expression-exogenous-cytokine-patient-derived-xenografts-via

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 ...

Loma Linda University

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 CD19(lo) 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 approximately 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.

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

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.

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

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.

Differences in mouse and human nonmemory B cell pools.

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.

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

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.

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

Ineavely Baez

Department of Pathology and Human Anatomy

Loma Linda University

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