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WO1993020185A1 - Procede permettant de faire proliferer in vitro des precurseurs de cellules dendritiques et utilisation de celles-ci pour produire des immunogenes - Google Patents

Procede permettant de faire proliferer in vitro des precurseurs de cellules dendritiques et utilisation de celles-ci pour produire des immunogenes Download PDF

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Publication number
WO1993020185A1
WO1993020185A1 PCT/US1993/003141 US9303141W WO9320185A1 WO 1993020185 A1 WO1993020185 A1 WO 1993020185A1 US 9303141 W US9303141 W US 9303141W WO 9320185 A1 WO9320185 A1 WO 9320185A1
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Prior art keywords
cells
dendritic
cell
antigen
csf
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PCT/US1993/003141
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English (en)
Inventor
Ralph M. Steinman
Kayo Inaba
Gerold Schuler
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Steinman Ralph M
Kayo Inaba
Gerold Schuler
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Application filed by Steinman Ralph M, Kayo Inaba, Gerold Schuler filed Critical Steinman Ralph M
Priority to AT93911581T priority Critical patent/ATE260971T1/de
Priority to EP93911581A priority patent/EP0633929B1/fr
Priority to DE69333433T priority patent/DE69333433T2/de
Priority to AU40461/93A priority patent/AU687733B2/en
Priority to JP51773893A priority patent/JP3649335B2/ja
Priority to CA2133409A priority patent/CA2133409C/fr
Publication of WO1993020185A1 publication Critical patent/WO1993020185A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/22—Immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/45—Bacterial antigens
    • A61K40/4524—Mycobacterium, e.g. Mycobacterium tuberculosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/25—Tumour necrosing factors [TNF]

Definitions

  • This invention relates to a method of culturing cells of the immune system.
  • a method is provide f° r culturing proliferating dendritic cell precursors an for their maturation in vitro to mature dendritic cells.
  • This invention also relates to dendritic cell modifie antigens which are T cell dependent, the method of makin them, and their use as immunogens.
  • Vaccines, methods o immunizing animals and humans using the mature dendriti cells of the invention, and the modified antigens are als described.
  • Dendritic cells process and present antigens most likely on abundant, newly synthesized MHC class II molecules, and then strong accessory and cell-cell adhesion functions are acquired (4-7) .
  • Dendritic cells can migrate via the blood a nd lymph to lymphoid organs (8-10) . There, presumably as the "interdigitating 1 * cells of the T-area (8,11-13), antigens can be presented to T cells in the recirculating pool (14) .
  • the progenitors of dendritic cells in the different compartments outlined above.
  • dendritic cells in delivering antigens in such a way that a strong immune response ensues i.e., "immunogenicity", is widely acknowledged, but the use of these cells is hampered by the fact that there are very few 0 in any given organ.
  • white cells In human blood, for example, about 0.1% of the white cells are dendritic cells (25) and these have not been induced to grow until this time.
  • previous studies (20, 21) have not reported the development, in culture, of large numbers of dendritic cells from bone 5 marrow.
  • dendritic cells can process foreign antigens into peptides that immunologically active T cells must recognize (4,6,7,14) i.e., dendritic cells accomplish the phenomenon of "antige presentation", the low numbers of dendritic cells prohibit c their use in identifying immunogenic peptides.
  • Dendritic cells in spleen (15) and afferent lymph (16,17) are not in the cell cycle but arise from a proliferating precursor.
  • dendritic cells emanate from the bone marrow (15,16,18,19), yet it has been difficult to generate these cells in culture except for two 5 reports describing their formation in small numbers (20,21) .
  • Steinman, R. The Dendritic Cell System and Its Role In Immunogenicity", Ann. Rev. Immunol.. 0 9:271-96 (1991).
  • polysaccharide generally elicit a T-cell independent immune response. There is no memory response and therefore no protection t subsequent infection with the polysaccharide antigen. 0 Proteins, however, do elicit a T-cell dependent response i infants.
  • conjugate vaccines containin a polysaccharide covalently coupled to a protein convert the polysaccharide T-independent response to a T-dependen response. Unfortunately, little is known concerning th 5 sites on proteins which confer their T-cell dependen character, therefore hampering the design of more specifi immunogens.
  • dendritic cells play a crucial role i the initiation of T-cell dependent responses.
  • Dendriti o cells bind and modify antigens in a manner such that th modified antigen when presented on the surface of th dendritic cell can activate T-cells to participate in th eventual production of antibodies.
  • the modification o antigens by dendritic cells may, for example, included 5 fragmenting a protein to produce peptides which have region which specifically are capable of activating T-cells.
  • MHC Q The events whereby cells fragment antigens int peptides, and then present these peptides in associatio with products of the major histocompatibility complex, (MHC Q are termed "antigen presentation".
  • the MHC is a region o highly polymorphic genes whose products are expressed on th surfaces of a variety of cells. MHC antigens are th principal determinants of graft rejection.
  • Dendritic cells are specialized antigen presenting cells in the immune response of whole animals (14,31). Again however, the ability to use dendritic cells to identify and extract the " immunogenic peptides is hampered by the small numbers of these specialized antigen presenting 5 cells.
  • Particle uptake is a specialized activity of mononuclear and polymorphonuclear phagocytes. Dead cells, immune complexes, and microorganisms all are avidly internalized. Following fusion with hydrolase-rich o lysosomes, the ingested particles are degraded (60,61). This degradation must be to the level of permeable amino acids (62,63) and saccharides, otherwise the vacuolar apparatus would swell with indigestible materials (64,65). Such clearance and digestive functions of phagocytes 5 contribute to wound healing, tissue remodeling, and host de ense.
  • APCs antigen presenting cells
  • processing requires the generation of peptides at least 8-18 amino acids in length (66,67), while scavenging entails digestion to amino acids (62,63).
  • presentation requires the binding of peptides to MHC class II products (6,68), whereas scavenging does not require MHC 5 products.
