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1.
Cancer Discov ; 12(4): 1106-1127, 2022 04 01.
Article in English | MEDLINE | ID: mdl-35046097

ABSTRACT

Remodeling of the microenvironment by tumor cells can activate pathways that favor cancer growth. Molecular delineation and targeting of such malignant-cell nonautonomous pathways may help overcome resistance to targeted therapies. Herein we leverage genetic mouse models, patient-derived xenografts, and patient samples to show that acute myeloid leukemia (AML) exploits peripheral serotonin signaling to remodel the endosteal niche to its advantage. AML progression requires the presence of serotonin receptor 1B (HTR1B) in osteoblasts and is driven by AML-secreted kynurenine, which acts as an oncometabolite and HTR1B ligand. AML cells utilize kynurenine to induce a proinflammatory state in osteoblasts that, through the acute-phase protein serum amyloid A (SAA), acts in a positive feedback loop on leukemia cells by increasing expression of IDO1-the rate-limiting enzyme for kynurenine synthesis-thereby enabling AML progression. This leukemia-osteoblast cross-talk, conferred by the kynurenine-HTR1B-SAA-IDO1 axis, could be exploited as a niche-focused therapeutic approach against AML, opening new avenues for cancer treatment. SIGNIFICANCE: AML remains recalcitrant to treatments due to the emergence of resistant clones. We show a leukemia-cell nonautonomous progression mechanism that involves activation of a kynurenine-HTR1B-SAA-IDO1 axis between AML cells and osteoblasts. Targeting the niche by interrupting this axis can be pharmacologically harnessed to hamper AML progression and overcome therapy resistance. This article is highlighted in the In This Issue feature, p. 873.


Subject(s)
Kynurenine , Leukemia, Myeloid, Acute , Animals , Humans , Kynurenine/metabolism , Kynurenine/therapeutic use , Leukemia, Myeloid, Acute/drug therapy , Mice , Osteoblasts/metabolism , Signal Transduction , Tumor Microenvironment
2.
Mol Ther ; 29(4): 1625-1638, 2021 04 07.
Article in English | MEDLINE | ID: mdl-33515514

ABSTRACT

Ongoing clinical trials for treatment of beta-globinopathies by gene therapy involve the transfer of the beta-globin gene, which requires integration of three to four copies per genome in most target cells. This high proviral load may increase genome toxicity, potentially limiting the safety of this therapy and relegating its use to total body myeloablation. We hypothesized that introducing an additional hypersensitive site from the locus control region, the complete sequence of the second intron of the beta-globin gene, and the ankyrin insulator may enhance beta-globin expression. We identified a construct, ALS20, that synthesized significantly higher adult hemoglobin levels than those of other constructs currently used in clinical trials. These findings were confirmed in erythroblastic cell lines and in primary cells isolated from sickle cell disease patients. Bone marrow transplantation studies in beta-thalassemia mice revealed that ALS20 was curative at less than one copy per genome. Injection of human CD34+ cells transduced with ALS20 led to safe, long-term, and high polyclonal engraftment in xenograft experiments. Successful treatment of beta-globinopathies with ALS20 could potentially be achieved at less than two copies per genome, minimizing the risk of cytotoxic events and lowering the intensity of myeloablation.


Subject(s)
Anemia, Sickle Cell/genetics , Bone Marrow Transplantation , Genetic Therapy , beta-Globins/genetics , beta-Thalassemia/genetics , Anemia, Sickle Cell/blood , Anemia, Sickle Cell/pathology , Anemia, Sickle Cell/therapy , Animals , Gene Expression/genetics , Genetic Vectors/genetics , Genetic Vectors/pharmacology , Hemoglobins/genetics , Heterografts , Humans , Lentivirus/genetics , Locus Control Region/genetics , Mice , Transduction, Genetic , beta-Globins/therapeutic use , beta-Thalassemia/blood , beta-Thalassemia/pathology , beta-Thalassemia/therapy
3.
Res Vet Sci ; 134: 137-146, 2021 Jan.
Article in English | MEDLINE | ID: mdl-33383491

