ABSTRACT
BACKGROUND: Relapse remains a challenge after transplantation in pediatric patients with hematological malignancies. Myeloablative regimens used for disease control are associated with acute and long-term adverse effects. We used a CD45RA-depleted haploidentical graft for adoptive transfer of memory T cells combined with NK-cell addback and hypothesized that maximizing the graft-versus-leukemia (GVL) effect might allow for reduction in intensity of conditioning regimen. METHODS: In this phase II clinical trial (NCT01807611), 72 patients with hematological malignancies (complete remission (CR)1: 25, ≥ CR2: 28, refractory disease: 19) received haploidentical CD34 + enriched and CD45RA-depleted hematopoietic progenitor cell grafts followed by NK-cell infusion. Conditioning included fludarabine, thiotepa, melphalan, cyclophosphamide, total lymphoid irradiation, and graft-versus-host disease (GVHD) prophylaxis consisted of a short-course sirolimus or mycophenolate mofetil without serotherapy. RESULTS: The 3-year overall survival (OS) and event-free-survival (EFS) for patients in CR1 were 92% (95% CI:72-98) and 88% (95% CI: 67-96); ≥ CR2 were 81% (95% CI: 61-92) and 68% (95% CI: 47-82) and refractory disease were 32% (95% CI: 11-54) and 20% (95% CI: 6-40). The 3-year EFS for all patients in morphological CR was 77% (95% CI: 64-87) with no difference amongst recipients with or without minimal residual disease (P = 0.2992). Immune reconstitution was rapid, with mean CD3 and CD4 T-cell counts of 410/µL and 140/µL at day + 30. Cumulative incidence of acute GVHD and chronic GVHD was 36% and 26% but most patients with acute GVHD recovered rapidly with therapy. Lower rates of grade III-IV acute GVHD were observed with NK-cell alloreactive donors (P = 0.004), and higher rates of moderate/severe chronic GVHD occurred with maternal donors (P = 0.035). CONCLUSION: The combination of a CD45RA-depleted graft and NK-cell addback led to robust immune reconstitution maximizing the GVL effect and allowed for use of a submyeloablative, TBI-free conditioning regimen that was associated with excellent EFS resulting in promising long-term outcomes in this high-risk population. The trial is registered at ClinicalTrials.gov (NCT01807611).
Subject(s)
Hematologic Neoplasms , Hematopoietic Stem Cell Transplantation , Killer Cells, Natural , Memory T Cells , Transplantation Conditioning , Transplantation, Haploidentical , Humans , Female , Male , Killer Cells, Natural/transplantation , Killer Cells, Natural/immunology , Child , Adolescent , Transplantation, Haploidentical/methods , Child, Preschool , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cell Transplantation/adverse effects , Transplantation Conditioning/methods , Hematologic Neoplasms/therapy , Graft vs Host Disease/prevention & control , Graft vs Host Disease/etiology , Infant , Young Adult , Adult , Treatment Outcome , Graft vs Leukemia EffectABSTRACT
Lentiviral vector (LV)-based gene therapy holds promise for a broad range of diseases. Analyzing more than 280,000 vector integration sites (VISs) in 273 samples from 10 patients with X-linked severe combined immunodeficiency (SCID-X1), we discovered shared LV integrome signatures in 9 of 10 patients in relation to the genomics, epigenomics, and 3D structure of the human genome. VISs were enriched in the nuclear subcompartment A1 and integrated into super-enhancers close to nuclear pore complexes. These signatures were validated in T cells transduced with an LV encoding a CD19-specific chimeric antigen receptor. Intriguingly, the one patient whose VISs deviated from the identified integrome signatures had a distinct clinical course. Comparison of LV and gamma retrovirus integromes regarding their 3D genome signatures identified differences that might explain the lower risk of insertional mutagenesis in LV-based gene therapy. Our findings suggest that LV integrome signatures, shaped by common features such as genome organization, may affect the efficacy of LV-based cellular therapies.
