WO2006049439A1 - Methode permettant de produire un lymphocyte t cd4 xenogenique et modele animal produisant ce lymphocyte t cd4 xenogenique - Google Patents

Methode permettant de produire un lymphocyte t cd4 xenogenique et modele animal produisant ce lymphocyte t cd4 xenogenique Download PDF

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WO2006049439A1
WO2006049439A1 PCT/KR2005/003693 KR2005003693W WO2006049439A1 WO 2006049439 A1 WO2006049439 A1 WO 2006049439A1 KR 2005003693 W KR2005003693 W KR 2005003693W WO 2006049439 A1 WO2006049439 A1 WO 2006049439A1
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animal
mhc
cells
rag
ciita
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PCT/KR2005/003693
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English (en)
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Seong-Hoe Park
Eun-Young Choi
Kyeong-Cheon Jung
Hyo-Jin Park
Seong-Pyo Park
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Dinona Inc.
Seoul National University Industry Foundation
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Priority claimed from KR1020050104826A external-priority patent/KR100666607B1/ko
Publication of WO2006049439A1 publication Critical patent/WO2006049439A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • 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
    • C12N2517/00—Cells related to new breeds of animals

Definitions

  • the present invention relates to a method of producing CD4 T-cells and an animal model producing CD4 T-cell. More specifically, the method concerns the production of CD4 T cells recognizing antigen presented by xenogenic major histocompatibility complex class (MHC-II) by transplantation of biological material containing cells having T cell differentiation capability and expressing xenogenic MHC II into immunodef ⁇ cient animal, an animal model with the ability to produce the T cells, and clinical applications thereof.
  • MHC-II major histocompatibility complex class
  • the human body defends itself from exposure to foreign substances by immunological reactions.
  • the main component of the immune reactions is the lymphocytes, particularly the CD4 + CD8 " T cells (CD4 T-cells), CD4 CD8 + T cells (CD8 T cells) and B-cells, which mediate antigen-specific immunity.
  • B cells mainly produce antibodies to antigens, and CD8 T cells specifically destroy infected cells.
  • CD4 T cells also known as helper T cells, serve a central role in the immune system by inducing and regulating activities of CD8 T cells and B cells. Malfunction of CD4 T cells is well known in immune- related diseases such as infection, acquired immune deficiency syndrome (AIDS), autoimmune disease and aging.
  • AIDS acquired immune deficiency syndrome
  • B cells T cells can only recognize its specific MHC-peptide complex.
  • the T cell receptor (TCR) of T cells acquires the ability to recognize the specific peptide-MHC complex by positive selection of T cell precursors in the thymic cortex.
  • T cells which recognize self antigens in the form of peptide-MHC complex as presented by thymic medullary epithelial cells and dendritic cells are negatively selected.
  • thymocytes recognizing MHC II differentiate to CD4 T cells and those recognizing MHC I differentiate to CD8 T cells.
  • This process results in a T cell repertoire consisting of about 2.5 x 10 7 clonotypic T cells in the peripheral lymphoid organs of human beings.
  • T cells With aging, production of T cells in the thymus declines, leading to reduced diversity of TCRs in the peripheral tissues and gradual loss of T cell function, which can increase susceptibility to infection, cancer, and autoimmune disease. Similarly, human immunodeficiency virus (HIV) impairs the immune system by selective destruction of CD4 T cells. Therefore, it is possible to treat aging- associated immune dysfunction, infection, autoimmune disease, and AIDS and to stimulate anti-tumor activity by replenishment with T cells having high TCR diversity.
  • HIV human immunodeficiency virus
  • T cells are extremely species-specific, and therefore it is very difficult to produce self MHC-restricted T cells in other organisms or animals.
  • T cell development occurs in the thymus by positive selection via recognition of MHC II molecules present on thymic cortex epithelial cells. Therefore, it was believed that transplantation of human hematopoietic stem cells into an animal host will either lead to failure of T cell development or lead to development of CD4 T cells that recognize MHC II molecules of the host animal. Consequently, it was unlikely that such CD4 T cells would have any therapeutic value in humans.
