WO2010071836A1 - Maturation de cellule nk et t à médiation par il-15 - Google Patents

Maturation de cellule nk et t à médiation par il-15 Download PDF

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WO2010071836A1
WO2010071836A1 PCT/US2009/068826 US2009068826W WO2010071836A1 WO 2010071836 A1 WO2010071836 A1 WO 2010071836A1 US 2009068826 W US2009068826 W US 2009068826W WO 2010071836 A1 WO2010071836 A1 WO 2010071836A1
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cells
lymphocytes
human
maturation
adaptive
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PCT/US2009/068826
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English (en)
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James Di Santo
Nicholas Huntington
Yannick Jacques
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Inserm
Cnrs
Institut Pasteur
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • 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
    • A01K2207/00—Modified animals
    • A01K2207/10—Animals modified by protein administration, for non-therapeutic purpose
    • 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
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of 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
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0387—Animal model for diseases of the immune system

Definitions

  • the present invention relates to a transgenic animal model system basecl on the development of transgenic mice bearing components of the human immune system, including large numbers of innate lymphocytes such as mature human natural killer (NK) cells, ⁇ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes.
  • the invention further relates to the utilization of said model system to screen for, or identify, compounds that modulate (increase or decrease) the number and/or activity or maturation of natural killer (NK) cells or other innate lymphocytes.
  • Such compounds may be used in immunotherapies for treatment of pathogenic diseases, cancer, autoimmune, infectious and inflamatory diseases, immunideficiency and for treatment of transplant patients.
  • the invention further relates to the use of the animal model system of the invention, wherein the immune system of the animal has been stimulated by IL-15, to generate antibodies, including monoclonal antibodies, against any antigen of interest.
  • the present invention is based on the discovery that innate cells in general and NK cell differentiation and expression of killer-like inhibitory receptors (KIRs) in particular as well as adaptive CD4 and CD 8 T cells in humans are all regulated by IL-15 mediated signal transduction that operates in a species-specific fashion where human IL- 15 -responsive cells require human IL-15/IL- 15R ⁇ complexes.
  • KIRs killer-like inhibitory receptors
  • Natural Killer cells participate in host protection by eliminating cells with altered expression of self-MHC-I which can result from viral infection or transformation (1). While a role for viral and stress-induced ligands in NK cells activation is appreciated, the best described regulatory mechanism of NK cell activity is the expression of inhibitory receptors for self MHC-I ligands by mature NK cells with high cytotoxic potential. In man, killer-like inhibitory receptors (KIRs) recognizing classical MHC-I molecules HLA-A, -B or -C are expressed on the predominate peripheral NK cell (CD56 lo CD16 + ) subset which possess abundant intracellular perforin and granzymes and display spontaneous cytotoxicity (2, 3).
  • KIRs killer-like inhibitory receptors
  • NK cells expressing at least one KIR recognizing self MHC-I have a lower threshold of activation and appear more functional than NK cells expressing no KIRs or those only expressing KERs recognizing non-self MHC-I ligands (8, 9).
  • This phenomenon termed “licensing” or “disarming” has been well characterized in mice (10) and suggests a role for KIR-self MHC-I interactions during human NK cell development.
  • KIR + NK cells are present in these patients indicating that normal MHC-I expression itself is not required for KIR expression.
  • KBR transporter associated with antigen processing
  • NK cell development requires IL-lSR ⁇ expressing cells to chaperon IL- 15 to the surface where it is bioactive and significantly more potent in inducing activation and proliferation of IL- 15 -responsive cells. This concept is called IL-15/IL- 15R ⁇ "trans-presentation" (14, 15).
  • Both WO2007/001677 and WO2007/046006 disclose that combinations of IL-15 and IL-lSR ⁇ , or complexes of fragments thereof, are capable of modulating an immune response in a host.
  • IL-15 appears to have a minimal effect on NK cell homeostasis in vivo (16, 17).