  • antigen presentation can function at a ° low capacity, since only a few hundred molecules of ligand need to be generated for successful stimulation of certain T-T hybrids (69,70) and primary T cell populations (71).
  • This invention provides a method of producing a population of dendritic cell precursors from proliferating cell cultures.
  • the method comprises (a) providing a tissue source comprising dendritic cell precursors; (b) treating the tissue source from (a) to increase the proportion of dendritic cell precursors to obtain a population of cells suitable for culture in vitro; (c) culturing the tissue source on a substrate in a culture medium comprising GM-CSF, or a biologically active derivative of GM-CSF, to obtain proliferating nonadherent cells and cell clusters; (d) subculturing the nonadherent cells and cell clusters to produce cell aggregates comprising proliferating dendritic cell precursors; and (e) serially subculturing the cell aggregates one or more times to enrich the proportion of dendritic cell precursors.
  • This invention also provides a method of producing in vitro mature dendritic cells from proliferating cell cultures.
  • the method comprises (a) providing a tissue source comprising dendritic cell precursor cells; (b) treating the tissue source from (a) to increase the proportion of dendritic cell precursors in order to obtain a population of cells suitable for culture in vitro; (c) culturing the tissue source on a substrate in a culture medium comprising GM-CSF, or a biologically active derivative of GM-CSF, to obtain non-adherent cells and cell clusters; (d) subculturing the nonadherent cells and cell clusters to produce cell aggregates comprising proliferating dendritic cell precursors; (e) serially subculturing the cell aggregates one or more times to enrich the proportion of dendritic cell precursors; and (f) continuing to culture the dendritic cell precursors for a period of time sufficient to allow them to mature into mature dendritic cells.
  • This invention also provides a method of increasing the proportion of dendritic cells present in the tissue source by pretreating the individual with a substance to stimulate he atopoiesis.
  • the pretreatment step comprises killing cells expressin antigens which are not expressed on dendritic precursor cells by contacting the bone marrow with antibodies specific for antigens not present on dendritic precursor cells in a medium comprising complement. Removal of undesirable non- dendritic cell precursors may also be accomplished b adsorbing the undesirable non-dendritic or their precurso cells onto a solid support.
  • This invention also provides a method of preparing antigens from dendritic cell precursors comprising providing precursor dendritic cells from a population of precursor cells capable of proliferating, contacting the precursor cells with antigen for a period of time sufficient to allow the dendritic cell precursors to phagocytose the antigen and obtain antigen-containing dendritic cell precursors; culturing the antigen containing-dendritic cell precursors under conditions and for a period of time sufficient for the antigen to be processed and presented by dendritic cell precursors.
  • the antigens processed by the dendritic cell precursors as a result of phagocytosis may themselves be used alone or in combination with adjuvants including dendritic cell precursors to evoke an immune response in an individual to the antigen.
  • the invention provides self- peptide antigens produced by pulsing the dendritic cells of
  • This invention also provides a method of treating autoimmune diseases by treating an individual with 0 therapeutically effective amounts of self-peptides produced according to the method of the invention to induce tolerance to the self-proteins.
  • the treatment of autoimmune diseases comprising administering to an individual in need of treatment a 5 therapeutically effective amount of antigen-activate dendritic cells where the antigen is a self-protein o autoantigen is also provided.
  • a further objective of the invention is to provide a method of immunizing individuals with T-cell dependen antigens for the prevention and treatment of disease.
  • Fig. 3 FACS analyses of dendritic cell precursors tha could be dislodged by Pasteur pipetting of proliferatin aggregates, and dendritic cells released spontaneously i culture.
  • the mAb are: Ml/42 anti-MHC class I [ATCC # TI 126]; NLDC145 anti-interdigitating cell (13); M5/114 anti MHC class II [ATCC # TIB 120]; 33D1 anti-dendritic cel [ATCC # TIB 227]; B5-5 anti-thy-1.
  • the staining with anti MHC mAbs is bimodal, but the released cell fraction o dendritic cells is richest in expression of MHC class I an II.
  • Fig. 6 Physical properties of the MLR stimulating cells that develop in GM-CSF supplemented bone marrow cultures [see text] .
  • Fig. 8 Detailed cell cytofluorometric phenotype analysi 0 of the la-positive cells released from the growing dendriti cell aggregates. Contaminating, la-negative granulocyte were gated out on the basis of lower forward light scatter so that one could examine the expression of many surfac antigens on the larger cells using rat and hamster anti 5 mouse mAbs (7,17) as indicated.
  • Fig. 9 Quantitation of developing cells that bear th dendritic cell restricted granule antigens 2A1 and M342.
  • Fig. 10 Progenitor-progeny relationships in growin dendritic cells. Growing aggregates were separated at d 5 from bone marrow cultures and pulsed with 3 H-TdR at 0. ⁇ Ci/ l, 3 x 10 5 cells/well, for 12h. All wells were replaced with fresh medium and returned to culture for 1, 2, or 3 days of chase. The yields of released cells during the chase were 2.0, 2.9, and 3.0 x 10 s respectively per well. The content of Ia + cells was 28% after the pulse, and 47%, 55%, and 62% on days 1, 2, and 3 respectively. The data are shown as percentage of cells that were radiolabeled, with the filled in bars being cells that express the 2A1 granule cell antigen of mature dendritic cells.
  • A An aggregate of developing dendritic cells cytospun after a 2Oh exposure to 2u latex spheres. Many cells in the aggregate are labeled with the uniform latex particles [arrows] .
  • the sorted cells were cytospun, stained with auramine-rhodamine to visualize the cell- associated BCG, and double labeled with a different mAb an immunoperoxidase.
  • the left and right panels of each pair are phase contrast and acid fast views respectively. Arrows on the left indicate the location of the bacilli on th right.
  • the label for class II, [I-A and I-E, M5/114] outlines the cell processes better than the dendritic cell restricted NLDC-145 antibody.