  • an object of the present invention is to provide a method of production for self CD4 T cells educated by self MHC II- expressing T cells in xenogenic organism, CD4 T cells and an animal model for producing the CD4 T cells, which can be used in the treatment of infection, autoimmune disease, AIDS and malignant tumors.
  • Another embodiment of the present invention is to provide a method of producing a xenogenic T cell in an animal comprising the steps of: transplanting xenogenic biological material containing cells capable of differentiating into T cell and expressing endogenous MHC II of a xenogenic donor into an immunodeficient animal which are incapable of expressing endogenous MHC II and therefore unable to mount an immune response to xenogenic T cell,
  • the still embodiment of the present invention is to provide CD4 T cells prepared by the method.
  • the further embodiment of the present invention is to provide an animal which produces a xenogenic (donor) CD4 T cell being capable of recognizing an antigen presented by MHC II of a xenogenic donor, prepared by transplantation of biological material containing a cell which have T cell differentiation capability and expressing its endogenous MHC II of the xenogenic donor into an immunodef ⁇ cient animal which doest not express endogenous MHC II, and is incapable of mounting an immune response to xenogenic T cells.
  • Figure 1 shows a DNA construct that induces expression of CIITA (MHC
  • Figure 2 shows expression levels of MHC II for transgenic mice (Plck- CIITA Tg) transfected with DNA construct of Figure 1 and for normal B6 mouse as analyzed by flow cytometry.
  • FIG. 3 shows the development of mouse thymocytes in Plck- CIITA tg+ CIITA 0/0 mouse as compared to normal (CIITA + ' 0 ) and CIITA-deficient mouse (CIITA 0/0 ).
  • Figure 4 shows the development of mouse thymocytes in normal (MHC
  • MHC II +/0 MHC II deficient (MHC II 0/0 )
  • Plck-CIITA tg+ MHC II 0/0 mouse as analyzed by flow cytometry.
  • Figure 5 shows development of mature CD4 and CD8 T cells in the peripheral blood of MHC II gene deficient mouse transplanted with bone marrow of both Plck-CIITA Tg and B6. PL mice as compared to control (both wild type B6 and B6.PL bone marrow).
  • Figure 6 shows the diversity of TCR receptors of normal B6 mouse, Plck- CIITA Tg, and Plck-CIITA tg+ CIITA 0/0 mice as analyzed by flow cytometry.
  • Figure 7 shows a comparison of CD4 + CD8 " T cell function in the periphery of normal B6, Plck-CIITA Tg, and PlCk-CIITA ⁇ + CIITA 0 ' 0 mice.
  • Figure 8 shows rat T cells within the blood of RAG-I, IL-2 receptor ⁇ chain, MHC II gene deficient (RAGr'TL ⁇ R ⁇ 'TVlHC II " ' " ) mouse at 15 weeks after rat bone marrow transplantation as analyzed by flow cytometry.
  • Figure 9 shows human T-cells in the blood of RAG 1 " ' " 11.-2RY 7 TVIHC II " ' " mouse at 18 weeks after human bone marrow transplantation as analyzed by flow cytometry.
  • Il-restricted CD4 T cells using xenogenic animals Il-restricted CD4 T cells using xenogenic animals.
  • xenogenic animal means another organism or taxonomical species as classified differently from the donor of the CD4 T cells. For example, a xenogenic animal would show an immunological reaction to the donor's transplant of blood, lymphocytes, or organs.
  • immunodef ⁇ cient animal means a mutant, either man-made or natural, that has been rendered incapable of immune reaction.
  • the appropriate immunodeficient animal for the invention is an animal lacking T cells, B cells, NK cells, and NK-T cell, and therefore does not mount an immunological response against xenogenic T cells.
  • the aforementioned animal does not express endogenous MHC II proteins, so that MHC II is not expressed or MHC II expression itself is blocked in the thymic_epithelia of such animals.
  • the method of inhibiting expression of MHC II molecules any of the following is possible: where the gene encoding the MHC II molecule is lacking; where MHC II gene transcription is unfunctional; where MHC II mRNA translation is unfunctional; or most preferably, the MHC II gene is knocked out.
  • An example of mice incapable of MHC II gene transcription, the gene encoding CIITA (MHC class II transactivator) is missing, but examples of such animals are not limited thereto.