  • NK cells are dramatically reduced in patients carrying mutations in the common gamma chain ( ⁇ c ) cytokine receptor (used in IL-15/-7/-4/-9/-2 and -21 signal transduction), Jak3 or the shared IL-2/15R ⁇ , while they are present in IL-7R ⁇ -deficient patients suggesting that IL-15 may regulate human NK cell development (18, 19).
  • ⁇ c common gamma chain
  • Jak3 the shared IL-2/15R ⁇
  • H ⁇ S human immune system
  • a recently developed HIS-mouse model is the engraftment of newborn Balb/c Rag2 ⁇ /" ⁇ c "/” mice with human hematopoietic stem cells (HSC) from fetal liver, cord blood or adult bone marrow (20, 21).
  • HSC human hematopoietic stem cells
  • Balb/c Rag2 "/" ⁇ c "/” HIS mice represent a practical HIS model with high human chimerism, most lymphocyte lineages generated, adaptive immune responses occasionally evoked and unlike earlier models do not develop thymomas (20-23).
  • T cell homeostasis involves the generation of new T cells in the thymus, the survival and proliferation of T cells in the periphery and the differentiation from na ⁇ ve to effector or memory [51].
  • TCR T cell receptor
  • pMHC self-peptide + MHC
  • cytokines cytokines
  • IL- 15 is a peculiar cytokine because it requires expression of the IL- 15R ⁇ chain by the same cell in order to be functional.
  • IL- 15 such as monocytes, dendritic cells and stromal cells must also express the IL-15R ⁇ in order to trans-present IL- 15 to IL- 15 responsive cells (expressing the IL-2R ⁇ / ⁇ c complex), and both IL- 15 and IL-15R ⁇ are up-regulated on myeloid cells following inflammation, thereby increasing IL- 15 bioavalability [63-65].
  • mice lacking these proteins have reduced numbers of memory CD8 T cells (most notably the CD122 hl subset), NK T cells and ⁇ / ⁇ T cells in the periphery [66 f 67].
  • memory phenotype CD8 T cells fail to proliferate and survive when adoptively transferred into IL- 15 deficient mice demonstrating a direct role for IL- 15 in their homeostasis [60, 68].
  • H ⁇ S human immune system
  • the mouse thymus is seeded with human T cell progenitors that are able to be positively selected and differentiate into mature CD4 and CD8 T cells capable of migrating to secondary lymphoid organs indicating that a level of cross-reactivity between murine-derived IL-7 and pMHC exist [69-71],
  • the number of peripheral T cells observed in Balb/c Rag2 "A ⁇ c "A HIS mice is limiting and despite exhibiting an abnormally high turnover rate, T cells fail to accumulate with time suggesting T cell survival and homeostasis is not optimal [69-73].
  • HIS Balb/c Rag2 "y" ⁇ c "A mice engrafted with fetal liver HSCs a new in vivo role for human IL- 15 trans-presentation in human T cell development and homeostasis has been identified.
  • Human IL- 15 trans-presentation in HIS mice increases na ⁇ ve CD4 and CD8 T cells, accelerates thymopoiesis providing increased peripheral T cells after hematopoeitic stem cell transplantation and results in higher immunoglobulin production against specific antigens after immunization.
  • This discovery provides a HIS model system for use in identification or generation of novel drags or antibodies for use in immunotherapies designed to treat a variety of different diseases and disorders.
  • the present invention relates to a transgenic animal model system based on the devlopment of mice bearing components of the human immune system, including innate lymphocytes such as mature human natural killer (NK) cells, ⁇ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes.
  • innate lymphocytes such as mature human natural killer (NK) cells, ⁇ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes.
  • the invention relates to immunodeficient mice which are genetically deprived of T, B lymphocytes and NK cells and which have been engrafted with human hematopoeitic stem cells.
  • the transgenic mice may further be transgenic for expression of human HLA class I and/or HLA class II molecules.
  • mice In such transgenic mice, the maturation of innate lymphocytes in general and NK cells in particular, as well as adaptive CD4 and CD8 T lymphocytes, is induced through administration of IL- 15 /IL-15R ⁇ agonists, thereby providing humanized mice having large numbers of innate human lymphocytes in general and human NK cells in particular and adaptive CD4 and CD 8 T lymphocytes.
