Early-life infection depletes preleukemic cells in a mouse model of hyperdiploid B-cell acute lymphoblastic leukemia.

Epidemiological studies report opposing influences of infection on childhood B cell acute lymphoblastic leukemia (B-ALL). Although infections in the first year of life appear to exert the largest impact on leukemia risk, the effect of early pathogen exposure on the fetal preleukemia cells (PLC) that lead to B-ALL has yet to be reported. Using cytomegalovirus as a model early-life infection, we show that virus exposure within one week of birth induces profound depletion of transplanted B-ALL cells in two mouse models and of in situ-generated PLC in Eu-ret mice. The age-dependent depletion of PLC results from an elevated STAT4-mediated cytokine response in neonates, with high levels of IL-12p40-driven IFN-g production inducing PLC death. Similar PLC depletion can be achieved in adult mice by impairing viral clearance. These findings provide mechanistic support for an inhibitory effect of early-life infection on B-ALL progression and could inform development of therapeutic or preventative approaches.

A Comparative Study of B Cell Blast Isolation Methods from Bone Marrow Aspirates of Pediatric Leukemia Patients.

Density gradient centrifugation is a conventional technique widely utilized to isolate bone marrow mononuclear cells (BM-MNC) from bone marrow (BM) aspirates obtained from pediatric B-cell acute lymphoblastic leukemia (B-ALL) patients. Nevertheless, this technique achieves incomplete recovery of mononuclear cells and is relatively time-consuming and expensive. Given that B-ALL is the most common childhood malignancy, alternative methods for processing B-ALL samples may be more cost-effective. In this pilot study, we use several readouts, including immune phenotype, cell viability, and leukemia-initiating capacity in immune-deficient mice, to directly compare the density gradient centrifugation and buffy coat processing methods. Our findings indicate that buffy coat isolation yields comparable BM-MNC product in terms of both immune and leukemia cell content and could provide a viable, lower cost alternative for biobanks processing pediatric leukemia samples.

The Eµ-Ret mouse is a novel model of hyperdiploid B-cell acute lymphoblastic leukemia.

The presence of supernumerary chromosomes is the only abnormality shared by all patients diagnosed with high-hyperdiploid B cell acute lymphoblastic leukemia (HD-ALL). Despite being the most frequently diagnosed pediatric leukemia, the lack of clonal molecular lesions and complete absence of appropriate experimental models have impeded the elucidation of HD-ALL leukemogenesis. Here, we report that for 23 leukemia samples isolated from moribund Eµ-Ret mice, all were characterized by non-random chromosomal gains, involving combinations of trisomy 9, 12, 14, 15, and 17. With a median gain of three chromosomes, leukemia emerged after a prolonged latency from a preleukemic B cell precursor cell population displaying more diverse aneuploidy. Transition from preleukemia to overt disease in Eµ-Ret mice is associated with acquisition of heterogeneous genomic abnormalities affecting the expression of genes implicated in pediatric B-ALL. The development of abnormal centrosomes in parallel with aneuploidy renders both preleukemic and leukemic cells sensitive to inhibitors of centrosome clustering, enabling targeted in vivo depletion of leukemia-propagating cells. This study reveals the Eµ-Ret mouse to be a novel tool for investigating HD-ALL leukemogenesis, including supervision and selection of preleukemic aneuploid clones by the immune system and identification of vulnerabilities that could be targeted to prevent relapse.

Systemic delivery of mutant huntingtin lowering antisense oligonucleotides to the brain using apolipoprotein A-I nanodisks for Huntington disease.