  • the aforementioned animals can be produced by mating immunodef ⁇ cient animals with animals whose MHC II expression or function has been blocked.
  • MHC II denotes all types of MHC II molecules naturally expressed in immunodef ⁇ cient animals. More specifically, I-A and I-E are found in mice and HLA-DP, HLA-DR and HLA-DQ in humans.
  • the immunodef ⁇ cient animal lacks at least one gene selected from the group consisting of recombination activating gene (RAG)-I, RAG-2 and interleukin-2 receptor ⁇ chain (IL-2R ⁇ ), or is severe combined immunodeficiency (SCID) animal, which cannot mount an immune response to xenogenic cells.
  • RAG recombination activating gene
  • IL-2R ⁇ interleukin-2 receptor ⁇ chain
  • SCID severe combined immunodeficiency
  • immunodeficient animals are mouse, rat or pig, but not limited thereto.
  • immunodeficient animals include SCID mice, RAG-I knock-out, RAG-2 knock-out, RAG-I or RAG-2 and IL-2R ⁇ gene knock-out animals, but are not limited thereto.
  • transgenic animals, animal mating and immunodeficient animals can be readily produced or performed through conventional methods by those skilled in the technical field, and may also be obtained by purchase or gift.
  • knock-outs of RAG-I and IL-2R ⁇ , and MHC-II were purchased and mated, resulting in mice deficient in RAG-I, IL-2 ⁇ and MHC-II (RAGl "/' IL-2Rf /' MHC II " ' " ).
  • the animal of the present invention does not express endogenous MHC II and is incapable of mounting an immune response, therefore it can serve as an animal model for producing CD4 T cells capable of recognizing antigen as presented by self MHC II molecules of a xenogenic organism whose biological sample containing cells expressing MHC II and which can differentiate into T cells, has been transplanted into the animal model.
  • the method of producing xenogenic T cells involves transplanting biological material including precursor cells with the potential to differentiate to MHC 1I + thymocytes, into an immunodeficient host followed by separation of developed CD4 T cells capable of recognizing antigen as presented by self MHC II from the blood, spleen and lymph nodes of the host.
  • the aforementioned xenogenic T cells can be either immature T cells in the thymus or mature T cells, particularly the thymic immature CD4 single positive T cells expressing MHC II on the cell surface.
  • the aforementioned biological material can be hematopoietic stem cells from bone marrow, or cord blood, or hematopoietic stem cells differentiated from embryonic stem cells, but are not limited thereto.
  • the aforementioned xenogenic animal can be human, rat, pig, monkey, or non-human primate.
  • the aforementioned transplantation of biological material can be performed by any conventional method known in the technical field of the present invention.
  • the transplantation can be performed by intravenous (IV) injection, specifically by tail IV injection of bone marrow cells depleted of T cells (5 x 10 6 cells), CD34-positive cells of bone marrow or cord blood origin (1-5 x 10 5 cells) in 4-12 week old mice.
  • IV intravenous
  • the aforementioned transplantation is performed only once, but second and third transplantation can be performed with a time interval of 1 to 4 weeks. The time interval is preferably controlled depending on the animal.
  • CD 4 T cells developed from transplanted hematopoietic stem cells can be isolated from blood, spleen or lymph nodes.
  • the method for separating CD4 T cells derived from blood, spleen or lymph node from xenogenic animals may involve using antibodies with magnetic or fluorescent tags to CD4 T cells, but is not restricted thereto.
  • the aforementioned cells can be sorted magnetically following reaction with anti-CD4 antibodies conjugated with magnetic beads (Milteny Biotech, Auburn, CA), or can be sorted by flow cytometry after reacting the aforementioned cells with anti-CD4 antibodies conjugated with fluorescent tags.
  • this invention transplanted rat and human bone marrow cells into RAG-I, IL-2 ⁇ and MHC II deficient mice H ' ' " ) and demonstrated that CD4 T cells of a rat or human donor could be produced. 15 Weeks after injection of 5 x 10 6 rat bone marrow cells resulted in CD4 T cells totaling 37.5% of rat lymphocytes within the mouse.