  • Such mice provide a humanized immune system model to study activity of innate human lymphocytes in general and human NK cells in particular and adaptive CD4 and CD8 T lymphocytes in vivo.
  • the transgenic animal model system of the invention provides a system for identification of novel drugs for use in immunotherapies designed to treat a variety of different diseases and disorders including but not limited to infectious disorders, cancers, autoimmune and inflammatory diseases, immunodeficiency and for treatment of transplant patients.
  • the animal model system of the invention provides a means for assaying the efficacy, toxicity, or side effects of newly developed immunotherapies including, for example, that of vaccines.
  • the animal model system of the invention may also be used to determine the mechanism of action of such immunotherapies.
  • the transgenic animal model system of the invention also provides a system for generating new therapeutics.
  • IL-15 stimulated HIS mice of the invention may be used to generate, for example, new monoclonal antibodies against any antigen of interest following immunization.
  • the present invention is based on the discovery that IL- 15/IL- 15R ⁇ trans- presentation promotes NK cell maturation, innate lymphocyte development as well as CD4 and CD8 T cell homeostasis. Accordingly, the transgenic animals of the invention may be used for screening for compounds that modulate the activity, or level of expression, of IL- 15 and/or IL- 15 Ra, thereby regulating the maturation of NK cells, CD4 T cells and CD8 T cells. Such compounds may be used in immunotherapies for treatment of pathogenic diseases, cancer, autoimmune, infectious and inflamatory diseases and for treatment of transplant patients. In a preferred embodiment of the invention, complexes of IL-15/IL-15R ⁇ that act as agonist on IL-15-responsive human cells are used. The composition can be administered as a therapeutic to treat an existing condition or as a prophylactic in advance of developing a condition.
  • FIG. 1 NK cells develop and populate to various lymphoid tissue in HIS-mice.
  • A 8 weeks following CD34 + CD38 ⁇ HSC engraftment, various organs from HIS -mice were analyzed for human NK cell reconstitution by flow cytometry (human CD45; hCD45). FACS plots are representative. Values represent mean percentage ⁇ s.e.m of 8 mice.
  • B Cellularity of the indicated organs was enumerated and the number of hCD45 + cells determined based of flow cytometry data. Numbers of cells in bone marrow (BM) are per femur and blood are per ml. Values represent mean ⁇ s.e.m. of 8 mice.
  • Human NK cells were analyzed in the thymus (C) and (D) spleen or bone marrow by flow cytometry using antibodies against the indicated human antigens. Events shown were pre-gated on hCD45 + . FACS plots are representative of 8 HIS-mice, with a total of 3 different donor HSCs represented.
  • E 5 x 10 4 CD56 + NK cells were purified from spleen and bone marrow of HIS-mice and stimulated in vitro with IL-12 and IL- 18 or 5 x 10 5 K562 AML cells in the presence of IL-15 for 18h.
  • A HIS mice were injected intra-peritoneally (every 5 days for 15 days) with human IL-15, IL- 15/IL-l 5Ra-Fc or PBS commencing 6 weeks after reconstitution. 3 days after the last injection, bone marrow was analyzed for human NK cells reconstitution by flow cytometry using antibodies against the indicated human antigens.
  • B HIS-mice treated as in (A) were injected intra- peritoneally with 1 mg BrdU daily on the last 2 days before being sacrificed.
  • hCD45 + NKp46 + cells from bone marrow were analyzed for intracellular proteins and incorporation of BrdU by flow cytometry. Facs plots are representative of 2 individual experiments using mice engrafted with and 2 different CD34 + HSC sources.
  • C HIS mice were injected intra-peritoneally (once per week for 4 weeks) with human IL- 15/IL-l 5Ra-Fc, RLI or PBS commencing 6 weeks after reconstitution. 7 days after the last injection, mice were sacrificed and thymus, spleen, liver and bone marrow were analyzed for human NK cells by flow cytometry.
