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WO1998015615A1 - Procede de production d'un micro-environnement thymique favorisant le developpement de cellules dendritiques - Google Patents

Procede de production d'un micro-environnement thymique favorisant le developpement de cellules dendritiques Download PDF

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WO1998015615A1
WO1998015615A1 PCT/US1997/018317 US9718317W WO9815615A1 WO 1998015615 A1 WO1998015615 A1 WO 1998015615A1 US 9718317 W US9718317 W US 9718317W WO 9815615 A1 WO9815615 A1 WO 9815615A1
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cells
thymic
dendritic cells
cell
dendritic
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WO1998015615A9 (fr
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Barton F. Haynes
Dhavalkumar D. Patel
Clayton A. Smith
G. Diego Miralles
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Duke University
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Duke University
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Priority to AU48149/97A priority patent/AU4814997A/en
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    • C12N2502/00—Coculture with; Conditioned medium produced by
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"

Definitions

  • the present invention relates, in general, to thymic microenvironment and, in particular, to a method of producing a thymic microenvironment in vitro that supports the development of dendritic cells from - hematopoietic pregenitor cells.
  • the invention further relates to a method of treating congenital and acquired immunodeficiencies, including malignant and autoimmune diseases and traditional T cell immunodeficiency diseases such as occurs in AIDS.
  • Dendritic cells are antigen presenting cells (APC) distributed widely in lymphoid and non-lymphoid tissues (Steinman et al, Advances in Experimental Medicine & Biology 329:1-9 (1993); Steinman, Experimental Hematology 24:859-62 (1996); Young et al, Stem Cells 14:376-87 (1996); Steinman et al, Immunological Reviews 156:25-37 (1997)).
  • APC antigen presenting cells
  • Dendritic cells posses a distinct morphologic appearance, express high levels of MHC class I and II and have a potent ability to process antigens and activate T cells (Wettendorff et al, Adv. Exp. Med. Biol . 378:371-374 (1995); u et al, J. Exp. Med. 184:903-11 (1996); Short an et al, Ann. Rev. Immunol. 14:29-47 (1996); Res et al, J. Exp. Med.
  • Dendritic cells developing within the thymus appear to be biologically distinct from extrathymic dendritic cells (Shorman et al, Ciba Found. Sy p. 204:130-41 (1997); Shortman et al. Adv. Exp. Med. Biol. 378:21-9 (1995); Saunders et al, J. Exp. Med. 184:2185- 96 (1996); Ardavin et al, Nature 362:761-3 (1993); Ardavin et al, Immunology Letters 38:19-25 (1993); Ardavin et al, Eur. J. Immunol. 22:859-62 (1992); Suss et al, J. Exp. Med.
  • thymic dendritic cells express molecules normally considered as markers of lymphoid cells (Steinman et al, Immunological Reviews 156:25-37
  • intrathymic dendritic cells may also be governed by different cytokines than those important in the development of extrathymic dendritic cells.
  • Saunders et al demonstrated that murine "low CD4" thymic precursors developed into thymic type dendritic cells in vitro with a combination of TNF- , IL-l ⁇ , IL-3, IL- 7, and SCF (Saunders et al, J. Exp. Med. 184:2185-96 (1996)).
  • the generation of dendritic cells from peripheral blood and bone marrow progenitors required GM-CSF (Caux et al, Blood Cell Biochemistry
  • Thymic dendritic cells may also have different functional properties than extrathymic dendritic cells.
  • thymic dendritic cells are believed to participate in the process of T cell negative selection and tolerance induction within the thymus (Steinman et al, Immunological Reviews 156:25-37 (1997); Shortman et al, Adv. Exp. Med. Biol.
  • CD34 + CD38 di thymocytes could dif erentiate into both T and NK cells in FTOC and develop into dendritic cells when cultured in vitro with GM-CSF and TNFc (Res et al, Blood 87:5196-206 (1996)).
  • GM-CSF and TNFc Res et al, Blood 87:5196-206 (1996).
  • the role that the thymus played in mediating the development of dendritic cells from intrathymic or extrathymic progenitors remained unclear because extrathymic culture of
  • CD34 + CD38 d ⁇ m cells was required to generate dendritic cells. Consequently, certain aspects of human thymic dendritic cell biology remain uncharacterized.
  • Prior to the present invention no experimental systems existed, other than FTOC, that generated thymic dendritic cells in vi tro in a thymic microenvironment.
  • the present inveniton provides systems that can be used to generate thymic dendritic cells from hematopoietic progenitors.
  • the present invention provides a method of producing, in vitro, a thymic microenvironment and a method of generating dentritic cells from hematopoietic progenitor cells using same.
  • FIG. 1A-F Generation of CDla + cells from
  • CD34 + CD38 ⁇ lin- and CD34+CD38+ lin- cells were separated from lin- UCB cells using fluorescence activated cell sorting (FACS) using the gates shown in Fig. 1A.
  • FACS fluorescence activated cell sorting
  • a post sort analysis of the sorted CD34 + CD38 + lin- cells (gate Rl) is shown in Fig. IB, and of the sorted CD34 + CD38 + lin- cells
  • CD34 + CD38 ⁇ lin- cells (gate R2) is shown in Fig. IC. After 21 day co-culture with mixed thymic stromal cells
  • Data presented are representative of 5 experiments .