  • Fig 14 Electron microscopy of BCG in dendritic cells. As in Fig 2, BCG was added to GM-CSF stimulated d6 bon marrow cultures for a day. After washing and 2 more days o culture, the released cells were processed for electro microscopy. A,B. Low power views to show the typical dendriti cells with numerous processes and a few phagocytosed BC [arrows] .
  • T cells were purified from lymph nodes that drain paws that had been primed with complete [CFA] or incomplete [IFA]
  • Mature dendritic cells are d8 bone marrow cultures, and immature dendritic cells are from d5-6 cultures.
  • Fig 16 Antigen presentation to naive lymph node T cells in situ.
  • Growing cultures of bone marrow dendritic cells were pulsed with BCG at d5-6, and used immediately or after a 2d chase culture to activate T cells.
  • the populations were injected into the paws of naive mice without artificial adjuvants.
  • Five days later the draining lymph nodes were taken and stimulated in vitro with graded doses of PPD or BSA (the dendritic cells had been grown with fetal calf serum) , the BSA to serve as a nonparticulate antigen.
  • Data are means and standard errors for groups of 5 mice, each studied separately. Control lymph nodes not exposed to BCG pulsed dendritic cells did not respond to PPD or to BSA ( ⁇ 2000 cpm) .
  • Fig 17 Antigen presentation to naive spleen cells in situ.
  • Growing cultures of bone marrow dendritic cells were pulsed with BCG at d5-6 (immature) , at d7-8 (mature) , or at d5-6 followed by a 2d chase.
  • 10 6 cells of each group were injected i.v. into groups of mice. 5 or 10 days later, the spleen cells were cultured in vitro with graded doses of PPD or BSA as antigen. Since the dendritic cells were cultured in FCS, the use of BSA serves as control to ensure that all dendritic cell populations were comparably immunogenic in vivo. Unprimed spleen did not respond to either BSA or PPD.
  • Figs 18A, B and C Mixed Leukocyte Reaction (MLR) assay of human dendritic cells produced according to the metho described in Example 6. Graded doses of irradiated cell (30 to 30,000 in serial 3 fold dilutions) were added to 2 10 5 accessory cell-depleted T cells.
  • MLR Mixed Leukocyte Reaction
  • tissue source comprising dendritic cell precursors whic precursor cells are capable of proliferating and maturing i vitro into dendritic cells when treated according to the method of the invention.
  • Such precursor cells are nonadherent and typically do not label with mAb markers found on mature dendritic cells such as Ia antigens, 2A1 and M342 antigens (34, 44) and the NLDC145 interdigitating cell source antigen (13) .
  • tissue sources are spleen, afferent lymph, bone marrow and blood. More preferred tissue sources are bone marrow and blood. Blood is also a preferred tissue source of precursor cells because it is easily accessible and could be obtained in relatively large quantities.
  • hematopoietic factor to be administered may be determined by one skilled in the art by monitoring the cell differential of individuals to whom the factor is being administered.
  • dosages of factors such as G-CSF and GM-CSF will b e similar to the dosage used to treat individuals recovering from treatment with cytotoxic agents.
  • GM-CSF or G-CSF is administered for 4 to 7 days at standard doses prior to removal of source tissue to increase the proportion of dendritic cell precursors.
  • the tissue source may be treated prior to culturing to enrich the proportion of dendritic precursor cells relative to other cell types. Such pretreatment may also remove cells whic may compete with the proliferation of dendritic precurso cells or inhibit their proliferation or survival Pretreatment may also be used to make the tissue source mor suitable for in vitro culture.
  • the method of treatment wil likely be tissue specific depending on the particular tissu source. For example, spleen or bone marrow if used as tissue source would first be treated so as to obtain singl cells followed by suitable cell separation techniques t separate leukocytes from other cell types. Treatment o blood would involve cell separation techniques to separat leukocytes from other cells types including red blood cell (RBCs) which are toxic.
  • RBCs red blood cell
  • B cells ar removed prior to culturing of bone marrow in GM-CSF.
  • Whil B cells and pre-B cells do not grow in response to GM-CSF they represent approximately 50% of the initial marro suspension and thereby preclude the use of staining wit anti-la monoclonal antibodies to quickly enumerate dendriti cells.
  • granulocytes are GM-CSF responsive an readily proliferate in the presence of GM-CSF. As such, th B cells and granulocytes mask the presence of dendritic cel precursors.
  • pretreatment cells which compete and mask the proliferation of precursor dendritic cells are killed.
  • Such pretreatment comprises killing cells expressing antigens which are not expressed on dendritic precursor cells by contacting bone marrow with antibodies specific for antigens not present on dendritic precursor cells in a medium comprising complement.
  • Another form of pretreatment to remove undesirable cells suitable for use with this invention is adsorbing the undesirable precursor cells or their precursors onto a solid support using antibodies specific for antigens expressed on the undesirable cells.
  • Several methods of adsorbing cells to solid supports of various types are known to those skilled in the art and are suitable for use with this invention. For example, undesirable cells may be removed by "panning" using a plastic surface such as a petri dish.
  • Any isotonic solution commonly used in tissue cultur may be used as the medium for separating blood leukocyte from platelets and red blood cells.
  • suc isotonic solutions are phosphate buffered saline.
  • RPMI 1640 is preferred.
  • Cells obtained from treatment of the tissue source are cultured to form a primary culture on an appropriate substrate in a culture medium supplemented with GM-CSF or a 5 GM-CSF derivative protein or peptide having an amino acid sequence which sequence maintains biologic activity typical of GM-CSF.
  • the appropriate substrate may be any tissue culture compatible surface to which cells may adhere.
  • the substrate is commercial plastic treated for use in tissue culture. Examples include various flasks, roller bottles, petri dishes and multi-well containing plates made for use in tissue culture. Surfaces treated with a substance, for example collagen or poly-L-lysine, or antibodies specific for a particular cell type to promote 5 cell adhesion may also be used provided they allow for the differential attachment of cells as described below.
