ABSTRACT
Using an acute promyelocytic leukemia (APL) preclinical model, we show that oncogene-specific PCR (Polymerase Chain Reaction)-based assays allow to evaluate the efficacy of immunotherapy combining all-trans retinoic acid (ATRA) and a DNA-based vaccine targeting the promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) oncogene. Kaplan-Meier survival analysis according to the peripheral blood PML-RARα normalized copy number (NCN) clearly shows that ATRA + DNA-treated mice with an NCN lower than 10 (43%) formed the group with a highly significant (p < 0.0001) survival advantage. Furthermore, a PCR assay was used to assess various tissues and organs for the presence of PML-RARα-positive cells in long-term survivors (n = 15). As expected, the majority of mice (n = 10) had no measurable tissue level of PML-RARα. However, five mice showed a weak positive signal in both the brain and spleen (n = 2), in the brain only (n = 2) and in the spleen only (n = 1). Thus tracking the oncogene-positive cells in long-term survivors reveals for the first time that extramedullary PML-RARα-positive cell reservoirs such as the brain may persist and be involved in relapses.
Subject(s)
Immunotherapy , Leukemia, Promyelocytic, Acute/therapy , Oncogene Proteins, Fusion/metabolism , Tretinoin/therapeutic use , Vaccines, DNA/therapeutic use , Animals , Brain/cytology , Gene Dosage , Kaplan-Meier Estimate , Leukemia, Promyelocytic, Acute/mortality , Mice , Mice, Transgenic , Neoplasm Proteins/therapeutic use , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/immunology , Spleen/cytology , Treatment OutcomeABSTRACT
DNA vaccination and all-trans retinoic acid (ATRA) result in a survival advantage in a mouse model of acute promyelocytic leukemia (APL). Depletion of CD4(+) or CD8(+) cells abolished this effect. CD4(+) depletions of long-term survivors resulted in relapse and death within 3 months, thus demonstrating the need of both CD4(+) and CD8(+) subsets for the generation of DNA-driven antileukemic immune responses and underscoring a crucial role of CD4(+) cells in the maintenance of durable remissions. Degranulation and cytotoxic carboxyfluorescein diacetate succinimidyl ester-based assays showed major histocompatibility complex-restricted APL-specific T cell-mediated immune responses. Sorted APL-specific CD8(+)CD107a(+) T cells showed an increase of antileukemic activity. Effectors from ATRA + DNA-treated mice were shown to secrete interferon-gamma when stimulated with either APL cells or peptides from the promyelocytic leukemia-RARalpha vaccine-derived sequences as detected by ELISpot assays. Our results demonstrate that DNA vaccination with ATRA confers the effective boosting of interferon-gamma-producing and cytotoxic T cells in the leukemic mice.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Immunity, Cellular , Leukemia, Promyelocytic, Acute/therapy , Tretinoin/administration & dosage , Vaccines, DNA/administration & dosage , Animals , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/pathology , Combined Modality Therapy , Disease Models, Animal , Humans , Immunity, Cellular/drug effects , Immunity, Cellular/genetics , Leukemia, Promyelocytic, Acute/immunology , Leukemia, Promyelocytic, Acute/pathology , Lymphocyte Activation/drug effects , Lymphocyte Activation/genetics , Mice , Oncogene Proteins, Fusion/administration & dosage , Oncogene Proteins, Fusion/genetics , Survival Analysis , Treatment Outcome , Tumor Cells, Cultured , Vaccines, DNA/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Myelodysplastic syndromes (MDS) are clonal stem cell hematologic disorders that evolve to acute myeloid leukemia (AML) and thus model multistep leukemogenesis. Activating RAS mutations and overexpression of BCL-2 are prognostic features of MDS/AML transformation. Using NRASD12 and BCL-2, we created two distinct models of MDS and AML, where human (h)BCL-2 is conditionally or constitutively expressed. Our novel transplantable in vivo models show that expression of hBCL-2 in a primitive compartment by mouse mammary tumor virus-long terminal repeat results in a disease resembling human MDS, whereas the myeloid MRP8 promoter induces a disease with characteristics of human AML. Expanded leukemic stem cell (Lin(-)/Sca-1(+)/c-Kit(+)) populations and hBCL-2 in the increased RAS-GTP complex within the expanded Sca-1(+) compartment are described in both MDS/AML-like diseases. Furthermore, the oncogenic compartmentalizations provide the proapoptotic versus antiapoptotic mechanisms, by activating extracellular signal-regulated kinase and AKT signaling, in determination of the neoplastic phenotype. When hBCL-2 is switched off with doxycycline in the MDS mice, partial reversal of the phenotype was observed with persistence of bone marrow blasts and tissue infiltration as RAS recruits endogenous mouse (m)BCL-2 to remain active, thus demonstrating the role of the complex in the disease. This represents the first in vivo progression model of MDS/AML dependent on the formation of a BCL-2:RAS-GTP complex. The colocalization of BCL-2 and RAS in the bone marrow of MDS/AML patients offers targeting either oncogene as a therapeutic strategy.