Subject(s)
Genetic Vectors , X-Linked Combined Immunodeficiency Diseases , Humans , Genetic Vectors/genetics , Genetic Therapy , Retroviridae/genetics , X-Linked Combined Immunodeficiency Diseases/genetics , X-Linked Combined Immunodeficiency Diseases/therapy , T-LymphocytesABSTRACT
CRISPR-Cas9-based therapeutic genome editing approaches hold promise to cure a variety of human diseases. Recent findings demonstrate pre-existing immunity for the commonly used Cas orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans, which threatens the success of this powerful tool in clinical use. Thus, a comprehensive investigation and potential risk assessment are required to exploit the full potential of the system. Here, we investigated existence of immunity to SpCas9 and SaCas9 in control rhesus macaques (Macaca mulatta) alongside monkeys transplanted with either lentiviral transduced or CRISPR-SpCas9 ribonucleoprotein (RNP)-edited cells. We observed significant levels of Cas9 antibodies in the peripheral blood of all transplanted and non-transplanted control animals. Transplantation of ex vivo transduced or SpCas9-mediated BCL11A enhancer-edited cells did not alter the levels of Cas9 antibodies in rhesus monkeys. Following stimulation of peripheral blood cells with SpCas9 or SaCas9, neither Cas9-specific T cells nor cytokine induction were detected. Robust and durable editing frequencies and expression of high levels of fetal hemoglobin in BCL11A enhancer-edited rhesus monkeys with no evidence of an immune response (>3 years) provide an optimistic outlook for the use of ex vivo CRISPR-SpCas9 (RNP)-edited cells.
ABSTRACT
Autologous chimeric antigen receptor (CAR) T cells targeting the CD19 antigen have demonstrated a high complete response rate in relapsed/refractory B-cell malignancies. However, autologous CAR T cell therapy is not an option for all patients. Here we optimized conditions for clinical-grade manufacturing of allogeneic CD19-CAR T cells using CD45RA-depleted donor memory T cells (Tm) for a planned clinical trial. Tm were activated using the MACS GMP T Cell TransAct reagent and transduced in the presence of LentiBOOST with a clinical-grade lentiviral vector that encodes a 2nd generation CD19-CAR with a 41BB.zeta endodomain. Transduced T cells were transferred to a G-Rex cell culture device for expansion and harvested on day 7 or 8 for cryopreservation. The resulting CD19-CAR(Mem) T cells expanded on average 34.2-fold, and mean CAR expression was 45.5%. The majority of T cells were CD4+ and had a central memory or effector memory phenotype, and retained viral specificity. CD19-CAR(Mem) T cells recognized and killed CD19-positive target cells in vitro and had potent antitumor activity in an ALL xenograft model. Thus we have successfully developed a current good manufacturing practice-compliant process to manufacture donor-derived CD19-CAR(Mem) T cells. Our manufacturing process could be readily adapted for CAR(Mem) T cells targeting other antigens.
Subject(s)
Neoplasms , Receptors, Antigen, T-Cell , Humans , Antigens, CD19/genetics , Immunotherapy, Adoptive/methods , T-Lymphocytes , Cyclic GMP/metabolismABSTRACT
Current chimeric antigen receptor-modified (CAR) T-cell products are evaluated in bulk, without assessing functional heterogeneity. We therefore generated a comprehensive single-cell gene expression and T-cell receptor (TCR) sequencing data set using pre- and postinfusion CD19-CAR T cells from blood and bone marrow samples of pediatric patients with B-cell acute lymphoblastic leukemia. We identified cytotoxic postinfusion cells with identical TCRs to a subset of preinfusion CAR T cells. These effector precursor cells exhibited a unique transcriptional profile compared with other preinfusion cells, corresponding to an unexpected surface phenotype (TIGIT+, CD62Llo, CD27-). Upon stimulation, these cells showed functional superiority and decreased expression of the exhaustion-associated transcription factor TOX. Collectively, these results demonstrate diverse effector potentials within preinfusion CAR T-cell products, which can be exploited for therapeutic applications. Furthermore, we provide an integrative experimental and analytic framework for elucidating the mechanisms underlying effector development in CAR T-cell products. SIGNIFICANCE: Utilizing clonal trajectories to define transcriptional potential, we find a unique signature of CAR T-cell effector precursors present in preinfusion cell products. Functional assessment of cells with this signature indicated early effector potential and resistance to exhaustion, consistent with postinfusion cellular patterns observed in patients. This article is highlighted in the In This Issue feature, p. 2007.
