Dendritic cells derived from multiple myeloma patients efficiently internalize different classes of myeloma protein.
Myeloma protein is a unique tumor antigen that can be used to devise tumor-specific vaccination strategies. As dendritic cells (DCs) are extremely potent at inducing T-cell responses, clinical protocols have been designed using myeloma protein-pulsed DCs to elicit anti-tumor cell responses in vivo. To optimize antigen pulsing of DCs, we investigated mechanisms of antigen uptake and evaluated various laboratory parameters including class of myeloma protein, antigen exposure time, and DC maturational stage.DCs were generated by culturing peripheral blood stem cells from myeloma patients in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Myeloma proteins were labeled with fluorescein isothiocyanate (FITC) and internalization of protein by DCs was measured by flow cytometry.IgG, IgA, and free-kappa light chain myeloma proteins were all rapidly internalized by DCs in a time-dependent fashion. Maturation of DCs with tumor necrosis factor-alpha (TNF-alpha) resulted in diminished uptake. Endocytosis of myeloma protein by DCs was primarily mediated by fluid-phase macropinocytosis based on morphology, nonsaturable uptake kinetics, and sensitivity to drugs that inhibit membrane ruffling. Pulse-chase experiments revealed that the majority of internalized myeloma protein disappeared within 4 hours but was retained in the presence of chloroquine, indicating antigen processing had occurred. Cultured DCs from myeloma patients are functional and can efficiently endocytose different classes of myeloma protein by the mechanism of macropinocytosis. This demonstrates the feasibility of using all classes of myeloma protein for producing DC vaccines, and defines culture conditions for optimizing antigen loading of DCs for induction of anti-myeloma responses.
Butch
A W
AW
Department of Pathology and Laboratory Medicine, UCLA Medical Center, 10833 Le Conte Ave., Mailroom A2-179 CHS, Los Angeles, CA 90095-1732, USA. abutch@mednet.ucla.edu
Kelly
K A
KA
Munshi
N C
NC
D017951:Antigen Presentation / Q000502:physiology*; D000951:Antigens, Neoplasm / Q000276:immunology / Q000378:metabolism*; D019496:Cancer Vaccines; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects / Q000502:physiology; D002738:Chloroquine / Q000494:pharmacology; D003713:Dendritic Cells / Q000502:physiology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D019650:Hematopoietic Stem Cell Mobilization; D006412:Hematopoietic Stem Cells / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007070:Immunoglobulin A / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D007145:Immunoglobulin kappa-Chains / Q000378:metabolism; D015847:Interleukin-4 / Q000494:pharmacology; D009101:Multiple Myeloma / Q000276:immunology / Q000473:pathology*; D009194:Myeloma Proteins / Q000276:immunology / Q000378:metabolism*; D015388:Organelles / Q000187:drug effects / Q000502:physiology; D010873:Pinocytosis / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology
D000951:Antigens, Neoplasm; D019496:Cancer Vaccines; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007145:Immunoglobulin kappa-Chains; D009194:Myeloma Proteins; D011994:Recombinant Proteins; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D002738:Chloroquine