Efficient delivery of therapeutics to the central nervous system (CNS) remains a major challenge for the treatment of neurological diseases. Huntington disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion mutation in the HTT gene which codes for a toxic mutant huntingtin (mHTT) protein. Pharmacological reduction of mHTT in the CNS using antisense oligonucleotides (ASO) ameliorates HD-like phenotypes in rodent models of HD, with such therapies being investigated in clinical trials for HD. In this study, we report the optimization of apolipoprotein A-I nanodisks (apoA-I NDs) as vehicles for delivery of a HTT-targeted ASO (HTT ASO) to the brain and peripheral organs for HD. We demonstrate that apoA-I wild type (WT) and the apoA-I K133C mutant incubated with a synthetic lipid, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, can self-assemble into monodisperse discoidal particles with diameters <20 nm that transmigrate across an in vitro blood-brain barrier model of HD. We demonstrate that apoA-I NDs are well tolerated in vivo, and that apoA-I K133C NDs show enhanced distribution to the CNS and peripheral organs compared to apoA-I WT NDs following systemic administration. ApoA-I K133C conjugated with HTT ASO forms NDs (HTT ASO NDs) that induce significant mHTT lowering in the liver, skeletal muscle and heart as well as in the brain when delivered intravenously in the BACHD mouse model of HD. Furthermore, HTT ASO NDs increase the magnitude of mHTT lowering in the striatum and cortex compared to HTT ASO alone following intracerebroventricular administration. These findings demonstrate the potential utility of apoA-I NDs as biocompatible vehicles for enhancing delivery of mutant HTT lowering ASOs to the CNS and peripheral organs for HD.

Targetable lesions and proteomes predict therapy sensitivity through disease evolution in pediatric acute lymphoblastic leukemia.

Childhood acute lymphoblastic leukemia (ALL) genomes show that relapses often arise from subclonal outgrowths. However, the impact of clonal evolution on the actionable proteome and response to targeted therapy is not known. Here, we present a comprehensive retrospective analysis of paired ALL diagnosis and relapsed specimen. Targeted next generation sequencing and proteome analysis indicate persistence of actionable genome variants and stable proteomes through disease progression. Paired viably-frozen biopsies show high correlation of drug response to variant-targeted therapies but in vitro selectivity is low. Proteome analysis prioritizes PARP1 as a pan-ALL target candidate needed for survival following cellular stress; diagnostic and relapsed ALL samples demonstrate robust sensitivity to treatment with two PARP1/2 inhibitors. Together, these findings support initiating prospective precision oncology approaches at ALL diagnosis and emphasize the need to incorporate proteome analysis to prospectively determine tumor sensitivities, which are likely to be retained at disease relapse.

Age and ligand specificity influence the outcome of pathogen engagement on preleukemic and leukemic B-cell precursor populations.

Common infections have long been proposed to play a role in the development of pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, epidemiologic studies report contradictory effects of infection exposure on subsequent B-ALL risk, and no specific pathogen has been definitively linked to the disease. A unifying mechanism to explain the divergent outcomes could inform disease prevention strategies. We previously reported that the pattern recognition receptor (PRR) ligand Poly(I:C) exerted effects on B-ALL cells that were distinct from those observed with other nucleic acid-based PRR ligands. Here, using multiple double-stranded RNA (dsRNA) moieties, we show that the overall outcome of exposure to Poly(I:C) reflects the balance of opposing responses induced by its ligation to endosomal and cytoplasmic receptors. This PRR response biology is shared between mouse and human B-ALL and can increase leukemia-initiating cell burden in vivo during the preleukemia phase of B-ALL, primarily through tumor necrosis factor α signaling. The age of the responding immune system further influences the impact of dsRNA exposure on B-ALL cells in both mouse and human settings. Overall, our study demonstrates that potentially proleukemic and antileukemic effects can each be generated by the stimulation of pathogen recognition pathways and indicates a mechanistic explanation for the contrasting epidemiologic associations reported for infection exposure and B-ALL.

Adaptation of the Th-MYCN Mouse Model of Neuroblastoma for Evaluation of Disseminated Disease.