Subject(s)
Receptors, Chimeric Antigen , T-Lymphocytes , Antigens, CD19 , Humans , Immunotherapy, Adoptive/methods , Receptors, Antigen, T-Cell/genetics , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolismABSTRACT
Individuals with age-related clonal hematopoiesis (CH) are at greater risk for hematologic malignancies and cardiovascular diseases. However, predictive preclinical animal models to recapitulate the spectrum of human CH are lacking. Through error-corrected sequencing of 56 human CH/myeloid malignancy genes, we identified natural CH driver mutations in aged rhesus macaques matching genes somatically mutated in human CH, with DNMT3A mutations being the most frequent. A CH model in young adult macaques was generated via autologous transplantation of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated gene-edited hematopoietic stem and progenitor cells (HSPCs), targeting the top human CH genes with loss-of-function (LOF) mutations. Long-term follow-up revealed reproducible and significant expansion of multiple HSPC clones with heterozygous TET2 LOF mutations, compared with minimal expansion of clones bearing other mutations. Although the blood counts of these CH macaques were normal, their bone marrows were hypercellular and myeloid-predominant. TET2-disrupted myeloid colony-forming units isolated from these animals showed a distinct hyperinflammatory gene expression profile compared with wild type. In addition, mature macrophages purified from the CH macaques showed elevated NLRP3 inflammasome activity and increased interleukin-1ß (IL-1ß) and IL-6 production. The model was used to test the impact of IL-6 blockage by tocilizumab, documenting a slowing of TET2-mutated expansion, suggesting that interruption of the IL-6 axis may remove the selective advantage of mutant HSPCs. These findings provide a model for examining the pathophysiology of CH and give insights into potential therapeutic interventions.
Subject(s)
Clonal Hematopoiesis , Dioxygenases , Humans , Young Adult , Animals , Aged , Clonal Hematopoiesis/genetics , Hematopoiesis/genetics , Interleukin-1beta/genetics , Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Macaca mulatta , CRISPR-Associated Protein 9 , Interleukin-6/genetics , Clone Cells , DNA-Binding Proteins/genetics , Dioxygenases/geneticsABSTRACT
T cells expressing CD19-specific chimeric antigen receptors (CD19-CARs) have potent antileukemia activity in pediatric and adult patients with relapsed and/or refractory B-cell acute lymphoblastic leukemia (B-ALL). However, not all patients achieve a complete response (CR), and a significant percentage relapse after CD19-CAR T-cell therapy due to T-cell intrinsic and/or extrinsic mechanisms. Thus, there is a need to evaluate new CD19-CAR T-cell products in patients to improve efficacy. We developed a phase 1/2 clinical study to evaluate an institutional autologous CD19-CAR T-cell product in pediatric patients with relapsed/refractory B-ALL. Here we report the outcome of the phase 1 study participants (n = 12). Treatment was well tolerated, with a low incidence of both cytokine release syndrome (any grade, n = 6) and neurotoxicity (any grade, n = 3). Nine out of 12 patients (75%) achieved a minimal residual disease-negative CR in the bone marrow (BM). High disease burden (≥40% morphologic blasts) before CAR T-cell infusion correlated with increased side effects and lower response rate, but not with CD19-CAR T-cell expansion. After infusion, CD8+ CAR T cells had a proliferative advantage over CD4+ CAR T cells and at peak expansion, had an effector memory phenotype with evidence of antigen-driven differentiation. Patients that proceeded to allogeneic hematopoietic cell transplantation (AlloHCT) had sustained, durable responses. In summary, the initial evaluation of our institutional CD19-CAR T-cell product demonstrates safety and efficacy while highlighting the impact of pre-infusion disease burden on outcomes. This trial was registered at www.clinicaltrials.gov as #NCT03573700.