ABSTRACT

"Humanized" immunodeficient mice generated via the transplantation of CD34+ human hematopoietic stem cells (hHSC) are an important preclinical model system. The triple transgenic NOD.Cg-PrkdcscidIl2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS) mouse line is increasingly used as recipient for CD34+ hHSC engraftment. NSGS mice combine the features of the highly immunodeficient NSG mice with transgenic expression of the human myeloid stimulatory cytokines GM-CSF, IL-3, and Kit ligand. While generating humanized NSGS (huNSGS) mice from two independent cohorts, we encountered a fatal macrophage activation syndrome (MAS)-like phenotype resulting from the transplantation of CD34+ hHSC. huNSGS mice exhibiting this phenotype declined clinically starting at approximately 10 weeks following CD34+ hHSC engraftment, with all mice requiring euthanasia by 16 weeks. Gross changes comprised small, irregular liver, splenomegaly, cardiomegaly, and generalized pallor. Hematological abnormalities included severe thrombocytopenia and anemia. Pathologically, huNSGS spontaneously developed a disseminated histiocytosis with infiltrates of activated macrophages and hemophagocytosis predominantly affecting the liver, spleen, bone marrow, and pancreas. The infiltrates were chimeric with a mixture of human and mouse macrophages. Immunohistochemistry suggested activation of the inflammasome in both human and murine macrophages. Active Epstein-Barr virus infection was not a feature. Although the affected mice exhibited robust chimerism of the spleen and bone marrow, the phenotype often developed in the face of low chimerism of the peripheral blood. Given the high penetrance and early lethality associated with the MAS-like phenotype here described, we urge caution when considering the use of huNSGS mice for the development of long-term studies.


Subject(s)
Macrophage Activation Syndrome/pathology , Macrophages/microbiology , Animals , Antigens, CD34 , DNA-Activated Protein Kinase/immunology , Epstein-Barr Virus Infections/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Herpesvirus 4, Human , Histiocytosis/immunology , Humans , Interleukin Receptor Common gamma Subunit/immunology , Macrophage Activation Syndrome/immunology , Male , Mice , Mice, Inbred NOD , Mice, SCID , Recombinant Proteins/immunology , Stem Cell Factor/immunology
4.
Nat Med ; 26(11): 1776-1787, 2020 11.
Article in English | MEDLINE | ID: mdl-32868878

ABSTRACT

An effective strategy to cure HIV will likely require a potent and sustained antiviral T cell response. Here we explored the utility of chimeric antigen receptor (CAR) T cells, expressing the CD4 ectodomain to confer specificity for the HIV envelope, to mitigate HIV-induced pathogenesis in bone marrow, liver, thymus (BLT) humanized mice. CAR T cells expressing the 4-1BB/CD3-ζ endodomain were insufficient to prevent viral rebound and CD4+ T cell loss after the discontinuation of antiretroviral therapy. Through iterative improvements to the CAR T cell product, we developed Dual-CAR T cells that simultaneously expressed both 4-1BB/CD3-ζ and CD28/CD3-ζ endodomains. Dual-CAR T cells exhibited expansion kinetics that exceeded 4-1BB-, CD28- and third-generation costimulated CAR T cells, elicited effector functions equivalent to CD28-costimulated CAR T cells and prevented HIV-induced CD4+ T cell loss despite persistent viremia. Moreover, when Dual-CAR T cells were protected from HIV infection through expression of the C34-CXCR4 fusion inhibitor, these cells significantly reduced acute-phase viremia, as well as accelerated HIV suppression in the presence of antiretroviral therapy and reduced tissue viral burden. Collectively, these studies demonstrate the enhanced therapeutic potency of a novel Dual-CAR T cell product with the potential to effectively treat HIV infection.


Subject(s)
CD4 Antigens/immunology , HIV Infections/therapy , Immunotherapy, Adoptive , Receptors, Chimeric Antigen/immunology , Animals , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Monoclonal, Humanized/pharmacology , Bone Marrow/immunology , Bone Marrow/virology , CD3 Complex/antagonists & inhibitors , CD4 Antigens/administration & dosage , Gene Expression Regulation/immunology , HIV Envelope Protein gp41/antagonists & inhibitors , HIV Envelope Protein gp41/immunology , HIV Infections/immunology , HIV Infections/pathology , HIV Infections/virology , HIV-1/immunology , HIV-1/pathogenicity , Humans , Liver/immunology , Liver/virology , Mice , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/immunology , Protein Domains/immunology , Receptors, CXCR4/antagonists & inhibitors , Receptors, CXCR4/immunology , Receptors, Chimeric Antigen/administration & dosage , T-Lymphocytes/immunology , Thymus Gland/immunology , Thymus Gland/virology , Tumor Necrosis Factor Receptor Superfamily, Member 9/antagonists & inhibitors
5.
Mol Ther ; 28(7): 1585-1599, 2020 07 08.
Article in English | MEDLINE | ID: mdl-32454027