  • the method of producing xenogenic CD4 T cells in this invention is based on the fact that MHC II molecules present on the surface of immature T cells (thymocytes) can educate other immature T cells, and therefore positively select T cells that can recognize self MHC II molecules. This method is possible under the premise that education by MHC II molecules of the host is specifically blocked. Also, dendritic cells derived from donor cells can induce typical negative selection of self-reactive T cells.
  • the produced CD4 T cells are capable of recognizing diverse antigens and therefore have therapeutic value for treating diseases caused by deterioration of the immune system, i.e. leukemia, AIDS, autoimmune disease and tumors, and aging-associated immune dysfunction.
  • the present invention relates to the animal model producing xenogenic CD4 T cells which is deficient in MHC II expression in all cell surfaces, does not induce immune response to xenogenic cells, and is transplanted with biological material containing xenogenic progenitor cells that are capable of differentiating into thymocytes expressing MHC II, ultimately producing the T cells of interest.
  • FIG. 1 shows the vector structure that consists of the proximal promoter of lck, CIITA cDNA, and the poly A tail of hGH (human growth factor).
  • CIITA is a critical transactivator required for the expression of MHC class II genes in most cells, and the proximal promoter of lck is known to be responsible for the specific expression of the target gene in thymocytes and mature T cells.
  • Human CIITA cDNA construct shown in Fig. 1 was also introduced in the vector containing human CD2 promoter (a generous gift from Dr. Dimitris Kioussis, National Institute for Medical Research, London, UK) to produce transgenic mice designated as CD2-CIITATg.
  • the transgene was injected into the pronuclei of fertilized eggs of B6 mice (C57BL/6). The presence of the inserted human CIITA sequence was confirmed by polymerase chain reaction (PCR) of genomic DNA and flow cytometry of peripheral blood of transgenic mice.
  • the transgenic mouse was designated as Plck-
  • Fresh cell suspensions of thymocytes, splenocytes, lymph node cells, and peripheral blood leukocytes of the transgenic mouse were prepared in phosphate buffered saline (PBS) solution including BSA (bovine serum albumin).
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • the erythrocytes removed by lysis with RBC lysis buffer (DiNonA Inc.) for 5 minutes, centrifuged at 1,000 rpm for 10 minutes to obtain cell pellet, and then suspended in phosphate buffered saline solution containing 5% BSA to produce 1 X 10 6 cell.
  • the suspended solution and antibodies were incubated for 30 min at 4°C, and then centrifuged at 1,000 rpm for 10 minutes to obtain cell pellets. The pellet was washed twice with PBS to remove the unreacted antibody. Finally 200 ⁇ l of PBS was added to the cell pellet after centrifugation, and the flow cytometric analysis was performed using FACS Calibur (Becton-Dickinson, Mountain View, CA).
  • Anti- mouse antibodies used in this study were as follows: anti-I-A b (AF6- 120.1), anti-I- AJl-E (2G9), and anti-CD3 (145-2C11) were purchased from Pharmingen (San Diego, CA), and anti-CD4 (GK 1.5), anti-CD8a (53-6.7), anti-B220 (RA3-B2), and anti-Mac-1 (Ml/70) mAbs were purchased from DiNonA Inc (Seoul, Korea).
  • FIG 2 the expression of MHC class II molecules on the surface of thymocytes and splenocytes of Plck-CIITA Tg mice is compared with that of wild type B6 mice.
  • the thick solid line represents the staining of cells from Plck-CIITA Tg with anti-MHC class II antibody
  • the thin solid line represents the staining of cells from wild type B6 mice with anti-MHC class II antibody.
  • Thymocytes are classified into four subsets according to the expression pattern of CD4 and CD8 (that is, CD4 " CD8 " double negative thymocytes, CD4 + CD8 + double positive thymocytes, and CD4 + CD8 " or CD4 " CD8 + single positive thymocytes).
  • Splenocytes are classified into CD3 + mature T cells, B220 + B cells and MaC-I + macrophages.
  • Plck-CIITA Tg mice MHC class II molecules (I-A b ) are expressed on CD4 + CD8 + DP thymocytes, and expression persisted in the mature CD4 and CD8 T cells of the spleen and lymph nodes, while all the different types of immature and mature T cells from wild-type littermates did not show persistent expression of I-A b .