Subject(s)
Burkitt Lymphoma , Lymphoma, B-Cell , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Receptors, Chimeric Antigen , Humans , Antigens, CD19 , CD8-Positive T-Lymphocytes , Cost of Illness , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , T-LymphocytesABSTRACT
Pediatric patients with high-risk hematologic malignancies who experience relapse after a prior allogeneic hematopoietic cell transplant (HCT) have an exceedingly poor prognosis. A second allogeneic HCT offers the potential for long-term cure but carries high risks of both subsequent relapse and HCT-related morbidity and mortality. Using haploidentical donors for HCT (haploHCT) can expand the donor pool and potentially enhance the graft-versus-leukemia effect but is accompanied by a risk of graft-versus-host disease (GVHD). The goal of this protocol was to intensify the antileukemia effect of haploHCT for pediatric patients with hematologic malignancies that relapsed after prior allogeneic HCT, while limiting regimen-associated toxicities. This phase II clinical trial evaluated a sub-myeloablative preparative regimen consisting of anti-thymocyte globulin, clofarabine, cytarabine, busulfan, and cyclophosphamide, in combination with plerixafor to sensitize leukemic blasts. Participants received a mobilized peripheral blood unmanipulated haploidentical donor graft with one dose of post-transplant cyclophosphamide as GVHD prophylaxis, followed by natural killer (NK) cell addback. Here we report the clinical outcomes and immune reconstitution of 17 participants treated on the study and 5 additional patients treated on similar single-patient treatment plans. Of the 22 participants analyzed, 12 (55%) had active disease at the time of HCT. The regimen provided robust immune reconstitution, with 21 participants (95%) experiencing neutrophil engraftment at a median of 14 days after HCT. In this high-risk population, the overall survival was 45% (95% confidence interval [CI], 24%-64%), with a 12-month event-free survival of 31% (95% CI, 14%-51%) and cumulative incidence of relapse at 12 months of 50% (95% CI, 27%-69%). Four participants (18%) remain in remission at >5 years follow-up. Expected HCT-related organ-specific toxicities were observed, and 13 participants (59%) experienced acute or chronic GVHD. This intensified but sub-myeloablative regimen, followed by a high-dose unmanipulated haploidentical graft, post-transplantation cyclophosphamide, and NK cell infusion, resulted in adequate immune reconstitution but failed to overcome the elevated risks of relapse and treatment-related morbidity in this high-risk population.
Subject(s)
Hematologic Neoplasms , Hematopoietic Stem Cell Transplantation , Child , Cyclophosphamide/therapeutic use , Graft vs Host Disease/prevention & control , Hematologic Neoplasms/epidemiology , Hematopoietic Stem Cell Transplantation/adverse effects , Heterocyclic Compounds , Humans , RecurrenceABSTRACT
CD19-CAR T cell therapy has evolved into the standard of care for relapsed/refractory B cell acute lymphoblastic leukemia (ALL); however, limited persistence of the CAR T cells enables tumor relapse for many patients. To gain a deeper understanding of the molecular characteristics associated with CAR T cell differentiation, we performed longitudinal genome-wide DNA methylation profiling of CD8+ CD19-CAR T cells post-infusion in ALL patients. We report that CAR T cells undergo a rapid and broad erasure of repressive DNA methylation reprograms at effector-associated genes. The CAR T cell post-infusion changes are further characterized by repression of genes (e.g., TCF7 and LEF1) associated with memory potential and a DNA methylation signature (e.g., demethylation at CX3CR1, BATF, and TOX) demarcating a transition toward exhaustion-progenitor T cells. Thus, CD19-CAR T cells undergo exhaustion-associated DNA methylation programming, indicating that efforts to prevent this process may be an attractive approach to improve CAR T cell efficacy.