High-risk neuroblastoma remains a profound clinical challenge that requires eradication of neuroblastoma cells from a variety of organ sites, including bone marrow, liver, and CNS, to achieve a cure. While preclinical modeling is a powerful tool for the development of novel cancer therapies, the lack of widely available models of metastatic neuroblastoma represents a significant barrier to the development of effective treatment strategies. To address this need, we report a novel luciferase-expressing derivative of the widely used Th-MYCN mouse. While our model recapitulates the non-metastatic neuroblastoma development seen in the parental transgenic strain, transplantation of primary tumor cells from disease-bearing mice enables longitudinal monitoring of neuroblastoma growth at distinct sites in immune-deficient or immune-competent recipients. The transplanted tumors retain GD2 expression through many rounds of serial transplantation and are sensitive to GD2-targeted immune therapy. With more diverse tissue localization than is seen with human cell line-derived xenografts, this novel model for high-risk neuroblastoma could contribute to the optimization of immune-based treatments for this deadly disease.

Inflammatory Immune Responses Trigger Rejection of Allogeneic Fibroblasts Transplanted into Mouse Skin.

Fibroblasts, or their homolog stromal cells, are present in most tissues and play an essential role in tissue homeostasis and regeneration. As a result, fibroblast-based strategies have been widely employed in tissue engineering. However, while considered to have immunosuppressive properties, the survival and functionality of allogeneic fibroblasts after transplantation remain controversial. Here, we evaluated innate and adaptive immune responses against allogeneic fibroblasts following intradermal injection into different immune-deficient mouse strains. While allogeneic fibroblasts were rejected 1 week after transplantation in immunocompetent mice, rejection did not occur in immunodeficient γ chain-deficient NOD-SCID (NSG) mice. T-cell- and B-cell-deficient RAG1 knockout mice showed greater loss of fibroblasts by day 5 after transplantation compared with NSG mice (P ≤ 0.05) but prolonged persistence compared with wild-type recipient (P ≤ 0.005). Loss of fibroblasts correlated with the expression of proinflammatory chemokine genes and infiltration of myeloid cells in the transplantation site. Depletion of macrophages and neutrophils delayed rejection, revealing the role of innate immune cells in an early elimination of fibroblasts that is followed by T-cell-mediated rejection in the second week. These findings indicate that the application of allogeneic fibroblasts in tissue engineering products requires further improvements to overcome cell rejection by innate and adaptive immune cells.

A cross-standardized flow cytometry platform to assess phenotypic stability in precursor B-cell acute lymphoblastic leukemia (B-ALL) xenografts.

With the continued poor outcome of relapsed acute lymphoblastic leukemia (ALL), new patient-specific approaches for disease progression monitoring and therapeutic intervention are urgently needed. Patient-derived xenografts (PDX) of primary ALL in immune-deficient mice have become a powerful tool for studying leukemia biology and therapy response. In PDX mice, the immunophenotype of the patient's leukemia is commonly believed to be stably propagated. In patients, however, the surface marker expression profile of the leukemic population often displays poorly understood immunophenotypic shifts during chemotherapy and ALL progression. We therefore developed a translational flow cytometry platform to study whether the patient-specific immunophenotype is faithfully recapitulated in PDX mice. To enable valid assessment of immunophenotypic stability and subpopulation complexity of the patient's leukemia after xenotransplantation, we comprehensively immunophenotyped diagnostic B-ALL from children and their matched PDX using identical, clinically standardized flow protocols and instrument settings. This cross-standardized approach ensured longitudinal stability and cross-platform comparability of marker expression intensity at high phenotyping depth. This analysis revealed readily detectable changes to the patient leukemia-associated immunophenotype (LAIP) after xenotransplantation. To further investigate the mechanism underlying these complex immunophenotypic shifts, we applied an integrated analytical approach that combined clinical phenotyping depth and high analytical sensitivity with unbiased high-dimensional algorithm-based analysis. This high-resolution analysis revealed that xenotransplantation achieves patient-specific propagation of phenotypically stable B-ALL subpopulations and that the immunophenotypic shifts observed at the level of bulk leukemia were consistent with changes in underlying subpopulation abundance. By incorporating the immunophenotypic complexity of leukemic populations, this novel cross-standardized analytical platform could greatly expand the utility of PDX for investigating ALL progression biology and assessing therapies directed at eliminating relapse-driving leukemic subpopulations.