  • Cells are preferably plated at an initial cell density of about 7.5 X 10 5 cells per cm 2 . At this dose, the surface is not fully covered by cells, but there are no big spaces (2-3 0 cell diameters) either.
  • the la-negative marrow 5 nonlymphocytes comprising dendritic cell precursors are preferably cultured in high numbers, about 10 6 /well (5 x 10 5 cells/cm 2 ) Liquid marrow cultures which are set up for purposes other than culturing dendritic cell precursors are typically seeded at 1/lOth this dose, but it is then 0 difficult to identify and isolate the aggregates of developing dendritic cells.
  • cytokines are produced from the human gene usin reco binant techniques (rhu) .
  • TNF ⁇ at concentrations fro about 10-50 U/ml may be used to increase dendritic cell yields several fold.
  • the primary cultures from the tissue source are allowe to incubate at about 37°C under standard tissue cultur conditions of humidity and pH until a population of cell has adhered to the substrate sufficiently to allow for th separation of nonadherent cells.
  • the dendritic cel precursor in blood initially is nonadherent to plastic, i contrast to monocytes, so that the precursors can b separated after overnight culture.
  • Monocytes an fibroblasts are believed to comprise the majority o adherent cells and usually adhere to the substrate withi about 6 to about 24 hours.
  • the nonadherent cells from the primary culture are subcultured by transferring them to new culture flasks at a density sufficient to allow for survival of the cells and which results in the development over time of clusters of growing cells that are loosely attached to the culture surface or to the firmly adherent cells on the surface. These clusters are the nidus of proliferating dendritic cell precursors.
  • culture flasks refers to any vessel suitable for culturing cells. It is desirable to subculture all of the nonadherent cells from the primary culture at a density of between about 2 X 10 5 cells and 5 X - 5 cells per cm 2 . Preferably at about 2.5 X 10 5 per cm 2 .
  • the dendritic cells besides being identified by thei stellate shape may also be identified by detecting thei expression of specific antigens using monoclonal antibodies.
  • a panel of monoclonal antibodies may be used t identify and characterize the cells in the GM-CSF expande cultures. The monoclonal antibodies are reviewed elsewher (23, 24 which are incorporated herein by reference).
  • the pulse and chase protocol which may be used to charge developing dendritic cells with organisms according to our invention allows the two broad components of immunostimulation to take place sequentially. These components are a) antigen capture and presentation, here the capture of particulates by immature dendritic cells, and b) development of potent accessory or immunostimulatory functions during the chase period. The situation is comparable to that seen in the handling of soluble proteins (4,6) and particles (74) by epidermal Langerhans cells. Each of the two broad components of APC function entails many subcomponents.
  • the novel antigens of the invention are prepared b combining substances to be modified or other antigens wit the dendritic cells prepared according to the method of th invention.
  • the dendritic cells process or modify antigen in a manner which promotes the stimulation of T-cells by th processed or modified antigens.
  • Such dendritic cel modified antigens are advantageous because they can be mor specific and have fewer undesirable epitopes than non- modified T-dependent antigens.
  • the dendritic cell modified antigens may be purified by standard biochemical methods.
  • MHC major histocompatibility complex
  • dendritic cells are pulsed with the endogenous antigen t produce the relevant "self-peptide".
  • the relevant self peptide is different for each individual because MH products are highly polymorphic and each individual MH molecules might bind different peptide fragments. Th "self-peptide” may then be used to design competing peptide or to induce tolerance to the self protein in the individua in need of treatment.
  • B. Blood Preparation Blood was obtained by cardiac puncture or from the carotid artery. The blood was diluted in, or allowed to drip into, RPMI-1640 with 100 u/ml heparin [about 2 ml/mouse]. Blood cells were pelleted at 1000 rpm at 4°, resuspended in RPMI 1640, and sedi ented again.
  • the adherent cells did not develop dendritic cell colonies, but during the next week, the nonadherent populations exhibited three changes. First, most of the lymphocytes and granulocytes died or could be removed by washing. Second, the surface of the well became covered with a monolayer of tightly adherent cells that included macrophages and fibroblasts. Third, affixed to scattered sites on the monolayer, there developed small aggregates of cells. The cultures were fed with GM-CSF (30 u/ml) at day 6-7 and then every 3 days by aspirating 0.5- 0.75 ml of the medium and adding back an equal volume of fresh medium with GM-CSF. The aggregates continued t expand in number and size. At about day 10, the cells were ready to be subcultured. Any residual loose cells could b rinsed off prior to dislodging the aggregates into fres medium and GM-CSF. About 0.8-1 million dislodged cells pe original well were divided into 3 subculture wells.
  • the cytospin cells were stained for specific antigens with mAb and immunoperoxidase as above. Also, the slides were dipped in photographic emulsion [Kodak autoradiography emulsion type NTB2 #165-4433] for exposure [5 days] prior to development, staining with Giemsa, and mounting in Permount. For pulse chase experiments, a lower dose of 3H-TdR was used to maintain cell viability, but the cells were handled similarly otherwise. The pulse was applied at 0.1 uCi/ml for 2h or for 16h, the latter to provide higher initial labeling indices. The cells were washed and chased for 1-3 days prior to harvesting and analysis as above with immunoperoxidase, autoradiography, and Giemsa staining.
  • Pulse chase experiments were then done to establish that labeled cells in the aggregate were giving rise to typical dendritic cells.
  • Cultures were first exposed to a low dose of 3H-TdR, either for 2h or for 16h, the latter to label a larger percentage of the cells in the aggregates.
  • the wells were washed free of radiolabel, and then the aggregates were dislodged and separated from free cells by lg sedimentation.
  • the aggregates were transferred to fresh medium without radiolabel, and over the next 1-3 days of culture, many dendritic cells were released into the medium. When the "chased" cultures were examined, several findings were apparent.
  • a second specialized feature of dendritic cells i their capacity to home to the T areas of peripheral lymphoi tissues (8,10).
