ABSTRACT
Flt3 ligand mobilizes dendritic cells (DCs) into blood, allowing generation in vivo of large numbers of DCs for immunotherapy. These immature DCs can be rapidly activated by soluble CD40 ligand (CD40L). We developed a novel overnight method using these cytokines to produce DCs for cancer immunotherapy. Flt3 ligand-mobilized DCs (FLDCs) were isolated, activated with CD40L, loaded with antigenic peptides from influenza matrix protein, hepatitis B core antigen, NY-ESO-1, MAGE-A4, and MAGE-A10, and injected into patients with resected melanoma. Three injections were given at 4-week intervals. Study end points included antigen-specific immune responses (skin reactions to peptides alone or peptide-pulsed FLDCs; circulating T-cell responses), safety, and toxicity. No patient had a measurable tumor. Six patients were entered. FLDCs were obtained, enriched, and cultured under Good Manufacturing Practice grade conditions. Overnight culture with soluble CD40L caused marked up-regulation of activation markers (CD83 and HLA-DR). These FLDCs were functional and able to stimulate antigen-specific T cells in vitro. No significant adverse events were attributable to FLDCs. Peptide-pulsed FLDCs caused strong local skin reactions up to 60 mm diameter with intense perivascular infiltration of T cells, exceeding those seen in our previous peptide-based protocols. Antigen-specific blood T-cell responses were induced, including responses to an antigen for which the patients were naive (hepatitis B core antigen) and MAGE-A10. MAGE-A10-specific T cells with a skewed T-cell receptor repertoire were detected in 1 patient in blood ex vivo and from tumor biopsies. Vaccination with FLDCs pulsed with peptides is safe and primes immune responses to cancer antigens.
Subject(s)
CD40 Ligand/immunology , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/therapeutic use , Dendritic Cells/immunology , Melanoma/therapy , Membrane Proteins/immunology , Skin Neoplasms/therapy , Adult , Antigen Presentation , CD8 Antigens/immunology , Cancer Vaccines/adverse effects , Cancer Vaccines/immunology , Cells, Cultured , Cytotoxicity, Immunologic , Female , Humans , Immunotherapy, Adoptive , Leukapheresis , Lymphocyte Activation , Male , Melanoma/immunology , Middle Aged , Peptides/immunology , Peptides/therapeutic use , Pilot Projects , Receptors, Antigen, T-Cell/metabolism , Skin Neoplasms/immunologyABSTRACT
Stem cells are a central feature of metazoan biology. Haematopoietic stem cells (HSCs) represent the best-characterized example of this phenomenon, but the molecular mechanisms responsible for their formation remain obscure. The stem cell leukaemia (SCL) gene encodes a basic helix-loop-helix (bHLH) transcription factor with an essential role in specifying HSCs. Here we have addressed the transcriptional hierarchy responsible for HSC formation by characterizing an SCL 3' enhancer that targets expression to HSCs and endothelium and their bipotential precursors, the haemangioblast. We have identified three critical motifs, which are essential for enhancer function and bind GATA-2, Fli-1 and Elf-1 in vivo. Our results suggest that these transcription factors are key components of an enhanceosome responsible for activating SCL transcription and establishing the transcriptional programme required for HSC formation.
Subject(s)
DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic/genetics , Hematopoietic Stem Cells/physiology , Transcription Factors/metabolism , Transcription, Genetic , Xenopus Proteins , Amino Acid Sequence , Animals , Basic Helix-Loop-Helix Transcription Factors , Embryo, Mammalian/physiology , Embryo, Nonmammalian , GATA2 Transcription Factor , Gene Expression Regulation, Developmental , Genes, Reporter , Helix-Loop-Helix Motifs/genetics , Humans , Macromolecular Substances , Mice , Mice, Transgenic , Molecular Sequence Data , Multiprotein Complexes , Protein Binding , Proto-Oncogene Protein c-fli-1 , Proto-Oncogene Proteins/metabolism , Sequence Alignment , T-Cell Acute Lymphocytic Leukemia Protein 1 , Trans-Activators/metabolism , Xenopus laevis/physiologyABSTRACT
The stem cell leukemia (SCL) gene encodes a basic helix-loop-helix transcription factor with a critical role in the development of both blood and endothelium. Loss-of-function studies have shown that SCL is essential for the formation of hematopoietic stem cells, for subsequent erythroid development and for yolk sac angiogenesis. SCL exhibits a highly conserved pattern of expression from mammals to teleost fish. Several murine SCL enhancers have been identified, each of which directs reporter gene expression in vivo to a subdomain of the normal SCL expression pattern. However, regulatory elements necessary for SCL expression in erythroid cells remain to be identified and the size of the chromosomal domain needed to support appropriate SCL transcription is unknown. Here we demonstrate that a 130-kilobase (kb) yeast artificial chromosome (YAC) containing the human SCL locus completely rescued the embryonic lethal phenotype of scl(-/-) mice. Rescued YAC(+) scl(-/-) mice were born in appropriate Mendelian ratios, were healthy and fertile, and exhibited no detectable abnormality of yolk sac, fetal liver, or adult hematopoiesis. The human SCL protein can therefore substitute for its murine homologue. In addition, our results demonstrate that the human SCL YAC contains the chromosomal domain necessary to direct expression to the erythroid lineage and to all other tissues in which SCL performs a nonredundant essential function.