  • Figures 2A-2T Phenotype of CDla + cells derived from CD34 + CD38 ⁇ lin- UCB cells cultured on thymic stromal monolayers for 21 days.
  • Cells were processed for 2- or 3-color staining with CDla conjugated to FITC or PE and monoclonal antibodies labeled with complementary fluorescent molecules (FITC, PE, or Cy) as indicated. Histograms show fluorescent intensity of test (thick line) and isotype-matched control (thin line) antibodies gated on CDla + cells. Results are representative of more than 3 experiments for each antibody tested.
  • CDla + CD14 ⁇ cells generated in vi tro from CD34+38-lin- or CD34 + CD38+lin- UCB cells on thymic stromal cell monolayers are good stimulators in allogeneic mixed lymphocyte reactions (MLR) .
  • CD34 + CD38"lin-UCB progenitors CD34 + CD38"lin-UCB progenitors.
  • CDla + CD14 ⁇ cells induce significantly more proliferation in MLR than CDla ⁇ CD14 + cells (p ⁇ 0.001 at all stimulator : responder ratios).
  • Fig. IB shows the thymidine uptake induced by CDla + CD14 ⁇ and CDla ⁇ CD14 + cells derived from more mature CD34+CD38 + lin- UCB progenitors.
  • CDla _ CD14 + cells generated from CD34+CD38 _ lin- progenitors are not potent stimulators in MLR.
  • FIGS. 4A-4F DCs grown on thymic stroma acquire markers of differentiated DCs in response to TNF- ⁇ .
  • FIGS 5A-5D Human thymic epithelial (TE) cells and thymic fibroblasts (TF) cultured in an artificial capillary system aggregate into nodules that recapitulate the human thymic microenvironment.
  • Human TE cells and TF from postnatal thymus were cultured in vi tro and mixed at a ratio of 95 TE : 5 TF in an artificial capillary system.
  • FIG. 5D photomicrographs of a representative nodule stained by indirect immunofluorescence with anti- keratin mAb AE-3 (Fig. 5A and Fig. 5C) and anti-stromal mAb TE7 (Fig. 5B and Fig. 5D) .
  • the arrowheads point to the fibrous capsule surrounding the nodule and the arrows point to a rest of thymic epithelium forming a rosette in sequential sections.
  • Data are representative of 10 experiments.
  • FIGS. 6A and 6B Umbilical cord blood progenitor cells differentiate into CDla + cells with dendritic morphology in thymic nodules in vi tro . Shown is the reactivity of anti-CDla mAb Nal/34 in sections of thymic stromal nodules (Fig. 6A) cultured in media alone or (Fig. 6B) co-cultured with lin(-) umbilical cord blood (UCB) cells for 4 weeks. Note that the thymic stromal nodules co-cultured with UCB cells contain numerous CDla + cells with a dendritic morphology while the thymic stromal nodules cultured in media alone do not contain any CDla + cells. Dendritic processes of a single CDla + cell in Fig. 6B are indicated by arrows. The CDla "1" cells were cytoplasmic
  • CD3-, CD33 lQ and CD83- which is consistent with the interpretation that these cells are early dendritic- lineage cells. Data are representative of 3 experiments.
  • the present invention relates to an in vi tro method of producing a thymic microenvironment from cultured thymic cells and to a method of reconstituting the immune system of a mammal, for example, a mammal suffering from an immunodeficiency, using same.
  • the invention also relates to a method of generating thymic dendritic cells from hematopoietic cells using such a thymic microenvironment.
  • Thymic epithelial cells suitable for use in the production of the thymic microenvironment of the present invention can be cultured from either fresh or frozen fetal or postnatal thymic tissues.
  • the thymic epithelial cells can be cultured and isolated as described, for example, by Singer et al, Human Immunol. 13:161 (1985) and Singer et al, J. Invest. Dermatol. 92:166 (1989) .
  • Culture medium can contain any of a variety of growth factors, such as EGF, FGF, IGF, and TGF ⁇ / ⁇ and insulin, and/or cytokines, such as IL-6, IL-8 and IFN- ⁇ .
  • Cells so derived can be used immediately or stored frozen, for example, in a medium containing a cryoprotectant such as DMSO.
  • the present invention relates, in one embodiment, to a method of producing a thymic microenvironment from thymic stroma, particularly, human thymic stroma, and to a method of using same to support the development of primative hematopoietic stem cells into dendritic cells.
  • thymic stromal cells thymic fibroblasts and thymic epithelial cells obtained from human thymus tissue as decribed above are depleted of T cells, for example, by adding hydrocortisone to the culture medium (Singer et al, Human Immunol. 13:161 (1985)) or other T cell depleting agent such as deoxyguanosine (Hong, Clin.
  • thymic fibroblasts Reduction in the number of thymic fibroblasts relative to thymic epithelial cells can be effected, for example, by complement-mediated lysis and/or growth on a feeder layer of irradiated NIH 3T3 fibroblasts.