  • Dendritic cells or other cell types were labeled at 2-10 x 10 6 /ml with carboxyfluorescein for 10 mi on ice [Molecular Probes C-1157; 30 uM final concentration in Hanks balanced salt solution (HBSS) with 5% FCS], washed in RPMI 1640, and injected in a volume of 50 ul RPMI-1640 into the foot pads.
  • HBSS Hanks balanced salt solution
  • FCS 5% FCS
  • the sections were applied to multiwell slides [Carlson Scientific microslides #111006], stored at -20°C, dried in a desiccator 30' prior to use [or left at room temp overnight], fixed in acetone, and stained with a peroxidase conjugated rabbit anti-FITC antibody [Dakopatts, P404].
  • dendritic cells that had been generated with GM-CSF from blood, homing to the T area was observed with injections of 200,000 cells. The selective localization to the T areas was confirmed by double labeling the specimens with mAb that stain B cells or T cells. Therefore dendritic cells produced in culture have the key functional features of this lineage: homing to the T-dependent regions and strong accessory activity.
  • the surface phenotype of the blood cell that gives rise to the dendritic cell colonies was assessed by treating the starting population with antibodies and complement. Treatment with either 33D1 anti-dendritic cell, anti-MH class II, or anti thy-1 did not eliminate the colony formin unit [not shown] . Instead, removal of thy-l + or Ia + cell enriched colony numbers several fold. CSF's other than GM CSF were also tested, either at the start of the 1-3 wee culture, or upon transfer of 2-3 week old aggregates to for veiled cells. None of the CSF's tested, i.e., IL-3, M-CSF G-CSF, SCF, supported the formation of colonies or matur dendritic cells.
  • dendritic colonie are very much dependent upon GM-CSF.
  • Dendritic cel precursors were not observed from neonatal epidermis, whi contains mainly la" Langerhans cells (29) .
  • the precursors to the aggregates of proliferating dendritic cells were not typical monocytes or dendritic cells, because the number of aggregates that developed could 5 be increased substantially if one depleted monocytes by adherence or la-positive cells with antibody and complement. Without wishing to be bound by theory, we tentatively conclude that blood contains an la-negative precursor that forms a proliferating aggregate. In the aggregate, o dendritic cells mature and are released as nonproliferating progeny.
  • the labeled progeny In pulse chase protocols, large numbers of labeled progeny were released from the aggregates, and these released cells were nonadherent, motile, and strongly stimulatory in the MLR. After combined autoradiography and immunoperoxidase labeling, the labeled progeny carried the granular antigens, the NLDC-145 antigen, and very high levels of MHC class II. Each of these cytologic and antigenic markers are largely restricted to dendritic cells.
  • the functional maturation that occurred in the ° proliferating aggregate is striking.
  • the dendritic cells that were generated in culture were potent MLR stimulators. 100 dendritic cells induced a much stronger primary MLR than 100,000 blood leukocytes. The increase in stimulating activity per la-positive cell was at least 2 logs between the time that the aggregates first appeared and the time that typical dendritic cells were released in large numbers. Over this time period, cell recovery increased 5-10 fold. Also the dendritic cell progeny homed in a precise way to the T cell area of lymph node, another functional property that was not detectable in blood cells [data not shown].
  • mice Female BALB/C, male DBA/ 2 , and female C57BL/6 mice, 7 wks old, were purchased from Japan SLC [Hamamatsu, Shizuoka, Japan].
  • the culture medium was RPMI-1640 [Nissui, Tokyo, Japan; GIBCO, Grand Island, NY] supplemented with 5% FCS, 50 ⁇ M 2-Mercaptoethanol, and 20 ⁇ g/ml genta icin.
  • Murine rGM- CSF 10 8 U/mg protein] was kindly provided by Kirin Brewery 5 Co [Maebashi, Gumma, Japan].
  • Kirin Brewery 5 Co [Maebashi, Gumma, Japan].
  • a panel of rat and hamster mAbs to mouse leukocyte antigens is described elsewhere (23, 24) .
  • FITC- and peroxidase-conjugated mouse anti-rat IgG were purchased from Boehringer Mannheim [Indianapolis, IN] and FITC- and peroxidase-conjugated goat anti-hamster lg [ ⁇ 0 and L-chain] were from Jackson Immunoresearch Lab [Westgrove, PA] and Caltag [San Francisco, CA] respectively.
  • Bone marrow cultures After removing all muscle tissues with gauze from the mouse femurs and tibias, the 5 bones were placed in a 60 mm dish with 70% alcohol for 1 min, washed twice with PBS, and transferred into a fresh dish with RPMI-1640. Both ends of the bones were cut with scissors in the dish, and then the marrow was flushed out using 2 ml of RPMI-1640 with a syringe and 25G needle. The tissue was suspended, passed through nylon mesh to remove small pieces of bone and debris, and red cells were lysed with ammonium chloride. After washing, lymphocytes and la- positive cells were killed with a cocktail of mAbs and rabbit complement for 60 min at 37°C.
  • the mAbs were GK 1.5 anti-CD4, HO 2.2 anti-CD8, B21-2 anti-la, and RA3-3A1/6.1 anti-B220/CD45R all obtained from the ATCC [TIB 207, 150, 229, and 146 respectively].
  • 7.5-10 x 10 5 cells were placed in 24 well plates [Nunc, Naperville, IL] in 1 ml of medium supplemented with 500-1000 U/ml rGM-CSF. The cultures were usually fed every 2d for about 2 to 10 days, by gently swirling the plates, aspirating 3/4 of the medium, and adding back fresh medium with GM-CSF.