  • Plck-CIITA Ts+ CIITA 0/c mice in which thymocytes act as the only thymic antigen presenting cells, were produced by backcrossing Plck- CIITA Tg mice to CIITA-deficient mice (CIITA 0 ' 0 ; 66.12982-CZIa""' 0 ' 1 ""'!; The Jackson Laboratory, Bar Harbor, ME), and thymic differentiation was evaluated.
  • MHC class II expression is near-totally suppressed, and it had been known that the development of mature CD4 T cells is severely defected in theses mice (Chang et al, 1996, Immunity, 4, 167-178).
  • MHC class II expression in Plck-CIITA Tg+ CIITA o/o mice is restricted to thymocytes and mature T cells, and thymocytes do not develop into mature CD4 T cells via thymocyte- thymic epithelial cell interaction.
  • Figure 3 shows the development status of thymocytes in Plck- CIITA tg+ CIITA 0/0 and wild type (CIITA + ' 0 ) mice. While differentiation into CD4 SP cells was almost completely abrogated by the lack of MHC II-expressing cells in the thymi of the CIITA 0/0 control mice (0.8%), a substantial fraction of the thymocytes (8.5%) developed into CD4 SP cells in the Plck-CIITA Ts+ CIITA 0/0 mice. These results imply that thymocytes could be positively selected by thymocyte- thymocyte interaction.
  • MHC II function for T-cell interaction To verify that MHC molecules on the surface of thymocytes are essential for the generation of CD4 SP thymocytes in Plck-CHTA tg+ CIITA 0/0 mice, we generated MHC class II-deficient mice (MHC II 0/0 ) in which the expression of the CIITA transgene was limited to T-lineage cells.
  • FIG. 4 shows the results of flow cytometric analysis of thymocytes from wild-type (MHC 1I + ' 0 ), MHC class II-deficient (MHC II° /0 ) and Plck-CIITA lg+ MHC II 0/0 mice.
  • MHC class II-null mice fails to generate CD4 SP thymocytes, irrespective of whether they have the Plck-CIITA transgene (Plck-CIITA TB+ MHC
  • irradiation bone marrow (BM) chimeras were generated by injecting mixed BM cells from Plck-CIITA Tg and B6.PL mice into irradiated (800 cGy) MHC Il-deficient mice (Plck-CIITA Tg +B6.PL->MHC II 0/0 ). Thymocytes and T cells of B6.PL mice express Thy 1.1 antigen on their surface, while Thy 1.2 antigen is expressed on T-lineage cells of Plck-CIITA Tg and wild- type B6 mice. Thus, thymocyte and T cells derived from B6.PL BM could be distinguished from those from Plck-CIITA Tg and wild-type B6 mice.
  • Peripheral blood was taken from the retro-orbital venous plexus of the chimeric mice at 8 weeks after engraftment, and stained with anti-CD4, anti-CD8, anti-thyl .l and anti-thy 1.2 antibodies.
  • Figure 5 shows the result of flow cytometric analysis of peripheral blood after antibody staining.
  • Control chimeras (B6+B6.PL->MHC II 0/0 ), which are reconstituted with mixed BM from normal B6 and B6.PL mice, are unable to produce CD4 T cells of either the B6.PL (Thyl .l) or B6 (Thyl .2) BM origin.
  • B6.PL Thyl .l
  • B6 Thyl .2
  • a substantial fraction (17.3%) of mature peripheral CD4 T cells is generated in Plck-CHTA Tg +B6.PL-»MHC II 0/0 chimeras, in which positively selecting MHC II molecules are provided exclusively by immature thymocytes of Plck-CIITA Tg BM origin.
  • CD4 T cells in Plck-CIITA Tg +B6.PL ⁇ MHC II 0/0 chimeras are derived from both Thyl.2 + (Plck-CIITA T8 donor and MHC II 0/0 host) and Thyl . l + (B6.PL donor) BM. It is clear from the proper generation of Thy 1.I + CD4 cells that MHC class II- ⁇ ositive thymocytes could induce the positive selection of other thymocytes via thymocyte-thymocyte interaction.