ABSTRACT

HIV infection preferentially depletes HIV-specific CD4+ T cells, thereby impairing antiviral immunity. In this study, we explored the therapeutic utility of adoptively transferred CD4+ T cells expressing an HIV-specific chimeric antigen receptor (CAR4) to restore CD4+ T cell function to the global HIV-specific immune response. We demonstrated that CAR4 T cells directly suppressed in vitro HIV replication and eliminated virus-infected cells. Notably, CAR4 T cells containing intracellular domains (ICDs) derived from the CD28 receptor family (ICOS and CD28) exhibited superior effector functions compared to the tumor necrosis factor receptor (TNFR) family ICDs (CD27, OX40, and 4-1BB). However, despite demonstrating limited in vitro efficacy, only HIV-resistant CAR4 T cells expressing the 4-1BBζ ICD exhibited profound expansion, concomitant with reduced rebound viremia after antiretroviral therapy (ART) cessation and protection of CD4+ T cells (CAR-) from HIV-induced depletion in humanized mice. Moreover, CAR4 T cells enhanced the in vivo persistence and efficacy of HIV-specific CAR-modified CD8+ T cells expressing the CD28ζ ICD, which alone exhibited poor survival. Collectively, these studies demonstrate that HIV-resistant CAR4 T cells can directly control HIV replication and augment the virus-specific CD8+ T cell response, highlighting the therapeutic potential of engineered CD4+ T cells to engender a functional HIV cure.


Subject(s)
CD28 Antigens/chemistry , CD4-Positive T-Lymphocytes/transplantation , HIV Infections/therapy , HIV/physiology , Inducible T-Cell Co-Stimulator Protein/chemistry , Receptors, Chimeric Antigen/metabolism , Animals , CD28 Antigens/genetics , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cells, Cultured , Disease Models, Animal , Disease Progression , Drug Resistance, Viral , HIV/immunology , HIV Infections/immunology , HIV-1/immunology , Humans , Immunotherapy, Adoptive , Inducible T-Cell Co-Stimulator Protein/genetics , Mice , Protein Domains , Receptors, Chimeric Antigen/genetics , Treatment Outcome , Virus Replication
6.
Exp Hematol ; 76: 60-66.e2, 2019 08.
Article in English | MEDLINE | ID: mdl-31369790

ABSTRACT

Exosomes are virus-size membrane-bound vesicles of endocytic origin present in all body fluids. Plasma of AML patients is significantly enriched in exosomes, which carry a cargo of immunosuppressive molecules and deliver them to recipient immune cells, suppressing their functions. However, whether these exosomes originate from leukemic blasts or from various normal cells in the bone marrow or other tissues is unknown. In the current study, we developed an AML PDX model in mice and studied the molecular cargo and immune cell effects of the AML PDX exosomes in parallel with the exosomes from plasma of the corresponding AML patients. Fully engrafted AML PDX mice produced exosomes with characteristics similar to those of exosomes isolated from plasma of the AML patients who had donated the cells for engraftment. The engrafted leukemic cells produced exosomes that carried human proteins and leukemia-associated antigens, confirming the human origin of these exosomes. Furthermore, the AML-derived exosomes carried immunosuppressive proteins responsible for immune cell dysfunctions. Our studies of exosomes in AML PDX mice serve as a proof of concept that AML blasts are the source of immunosuppressive exosomes with a molecular profile that mimics the content and functions of the parental cells.


Subject(s)
Exosomes , Leukemia, Myeloid, Acute/pathology , Neoplastic Stem Cells/metabolism , Tumor Escape/physiology , Aged , Animals , Antigens, Neoplasm/blood , Female , Heterografts , Humans , Leukemia, Myelomonocytic, Acute/pathology , Lymphocyte Activation , Male , Mice , Mice, Inbred NOD , Mice, SCID , Middle Aged , Neoplasm Proteins/blood , Neoplasm Transplantation , T-Lymphocyte Subsets/immunology
7.
PeerJ ; 6: e4661, 2018.
Article in English | MEDLINE | ID: mdl-29682426