  • Th aggregates were subcultured at 2-3 x 10 5 /ml in fresh maxim with GM-CSF, typically for 1 day in 16 mm wells. Afte overnight culture, large numbers of typical dendritic cell were released. Adherent macrophages also expanded in thes cultures, but most remained firmly adherent to the cultur surface. ° C. Cytological Comparison of Dendritic Cell Precursors and Ia-ne ative. Bone Marrow Nonlymphocytes
  • dendritic profiles are in the released fraction [a hand lens is useful to detect cell 5 shape and contaminating granulocytes, in the Giemsa stain], while the adherent cells are for the most part typical vacuolated macrophages. Strong MHC class II expression occurs on all released cells but for a few typical granulocytes. Only a subset of the firmly adherent cells o express class II. Most, released cells express the 2A1 endocytic vacuole antigen, while the adherent cells are 2A1 weak or negative.
  • Cell surface and intracellular antigens Cell surface 5 staining utilized cytofluorography [FACScan; Becton
  • EDTA-PBS EDTA-PBS
  • EDTA-PBS EDTA-PBS
  • cytospins were fixed in acetone and stained with mAbs followed by peroxidase conjugated anti-rat or anti- hamster lg.
  • the peroxidase was visualized with diaminobenzidine, and the nuclei counterstained with Giemsa.
  • F. Mixed leukocyte reactions Cells from the bone marrow cultures were exposed to 15 Gy of X-ray irradiation and applied in graded doses to 3 x 10 5 syngeneic or allogeneic T cells in 96 well flat bottomed culture plates for 4d. The cells were prepared by passing spleen and lymph node suspensions through nylon wool and then depleting residual APCs with anti-la plus Jlld mAbs plus complement. 3H- thymidine uptake was measured at 80-94h after a pulse of 4 uCi/ml [222 GBq/mmol; American Radiolabeled Chemicals, Ine, St.Louis, MO].
  • G Aggregates of proliferating dendritic cells from mouse bone marrow supplemented with GM-CSF.
  • Attached to the surface of the culture wells were cells with the cytologic features of macrophages, and these also expanded in numbers during the first week of culture. These cells could be dislodged by pipetting after incubation at 37°C in the presence of 10 mM EDTA.
  • the nonadherent cells which were mainly granulocytes in the first 4 days, were obtained by gently swirling the plates and harvesting the cells.
  • the loosely adherent cells which contained the aggregates of presumptive dendritic cell precursors and dendritic cells at day 4 and later times, were dislodged by pipetting over the surface of firmly adherent stromal cells.
  • the most potent stimulating activity was in the adherent fraction.
  • the nonadherent fraction was very active. If one tested firmly adherent macrophages, there was no MLR stimulating activity [Fig 6B, open squares].
  • Cytospins were prepared to further compare the released dendritic cells with the firmly adherent stromal population.
  • Giemsa stain the cells that had released from the aggregates had the typical stellate shape of dendritic 5 cells, while the adherent cells were for the most part vacuolated macrophages.
  • Many of the dendritic cells had a perinuclear spot of nonspecific esterase stain, while the more adherent populations had abundant cytoplasmic esterase.
  • the released cells stained strongly for MHC class II 0 products, except for the contaminants with typical granulocyte nuclei.
  • the strongly adherent cells contained a subpopulation of class II positive cells.
  • the antibodies are termed M342 (34) and 2A1. Many of the dendritic cells had strong 2A1 stain, and a smaller number expressed M342. The adherent cells had a few profiles with weak 2A1.
  • the released cells had many large veils or lamellipodia extending from several directions of the cell body.
  • the cytoplasm had many mitochondria, few electron dense granules and lysosomes, but several electron lucent vesicles some with the cytologic features of multivesicular bodies.
  • the numerous cell processes extending from the dendritic cells were evident in the semi-thin sections of our preparations.
  • a bone marrow-derived dendritic cell at d5 of culture shows many cytoplasmic veils.
  • a close up of the perinuclear region shows profiles of smooth reticulum and vacuoles. There are few lysosomal or phagocytic structures.
  • mice BALB/C x DBA/2 Fl, C57BL/6 x DBA/2 Fl, and BALB/C male and female mice were purchased from the Trudeau Institute [Saranac Lake, NY] and Japan SLC [Hamamatsu] and used at 6-10 weeks of age.
  • Bone marrow cultures As described in Example 2 above, bone marrow was flushed from the femus and tibias, depleted of red cells with 0.83% ammonium chloride, and cultured in 24 well plates [Nunc, Napaville, IL and Corning #25820, Corning NY] at 10° cells/well in 1 ml of RPMI-1640 supplemented with 5% fetal calf serum, 20 ug/ml gentamicin, and 1000 U/ml of recombinant murine GM-CSF [Kiren Brewery, Maebashi, Gumma, Japan; 9.7 x 10 7 U/mg] .
  • cells in the washed cultures were dislodged and 3-4 x 10 6 cells transferred to a 60 mm Petri dish for a 1 or 2 day "chase" period in particle-free, fresh, GM-CSF supplemented medium.
  • Class Il-rich, mature dendritic cells developed during the chase as described in Example 2, and these were isolated by cell sorting [below].
  • particles were also administered to 7-8d bone marrow cultures that are rich in single nonproliferating mature dendritic cells.
  • BCG mycobacteria [Trudeau Institute, 1.5-2.5 x 10 8 CFU/ml; Kyowa Pharmaceutical Industries, Tokyo] were administered at approximately 10 7 live BCG per 16mm diameter well. Uptake was assessed following an "acid fast" stain using an auramine-rhodamine procedure that is more sensitive than Ziehl " Neelsen and facilitates organism counts. Colloidal carbon [Pellikan Ink, Hannover, Germany] was added at 1:2000 dilution. The carbon was identified as a black granular stain in specimens stained with Diff-Quik R [Baxter Healthcare Corp, Miami, FL] . Suspensions of 2u latex particles [0.5% v/v; Seradyn, Indianapolis, IN] were applied to the cultures at 50 ul/well, a dose which covers the surface of the culture well with beads.