Subject(s)
Antigens, CD19/immunology , CD8-Positive T-Lymphocytes/immunology , DNA Methylation , Gene Expression Regulation, Neoplastic , Immunotherapy, Adoptive/methods , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Receptors, Antigen, T-Cell/immunology , Adolescent , Adult , Child , Child, Preschool , Female , Humans , Infant , Male , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Young AdultABSTRACT
Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress γ-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and ß-thalassemia, although the optimal technical approaches and limiting toxicities are not yet fully defined. We disrupted an HBG1/HBG2 gene promoter motif that is bound by the transcriptional repressor BCL11A. Electroporation of Cas9 single guide RNA ribonucleoprotein complex into normal and SCD donor CD34+ hematopoietic stem and progenitor cells resulted in high frequencies of on-target mutations and the induction of HbF to potentially therapeutic levels in erythroid progeny generated in vitro and in vivo after transplantation of hematopoietic stem and progenitor cells into nonobese diabetic/severe combined immunodeficiency/Il2rγ-/-/KitW41/W41 immunodeficient mice. On-target editing did not impair CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages at 16 to 17 weeks after xenotransplantation. No off-target mutations were detected by targeted sequencing of candidate sites identified by circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), an in vitro genome-scale method for detecting Cas9 activity. Engineered Cas9 containing 3 nuclear localization sequences edited human hematopoietic stem and progenitor cells more efficiently and consistently than conventional Cas9 with 2 nuclear localization sequences. Our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a mechanism-based approach to induce HbF for treating hemoglobinopathies.
Subject(s)
Fetal Hemoglobin/genetics , Gene Editing , gamma-Globins/genetics , Anemia, Sickle Cell/genetics , Animals , Base Sequence , CRISPR-Cas Systems , Disease Models, Animal , Erythropoiesis/genetics , Gene Expression Regulation , Gene Targeting , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Hemoglobinopathies/genetics , Heterografts , Humans , Immunophenotyping , Mice , Models, Biological , Mutation , Promoter Regions, Genetic , RNA, Guide, Kinetoplastida , Sequence DeletionABSTRACT
Purpose: NKG2D ligands (NKG2DL) are expressed on various tumor types and immunosuppressive cells within tumor microenvironments, providing suitable targets for cancer therapy. Various immune cells express NKG2D receptors, including natural killer (NK) cells and CD8+ T cells. Interactions between NKG2DL and NKG2D receptors are essential for NK-cell elimination of osteosarcoma tumor-initiating cells. In this report, we used NKG2D-NKG2DL interactions to optimize an immunotherapeutic strategy against osteosarcoma. We evaluated in vitro and in vivo the safety and cytotoxic capacity against osteosarcoma cells of CD45RA- memory T cells expressing an NKG2D-4-1BB-CD3z chimeric antigen receptor (CAR).Experimental Design: CD45RA- cells from healthy donors were transduced with NKG2D CARs containing 4-1BB and CD3z signaling domains. NKG2D CAR expression was analyzed by flow cytometry. In vitro cytotoxicity of NKG2D-CAR+ CD45RA- T cells against osteosarcoma was evaluated by performing conventional 4-hour europium-TDA release assays. For the in vivo orthotopic model, 531MII YFP-luc osteosarcoma cells were used as targets in NOD-scid IL2Rgnull mice.Results: Lentiviral transduction of NKG2D-4-1BB-CD3z markedly increased NKG2D surface expression in CD45RA- cells. Genetic stability was preserved in transduced cells. In vitro, NKG2D-CAR+ memory T cells showed significantly increased cytolytic activity than untransduced cells against osteosarcoma cell lines, while preserving the integrity of healthy cells. NKG2D-CAR+ memory T cells had considerable antitumor activity in a mouse model of osteosarcoma, whereas untransduced T cells were ineffective.Conclusions: Our results demonstrate NKG2D-4-1BB-CD3z CAR-redirected memory T cells target NKG2DL-expressing osteosarcoma cells in vivo and in vitro and could be a promising immunotherapeutic approach for patients with osteosarcoma. Clin Cancer Res; 23(19); 5824-35. ©2017 AACR.