ABSTRACT

In 2015, as part of the Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative, we published a Registered Report (Shan et al., 2015) that described how we intended to replicate selected experiments from the paper "Androgen Receptor Splice Variants Determine Taxane Sensitivity in Prostate Cancer" (Thadani-Mulero et al., 2014). Here we report the results of those experiments. Growth of tumor xenografts from two prostate cancer xenograft lines, LuCaP 86.2, which expresses wild-type androgen receptor (AR) and AR variant 567, and LuCaP 23.1, which expresses wild-type AR and AR variant 7, were not affected by docetaxel treatment. The LuCaP 23.1 tumor xenografts grew slower than in the original study. This result is different from the original study, which reported significant reduction of tumor growth in the LuCaP 86.2. Furthermore, we were unable to detect ARv7 in the LuCaP 23.1, although we used the antibody as stated in the original study and ensured that it was detecting ARv7 via a known positive control (22rv1, Hörnberg et al., 2011). Finally, we report a meta-analysis of the result.

8.
Haematologica ; 103(6): 959-971, 2018 06.
Article in English | MEDLINE | ID: mdl-29545344

ABSTRACT

Patient-derived xenotransplantation models of human myeloid diseases including acute myeloid leukemia, myelodysplastic syndromes and myeloproliferative neoplasms are essential for studying the biology of the diseases in pre-clinical studies. However, few studies have used these models for comparative purposes. Previous work has shown that acute myeloid leukemia blasts respond to human hematopoietic cytokines whereas myelodysplastic syndrome cells do not. We compared the engraftment of acute myeloid leukemia cells and myelodysplastic syndrome cells in NSG mice to that in NSG-S mice, which have transgene expression of human cytokines. We observed that only 50% of all primary acute myeloid leukemia samples (n=77) transplanted in NSG mice provided useful levels of engraftment (>0.5% human blasts in bone marrow). In contrast, 82% of primary acute myeloid leukemia samples engrafted in NSG-S mice with higher leukemic burden and shortened survival. Additionally, all of 5 injected samples from patients with myelodysplastic syndrome showed persistent engraftment on week 6; however, engraftment was mostly low (<2%), did not increase over time, and was only transiently affected by the use of NSG-S mice. Co-injection of mesenchymal stem cells did not enhance human myelodysplastic syndrome cell engraftment. Overall, we conclude that engraftment of acute myeloid leukemia samples is more robust compared to that of myelodysplastic syndrome samples and unlike those, acute myeloid leukemia cells respond positively to human cytokines, whereas myelodysplastic syndrome cells demonstrate a general unresponsiveness to them.


Subject(s)
Cytokines/metabolism , Graft Survival/immunology , Immunocompromised Host , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/metabolism , Myelodysplastic Syndromes/immunology , Myelodysplastic Syndromes/metabolism , Animals , Bone Marrow Transplantation , Cytokines/blood , Disease Models, Animal , Female , Humans , Leukemia, Myeloid, Acute/therapy , Male , Mesenchymal Stem Cells/metabolism , Mice , Myelodysplastic Syndromes/therapy , Transplantation, Heterologous
10.
Elife ; 62017 06 27.
Article in English | MEDLINE | ID: mdl-28653617

ABSTRACT

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Fung et al., 2015), that described how we intended to replicate selected experiments from the paper "Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia" (Dawson et al., 2011). Here, we report the results of those experiments. We found treatment of MLL-fusion leukaemia cells (MV4;11 cell line) with the BET bromodomain inhibitor I-BET151 resulted in selective growth inhibition, whereas treatment of leukaemia cells harboring a different oncogenic driver (K-562 cell line) did not result in selective growth inhibition; this is similar to the findings reported in the original study (Figure 2A and Supplementary Figure 11A,B; Dawson et al., 2011). Further, I-BET151 resulted in a statistically significant decrease in BCL2 expression in MV4;11 cells, but not in K-562 cells; again this is similar to the findings reported in the original study (Figure 3D; Dawson et al., 2011). We did not find a statistically significant difference in survival when testing I-BET151 efficacy in a disseminated xenograft MLL mouse model, whereas the original study reported increased survival in I-BET151 treated mice compared to vehicle control (Figure 4B,D; Dawson et al., 2011). Differences between the original study and this replication attempt, such as different conditioning regimens and I-BET151 doses, are factors that might have influenced the outcome. We also found I-BET151 treatment resulted in a lower median disease burden compared to vehicle control in all tissues analyzed, similar to the example reported in the original study (Supplementary Figure 16A; Dawson et al., 2011). Finally, we report meta-analyses for each result.


