disease that includes contributions of the background genetic landscape that can influence disease progression or treatment outcome in health disparities diseases. PDX models are possible because most cytokines produced in the mouse are active on human cells, however mouse TSLP is species-specific. Thus classic PDX models do not provide TSLP that can activate the CRLF2 receptor that is overexpressed in CRLF2 B-ALL. To address this issue we engineered PDX mice to express physiological levels of human TSLP (+T PDX mice) and control -T mice that lacked human TSLP. In vivo TSLP activity was validated and +T PDX were successfully generated using leukemia cells from two Hispanic pediatric patients with CRLF2 B-ALL. To determine whether +T PDX mice provide a preclinical model of B-ALL that more closely mirrors patients than -T PDX mice, we compared RNAseq gene expression profiles of leukemia cells isolated from +T PDX and -T PDX mice to that from the original patient sample. The gene expression pattern in leukemia cells from +T mice was significantly closer to primary patient sample than that from -T mice. The +T PDX mice described here provide a novel in vivo preclinical model for evaluating efficacy of drugs, such as Mcl-1 inhibitor, in context of the background genetic landscape and physiological human TSLP present in patients. Citation Format: Kimberly J. Payne, Cornelia Stoian, Jacqueline S. Coats, Olivia Francis, Terry-Ann M. Milford, Ineavely Baez, Pierce J. McCarthy, George Mambo, Anna V.C. White, Mariah M.Z. Jackson, Juliette M. Personius, Veriah Vidales, Muhammad Omair Kamal, Shadi Farzin Gohar, Sinisa Dovat. A novel patient-derived xenograft model for evaluating therapies that target the CRLF2 signaling pathway to reduce health disparities for Hispanic children with leukemia. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B46.

Abstract A07: A novel patient-derived xenograft model to define the role of TSLP-induced CRLF2 signals and identify therapies for Ph-like B-ALL

A subset of high-risk B cell acute lymphoblastic leukemia (ALL) shows a gene expression profile similar to Philadelphia chromosome positive (Ph+) ALL and has been described as Ph-like ALL. Approximately 50% of Ph-like B-ALL is characterized by genetic alterations leading to overexpression of CRLF2 (CRLF2 B-ALL). CRLF2 B-ALL occurs 5 times more often in Hispanic and Native American children than others and is prevalent in adolescents and young adults. Biologically, CRLF2 acts as a receptor component for the cytokine, TSLP, which induces JAK2-STAT5 and PI3/AKT/mTOR pathway activation downstream of binding to CRLF2. While activating JAK mutations are associated with CRLF2 B-ALL, over half of CRLF2 B-ALL lack such mutations. Our data show that primary human bone marrow (BM) stromal cells express TSLP. Thus TSLP is present in the tumor microenvironment to provide TSLP-induced CRLF2 signals that could play a role in the initiation, maintenance and/or progression of CRLF2 B-ALL. Consistent with this, TSLP has been reported to increase in vitro production of human fetal B cell precursors. However studies of TSLP in B lymphopoiesis have been conducted almost exclusively in mice which show low homology (~40%) with respect to human TSLP and CRLF2. Further, phospho flow cytometry assays show that human, but not mouse TSLP activates CRLF2 signals in primary human CRLF2 B-ALL cells and cell lines as indicated by increased pSTAT5, pAKT and pS6. These data indicate that the mouse TSLP present in classic patient derived xenograft models (PDX) does not produce the TSLP-induced CRLF2 signals present in the patient. To address this challenge we engineered PDX mice to produce human TSLP (hTSLP) by transplanting them with stromal cells transduced to express hTSLP (+T mice). Control (T) mice were produced by transplantation with stroma transduced with a control vector. Supernatant from engineered +T stroma, but not T stroma, induced JAK/STAT5 and PI3K/AKT/mTOR pathway activation in human CRLF2 B-ALL cells. ELISA assays showed that serum levels of hTSLP in mice was proportional to numbers of stromal cells injected at weekly time points. Normal human serum levels of hTSLP (12-32 pg/ml) could be achieved in +T mice, while hTSLP was undetectable in T mice. Because TSLP has been shown to increase in vitro production of human B cell precursors, we evaluated the in vivo functionality of our model by comparing the production of normal B cell precursors in the BM of +T and T PDX mice generated with human umbilical cord blood CD34+ cells. Data from 3 different cord blood donors showed that production of B cell precursors is 3-5 fold increased in +T as compared to T mice. TSLP-induced increases were specific to B lineage cells, initiated in the earliest CD19+ B cell precursors, and maintained through later stages of B cell development. Next we evaluate the in vivo functionality of our model using primary CRLF2 B-ALL leukemia cells. Human CRLF2 B-ALL cells were isolated from the BM of PDX mice and whole genome microarray was performed. Evaluation of microarray data by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis showed that genes downstream of mTOR pathway activation were upregulated in +T as compared to T PDX mice, confirming hTSLP activity in the +T PDX mice. To determine whether +T PDX mice provide a preclinical model of B-ALL that more closely mirrors patients than T PDX mice, we compared RNAseq gene expression profiles of leukemia cells from +T and T PDX mice to that from original patient sample. The gene expression pattern in +T mice was significantly closer to primary patient sample than that from T mice. The +T and T PDX mice described here provide a novel preclinical model for studying the role of TSLP in the initiation, progression and maintenance of CRLF2 B-ALL and for evaluating drug efficacy in an in vivo model that more closely mirrors the in vivo environment present in patients. Citation Format: Olivia L. Francis, Terry-Ann Milford, Ineavely Baez, Jacqueline S. Coats, Christopher L. Morris, Ross Fisher, Ben Van Handel, Ruijun Su, Batul Suterwala, Muhammad Kamal, Shadi Farzin Gohar, Sinisa Dovat, Kimberly J. Payne. A novel patient-derived xenograft model to define the role of TSLP-induced CRLF2 signals and identify therapies for Ph-like B-ALL. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A07.