Subject(s)
NK Cell Lectin-Like Receptor Subfamily K/genetics , Osteosarcoma/therapy , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/immunology , Animals , Cell Line, Tumor , Female , Flow Cytometry , Gene Expression Regulation, Neoplastic/genetics , Humans , Immunotherapy , Killer Cells, Natural/immunology , Killer Cells, Natural/pathology , Lentivirus/genetics , Mice , NK Cell Lectin-Like Receptor Subfamily K/therapeutic use , Neoplastic Stem Cells/immunology , Osteosarcoma/genetics , Osteosarcoma/immunology , Osteosarcoma/pathology , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/therapeutic use , Signal Transduction/genetics , Signal Transduction/immunology , Transduction, Genetic , Xenograft Model Antitumor AssaysABSTRACT
The use of bone marrow-derived mesenchymal stromal cells (BMSC) in the treatment of alloimmune and autoimmune conditions has generated much interest, yet an understanding of the therapeutic mechanism remains elusive. We therefore explored immune modulation by a clinical-grade BMSC product in a model of human-into-mouse xenogeneic graft-versus-host disease (x-GVHD) mediated by human CD4(+) Th1 cells. BMSC reversed established, lethal x-GVHD through marked inhibition of Th1 cell effector function. Gene marking studies indicated BMSC engraftment was limited to the lung; furthermore, there was no increase in regulatory T cells, thereby suggesting a paracrine mechanism of BMSC action. BMSC recipients had increased serum CD73 expressing exosomes that promoted adenosine accumulation ex vivo. Importantly, immune modulation mediated by BMSC was fully abrogated by pharmacologic therapy with an adenosine A2A receptor antagonist. To investigate the potential clinical relevance of these mechanistic findings, patient serum samples collected pre- and post-BMSC treatment were studied for exosome content: CD73 expressing exosomes promoting adenosine accumulation were detected in post-BMSC samples. In conclusion, BMSC effectively modulate experimental GVHD through a paracrine mechanism that promotes adenosine-based immune suppression.
Subject(s)
Adenosine A2 Receptor Antagonists/pharmacology , Mesenchymal Stem Cells/immunology , Signal Transduction/immunology , Th1 Cells/immunology , Animals , Bone Marrow/drug effects , Bone Marrow/immunology , Coculture Techniques , Graft vs Host Disease/immunology , Humans , Mesenchymal Stem Cells/drug effects , Mice , Mice, Inbred NOD , Mice, SCID , Signal Transduction/drug effects , Th1 Cells/drug effectsABSTRACT
We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.
Subject(s)
Cell Tracking/methods , Fluorescent Antibody Technique/methods , Hematopoietic Stem Cells/chemistry , Hematopoietic Stem Cells/cytology , Microscopy, Confocal/methods , Microscopy, Fluorescence, Multiphoton/methods , Animals , Clone Cells/chemistry , Clone Cells/cytology , Female , Hematopoietic Stem Cell Transplantation/methods , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Mice , Mice, Inbred C57BLABSTRACT
Myeloid derived suppressor cells (MDSCs) play a critical role in suppression of immune responses in cancer and inflammation. Here, we describe how regulation of Bcl2a1 by cytokines controls the suppressor function of CD11b(+) Gr-1(high) granulocytic MDSCs. Coculture of CD11b(+) Gr-1(high) granulocytic MDSCs with antigen-stimulated T cells and simultaneous blockade of IFN-γ by the use of anti-IFN-γ blocking antibody, IFN-γ(-/-) effector T cells, IFN-γR(-/-) MDSCs or STAT1(-/-) MDSCs led to upregulation of Bcl2a1 in CD11b(+) Gr-1(high) cells, improved survival, and enhanced their suppressor function. Molecular studies revealed that GM-CSF released by antigen-stimulated CD8(+) T cells induced Bcl2a1 upregulation, which was repressed in the presence of IFN-γ by a direct interaction of phosphorylated STAT-1 with the Bcl2a1 promotor. Bcl2a1 overexpressing granulocytic MDSCs demonstrated prolonged survival and enhanced suppressor function in vitro. Our data suggest that IFN-γ/ STAT1-dependent regulation of Bcl2a1 regulates survival and thereby suppressor function of granulocytic MDSCs.