  • Monolayers of thymic stromal cultures prepared as described above can be used directly for dendritic cell production or the thymic stromal cells can be cultured, for example, in an artificial capillary system (eg optionally, with a coating of ProNectinTM F) , to provide 3-dimension cell aggregates or nodules (see Example 1) .
  • an artificial capillary system eg optionally, with a coating of ProNectinTM F
  • various zero gravity culture strategies or strategies providing for three-dimensional cell aggregation with low to no shear stress can also be used to optimize thymic cell growth.
  • Rotating-wall vessel (RWV) technology (Schwartz et al, J. Tiss. Cult. Meth.
  • Stem cells suitable for coculture with the thymic stromal cultures described above can be obtained from human umbilical cord blood, bone marrow and GCSF mobilized peripheral blood stem cells (Siena et al, Exp. Hem. 23:1463 (1995)) (G-PBSCs) .
  • Preferred stem cells are CD34 + CD38 " lin " or CD34 + CD38 + lin " .
  • Such cells can be isolated from the indicated sources using commercially available lineage depleting antibody cocktails and art recognized cell sorting techniques (see Example 1) .
  • Example 1 The data presented in Example 1 demonstrate that umbilical cord blood CD34 + CD38 " lin " and CD34 + CD34 + lin " hematopoeitic progenitor cells cocultured on thymic stromal monolayers in serum free medium develop into cells with phenotypic, morphologic and functional characteristics of thymic dendritic cells.
  • thymic nodules produced as indicated above can support development of dendritic cells from hematopoietic cell progenitors by incubating the nodules with the progenitor cells under conditions such that the progenitor cells migrate into and differentiate in the nodules.
  • thymic stromal cells used in the production of a thymic microenvironment as described herein can be genetically engineered so as to express various factors (eg secreted or surface bound factors) such as CD40 ligand and flt-3-ligand which can increase the yield and activity of the resulting thymic dendritic cells.
  • factors eg secreted or surface bound factors
  • Retroviral vectors can be used to effect the genetic manipulation, as can a variety of other engineering techniques.
  • the thymic stromal cells can be selected/designed to express specific MHC molecules.
  • the thymic stromal cells can be used as packaging systems for transfer of genetic materials into developing hematopoietic cells) (Liu et al, Cell 86:367 (1996) ) .
  • Such cells can be used to reconstitute an immune system that is superior to that of the recipient, for example, in its ability to defend against infection (specific MHC molecules to defend against infection with HIV) or its resistance to infection (CCR5 mutations in preventing infection with HIV) .
  • the present invention relates to immortalized human thymic epithelial cells and to a method of producing same.
  • the establishment of immortalized human thymic epithelial stroma and individual clonal lines derived from such stroma is useful for several reasons.
  • a readily available and consistent source of stroma which supports human thymic dendritic cell generation reduces intra-experimental variation and improves the logistics of generating thymic dendritic cells for study.
  • An immortalized thymic epithelial stromal line can also be extensively characterized, validated for clinical use and can be expanded to scale up the generation of thymic dendritic cells.
  • identification of cytokines ..
  • human thymic epithelial cells in thymus chunks can be immortalized via retroviral vector gene transfer of the papilloma virus E6E7 genes (Furukawa et al, Am. J. Pathol. 148:1763 (1996); LePoole et al, In Vitro Cell. & Devel . Biol. Animal 33:42 (1997)) in order to increase the number of passages these lines can be propagated.
  • One such line, designated TE750 was deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, USA, on October 10, 1997, under the terms of the Budapest
  • TE750 cells could support the generation of thymic dendritic cells
  • CDla + CD14 " HLA DR + cells were generated indicating that immortalized thymic epithelial can also support the development of thymic dendritic cells from primitive progenitors.
  • CD34 + CD38 " lin " cells were placed into the insert of Transwell cultures, separated from the thymic epithelial by a permeable membrane which precluded cell-cell contact.
  • the present invention relates to a method of treating or preventing an autoimmune disease.
  • This embodiment of the invention results from the fact that thymic dendritic cells play a critical role in the negative selection of autoreactive T cells. Accordingly, dendritic cells pulsed with antigens that incite autoimmune disease, for example, diabetes or multiple sclerosis (or other autoimmune disease) , can be used in treatment or prevention protocols.
  • insulin and other islet cell autoantigens are expressed in the thymus and the intrathymic expression of insulin mRNA is regulated by a known disease susceptibility locus for Type I diabetes (Atkinson et al, New Engl . J. Med. 331:1428 (1994); Pugliese et al, Nat. Genetics 15:293 (1997)).
  • Thymic expression of these antigens can mediate the negative selection and tolerance of islet cell reactive T cells. For tolerance to occur, these antigens must be expressed at very high levels in any thymic stromal cell type or they must be expressed by cells specializing in negative selection (preferably, dendritic cells) .
  • dendritic cells propagated and pulsed ex vivo with a diabetes inciting antigen can be used to prevent or treat Type I diabetes.
  • a diabetes inciting antigen such as insulin
  • dendritic cells expanded from progenitor cells of preferably a patient's own bone marrow and pulsed with diabetes inciting antigen (or encoding sequence) can be used to treat or prevent this disease.