  • TCR The T cell receptor
  • TCR V ⁇ The variable region of TCR ⁇ chain
  • CD4 SP thymocytes and peripheral CD4 T cells were typed for TCR V ⁇ usage, and compared to the corresponding cells in Plck-CIITA Tg or normal mice.
  • Thymocytes and splenocytes were extracted from each mouse, and stained with anti-CD4, anti-CD8, and anti-TCR V ⁇ .
  • FIG. 6 shows the results of flow cytometric analysis of thymocytes and splenocytes from wild type B6, Plck-CIITA Tg, and Plck-CIITA tg+ CIITA 0/0 mice.
  • the percentages of each TCR V ⁇ family in the CD4 SP thymocytes and mature CD4 T cell show marginal differences, the overall TCR V ⁇ profiles are similar for the three mouse types, which indicates that MHC II-expressing thymocytes are able to select CD4 T cells with diverse TCR V ⁇ subsets.
  • CD4 T cells selected on MHC II-positive thymocytes are functionally competent were addressed using mixed lymphocyte cultures in vitro.
  • the function of the CD4 T cells from Plck-CIITA T6+ CIITA 0/0 mice was compared with those from Plck-CIITA Tg and normal mice in mixed lymphocyte reactions.
  • CD4 T cells from spleens and lymph nodes of each mouse were isolated by magnetic cell sorting using MACS with anti-CD4 (GKl .5) microbeads.
  • the isolated CD4 T cells (1 x 10 5 ) were stimulated for 3 days with irradiated (3,000 cGy) B6 or BALB/c splenocytes (1 x 10 5 or 4 x 10 5 ) in DMEM medium (Gibco, Carlsbad, CA) that was supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT) and 50 ⁇ M ⁇ -mercaptoethanol.
  • FBS fetal bovine serum
  • the cultures were pulsed with 1 ⁇ Ci/well [ 3 H]-thymidine (Amersham Bioscience) for the final 16 hrs of incubation and the mean incorporation of thymidine in DNA were measured in quadruplicate wells by liquid scintillation counting.
  • Figure 7 shows the results of thymidine uptake after mixed lymphocyte reaction.
  • the CD4 T cells from Plck-CIITA Tg+ CIITA 0/0 mice give more vigorous responses to allogeneic antigen presenting cells (BALB/c) than do those from Plck- CIITA Tg or normal mice.
  • the CD4 T cells from Plck-CIITA Tg+ CIITA 0/0 mice show proliferative responses in the presence of syngeneic stimulator cells (B6), while the CD4 T cells from CIITA Tg or wild-type mice do not respond in this way.
  • B6 syngeneic stimulator cells
  • More vigorous response of CD4 T cells from Plck-CIITA Tg+ CIITA 0/0 mice may reflect the defect in negative selection by medullary thymic epithelial cells and dendritic cells, compared with Plck-CIITA Tg and wild type B6 mice.
  • Bone marrow cells were collected from both femur and tibia of SD rats, and low-density bone marrow cells were obtained after centrifugation (1,000 g, 30 min) onto a 28% bovine serum albumin cushion as previously described by Prakapas et al. (Prakapas et al., 1993, Immunol Lett, 37:63-71). Low-density bone marrow cells were suspended in PBS containing 5% fetal bovine serum, and incubated with biotin- conjugated anti-rat CD4 antibody (Pharmingen, San Diego, CA) at 4 ° C for 20 minutes.
  • biotin- conjugated anti-rat CD4 antibody Pieringen, San Diego, CA
  • mice After washing with PBS, cells were incubated with anti-biotin magnetic bead (Milteny Biotech, Auburn, CA) ) at 4 ° C for 20 minutes, and then mature CD4 T cells were depleted by magnetic cell sorting using MACS system (Milteny Biotech, Auburn, CA). All RAGr'TL-2R ⁇ " ' " MHC II " ' " mice were irradiated with 2.5 Gy using a cobalt radiation source 1 day before cell transfer. Five million rat bone marrow cells were intravenously inoculated into mice.
  • anti-biotin magnetic bead Milteny Biotech, Auburn, CA
  • Rat bone marrow-derived leukocyte population was determined by flow cytometry.
  • Figure 8 shows the results of flow cytometric analysis of peripheral blood taken at 15 weeks after bone marrow transplantation.