  • D. Isolation of mature dendritic cells by cell sorting As noted before in Example 2, the dendritic cells that are produced in GM-CSF stimulated bone marrow cultures express very high levels of surface MHC class II products [monoclonals B21-2, TIB 227 and M5/114, TIB 120 from the ATCC] as well as moderate levels of a dendritic cell- restricted antigen recognized by monoclonal NLDC-145. Immediately after the pulse with BCG, or after an additional 2 days of "chase" culture, the cells were stained with biotin B21-2 and FITC-streptavidin [Tago, Burlingame, CA].
  • Class Il-rich cells then were sorted [FACStar Plus, Becton Dickinson, Mountainview, CA] and cytospun onto glass slides [Shandon Inst. Sewicky, PA] .
  • the sorted cells were stained with Diff Quick® which outlines the stellate shape of dendritic cells in cytospins and allows enumeration of profiles containing perinuclear depots of internalized colloidal carbon or latex spheres.
  • the cytospins were fixed in absolute acetone for 10 min at room temperature and stained with M5/114 anti-class II, NLDC-145 anti-dendritic cell, or RA3-6B2 anti-B220 or anti-B cell [the latter as a control] followed by POX conjugated mouse anti-rat lg [Boehringer Mannheim, Indianapolis,IN] and diaminobenzidine tetraHCl [Polyscience Ine, Warrington, PA].
  • the preparations were then double labeled for acid-fast bacilli with auramine rhodamine. Virtually all the cells in the preparation were rich in NLDC-145 and MHC class II products.
  • the number of BCG bacilli in at least 400 cells were enumerated.
  • mice were primed with complete Freunds' adjuvant [CFA, SIGMA, St.Louis, MO; 25 ul in the fore and rear paws] or as a control, mycobacteria- free incomplete Freunds' [ICFA]. 7-14d later, the draining lymph nodes were dissociated into a single cell suspension and depleted of APCs with mAbs to MHC class II, B220, and heat stable antigens [M5/114 anti-la, RA3-6B2 anti-B220, and Jlld anti-HSA; TIB 120, 146, and 183 from the ATCC respectively] and rabbit complement.
  • CFA complete Freunds' adjuvant
  • SIGMA St.Louis, MO
  • ICFA mycobacteria- free incomplete Freunds'
  • 3xl0 5 of these APC- depleted, primed T cells were cultured in 96 well flat- bottomed microtest wells [Corning #25860] in RPMI-1640 medium supplemented with 0.5% mouse serum and 50 uM 2- mercaptoethanol.
  • Graded doses of BCG-pulsed, bone marrow or spleen APCs were added.
  • 1 uCi of 3H-thymidine [NEN, Boston, MA; 20 Ci/mmol; 4 uCi/ml] uptake was added to monitor DNA synthesis at 72-88h. Data shown are means of triplicates in which standard errors were ⁇ 15% of the mean.
  • APCs that had been pulsed with antigen in vitro were administered in vivo to unprimed CxD2 Fl mice.
  • To prime T cells in draining lymph node 2x10 s dendritic cells were injected into the paws, and lymph node cells were prepared 5d later.
  • To prime T cells in spleen 10 6 cells were injected i.v. , and splenocytes were prepared 5 or lOd later.
  • bulk lymph node or spleen cells were cultured as above and challenged with graded doses of protein antigens, either purified protein derivative [PPD, from Statenserum Institute, Copenhagen, Denmark, or from Dr.
  • proliferating cell aggregates When mouse bone marrow or blood is stimulated with GM- CSF, proliferating cell aggregates appear, and these give rise to large numbers of typical immunostimulatory dendritic cells.
  • the proliferating aggregates are best identified by washing away the majority of nonadherent granulocytes that are also induced by GM-CSF in the cultures.
  • the time point when the aggregates were first sizable [5-10 cells wide] we applied different particles over a 20-22h period.
  • Fig 12A Aggregates that had been exposed to particles were recultured an additional 2 days. During this time, large numbers of cells were released into suspension. These primarily were mature dendritic cells with characteristic stellate shapes and high levels of MHC class II and NLDC-145 antigens. When the released cells were examined by light microscopy, many contained latex spheres and often around a clear perinuclear zone or centrosphere [Fig 12B] . We also studied colloidal carbon uptake in a similar manner.
  • Live BCG mycobacteria were administered as the phagocytic meal over a 20-22h period using the protocol for administering latex particles described above.
  • Cell- associated bacilli were visualized by a sensitive fluorescent acid-fast stain.
  • the developing dendritic cell aggregates contained many organisms.
  • the cells were resuspended and sorted those cells with high levels of MHC class II products.
  • the BCG pulse about 20% of the sorted cells contained acid fast bacilli [Table 1] .
  • the majority of MHC class II-weak cells were not studied further because of excessive stickiness during cell sorting.
  • Quantitative data of dendritic cells containing BCG Mouse bone marrow cultures were stimulated in 16mm wells for 5d with GM-CSF, washed, and exposed to BCG organisms for 20h. The cultures were washed again and either examined immediately, or pooled and transferred to a 60 mm dish for an additional 2d chase culture. The dendritic cells in the cultures were selected as la-rich cells using a fluorescent activated cell sorter and then cytospun onto glass slides for staining for acid fast bacilli. During the chase period, the percentage of la-rich cells in the cultures increased 2-2.5 fold, and the total number of cells increased 2 fold, resulting in a 4-5 fold increase in the number of la-rich cells.
  • the percentage of dendritic cells containing BCG also rose to 50% [Table 1, Fig. 13].
  • Double labeling experiments verified that cells with acid fast bacilli expressed MHC class II and the dendritic cell-restricted NLDC 145 antigen Fig. 13. Because the total number of MHC class II and NLDC- 145 positive cells had increased 4-fold in just 2d, it is likely that these BCG-laden dendritic cells were derived from less mature but phagocytic progenitors in the aggregates.
  • BSA bovine serum albumin
  • the surface markers of the primed cells were tested by antibody and complement mediated lysis of the populations prior to measuring 3H-thymidine uptake [data not shown] .