  • Such cells can be administered intravenously or intrathy ically .
  • a similar approach can be used to treat other autoimmune diseases.
  • dendritic cells produced as described above can be pulsed with myelin basic protein.
  • the present invention relates to a method of producing a cancer vaccine using dendritic cells prepared as described herein.
  • dendritic cells generated or isolated from the spleen or bone marrow, when pulsed with tumor antigens in vi tro and inoculated into tumor bearing animals, serve as extremely effective cancer vaccines (Porgador et al, J. Exp. Med. 182:255 (1995); Mayordomo et al, Nat. Med. 1:1297 (1995); Boczkowski et al, J. Exp. Med. 184:465 (1996); Alijagic et al, Eur. J. Immunol. 25:3100 (1995); Bernhard et al. Can. Res.
  • the dendritic cells have either been isolated or generated from peripheral blood mononuclear cells (Morse et al, Ann. Surg. 226:6 (1997); Engelman, Biol. Bone Marrow Transp. 2:115 (1996)). Recently, several methods have been described for improving the yield, safety, and efficiency of generating dendritic cells from the peripheral blood including using serum free media, adding macrophage conditioned media, and collecting peripheral blood mononuclear cells after treatment with chemotherapy and/or cytokines (Morse et al, Ann. Surg. 226:6 (1997); Bender et al, J. Immunol. Meth. 196:121 (1996); Romani et al, J. Immunol. Meth.
  • Immature dendritic cells are preferred as the cellular platform in a dendritic cell vaccine (Morse et al, Ann. Surg. 226:6 (1997); Romani et al, J. Immunol. Meth.
  • immature dendritic cells produced in accordance with the present invention are pulsed with a tumor antigen (eg, MAGE, CEA, and her-2/neu (Boon et al, J. Exp. Med. 183:725 (1996)), or nucleic acid (RNA or DNA) encoding same using, for example, standard techniques.
  • a tumor antigen eg, MAGE, CEA, and her-2/neu (Boon et al, J. Exp. Med. 183:725 (1996)
  • RNA or DNA nucleic acid
  • the pulsed cells can be used in vaccination therapies to treat existing tumors or prevent tumor development in individuals at increased risk (Boon et al, J. Exp. Med. 183:725 (1996); Hsu et al. Nature Med. 2:52 (1996)).
  • the thymic microenvironments produced as described above can be used in thymic transplantation for treating congenital and acquired immunodeficiencies including, but not limited to, DiGeorge syndrome, Ataxia-Telangiectasia and Nezelof's disease (congenital immunodeficiencies) and acquired immunodeficiency syndromes, such as AIDS and cancer after ablative chemotherapy (Mackall et al, N. Engl . J. Med. 332:143 (1995)).
  • the present thymic microenvironments can be transplanted into patients by a variety of methods including implantation into the omentum or readily accessible muscles including, but not limited to, the forearm, thigh and calf muscles.
  • Thymic stromal cultures Thymic epithelial (TE) cells and thymic fibroblasts (TF) were cultured by an explant technique and propagated in enriched medium containing 67% DMEM (Gibco BRL, Grand Island, NY) , 22% F12 (Gibco BRL) , 5% Fetal Clone II serum (HyClone, Logan, Utah), 0.4 ⁇ g/ml hydrocortisone, 5 ⁇ g/ml insulin, 11 ng/ml recombinant human epidermal growth factor (Collaborative Biomedical, Bedford MA), 0.18 ⁇ M adenine, 10 M cholera toxin (ICN Biomedicals, Aurora, OH), 0.25 ⁇ g/ml fungizone and 50 ⁇ g/ml genatmicin (TE medium) on irradiated NIH 3T3 fibroblast feeder layers as described (Singer et al, J.
  • Thymic stromal cells thymic fibroblasts and TE cells
  • Thymic stromal cells thymic fibroblasts and TE cells
  • Contaminating thymic fibroblasts were removed from TE cell monolayers by treatment with 0.02% EDTA in PBS followed by complement-mediated lysis with mAb 1B10, which binds to a cell-surface antigen on human fibroblasts (Singer et al, J. Invest. Dermatol. 92:166-176 (1989)).
  • TE cell preparations were >95% positive for the keratin marker AE-3 and negative for CDla, CD7 and CD14.
  • TF Thymic fibroblasts
  • Non-agglutinated white blood cells were harvested and residual red cells were hemolysed at 37°C in 0.17 M NH4CI containing 10 mM Tris-HCl, pH 7.2 and 200 mM EDTA.