Subject(s)
Antineoplastic Agents/administration & dosage , Chromatin/metabolism , Heterocyclic Compounds, 4 or More Rings/administration & dosage , Leukemia, Biphenotypic, Acute/drug therapy , Nerve Tissue Proteins/antagonists & inhibitors , Receptors, Cell Surface/antagonists & inhibitors , Animals , Cell Line, Tumor , Disease Models, Animal , Heterografts , Humans , Mice , Nerve Tissue Proteins/metabolism , Protein Binding , Receptors, Cell Surface/metabolism , Treatment Outcome
11.
Nat Commun ; 8: 14630, 2017 03 02.
Article in English | MEDLINE | ID: mdl-28251988

ABSTRACT

Monoclonal antibodies are one of the fastest growing classes of pharmaceutical products, however, their potential is limited by the high cost of development and manufacturing. Here we present a safe and cost-effective platform for in vivo expression of therapeutic antibodies using nucleoside-modified mRNA. To demonstrate feasibility and protective efficacy, nucleoside-modified mRNAs encoding the light and heavy chains of the broadly neutralizing anti-HIV-1 antibody VRC01 are generated and encapsulated into lipid nanoparticles. Systemic administration of 1.4 mg kg-1 of mRNA into mice results in ∼170 µg ml-1 VRC01 antibody concentrations in the plasma 24 h post injection. Weekly injections of 1 mg kg-1 of mRNA into immunodeficient mice maintain trough VRC01 levels above 40 µg ml-1. Most importantly, the translated antibody from a single injection of VRC01 mRNA protects humanized mice from intravenous HIV-1 challenge, demonstrating that nucleoside-modified mRNA represents a viable delivery platform for passive immunotherapy against HIV-1 with expansion to a variety of diseases.


Subject(s)
Antibodies, Neutralizing/genetics , HIV-1/drug effects , Nucleosides/chemistry , RNA, Messenger/administration & dosage , Animals , Antibodies, Monoclonal/genetics , Broadly Neutralizing Antibodies , Drug Administration Schedule , Female , HIV Antibodies/biosynthesis , HIV Infections/immunology , HIV Infections/therapy , HIV-1/immunology , Humans , Immunization, Passive , Lipids/chemistry , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Nanoparticles/chemistry , RNA, Messenger/chemistry , RNA, Messenger/pharmacology , RNA, Messenger/therapeutic use
12.
Elife ; 42015 Sep 01.
Article in English | MEDLINE | ID: mdl-26327698

ABSTRACT

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered report describes the proposed replication plan of key experiments from 'Inhibition of bromodomain and extra terminal (BET) recruitment to chromatin as an effective treatment for mixed-lineage leukemia (MLL)-fusion leukemia' by Dawson and colleagues, published in Nature in 2011 (Dawson et al., 2011). The experiments to be replicated are those reported in Figures 2A, 3D, 4B, 4D and Supplementary Figures 11A-B and 16A. In this study, BET proteins were demonstrated as potential therapeutic targets for modulating aberrant gene expression programs associated with MLL-fusion leukemia. In Figure 2A, the BET bromodomain inhibitor I-BET151 was reported to suppress growth of cells harboring MLL-fusions compared to those with alternate oncogenic drivers. In Figure 3D, treatment of MLL-fusion leukemia cells with I-BET151 resulted in transcriptional suppression of the anti-apoptotic gene BCL2. Figures 4B and 4D tested the therapeutic efficacy of I-BET151 in vivo using mice injected with human MLL-fusion leukemia cells and evaluated disease progression following I-BET151 treatment. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.


Subject(s)
Chromatin/metabolism , Leukemia, Biphenotypic, Acute/therapy , Animals , Female , Humans , Male , Mice, SCID , Reproducibility of Results , Treatment Outcome
13.
PeerJ ; 3: e1232, 2015.
Article in English | MEDLINE | ID: mdl-26401448

ABSTRACT

The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative seeks to address growing concerns about reproducibility in scientific research by conducting replications of recent papers in the field of prostate cancer. This Registered Report describes the proposed replication plan of key experiments from "Androgen Receptor Splice Variants Determine Taxane Sensitivity in Prostate Cancer" by Thadani-Mulero and colleagues (2014) published in Cancer Research in 2014. The experiment that will be replicated is reported in Fig. 6A. Thadani-Mulero and colleagues generated xenografts from two prostate cancer cell lines; LuCaP 86.2, which expresses predominantly the ARv567 splice variant of the androgen receptor (AR), and LuCaP 23.1, which expresses the full length AR as well as the ARv7 variant. Treatment of the tumors with the taxane docetaxel showed that the drug inhibited tumor growth of the LuCaP 86.2 cells but not of the LuCaP 23.1 cells, indicating that expression of splice variants of the AR can affect sensitivity to docetaxel. The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative is a collaboration between the Prostate Cancer Foundation, the Movember Foundation and Science Exchange, and the results of the replications will be published by PeerJ.