Centrosome Amplification Is a Potential Molecular Target in Paediatric Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most common form of cancer in children, with most cases arising from fetal B cell precursor, termed B-ALL. Here, we use immunofluorescence analysis of B-ALL cells to identify centrosome amplification events that require the centrosome clustering pathway to successfully complete mitosis. Our data reveals that primary human B-ALL cells and immortal B-ALL cell lines from both human and mouse sources show defective bipolar spindle formation, abnormal mitotic progression, and cell death following treatment with centrosome clustering inhibitors (CCI). We demonstrate that CCI-refractory B-ALL cells exhibit markers for increased genomic instability, including DNA damage and micronuclei, as well as activation of the cyclic GMP-AMP synthase (cGAS)-nuclear factor kappa B (NF-κB) signalling pathway. Our analysis of cGAS knock-down B-ALL clones implicates cGAS in the sensitivity of B-ALL cells to CCI treatment. Due to its integral function and specificity to cancer cells, the centrosome clustering pathway presents a powerful molecular target for cancer treatment while mitigating the risk to healthy cells.

PDX models reflect the proteome landscape of pediatric acute lymphoblastic leukemia but divert in select pathways.

BACKGROUND: Murine xenografts of pediatric leukemia accurately recapitulate genomic aberrations. How this translates to the functional capacity of cells remains unclear. Here, we studied global protein abundance, phosphorylation, and protein maturation by proteolytic processing in 11 pediatric B- and T- cell ALL patients and 19 corresponding xenografts. METHODS: Xenograft models were generated for each pediatric patient leukemia. Mass spectrometry-based methods were used to investigate global protein abundance, protein phosphorylation, and limited proteolysis in paired patient and xenografted pediatric acute B- and T- cell lymphocytic leukemia, as well as in pediatric leukemia cell lines. Targeted next-generation sequencing was utilized to examine genetic abnormalities in patients and in corresponding xenografts. Bioinformatic and statistical analysis were performed to identify functional mechanisms associated with proteins and protein post-translational modifications. RESULTS: Overall, we found xenograft proteomes to be most equivalent with their patient of origin. Protein level differences that stratified disease subtypes at diagnostic and relapse stages were largely recapitulated in xenografts. As expected, PDXs lacked multiple human leukocyte antigens and complement proteins. We found increased expression of cell cycle proteins indicating a high proliferative capacity of xenografted cells. Structural genomic changes and mutations were reflected at the protein level in patients. In contrast, the post-translational modification landscape was shaped by leukemia type and host and only to a limited degree by the patient of origin. Of 201 known pediatric oncogenic drivers and drug-targetable proteins, the KMT2 protein family showed consistently high variability between patient and corresponding xenografts. Comprehensive N terminomics revealed deregulated proteolytic processing in leukemic cells, in particular from caspase-driven cleavages found in patient cells. CONCLUSION: Genomic and host factors shape protein and post-translational modification landscapes differently. This study highlights select areas of diverging biology while confirming murine patient-derived xenografts as a generally accurate model system.

Differential Depletion of Bone Marrow Resident B-ALL after Systemic Administration of Endosomal TLR Agonists.

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. While frontline chemotherapy regimens are generally very effective, the prognosis for patients whose leukemia returns remains poor. The presence of measurable residual disease (MRD) in bone marrow at the completion of induction therapy is the strongest predictor of relapse, suggesting that strategies to eliminate the residual leukemic blasts from this niche could reduce the incidence of recurrence. We have previously reported that toll-like receptor (TLR) agonists achieve durable T cell-mediated protection in transplantable cell line-based models of B cell precursor leukemia (B-ALL). However, the successful application of TLR agonist therapy in an MRD setting would require the induction of anti-leukemic immune activity specifically in the bone marrow, a site of the chemotherapy-resistant leukemic blasts. In this study, we compare the organ-specific depletion of human and mouse primary B-ALL cells after systemic administration of endosomal TLR agonists. Despite comparable splenic responses, only the TLR9 agonist induced strong innate immune responses in the bone marrow and achieved a near-complete elimination of B-ALL cells. This pattern of response was associated with the most significantly prolonged disease-free survival. Overall, our findings identify innate immune activity in the bone marrow that is associated with durable TLR-induced protection against B-ALL outgrowth.