  • the white cell fractions were brought to 6-8 XlO " ? cells/ml in PBS containing 4% fetal calf serum (FCS) and were depleted through the addition of a commercial antibody cocktail and magnetic colloid as per the manufacturer's instructions (CD34 + StemSep enrichment cocktail, StemCell Technologies,
  • DMEM Dulbecco's modified MEM
  • FACS fluorescence activated cell sorting
  • Antibody reagents mAbs to the following antigens were used for indirect immunofluorescence staining: P3x63/Ag8 (IgGl, from American Type Culture Collection (ATCC) , Rockville, MD) ; CDla (Nal/34, from Andrew McMichael) (McMichael et al, Eur. J. Immunol. 9:205-10 (1979)); CD2 (35.1, ATCC), CD3 (Leu4, Becton Dickinson, Mountain View, CA) , CD4 (Leu3a, ATCC), CD7 (3Ale) (Haynes et al, Proc. Natl. Acad. Sci. 76:5829-33
  • CD14 LeuM3 (Dimitriu-Bona et al, J. Immunol. 130:145-52 (1983)), AE3 (keratin from TT Sun) (Woodcock-Mitchell et al, J. Cell. Biol. 95:580-88 (1982)), 1B10 (fibroblasts) (Singer et al, J. Invest. Dermatol. 92:166-176 (1989)), M38 (C-terminal region of type I procollagen) (McDonal et al, J. Clin. Invest. 78:1237-44 (1986)), and fluorescein-conjugated goat anti-mouse Ig (Kirkegaard & Perry Laboratories,
  • CD2 leu5, FITC
  • CD3 leu4, PerCP
  • CD5 leul, PE
  • CD7 leu9, FITC
  • CD8 SKI, FITC
  • CDllc S-HCL-3, PE
  • CD14 MfP9
  • CDla T6, PE
  • CD4 T4, PE
  • CD83 HB15a, PE
  • CD3 UCHTl, Cy
  • CD3 CD3
  • CDla HI149, FITC
  • CD2 RPA-2.10, Cy
  • CD40 5C3, FITC
  • CD86 233KFUN-1)
  • FITC CD95
  • DX2, FITC HLA A,B,C (G46-2.6, FITC)
  • IgGl IgGl
  • Irrelevant isotype-matched mAbs were used as negative control. Quantitation of the surface staining was performed on a FACScan and a FACScalibur (Becton- Dickinson) using a 488 argon laser for fluorescence excitation. Data was analyzed using CellQuest software (Becton Dickinson) . In all experiments, cells stained with isotype-matched control antibodies were used to set cursors so that ⁇ 1% of the cells were considered positive. Microscopy: Sorted cells were centrifuged onto glass slides using a Shandon cytocentrifuge (Shandon Southern Instrument Co., Sewickley, PA) at 1000 RPM for 3 minutes.
  • Cytospins were air-dried and stained with Wright Giemsa stain and examined by light microscopy.
  • thymic nodules and sorted cells were fixed with 2% glutaraldehyde in 150 nM sodium cacodylate buffer plus 2.5 mM CaCl2, pH 7.2, washed, and embedded in 1% agar.
  • blocks were washed with cacodylate buffer followed by 200 mM sodium acetate, pH 5.2. Samples were stained en bloc for one hour with 1% uranyl acetate in sodium acetate buffer.
  • PBMCs (1.5 x 10 ⁇ ) obtained from healthy donors were cultured in RPMI 1640 supplemented with 10% FCS or 10% human AB serum in 96 well U-bottom tissue culture plates. Irradiated (3500 rads) sorted CDla+CDl4- and
  • CDla-CDl4+ cells were added in graded doses of 1.5x10 ⁇
  • thymic stromal microenvironment nodules Development of human thymic stromal microenvironment nodules : Cultured thymic stromal cells were co-cultured in an artificial capillary sytem (Cellmax; Cellco, Inc., Germantown, MD) with a coating of ProNectinTM F to promote adhesion of stromal cells to the capillaries. 20-100 x 10- thymic stromal cells (95% TE cells by reactivity with anti-keratin antibody AE-3 and 5% TF by reactivity with anti-procollagen antibody M38) were seeded per capillary module with an extracapillary space of 12 ml. TE medium (Singer et al, Hum. Immunol.
  • Sorted CD34 + 38 ⁇ lin- or CD34+CD38+ lin- cells at 10 3 to 10 4 cells/well were added onto 24 well plates containing approximately 10 micronodules/well and cultured in 1 mL of serum free media.
  • CD34+ cord blood cells differentia te into CDla+ cells on thymic stromal cell monolayers In order to determine whether human thymic stroma could support the development of DCs from hematopoietic progenitors,
  • CD34+CD38" lin- (R2 in Fig. 1A) and CD34 + CD38+lin- umbilical cord blood cells (Rl in Fig. 1A) were isolated by sterile cell sorting and co-culture with pre-established irradiated human thymic stromal monolayers (Herbein et al, Stem Cells (Dayt) 12:187-97 (1994), Terstappen et al. Blood 777:1218-27 (1991)). Prior to co-culture, the sorted populations had greater than 98% purity (Figure IB and IC) and were >98% CDla- ( Figure ID) .
  • UCB progenitors cultured in serum-free media alone did not expand nor change in morphology.
  • Immunophenotypic analysis of co- cultured cells revealed the presence of a number of CDla + CD14 ⁇ HLA-DR + cells ( Figures 1 and 2) similar to previous descriptions of human DCs (Caux et al, Blood Cell Biochemistry 7:263-301 (1996)). The percentage of
  • CDla+CD14- cells could be generated from both the CD34+CD38-lin- and CD34+CD38+lin-populations suggested that both of these cell types could develop into DCs in the thymic stroma monolayers.