Subject(s)
Apoptosis/immunology , CD11b Antigen/immunology , Interferon-gamma/immunology , Myeloid Cells/immunology , Proto-Oncogene Proteins c-bcl-2/immunology , Receptors, Chemokine/immunology , Animals , CD8-Positive T-Lymphocytes/immunology , Cell Line , Female , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Minor Histocompatibility Antigens , STAT1 Transcription Factor/immunology , Up-Regulation/immunologyABSTRACT
Retroviral vectors are an efficient and widely employed means of introducing an exogenous expression cassette into target cells. These vectors have been shown to integrate semi-randomly into the cellular genome, and can be associated with genotoxicity due to impact on expression of proximate genes. Therefore, efficient and accurate integration site analysis, while quantifying contributions of individual vector-containing clones, is desirable. Linear amplification-mediated polymerase chain reaction (LAM-PCR) is a widely used technique for identifying integrated proviral and host genomic DNA junctions. However, LAM-PCR is subject to selection bias inherent in the reliance of the assay on the presence of a restriction enzyme-cutting site adjacent to a retrievable integration site, and it is further limited by an inability to discriminate prior to sequencing between the flanking genomic DNA of interest and uninformative internal vector DNA. We report a modified restriction enzyme-free LAM-PCR (Re-free LAM-PCR) approach that is less time and labor intensive compared to conventional LAM-PCR, but in contrast to some other nonrestrictive methods, compares in efficiency and sensitivity, excludes retrieval of uninformative internal vector sequences, and allows retrieval of integration sites unbiased by the presence of nearby restriction sites. However, we report that Re-free LAM-PCR remains inaccurate for quantitation of the relative contributions of individual integration site-containing clones in a polyclonal setting, suggesting that bias in LAM-PCR retrieval of integration sites is not wholly explained by restriction enzyme-related factors.
Subject(s)
Genetic Therapy/methods , Genetic Vectors/genetics , Polymerase Chain Reaction/methods , Retroviridae/genetics , Virus Integration/genetics , Binding Sites/genetics , Blotting, Southern , Flow Cytometry , Genetic Vectors/therapeutic use , Restriction Mapping , Sensitivity and SpecificityABSTRACT
We previously reported the development of a lethal myeloid sarcoma in a non-human primate model utilizing retroviral vectors to genetically modify hematopoietic stem and progenitor cells. This leukemia was characterized by insertion of the vector provirus into the BCL2A1 gene, with resultant BCL2A1 over-expression. There is little information on the role of this anti-apoptotic member of the BCL2 family in hematopoiesis or leukemia induction. Therefore we studied the impact of Bcl2a1a lentiviral over-expression on murine hematopoietic stem and progenitor cells. We demonstrated the anti-apoptotic function of this protein in hematopoietic cells, but did not detect any impact of Bcl2a1a on in vitro cell growth or cell cycle kinetics. In vivo, we showed a higher propensity of HSCs over-expressing Bcl2a1a to engraft and contribute to hematopoiesis. Mice over-expressing Bcl2a1a in the hematologic compartment eventually developed an aggressive malignant disease characterized as a leukemia/lymphoma of B-cell origin. Secondary transplants carried out to investigate the primitive origin of the disease revealed the leukemia was transplantable. Thus, Bcl2a1 should be considered as a proto-oncogene with a potential role in both lymphoid and myeloid leukemogenesis, and a concerning site for insertional activation by integrating retroviral vectors utilized in hematopoietic stem cell gene therapy.
Subject(s)
Apoptosis/genetics , Cell Transformation, Neoplastic/genetics , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Proto-Oncogene Proteins c-bcl-2/genetics , Animals , Cell Line , Gene Expression , Hematopoiesis/genetics , Humans , Leukemia/genetics , Leukemia/pathology , Mice , Minor Histocompatibility Antigens , Proto-Oncogene Mas , Transduction, Genetic , Tumor Burden/geneticsABSTRACT
We demonstrate a methodology for tracing the clonal history of hematopoietic stem and progenitor cells (HSPCs) behavior in live tissues in 4 dimensions (4D). This integrates genetic combinatorial marking using lentiviral vectors encoding various fluorescent proteins (FPs) with advanced imaging methods. Five FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were concurrently used to create a diverse palette of color-marked cells. A key advantage of imaging using a confocal/2-photon hybrid microscopy approach is the simultaneous assessment of uniquely 5FP-marked cells in conjunction with structural components of the tissues at high resolution. Volumetric analyses revealed that spectrally coded HSPC-derived cells can be detected noninvasively in various intact tissues, including the bone marrow, for extensive periods of time after transplantation. Live studies combining video-rate multiphoton and confocal imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner. This methodology has applications in the understanding of clonal architecture in normal and perturbed hematopoiesis.