  • the percentage of rat MHC class I + cells in the peripheral blood mononuclear cells of RAG r'lL ⁇ R ⁇ ' ⁇ MHC IF ' ' mouse is 92.7% ( Figure 8, left panel).
  • Rat CD4 and CD8 T cells in rat MHC class I + population reach 37.5% and 20.1%, respectively ( Figure 8, right panel).
  • EXAMPLE 5 Production of human CD4 T cells in mice
  • Human CD4 T cells were generated in RAGr y" IL-2R ⁇ " ⁇ MHC H ' ' " mice after the xeno-transplantation of human hematopoietic stem cells.
  • Human cord blood cells were collected during normal full-term deliveries. Mononuclear cells were separated by Ficoll-Hypaque density-gradient centrifugation and suspended in PBS containing 5% fetal bovine serum.
  • Human CD34 + cells were isolated by incubation of cord blood cells with magnetic bead-conjugated anti-human CD34 antibody at 4°C for 20 minute, followed by magnetic sorting using MACS system.
  • mice All RAGl "/" IL-2R ⁇ "/' MHC II “7” mice were irradiated with 2.4 Gy using a cobalt radiation source 1 day before cell transfer.
  • One hundred thousand human CD34 + cells were intravenously inoculated into mice.
  • Peripheral blood was taken from the retro-orbital venous plexus every other week, and stained with anti-mouse CD45, anti-human CD45, anti-human CD4, and anti-human CD8 antibodies. Human leukocyte population was determined by flow cytometry.
  • Figure 9 shows the results of flow cytometric analysis of peripheral blood taken at 18 weeks after bone marrow transplantation.
  • the percentage of human CD45 + cells in the peripheral blood mononuclear cells of RAGl "/' IL-2R ⁇ "/" MHC II ' ⁇ mouse is 80.7% (Figure 9, left panel).
  • Human CD4 and CD8 T cells in human CD45 + population reach 5.5% and 5.6%, respectively ( Figure 9, right panel).
  • Figure 9, left panel Human CD4 and CD8 T cells in human CD45 + population reach 5.5% and 5.6%, respectively
  • this invention pertains to the production of CD4 T cells in xenogenic animals.
  • produced CD4 T cells are capable of recognizing diverse antigens and therefore have therapeutic value for treating diseases caused by deterioration of the immune system, i.e. aging, leukemia, AIDS, autoimmune disease and tumors.

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Abstract

L'invention concerne une méthode permettant de produire des lymphocytes T CD4 xénogéniques. Elle porte plus spécifiquement sur la production de lymphocytes T CD4+CD8' reconnaissant l'antigène présenté par le complexe majeur d'histocompatibilité de classe II (CMH-II) xénogénique, par transplantation d'un matériel biologique contenant des cellules capables de se différencier en lymphocytes T et exprimant un CMH-II xénogénique dans un animal immunodéficient. Du fait que la méthode décrite permet de fournir à l'animal et à l'homme des auto-immunocytes préparés par un animal xénogénique, elle peut servir au traitement des pathologies associées à un dysfonctionnement immunitaire telle que les maladies auto-immunes, le SIDA et les tumeurs malignes.
PCT/KR2005/003693 2004-11-03 2005-11-03 Methode permettant de produire un lymphocyte t cd4 xenogenique et modele animal produisant ce lymphocyte t cd4 xenogenique WO2006049439A1 (fr)

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KR1020050104826A KR100666607B1 (ko) 2004-11-03 2005-11-03 이종의 ｃｄ４ ｔ-세포 생산방법 및 이종의 ｃｄ４ｔ-세포를 생산하는 동물모델
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JP2015002719A (ja) * 2013-06-21 2015-01-08 全国農業協同組合連合会 免疫不全ブタ

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010126278A3 (fr) * 2009-04-27 2011-03-31 Sung Joo Kim Souris nsg humanisée, son procédé de production et son utilisation
US8604271B2 (en) 2009-04-27 2013-12-10 Samsung Life Welfare Foundation Humanized NSG mouse, method of producing the same and use thereof
JP2015002719A (ja) * 2013-06-21 2015-01-08 全国農業協同組合連合会 免疫不全ブタ

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