  • CDla + CDl4 ⁇ cells generated after 21 days of culture from both CD34+CD38- lin- and CD34 + CD38 + lin- umbilical cord cells were isolated by FACS and examined by light and electron microscopy.
  • CDla ⁇ CD14 + cells were also sorted from both cultures to serve as controls. Analysis of the sorted cells revealed a purity greater than 97%.
  • light microscopy CDla + CD14 ⁇ cells possessed a DC morphology with an irregular shape and multiple dendritic processes.
  • CDla + CDl4 ⁇ cells had euchromatic, lobulated or indented nuclei and a clear cytoplasm with rough endoplasmic reticulu and well-developed Golgi apparati. These cells did not contain Birbeck granules. These findings are consistent with these cells being mature thymic type DCs.
  • the control CDla ⁇ CD14 + cells from both precursor types had the morphologic appearance of macrophages, with indented nuclei and foamy cytoplasm and no evidence of cytoplasmatic dendritic projections.
  • Immunophenotype of CDla-t- cells expanded in thymic stroma In order to better characterize the DCs generated from UCB progenitors on thymic monolayers, extensive phenotypic evaluations were performed using multiparameter FACS analysis (Fig. 2) .
  • CDla + cells generated on thymic stroma from CD34+CD38-lin- UCB cells were negative for surface CD3, CD8, CD19, CD25, CD34, and CD95, expressed CD2, CD4, CDllc, CD13, CD16, CD33, CD38, CD40, CD45, CD49e, CD80, CD83, CD86, MHC class I and MHC class II.
  • CDla+CD14- and CDla -CD14+ cells genera ted in thymic stroma to act as antigen presenting cells : In order to determine whether the putative DCs generated on thymic stroma were able to activate T cells, CDla + CD14 " and CDla ⁇ CD14 + cells were sorted by
  • CDla + CD14- cells were much more potent stimulators in the MLRs than
  • CDla _ CD14+ cells (Fig. 3) .
  • CDla + CD14 ⁇ cells generated from CD34+CD38-lin- UCB cells were more potent stimulators of the MLR on a per cell basis than the CDla + CD14 _ cells generated from CD34+CD38+lin- cells (Fig. 3) . This suggests that more primitive progenitors may not only generate larger numbers of DCs but that these DCs may be qualitatively different from DCs generated from more mature progenitors .
  • TNF- ⁇ treatment induced expression of CDla, CD83, CD80 and CD86 on large numbers of cells derived from CD34+CD38-lin- progenitors (Fig. 45) . In addition, most of these cells displayed a dendritic cell morphology. While TNF- ⁇ treatment of co-cultures established with CD34 + CD38 + lin- cells caused an increase in the fraction of cells with mature DC markers, not all cells expressed DC markers and a significant number of CDla ⁇ CD33 + cells were also observed. This suggested that these cultures may have contained a significant fraction of non-DC myeloid cells. This was confirmed by light microscopic examination that revealed a number of myeloid lineage cells including neutrophils and macrophages at different stages of maturation in the CD34+CD38+lin- co-cultures treated with TNF- ⁇ .
  • thymic microenvironment nodules from cul tured thymic epithelial cells and thymic fibroblasts : Since thymic stromal monolayers do not have the full differentiation capacity of reaggregation cultures such as that seen with fetal thymic organ cultures (Barcena et al, J. Exp. Med. 180:123-32 (1994), Res et al. Blood 87:5196-206 (1996), Spits et al. Blood 85:2654-70 (1995)), and due to the difficulties of obtaining sufficient human fetal thymus for studies, a culture system was developed to form three-dimensional aggregates of cultured post natal TE cells and thymic fibroblasts.
  • the nodules contained TE cells (keratin positive) in a fibroblast matrix (identified by TE7) that was encapsulated by a layer of procollagen-positive fibroblasts.
  • the thymic stromal nodules contained numerous desmosomes and hemi- desmosomes indicating that the epithelial cells within the nodules are able to interconnect and form a network similar to that seen in normal thymus (Haynes et al, J. Exp. Med. 159:1149-68 (1984), Haynes et al, J. Immunol. 132:2678 (1984) ) .
  • TE cells in nodules did not terminally differentiate as determined by lack of reactivity with mAbs STE1, STE2 and 11.24 (CD44v9) (Patel et al, J. Clin. Immunol;. 15:80-92 (1995)), nor did they form Hassall's bodies. This pattern is similar to that seen in the thymic stroma of patients with severe combined immunodeficiency (reviewed in Haynes et al, J. Exp. Med. 159:1149-68 (1984), Patel et al, Int. Immunol. 6:247-254 (1996) ) .
  • CD34+ cord blood cells differentiate in thymic nodules into CDla+ cells with DC morphology To test the functional status of the thymic nodules, an evaluation was made as to whether umbilical cord blood hematopoietic cell progenitors migrate into and differentiate in the nodules in vitro .