14.
Immunity ; 34(2): 163-74, 2011 Feb 25.
Article in English | MEDLINE | ID: mdl-21349429

ABSTRACT

V(D)J gene segment recombination is linked to the cell cycle by the periodic phosphorylation and destruction of the RAG-2 protein at the G1-to-S cell cycle transition. To examine the function of this coupling, we constructed mice in which the phosphorylation site at threonine 490 of RAG-2 was mutated to alanine. The RAG-2(T490A) mutation uncoupled DNA cleavage from cell cycle and promoted aberrant recombination. Similar aberrant recombination products were observed in mice deficient in the Skp2 ubiquitin ligase subunit, which is required for periodic destruction of RAG-2. On a p53-deficient background, the RAG-2(T490A) mutation induced lymphoid malignancies characterized by clonal chromosomal translocations involving antigen receptor genes. Taken together, these observations provide a direct link between the periodic destruction of RAG-2 and lymphoid tumorigenesis. We infer that cell cycle control of the V(D)J recombinase limits the potential genomic damage that could otherwise result from RAG-mediated DNA cleavage.


Subject(s)
Cell Cycle , DNA-Binding Proteins/physiology , Gene Rearrangement , Genomic Instability , Lymphoma, Non-Hodgkin/genetics , Receptors, Antigen, T-Cell/genetics , Amino Acid Substitution , Animals , Base Sequence , DNA Breaks, Double-Stranded , DNA Repair , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Female , Gene Knock-In Techniques , Genes, p53 , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphorylation , Protein Processing, Post-Translational , S-Phase Kinase-Associated Proteins/genetics , Specific Pathogen-Free Organisms , T-Lymphocytes/metabolism , Translocation, Genetic
15.
Clin Cancer Res ; 15(21): 6602-8, 2009 Nov 01.
Article in English | MEDLINE | ID: mdl-19825945

ABSTRACT

PURPOSE: To investigate the impact of interferon-gamma-mediated upregulation of major histocompatibility complex class I expression on tumor-specific T-cell cytotoxicity and T-cell trafficking into neuroblastoma tumors in vivo. EXPERIMENTAL DESIGN: Restoration of major histocompatibility complex class I expression by interferon-gamma treatment enhances killing of neuroblastoma cells. To understand the potential of this approach in vivo, we developed a novel model of neuroblastoma in which NOD/scid/IL2R gamma(null) immunodeficient mice are engrafted with both human T cells and tumor cells. RESULTS: Here, we show enhanced killing of neuroblastoma cells by patient-derived, tumor-specific T cells in vitro. In addition, interferon-gamma treatment in vivo induces efficient upregulation of major histocompatibility complex class I expression on neuroblastoma tumor cells, and this is accompanied by significantly enhanced infiltration of T cells into the tumor. In a pilot clinical trial in patients with high-risk neuroblastoma, we similarly observed augmented T-cell trafficking into neuroblastoma nests in tumor biopsy specimens obtained from patients after 5 days of systemic interferon-gamma therapy. CONCLUSIONS: Interferon-gamma overcomes critical obstacles to the killing of human neuroblastoma cells by specific T cells. Together, these findings provide a rationale for the further testing of interferon-gamma as an approach for improving the efficacy of T cell-based therapies for neuroblastoma and other major histocompatibility complex class I-deficient malignancies. In addition, we describe a model that may expedite the preclinical screening of approaches aimed at augmenting T-cell trafficking into human tumors.


Subject(s)
Histocompatibility Antigens Class I/metabolism , Interferon-gamma/pharmacology , Lymphocytes, Tumor-Infiltrating/immunology , Neuroblastoma/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Cell Line, Tumor , Humans , Interferon-gamma/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Transplantation , Pilot Projects , Up-Regulation
16.
PLoS One ; 3(9): e3289, 2008 Sep 26.
Article in English | MEDLINE | ID: mdl-18818761