  • the proliferating cells were positive for thy-1, but negative for MHC class II, heat stable antigen, and B220.
  • Anti-CD4 hybridoma culture supernatant blocked proliferation more than 85% i.e., the primed cells were helper-type T cells.
  • Dendritic cells prepared according to the method described in Example 1 are plated at a concentration of approximately 1 x 10 s cells per well of a 24 well plastic culture plate. The cells are incubated in RPMI 1640 containing 5% fetal calf serum and GM-CSF (30 u/ml) . Antigen is added to the dendritic cell cultures and the cultures are incubated with antigen for approximately 4 hours or for sufficient time to allow the dendritic cells to handle the antigen in an immunologically relevant form, or in a form that can be recognized by T cells.
  • Such handling of the antigen by the dendritic cells involves the dendritic cells 1) acquiring, 2) processing, and 3) presenting the antigen to the T cells in a form which is recognized by the T cells. Following binding of the antigen to the dendritic cells the cells are collected from the culture and used to immunize syngeneic mice. The activated dendritic cells are injected subcutaneously into the mice in an amount sufficient to induce an immune response to the antigen.
  • Dendritic cells prepared as described in Example 1 are pulsed with a protein antigen for a time sufficient to allow the dendritic cells to acquire, process and present the modified antigen on the surface of the dendritic cells. The dendritic cells are then collected from the culture for extraction of the modified antigen.
  • the dendritic cells are solubilized with detergent to extract the modified antigen bound to MHC molecules.
  • the MHC molecules bound to modified antigen are purified by precipitation with antibodies which bind the MHC molecules such as MH2.
  • the modified antigens are extracted from the precipitate for analysis.
  • the isolated mononuclear cells were depleted of cells that were not dendritic cell progenitors. These contaminants were coated with monoclonal antibodies to CD3 and HLA-DR antigens and depleted on petri dishes coated with affinity-purified, goat anti-mouse IgG ("panning") .
  • Cells were cultured under the following conditions: 1) without presence of additional cytokines; 2) GM-CSF, 400 or 800 U/ml; 3) GM-CSF, 400 or 800 U/ml, plus IL-l ⁇ , 50 LAF units/ml for the last 24 h of culture; 4) GM-CSF, 400 or 800 U/ml, plus TNF ⁇ , 50 U/ml; 5) GM-CSF, 400 or 800 U/ml, plus TNF- ⁇ , 50 U/ml, plus IL-l ⁇ , 50 LAF units/ml for the last 24 h of culture; 6) GM-CSF, 400 or 800 U/ml, plus IL-3, 100 U/ml; 7) GM-CSF, 400 or 800 U/ml, plus IL-3, 100 U/ml, plus IL-l ⁇ , 50 LAF units/ml for the last 24 h.
  • GM-CSF is an essential cytokine. G-CSF, M-CSF, IL-3, or no cytokine do not permit the development of dendritic cell balls. GM-CSF at 400-800 U/ml is optimal, irregardless of whether donors had been treated with either GM-CSF or G-CSF to expand the number of myeloid progenitor cells in blood. Addition of TNF ⁇ at 10-50 U/ml usually but not always increased dendritic cell yields up to two-fold (cf. Caux et al..
  • Dendritic cells are the principal cells in mouse spleen bearing immunogenic fragments of foreign proteins. J. Exp. Med. 172:383.
  • HRP horseradish peroxidase

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Abstract

On décrit un procédé qui permet de faire proliférer en culture des précurseurs de cellules dendritiques. On décrit aussi un procédé qui permet de produire en culture des cellules dendritiques matures à partir de précurseurs de cellules dendritiques proliférantes. Les cultures de cellules dendritiques matures fournissent un moyen efficace de produire de nouveaux antigènes, dépendants des lymphocytes T, à savoir des antigènes modifiés par les cellules dendritiques ou des cellules dendritiques activées par un antigène, y compris des particules, ledit antigène étant traité et exprimé sur cette cellule dendritique activée par antigène. On peut utiliser les nouveaux antigènes inventés comme immunogènes destinés à des vaccins ou pour le traitement de maladies. On peut aussi utiliser ces antigènes pour traiter des maladies auto-immunes comme le diabète juvénile et la sclérose en plaques.
PCT/US1993/003141 1992-04-01 1993-04-01 Procede permettant de faire proliferer in vitro des precurseurs de cellules dendritiques et utilisation de celles-ci pour produire des immunogenes WO1993020185A1 (fr)

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AT93911581T ATE260971T1 (de) 1992-04-01 1993-04-01 Verfahren zur in vitro kultivierung dendritischer vorläuferzellen und deren verwendung zur immunogen herstellung
EP93911581A EP0633929B1 (fr) 1992-04-01 1993-04-01 PROCEDE PERMETTANT DE FAIRE PROLIFERER $i(IN VITRO) DES PRECURSEURS DE CELLULES DENDRITIQUES ET UTILISATION DE CELLES-CI POUR PRODUIRE DES IMMUNOGENES
DE69333433T DE69333433T2 (de) 1992-04-01 1993-04-01 Verfahren zur in vitro kultivierung dendritischer vorläuferzellen und deren verwendung zur immunogen herstellung
AU40461/93A AU687733B2 (en) 1992-04-01 1993-04-01 Method for in vitro proliferation of dendritic cell precursors and their use to produce immunogens
JP51773893A JP3649335B2 (ja) 1992-04-01 1993-04-01 樹枝状細胞前駆体のインビトロ増殖の方法およびその免疫原製造への使用
CA2133409A CA2133409C (fr) 1992-04-01 1993-04-01 Methode de proliferation in vitro de precurseurs de cellules dendritiques et facon d'utiliser lesdits precurseurs pour produire des immunogenes

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EP0765386A1 (fr) * 1994-06-14 1997-04-02 The Board Of Trustees Of The Leland Stanford Junior University Procedes pour l'activation de lymphocytes t in vivo par des cellules dendritiques a impulsions antigeniques
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