  • Lin- UCB cells were incubated with thymic nodules in a 24-well flat- bottom plate in serum free medium at 37°C. After 28 days of co-culture with thymic nodules, the nodules were analyzed for markers of T and NK cells (CDla, CD3, CD7), progenitor cells (CD33, CD34), myeloid cells (CD14) and DC (CDla, CD83) .
  • Nodules cultured in the absence of UCB progenitor cells were also analyzed. No CD3 or CD7 expressing cells were detected in the nodules by indirect immunofluorescence . However, there were numerous CDlakri'Jht cells with dendritic morphology in the nodules seeded with lin- UCB cells (Fig. 6B) , but not in the nodules cultured without UCB cells (Fig. 6A) . The CDla bright cells were CD33 1D and CD83-. Further, at day 0, lin ⁇ cells did not express
  • the slow turning lateral vessel (Synthecon, Inc., Friendswood, TX) can be used to assess the utility of low fluid shear forces (typically 0.81 dyn/cm 2 ; Tsao et al. The Physiologist 35:549 (1992)) on the growth and differentiation of TE cells and thymic fibroblasts (TF) .
  • the STLV, filled with GM (Table 1) can be inoculated with Cytodex-3 microcarrier beads
  • the vessel can be rotated at calculated shear forces ranging from 0.51 dyn/cm 2 to 0.92 dyn/cm 2 (Tsao et al, The Physiologist 35:549 (1992); Goodwin et al, Proc . Soc. Exp. Biol. Med. 202:181 (1993)) for 7d. Aliquots of beads can be removed every 1-2 days and cell number and viability enumerated by the method of Goodwin et al, Proc. Soc. Exp. Biol. Med. 202:181 (1993)). Aggregate formation can be assessed by visual inspection under light microscopy and scanning electron microscopy. To determine the status of epithelial cell differentiation, indirect immunofluorescent studies can be performed with mAbs STE1, STE2, A3D8 and 11.24
  • TE thymic epithelia
  • fibroblasts which divide more rapidly than TE cells in two-dimensional tissue culture systems. Growth of TF and TE cells may be different in a low gravity setting than on planar surfaces as has been demonstrated in Example 1.
  • the problem of fibroblast overgrowth can be circumvented by purging the system of TF by complement mediated lysis after treatment with the fibroblast-specific mAB 1B10 (Singer, J. Invest. Dermatol. 92:166 (1989)). Thymic fibroblasts are, however, stimulatory for TE cell growth.
  • TF and TE cells can be co-cultured at different ratios (1:1, 1:5 and 1:25) on Cytodex-3 microcarrier beads in the STLV to determine the optimal ratio of TF to TE cells.
  • Cell number, viability and differentiation status can be assessed as above.
  • TF cells can be differentiated from TE cells by reactivity with anti-keratin mAb AE-3 on cytopreps or sections or by flow cytometry using antibodies to CD104 and CD105.
  • TE cells express cytoplasmic keratins and CD104 while TF do not.
  • Tissue structure can be analyzed by transmission electron microscopy which can easily differentiate TF from TE cells, as TE cells (but not TF) contain tonofilaments and desmosomes.
  • EGF, insulin and IL-6 are growth factors for human TE cells and combinations of these factors may improve the growth of TE cells.
  • TGF-alpha is a growth factor for TE cells and that TGF-beta may be inhibitory for TE cell growth.
  • IL-8 may be an autocrine growth factor for TE cells or may influence the expression of surface molecules.
  • EFG, FGF, TGF-alpha, TGF-beta, IL-6 and IL-8 can be used alone or in combination to supplement TE cell and/or fibroblast growth. Not only may these factors affect growth and differentiation, they may (like IFN-gamma) affect the expression of adhesion and MHC molecules involved in T cell development. The effect of these factors can be determined by indirect immunofluorescence and flow cytometry on the expression of surface molecules expressed on TE cells.
  • thymic stromal grafts can be implanted under the renal capsule of SCID mice using techniques previously reported (Barry et al, J. Exp. Med. 173:167 (1991)). SCID mice can be treated with anti-asialo GM- 1 to abrogate endogenous NK cell activity. The ability of autologous and allogeneic thymocytes to migrate to the grafts can be tested by IP injection of 50 x 10 6 thymocytes. Migration to grafts can be assessed by IF using anti-human CD2, CD3, CD4, CD6, CD7 and CD8 mAbs
  • thymic stromal cell cultures grown in microgravity function to induce thymocyte differentiation in vi tro . Immature populations of autologous thymocytes, both triple negative (CD3-4-8-, CD7+) and double positive
  • CD3 l0 4+8+ CD3 l0 4+8+
  • FACS fluorescence activated cell sorting
  • the immature thymocytes can be injected into autologous stromal tissues (grown in vitro in microgravity) using a Narishigi micromanipulator, and as a control into thymocyte- depleted chunks of autologous thymus. After in vi tro culture, aliquots of tissues harvested at intervals up to 4 weeks can be analyzed for T cell differentiation by IF on frozen sections using mAbs to CD1, CD2, CD3, CD4, CD6, CD7 and CD8. * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

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Abstract

La présente invention a trait, de manière générale, à un micro-environnement thymique, et, en particulier, à un procédé de production d'un micro-environnement thymique. Cette invention se réfère en outre à une méthode de traitement d'immunodéficiences congénitales et acquises, au moyen d'un micro-environnement thymique produit in vitro. Sont inclus dans les immunodéficiences acquises des syndromes observés dans des affections malignes et des maladies auto-immunes, ainsi que dans des déficits immunitaires classiques des cellules T tels que dans le SIDA.