Abstract 3295: A novel patient-derived xenograft model for evaluating the role of TSLP in CRLF2 B-ALL

B-cell acute lymphoblastic leukemia (B-ALL) with genetic alterations leading to overexpression of CRLF2 (CRLF2 B-ALL) is associated with poor outcomes. CRLF2 B-ALL is 5 times more common in Hispanic children than others making it a significant biological component of pediatric cancer health disparities. CRLF2 is a component of the receptor complex that is activated by the cytokine, TSLP. Receptor signaling induces Jak/STAT5 and PI3/AKT/mTOR pathway activation and plays a role in the proliferation and differentiation B cell precursors. We found that primary human bone marrow (BM) stroma express TSLP providing an in vivo source of TSLP to stimulate CRLF2 B-ALL cells. Our goal was to develop patient-derived xenograft (PDX) models of CRLF2 B-ALL for studies to understand disease mechanisms and identify therapies to treat CRLF2 B-ALL and reduce the health disparities for Hispanic children with this disease. PDX models are possible because many cytokines produced in the mouse show cross species activity on human cells. However, available data suggests that mouse TSLP does not activate human CRLF2-mediated signals. Using phospho flow cytometry we show that mouse TSLP was unable to induce increases in pSTAT5, pAKT and pS6 observed in CRLF2 B-ALL cells stimulated with human TSLP. We developed a human TSLP +/- PDX model system by transplanting immune deficient NSG mice with HS-27 stroma transduced to express human hTSLP (hTSLP+ mice) or with control vector (hTSLP- mice). Human TSLP was present at normal human serum levels in hTSLP+ mice but undetectable in hTSLP- mice. To identify genes targeted by TSLP in CRLF2 B-ALL and verify pathway activation, we transplanted primary leukemia cells from a Hispanic patient into hTSLP+ and hTSLP- mice. Whole genome microarray was performed on CRLF2 B-ALL cells isolated from the BM of the hTSLP+ and hTSLP- PDX mice. Microarray identified 280 genes that are upregulated and 281 genes that are downregulated in vivo in leukemia cells from hTSLP+ as compared to hTSLP- PDX mice. Evaluation of microarray data by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis showed that genes downstream of mTOR pathway activation were upregulated in hTSLP+ as compared to hTSLP- mice, confirming hTSLP activity in the hTSLP+ PDX mice. Our next question was whether cells expanded in hTSLP+ vs. hTSLP- mice would exhibit changes in their ability to respond to TSLP. When we subjected PDX-expanded primary CRLF2 B-ALL cells to ex vivo TSLP stimulation ∼1/3 fewer gene targets were up- and downregulated in the leukemia cells expanded in hTSLP- mice as compared to cells from hTSLP+ mice. This suggests that CRLF2 B-ALL cells expanded in xenograft without TSLP lose some of their ability to respond to TSLP. The hTSLP+ CRLF2 B-ALL PDX mice described here provide a novel preclinical model for studying disease mechanisms and identifying therapies to target signaling pathways activated by TSLP in CRLF2 B-ALL and reduce cancer health disparities for this disease. Citation Format: Olivia L. Francis, Parveen Shiraz, Terry-Ann Milford, Ineavely Baez, Jacqueline S. Coats, Karina Mayagoitia, Elizabeth Ginelli, Katherine R. Salcedo-Concepcion, Shannalee Martinez, Xiaobing Zhang, Valeri Filippov, Ruijun J. Su, Ross Fisher, Christopher L. Morris, Sinisa Dovat, Kimberly J. Payne. A novel patient-derived xenograft model for evaluating the role of TSLP in CRLF2 B-ALL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3295. doi:10.1158/1538-7445.AM2015-3295