The nonmotor adaptor HMMR dampens Eg5-mediated forces to preserve the kinetics and integrity of chromosome segregation.

Mitotic spindle assembly and organization require forces generated by motor proteins. The activity of these motors is regulated by nonmotor adaptor proteins. However, there are limited studies reporting the functional importance of adaptors on the balance of motor forces and the promotion of faithful and timely cell division. Here we show that genomic deletion or small interfering RNA silencing of the nonmotor adaptor Hmmr/HMMR disturbs spindle microtubule organization and bipolar chromosome-kinetochore attachments with a consequent elevated occurrence of aneuploidy. Rescue experiments show a conserved motif in HMMR is required to generate interkinetochore tension and promote anaphase entry. This motif bears high homology with the kinesin Kif15 and is known to interact with TPX2, a spindle assembly factor. We find that HMMR is required to dampen kinesin Eg5-mediated forces through localizing TPX2 and promoting the formation of inhibitory TPX2-Eg5 complexes. In HMMR-silenced cells, K-fiber stability is reduced while the frequency of unattached chromosomes and the time needed for chromosome segregation are both increased. These defects can be alleviated in HMMR-silenced cells with chemical inhibition of Eg5 but not through the silencing of Kif15. Together, our findings indicate that HMMR balances Eg5--mediated forces to preserve the kinetics and integrity of chromosome segregation.

Cell Cycle-Dependent Tumor Engraftment and Migration Are Enabled by Aurora-A.

Cell-cycle progression and the acquisition of a migratory phenotype are hallmarks of human carcinoma cells that are perceived as independent processes but may be interconnected by molecular pathways that control microtubule nucleation at centrosomes. Here, cell-cycle progression dramatically impacts the engraftment kinetics of 4T1-luciferase2 breast cancer cells in immunocompetent BALB/c or immunocompromised NOD-SCID gamma (NSG) mice. Multiparameter imaging of wound closure assays was used to track cell-cycle progression, cell migration, and associated phenotypes in epithelial cells or carcinoma cells expressing a fluorescence ubiquitin cell-cycle indicator. Cell migration occurred with an elevated velocity and directionality during the S-G2-phase of the cell cycle, and cells in this phase possess front-polarized centrosomes with augmented microtubule nucleation capacity. Inhibition of Aurora kinase-A (AURKA/Aurora-A) dampens these phenotypes without altering cell-cycle progression. During G2-phase, the level of phosphorylated Aurora-A at centrosomes is reduced in hyaluronan-mediated motility receptor (HMMR)-silenced cells as is the nuclear transport of TPX2, an Aurora-A-activating protein. TPX2 nuclear transport depends upon HMMR-T703, which releases TPX2 from a complex with importin-α (KPNA2) at the nuclear envelope. Finally, the abundance of phosphorylated HMMR-T703, a substrate for Aurora-A, predicts breast cancer-specific survival and relapse-free survival in patients with estrogen receptor (ER)-negative (n = 941), triple-negative (TNBC) phenotype (n = 538), or basal-like subtype (n = 293) breast cancers, but not in those patients with ER-positive breast cancer (n = 2,218). Together, these data demonstrate an Aurora-A/TPX2/HMMR molecular axis that intersects cell-cycle progression and cell migration.Implications: Tumor cell engraftment, migration, and cell-cycle progression share common regulation of the microtubule cytoskeleton through the Aurora-A/TPX2/HMMR axis, which has the potential to influence the survival of patients with ER-negative breast tumors. Mol Cancer Res; 16(1); 16-31. ©2017 AACR.

Absolute lymphocyte counts at end of induction correlate with distinct immune cell compartments in pediatric B cell precursor acute lymphoblastic leukemia.