Subject(s)
Genes, bcl-2 , Genes, ras , Myelodysplastic Syndromes/genetics , Animals , Bone Marrow Transplantation , Cell Transplantation , Colony-Forming Units Assay , Disease Models, Animal , Disease Progression , Immunophenotyping , Leukemia/genetics , Leukemia, Myeloid/genetics , Mice , Mice, Transgenic , Microscopy, Confocal , Myelodysplastic Syndromes/pathology , Myelodysplastic Syndromes/physiopathology , SpleenABSTRACT
Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop acute myelogenous leukemia (AML). The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is loss of chromosomal material (genomic instability). Using our two-step mouse model for myeloid leukemic disease progression involving overexpression of human mutant NRAS and BCL2 genes, we show that there is a stepwise increase in the frequency of DNA damage leading to an increased frequency of error-prone repair of double-strand breaks (DSB) by nonhomologous end-joining. There is a concomitant increase in reactive oxygen species (ROS) in these transgenic mice with disease progression. Importantly, RAC1, an essential component of the ROS-producing NADPH oxidase, is downstream of RAS, and we show that ROS production in NRAS/BCL2 mice is in part dependent on RAC1 activity. DNA damage and error-prone repair can be decreased or reversed in vivo by N-acetyl cysteine antioxidant treatment. Our data link gene abnormalities to constitutive DNA damage and increased DSB repair errors in vivo and provide a mechanism for an increase in the error rate of DNA repair with MDS disease progression. These data suggest treatment strategies that target RAS/RAC pathways and ROS production in human MDS/AML.
Subject(s)
DNA Damage , DNA Repair , Genomic Instability , Leukemia, Myeloid/genetics , Reactive Oxygen Species/metabolism , Animals , Disease Models, Animal , Disease Progression , Genes, bcl-2 , Genes, ras , Leukemia, Myeloid/metabolism , Leukemia, Myeloid/pathology , Mice , Mice, TransgenicABSTRACT
Human cytomegalovirus has evolved multiple strategies to interfere with immune recognition by the host. A variety of mechanisms affect antigen presentation by major histocompatibility complex class I molecules resulting in a reduced class I cell-surface expression. This downregulation is expected to trigger natural killer (NK) cytotoxicity, requiring counteraction by the virus to establish long-term infection. Here we describe that the human cytomegalovirus gpUS6 protein, which has been demonstrated to downregulate the expression of human leukocyte antigen (HLA) class I and the presentation of cytotoxic T lymphocyte epitopes by blocking transporter associated with antigen presentation (TAP function), does not affect the ability of HLA-E to inhibit NK cell mediated lysis of K-562 cells by interaction with CD94/NKG2A expressed on NK cells. Cell surface expression and function of HLA-E is not altered although gpUS6 inhibits TAP-dependent peptide transport by 95%. Moreover, HLA-E molecules presenting HLA class I signal sequence-derived peptides are functionally detectable on transfected TAP-deficient RMA-S cells.
Subject(s)
ATP-Binding Cassette Transporters/antagonists & inhibitors , HLA Antigens/immunology , Histocompatibility Antigens Class I/immunology , Killer Cells, Natural/immunology , RNA-Binding Proteins/physiology , Viral Proteins/physiology , ATP Binding Cassette Transporter, Subfamily B, Member 2 , Animals , Antigen Presentation , Antigens, CD/immunology , Cytotoxicity, Immunologic , Gene Expression Regulation , Genes, MHC Class I , HLA Antigens/biosynthesis , HLA-A2 Antigen/chemistry , HLA-A2 Antigen/immunology , Humans , K562 Cells , Lectins, C-Type/immunology , Lymphocyte Activation , Mice , NK Cell Lectin-Like Receptor Subfamily C , NK Cell Lectin-Like Receptor Subfamily D , Peptide Fragments/immunology , Peptide Fragments/metabolism , Receptors, Immunologic/immunology , Receptors, Natural Killer Cell , Recombinant Fusion Proteins/immunology , Transfection , HLA-E AntigensABSTRACT
Nonclassical major histocompatibility complex (MHC) class I human leukocyte antigen E (HLA-E) and HLA-G molecules differ from classical ones by specific patterns of transcription, protein expression, and immunotolerant functions. The HLA-G molecule can be expressed as four membrane-bound (HLA-G1 to -G4) and three soluble (HLA-G5 to -G7) proteins upon alternative splicing of its primary transcript. In this study, we describe a new set of monoclonal antibodies (mAbs) called MEM-G/01, -G/04, -G/09, -G/13, MEM-E/02, and -E/06 recognizing HLA-G or HLA-E. The pattern of reactivity of these mAbs were analyzed on transfected cells by flow cytometry, Western blotting, and immunochemistry. MEM-G/09 and -G/13 mAbs react exclusively with native HLA-G1 molecules, as the 87G mAb. MEM-G/01 recognizes (similar to the 4H84 mAb) the denatured HLA-G heavy chain of all isoforms, whereas MEM-G/04 recognizes selectively denatured HLA-G1, -G2, and -G5 isoforms. MEM-E/02 and -E/06 mAbs bind the denatured and cell surface HLA-E molecules, respectively. These mAbs were then used to analyze the expression of HLA-G and HLA-E on freshly isolated cytotrophoblast cells, on the JEG-3 placental tumor cell line, and on cryopreserved and paraffin-embedded serial sections of trophoblast tissue. These new mAbs represent valuable tools to study the expression of HLA-G and HLA-E molecules in cells and tissues under normal and pathologic conditions.