Subject(s)
Clonal Evolution/physiology , Fluorescent Dyes/analysis , Hematopoietic Stem Cells/ultrastructure , Microscopy, Fluorescence, Multiphoton , Stem Cells/ultrastructure , Animals , Cell Tracking/methods , Cells, Cultured , Clone Cells/cytology , Female , Fluorescent Dyes/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Imaging, Three-Dimensional , Mice , Mice, Inbred C57BL , Microscopy, Confocal/methods , Microscopy, Fluorescence, Multiphoton/methods , NIH 3T3 Cells , Staining and Labeling/methods , Stem Cells/cytology , Stem Cells/metabolismABSTRACT
The ability to expand hematopoietic stem and progenitor cells (HSPCs) in vitro will enhance the success of a wide range of transplant-related therapies. PTEN (phosphatase and tensin homologue deleted on chromosome 10) has been implicated as a regulator of murine HSPC self-renewal, but little is understood about the role of PTEN in human HSPC regulation. We tested the impact of transient small interfering RNA (siRNA)-induced inhibition of PTEN expression in human CD34(+) cells on their cell cycle profile, their susceptibility to retroviral transduction, and their ability to self-renew and repopulate nonobese diabetic/severe combined immunodeficiency disease with interleukin-2 receptor γ-chain deficiency mice. Reduced PTEN messenger RNA and protein levels were confirmed in PTEN siRNA-treated CD34(+) cells compared with control siRNA-treated CD34(+) cells. Transient silencing of PTEN in CD34(+) cells promoted their entry into cell cycle, and increased their expansion in vitro compared with control siRNA-treated CD34(+) cells. When these cells were transduced with retroviral vectors, transduction efficiencies in the bulk CD34(+) cells transfected with PTEN siRNA were significantly higher compared with CD34(+) cells transfected with a control siRNA. Transient PTEN suppression in CD34(+) cells also increased their proliferation and engraftment potential in nonobese diabetic/severe combined immunodeficiency disease with interleukin-2 receptor γ-chain deficiency mice, and maintained their multilineage differentiation capacity in vivo. No mice developed myeloproliferative disorders or leukemias. Similar to findings with murine HSPC, PTEN may also promote quiescence of human HSPC. With optimization of technologies for transfer of siRNA in primary CD34(+) cells, this approach may facilitate investigations into the mechanisms underlying HSPC self-renewal, and could find clinical applications in gene therapy protocols.
Subject(s)
Antigens, CD34/metabolism , Gene Silencing , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , PTEN Phosphohydrolase/deficiency , Animals , Cell Cycle , Cell Proliferation , Humans , Mice , Mice, SCID , PTEN Phosphohydrolase/genetics , RNA, Small Interfering/geneticsABSTRACT
Cooperation of multiple mutations is thought to be required for cancer development. In previous studies, murine myeloid leukemias induced by transducing wild-type bone marrow progenitors with a SRY sex determining region Y-box 4 (Sox4)-expressing retrovirus frequently carried proviral insertions at Sfpi1, decreasing its mRNA levels, suggesting that reduced Sfpi1 expression cooperates with Sox4 in myeloid leukemia induction. In support of this hypothesis, we show here that mice receiving Sox4 virus-infected Sfpi1(ko/+) bone marrow progenitors developed myeloid leukemia with increased penetrance and shortened latency. Interestingly, Sox4 expression further decreased Sfpi1 transcription. Ectopic SOX4 expression reduced endogenous PU.1 mRNA levels in HL60 promyelocytes, and decreased Sfpi1 mRNA levels were also observed in the spleens of leukemic and preleukemic mice receiving Sox4 virus-infected wild-type bone marrow cells. In addition, Sox4 protein bound to a critical upstream regulatory element of Sfpi1 in ChIP assays. Such cooperation probably occurs in de novo human acute myeloid leukemias, as an analysis of 285 acute myeloid leukemia patient samples found a significant negative correlation between SOX4 and PU.1 expression. Our results establish a novel cooperation between Sox4 and reduced Sfpi1 expression in myeloid leukemia development and suggest that SOX4 could be an important new therapeutic target in human acute myeloid leukemia.