Several retrospective studies in children with B cell precursor (BCP) acute lymphoblastic leukemia (ALL) provided clinical evidence that higher absolute lymphocyte counts (ALC) early into treatment significantly correlated with improved relapse-free and overall survival. It still remains unknown, however, whether the predictive role of higher ALCs reflects general bone marrow recovery or a more specific attribute of immune function. To investigate this question, we implemented a prospective observational cohort study in 20 children with BCP ALL on day 29 (D29) of induction chemotherapy and immunophenotyped their lymphoid (T, B and natural killer cells) and myeloid (neutrophils, monocytes, dendritic cells) compartments. In a first evaluation of a cohort treated with Children's Oncology Group-based induction chemotherapy, the immune cell compartments were differentially depleted at D29. Neither gender, risk status, minimal residual disease, nor bone marrow recovery markers correlated with D29 ALC. In contrast, both CD3+ T cell and dendritic cell compartments, which did not correlate with age, significantly correlated with D29 ALC (p < 0.0001). In addition, subset complexity of cellular immune compartments was preserved at D29. This study reveals that D29 ALC significantly correlates with distinct immune cell compartments but not with bone marrow recovery markers, suggesting that higher D29 ALCs may contribute to leukemia control by inducing specific host immune activity.

Tumor Variant Identification That Accounts for the Unique Molecular Landscape of Pediatric Malignancies.

Precision oncology trials for pediatric cancers require rapid and accurate detection of genetic alterations. Tumor variant identification should interrogate the distinctive driver genes and more frequent copy number variants and gene fusions that are characteristics of pediatric tumors. Here, we evaluate tumor variant identification using whole genome sequencing (n = 12 samples) and two amplification-based next-generation sequencing assays (n = 28 samples), including one assay designed to rapidly assess common diagnostic, prognostic, and therapeutic biomarkers found in pediatric tumors. Variant identification by the three modalities was comparable when filtered for 151 pediatric driver genes. Across the 28 samples, the pediatric cancer-focused assay detected more tumor variants per sample (two-sided, P < .05), which improved the identification of potentially druggable events and matched pathway inhibitors. Overall, our data indicate that an assay designed to evaluate pediatric cancer-specific variants, including gene fusions, may improve the detection of target-agent pairs for precision oncology.

IFN-γ directly inhibits murine B-cell precursor leukemia-initiating cell proliferation early in life.

The early-life immune environment has been implicated as a modulator of acute lymphoblastic leukemia (ALL) development in children, with infection being associated with significant changes in ALL risk. Furthermore, polymorphisms in several cytokine genes, including IL-10 and IFN-γ, are associated with leukemia development. However, the mechanisms and timing of these influences remain unknown. Here, we use the Eµ-ret transgenic mouse model of B-cell precursor ALL to assess the influence of IFN-γ on the early-life burden of leukemia-initiating cells. The absence of IFN-γ activity resulted in greater numbers of leukemia-initiating cells early in life and was associated with accelerated leukemia onset. The leukemia-initiating cells from IFN-γ-knockout mice had reduced suppressor of cytokine signaling (SOCS-1) expression, were significantly more sensitive to IFN-γ, and exhibited more rapid expansion in vivo than their wild-type counterparts. However, sensitivity to this inhibitory pathway was lost in fully transformed IFN-γ-knockout leukemia cells. These results demonstrate that the influence of IFN-γ on ALL progression may not be mediated by selection of nascent transformed cells but rather through a general SOCS-mediated reduction in B-cell precursor proliferation. Thus, while cytokine levels may influence leukemia at multiple points during disease progression, our study indicates a significant early influence of basal, infection-independent cytokine production on leukemogenesis.

Y-box-binding protein 1 contributes to IL-7-mediated survival signaling in B-cell precursor acute lymphoblastic leukemia.