ABSTRACT

IL-18 has pleotropic effects on the activation of T cells during antigen presentation. We investigated the effects of human IL-18 on the engraftment and function of human T cell subsets in xenograft mouse models. IL-18 enhanced the engraftment of human CD8(+) effector T cells and promoted the development of xenogeneic graft versus host disease (GVHD). In marked contrast, IL-18 had reciprocal effects on the engraftment of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) in the xenografted mice. Adoptive transfer experiments indicated that IL-18 prevented the suppressive effects of Tregs on the development of xenogeneic GVHD. The IL-18 results were robust as they were observed in two different mouse strains. In addition, the effects of IL-18 were systemic as IL-18 promoted engraftment and persistence of human effector T cells and decreased Tregs in peripheral blood, peritoneal cavity, spleen and liver. In vitro experiments indicated that the expression of the IL-18Ralpha was induced on both CD4 and CD8 effector T cells and Tregs, and that the duration of expression was less sustained on Tregs. These preclinical data suggest that human IL-18 may have use as an adjuvant for immune reconstitution after cytotoxic therapies, and to augment adoptive immunotherapy, donor leukocyte infusions, and vaccine strategies.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Gene Expression Regulation , Interleukin-18/metabolism , T-Lymphocytes, Regulatory/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Female , Graft vs Host Disease/metabolism , Humans , Male , Mice , Mice, Inbred NOD , Mice, SCID , Models, Biological , Neoplasm Transplantation , T-Lymphocytes, Regulatory/metabolism
17.
J Immunol ; 181(4): 2855-68, 2008 Aug 15.
Article in English | MEDLINE | ID: mdl-18684977

ABSTRACT

The costimulatory requirements required for peripheral blood T regulatory cells (Tregs) are unclear. Using cell-based artificial APCs we found that CD28 but not ICOS, OX40, 4-1BB, CD27, or CD40 ligand costimulation maintained high levels of Foxp3 expression and in vitro suppressive function. Only CD28 costimulation in the presence of rapamycin consistently generated Tregs that consistently suppressed xenogeneic graft-vs-host disease in immunodeficient mice. Restimulation of Tregs after 8-12 days of culture with CD28 costimulation in the presence of rapamycin resulted in >1000-fold expansion of Tregs in <3 wk. Next, we determined whether other costimulatory pathways could augment the replicative potential of CD28-costimulated Tregs. We observed that while OX40 costimulation augmented the proliferative capacity of CD28-costimulated Tregs, Foxp3 expression and suppressive function were diminished. These studies indicate that the costimulatory requirements for expanding Tregs differ from those for T effector cells and, furthermore, they extend findings from mouse Tregs to demonstrate that human postthymic Tregs require CD28 costimulation to expand and maintain potent suppressive function in vivo.


Subject(s)
CD28 Antigens/metabolism , Lymphocyte Activation/immunology , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/immunology , Adoptive Transfer , Animals , CD28 Antigens/physiology , Cell Culture Techniques , Female , Graft vs Host Disease/immunology , Graft vs Host Disease/therapy , Humans , K562 Cells , Male , Mice , Mice, Inbred NOD , Mice, SCID , Signal Transduction/immunology , T-Lymphocytes, Regulatory/transplantation , Thymus Gland/cytology , Thymus Gland/immunology , Thymus Gland/metabolism
18.
J Clin Oncol ; 24(36): 5725-34, 2006 Dec 20.
Article in English | MEDLINE | ID: mdl-17179106

ABSTRACT

PURPOSE: Tumor immunosurveillance influences oncogenesis and tumor growth, but it remains controversial whether clinical failure of immunosurveillance is a result of lymphocyte dysfunction or tumor escape. In this study, our goal was to characterize the physiology of tumor immunosurveillance in children with high-risk neuroblastoma (HR-NBL). PATIENTS AND METHODS: Immunohistopathologic studies were carried out on 26 tumor samples from a cohort of HR-NBL patients diagnosed at Children's Hospital of Philadelphia for the 2-year period from May 2003 to May 2005. Blood from nine HLA-A2+ patients in this cohort was analyzed for T cells specific for the antiapoptotic protein survivin. RESULTS: Survivin protein was expressed by 26 of 26 tumors. In HLA-A2+ patients, circulating cytotoxic T lymphocytes (CTLs) specific for survivin were detected by peptide/major histocompatibility complex tetramer analysis in the blood of eight of nine children with HR-NBL at the time of diagnosis. Rather than being selectively rendered anergic in vivo, circulating survivin-specific CTLs were highly functional as shown by cytotoxicity and interferon gamma enzyme-linked immunospot assays in six of nine patients. Survivin-specific CD107a mobilization by T cells was found in five of five patients. By immunohistochemistry, tumor-infiltrating T cells were few or absent in 26 of 26 tumors. CONCLUSION: Children with HR-NBL harbor robust cellular immune responses to the universal tumor antigen survivin at the time of diagnosis, but intratumoral T cells are strikingly rare, suggesting a failure of cellular immunosurveillance. Efforts to develop novel therapies that increase T-cell trafficking into tumor nests are warranted.