PCT/US1997/018317 1996-10-10 1997-10-10 Procede de production d'un micro-environnement thymique favorisant le developpement de cellules dendritiques Ceased WO1998015615A1 (fr)

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EP97910881A EP0931138A4 (fr) 1996-10-10 1997-10-10 Procede de production d'un micro-environnement thymique favorisant le developpement de cellules dendritiques
CA002268284A CA2268284A1 (fr) 1996-10-10 1997-10-10 Procede de production d'un micro-environnement thymique favorisant le developpement de cellules dendritiques
JP51776698A JP2001503976A (ja) 1996-10-10 1997-10-10 樹状細胞の成熟を補助する胸腺微環境の生産法
AU48149/97A AU4814997A (en) 1996-10-10 1997-10-10 Method of producing a thymic microenvironment that supports the development of dendritic cells

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WO1999055866A1 (fr) * 1998-04-24 1999-11-04 Transkaryotic Therapies, Inc. Apport de proteines therapeutiques par implantation de cellules genetiquement modifiees dans l'epiploon
EP1048308A3 (fr) * 1999-04-30 2001-10-24 Medtronic, Inc. Système de perfusion pour créer des micro-environnements dans un corps vivant
US6440735B1 (en) 1998-03-31 2002-08-27 Geron Corporation Dendritic cell vaccine containing telomerase reverse transcriptase for the treament of cancer
US6495333B1 (en) 1998-09-22 2002-12-17 Becton Dickinson And Company Flow cytometric, whole blood dendritic cell immune function assay
WO2003105874A1 (fr) * 2002-06-14 2003-12-24 Monash University Cellules epitheliales thymiques a capacite progenitrice
US7351546B2 (en) 1998-09-22 2008-04-01 Becton, Dickinson And Company Flow cytometric, whole blood dendritic cell immune function assay
US7402307B2 (en) 1998-03-31 2008-07-22 Geron Corporation Method for identifying and killing cancer cells
US10617721B2 (en) 2013-10-24 2020-04-14 Ospedale San Raffaele S.R.L. Methods for genetic modification of stem cells

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AUPO723097A0 (en) * 1997-06-06 1997-07-03 Australian National University, The A method for culturing cells

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Title
EXPERIMENTAL CELL RESEARCH, 1995, Vol. 218, TSAO S-W. et al., "Characterization of Human Ovarian Surface Epithelial Cells Immortalized by Human Papilloma Viral Oncogenes (HPV-E6E7 ORFs)", pages 499-507. *
JOURNAL OF EXPERIMENTAL CELL RESEARCH, December 1996, Vol. 184, SAUNDERS D. et al., "Dendritic Cell Development in Culture from Thymic Precursor Cells in the Absence of Granulocyte/Macrophage Colony-Stimulating Factor", pages 2185-2196. *
NATURE MEDICINE, December 1995, Vol. 1, No. 12, MAYORDOMO J.I. et al., "Bone Marrow-Derived Dendritic Cells Pulsed with Synthetic Tumor Peptides Elicit Protective and Therapeutic Antitumor Immunity", pages 1297-1302. *
NATURE, 22 April 1993, Vol. 362, ARDAVIN C. et al., "Thymic Dendritic Cells and T Cells Develop Simultaneously in the Thymus from a Common Precursor Population", pages 761-763. *
See also references of EP0931138A4 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6440735B1 (en) 1998-03-31 2002-08-27 Geron Corporation Dendritic cell vaccine containing telomerase reverse transcriptase for the treament of cancer
US7402307B2 (en) 1998-03-31 2008-07-22 Geron Corporation Method for identifying and killing cancer cells
US7824849B2 (en) 1998-03-31 2010-11-02 Geron Corporation Cellular telomerase vaccine and its use for treating cancer
WO1999055866A1 (fr) * 1998-04-24 1999-11-04 Transkaryotic Therapies, Inc. Apport de proteines therapeutiques par implantation de cellules genetiquement modifiees dans l'epiploon
US6495333B1 (en) 1998-09-22 2002-12-17 Becton Dickinson And Company Flow cytometric, whole blood dendritic cell immune function assay
US7351546B2 (en) 1998-09-22 2008-04-01 Becton, Dickinson And Company Flow cytometric, whole blood dendritic cell immune function assay
EP1048308A3 (fr) * 1999-04-30 2001-10-24 Medtronic, Inc. Système de perfusion pour créer des micro-environnements dans un corps vivant
WO2003105874A1 (fr) * 2002-06-14 2003-12-24 Monash University Cellules epitheliales thymiques a capacite progenitrice
US10617721B2 (en) 2013-10-24 2020-04-14 Ospedale San Raffaele S.R.L. Methods for genetic modification of stem cells

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