Y-box-binding protein 1 (YB-1) is a regulatory protein that is associated with drug resistance and relapse in solid tumors. As YB-1 mediates some of its activity through growth factor receptor signaling dysregulation, the present study compared the expression of YB-1 and interleukin 7 (IL-7) receptor α (IL-7Rα) in pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) and normal BCP cells. The expression levels of IL-7Rα and YB-1 were higher in relapsed vs. diagnostic samples of primary BCP ALL; however, co-expression was also observed in a minor BCP cell population in samples from healthy donors. Functional crosstalk between YB-1 and IL-7R was detected: Overexpression of YB-1 increased surface levels of IL-7R in B cells, and the stimulation of BCP ALL cell lines and primary samples by IL-7 activated YB-1 by phosphorylation at S102 in a phosphatidylinositol 3-kinase-independent and MEK1/2-dependent manner. Targeted knockdown of YB-1 reduced IL-7-mediated protection against rapamycin, and an inhibitor of MEK1/2 potentiated rapamycin-mediated killing in the presence of IL-7. These data establish a novel link between two well-characterized pro-survival factors in acute leukemia, and suggest that YB-1 inhibition may represent a novel therapeutic strategy for increasing sensitivity to chemotherapy in patients with refractory acute B-cell leukemia.

Intravenous immunoglobulin skews macrophages to an anti-inflammatory, IL-10-producing activation state.

Intravenous Ig is used to treat autoimmune or autoinflammatory disorders, but the mechanism by which it exerts its immunosuppressive activity is not understood completely. To examine the impact of intravenous Ig on macrophages, we compared cytokine production by LPS-activated macrophages in the presence and absence of intravenous Ig. Intravenous Ig treatment induced robust production of IL-10 in response to LPS, relative to LPS stimulation alone, and reduced production of proinflammatory cytokines. This anti-inflammatory, intravenous Ig-induced activation was sustained for 24 h but could only be induced if intravenous Ig were provided within 1 h of LPS stimulation. Intravenous Ig activation led to enhanced and prolonged activation of MAPKs, Erk1/2, p38, and Erk5, and inhibition of each reduced intravenous Ig-induced IL-10 production and suppression of IL-12/23p40. IL-10 production occurred rapidly in response to intravenous Ig + LPS and was sufficient to reduce proinflammatory IL-12/23p40 production in response to LPS. IL-10 induction and reduced IL-12/23p40 production were transcriptionally regulated. IL-10 played a direct role in reducing proinflammatory cytokine production by macrophages treated with intravenous Ig + LPS, as macrophages from mice deficient in the IL-10R ß chain or in IL-10 were compromised in their ability to reduce proinflammatory cytokine production. Finally, intraperitoneal injection of intravenous Ig or intravenous Ig + LPS into mice activated macrophages to produce high levels of IL-10 during subsequent or concurrent LPS challenge, respectively. These findings identify IL-10 as a key anti-inflammatory mediator produced by intravenous Ig-treated macrophages and provide insight into a novel mechanism by which intravenous Ig may dampen down inflammatory responses in patients with autoimmune or autoinflammatory diseases.

Noninvasive bioluminescent imaging of primary patient acute lymphoblastic leukemia: a strategy for preclinical modeling.

The efficient engraftment in immune-deficient mice achieved with both acute lymphoblastic leukemia (ALL) cell lines and primary samples has facilitated identification of the antileukemia activity of a wide variety of agents. Despite widespread usage, however, little is known about the early ALL localization and engraftment kinetics in this model, limiting experimental read-outs primarily to survival and endpoint analysis at high disease burden. In this study, we report that bioluminescent imaging can be reproducibly achieved with primary human ALL samples. This approach provides a noninvasive, longitudinal measure of leukemia burden and localization that enhances the sensitivity of treatment response detection and provides greater insight into the mechanism of action of antileukemia agents. In addition, this study reveals significant cell line- and species-related differences in leukemia migration, especially early in expansion, which may confound observations between various leukemia models. Overall, this study demonstrates that the use of bioluminescent primary ALL allows the detection and quantitation of treatment effects at earlier, previously unquantifiable disease burdens and thus provides the means to standardize and expedite the evaluation of anti-ALL activity in preclinical xenograft studies.