Subject(s)
Immunologic Surveillance , Lymphocytes, Tumor-Infiltrating/immunology , Microtubule-Associated Proteins/metabolism , Neoplasm Proteins/metabolism , Neuroblastoma/immunology , Adolescent , Child , Child, Preschool , Cohort Studies , Female , Humans , Immunohistochemistry , Infant , Inhibitor of Apoptosis Proteins , Male , Risk Factors , Survivin
19.
Am J Physiol Regul Integr Comp Physiol ; 291(6): R1602-12, 2006 Dec.
Article in English | MEDLINE | ID: mdl-16873554

ABSTRACT

Progranulin (pgrn; granulin-epithelin precursor, PC-cell-derived growth factor, or acrogranin) is a multifunctional secreted glycoprotein implicated in tumorigenesis, development, inflammation, and repair. It is highly expressed in macrophage and monocyte-derived dendritic cells. Here we investigate its regulation in myeloid cells. All-trans retinoic acid (ATRA) increased pgrn mRNA levels in myelomonocytic cells (CD34(+) progenitors; monoblastic U-937; monocytic THP-1; progranulocytic HL-60; macrophage RAW 264.7) but not in nonmyeloid cells tested. Interleukin-4 impaired basal expression of pgrn in U-937. Differentiation agents DMSO, and, in U-937 only, phorbol ester [phorbol 12-myristate,13-acetate (PMA)] elevated pgrn mRNA expression late in differentiation, suggestive of roles for pgrn in more mature terminally differentiated granulocyte/monocytes rather than during growth or differentiation. The response of pgrn mRNA to ATRA differs in U-937 and HL-60 lineages. In U-937, ATRA and chemical differentiation agents greatly increased pgrn mRNA stability, whereas, in HL-60, ATRA accelerated pgrn mRNA turnover. The initial upregulation of pgrn mRNA after stimulation with ATRA was independent of de novo protein synthesis in U-937 but not HL-60. Chemical blockade of nuclear factor-kappaB (NF-kappaB) activation impaired ATRA-stimulated pgrn expression in HL-60 but not U-937, whereas in U-937 it blocked PMA-induced pgrn mRNA expression, suggestive of cell-specific roles for NF-kappaB in determining pgrn mRNA levels. We propose that: 1) ATRA regulates pgrn mRNA levels in myelomonocytic cells; 2) ATRA acts in a cell-specific manner involving the differential control of mRNA stability and differential requirement for NF-kappaB signaling; and 3) elevated pgrn mRNA expression is characteristic of more mature cells and does not stimulate differentiation.


Subject(s)
Gene Expression Regulation/physiology , Intercellular Signaling Peptides and Proteins/metabolism , Interleukin-4/administration & dosage , Myeloid Cells/cytology , Myeloid Cells/metabolism , Tretinoin/administration & dosage , Cell Differentiation/drug effects , Cell Line , Dose-Response Relationship, Radiation , Gene Expression Regulation/drug effects , Humans , Myeloid Cells/drug effects , Progranulins
20.
In Vitro Cell Dev Biol Anim ; 42(3-4): 58-62, 2006.
Article in English | MEDLINE | ID: mdl-16759149

ABSTRACT

To increase the efficiency of stable cell line establishment from primary ovarian cancer specimens, we simultaneously initiated cultures under multiple conditions, varying extracellular matrices and the inclusion of supplements (e.g., serum or serum albumin), while minimizing exposure to xenogeneic antigens (e.g., fetal calf serum). Primary cultures were initiated from 30 specimens; cell lines were established from 10 of these for a success rate of 33%. In some instances, multiple cell lines were established from the same specimen. Five lines were characterized extensively with respect to growth properties, antigen expression, and genomic alterations. Although these lines are all low-passage, marked heterogeneity was observed, even between lines derived from the same specimen. The culture approach outlined herein will facilitate generation of reagents useful for many aspects of ovarian cancer biology.


Subject(s)
Cell Culture Techniques , Cell Line , Ovarian Neoplasms/pathology , Animals , Cells, Cultured , Female , Humans , Tumor Cells, Cultured
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