  • mice were sacrificed 3 days after final treatment and were injected intra- peritoneally with 1 mg BrdU daily on the last 2 days before being sacrificed. Facs plots are representative of 2 individual experiments of 3-5 mice per treatment group engrafted with 2 different CD34 + HSC sources.
  • CD8 T cells in the spleen of RLI- treated HIS-mice were enumerated and analyzed for BrdU incorporation by flow cytometry. Facs plots are representative of 4 mice and gated on hCD45 + cells. Histograms are further gated on CD3 + CD8 + cells. Cellularities are mean ⁇ s.e.m of 4 mice.
  • NK cells were enumerated based on (A) and surface expression hCD45 and NK ⁇ 46 determined by flow cytometry.
  • NK cell subsets were analyzed by flow cytometry to detect surface expression of CD56 and CDl 6. FACS plots are pre-gated on hCD45 + cells and are representative of 3-4 mice in each group. Data in (A) and (D) are mean ⁇ s.e.m of 3-6 mice in each group. P values are given when statistically significant.
  • FIG. 4 Human IL-15/IL-l 5R ⁇ agonists promote NK cells differentiation in vivo. NK cell maturation and KIR expression was analyzed in thymus, bone marrow and spleen of HIS-mice treated as in (2C) by flow cytometry for surface expression of CD56, CD16 and KIRs (KIR-2DL2/3/ 1 /-2DS 1/2/4/- 3DL1/S1). HIS-mice were sacrificed 7 days after last treatment. Histograms are gated on hCD45 + CD56 !o CD16 + cells and are representative of 5 mice and 2 different CD34 + HSC sources. **P ⁇ 0.01, *P ⁇ 0.05.
  • FIG. 1 Lymphocyte reconstitution in HIS-mice. 8 weeks after engraftment with human fetal liver HSCs, the indicated organs from HIS -mice were harvested and analyzed for the indicated surface antigen expression by flow cytometry. FACS plots are gated on hCD45 + cells and are representative of at least 8 HIS-mice.
  • Splenic DX5 + murine NK cells were labeled with CFSE and cultured in 30ng/ml murine IL-15 for 72 hours. Cells were the analyzed for surface expression of NKLl and DX5 by flow cytometry.
  • 5 x 104 CD56 + NK cells purified from human peripheral blood were labeled with CFSE and cultured for 3 days in media alone or with 2 x 104 pre-activated human (same donor peripheral blood derived) or murine myeloid cells(derived from Rag2 "/" ⁇ c "/” bone marrow).
  • Myeloid cells were purified by a ⁇ ti-PE magnetic beads against anti-CD 1 lb/CDl lc/F480-PE for mouse and anti- CD 14/CD83/CD116-PE for human and cultured over night 5mg/ml LPS and 10ng/ml IL-4.
  • FIG. 8 Trans-presented IL- 15 enhances human CD4 + and CD8 + T cell development and reconstitution of lymphoid organs in HIS mice.
  • A Experimental scheme. Newborn Rag2 * ⁇ ⁇ c "A mice were irradiated with 3.3 Gy injected intra-hepalic (i.h.) with 5x10 4 CD34 + CD38 " human fetal liver cells. At 8,9,10 and 11 weeks of age, HIS mice were injected intra-peritoneally (i.p) with 2.5 ⁇ g IL-15- ⁇ L-15R ⁇ fusion protein (RLI) or PBS. Mice were sacrificed and analysed at 12 weeks.
  • RLI 2.5 ⁇ g IL-15- ⁇ L-15R ⁇ fusion protein
  • FIG. 9 Trans-presented IL- 15 treatment in vivo results in minimal phenotypic alteration to expanded T cell pool in primary and secondary lymphoid organs.
  • A Thymus, spleen and mesenteric lymph nodes (mLN) from HIS mice treated as in Fig 8A were analysed by flow cytometry for T cell reconstitution by CD27 and CD3.
  • B Human T cells (hCD45 + CD3 + ) were further analysed for surface antigens associated with activation (CD69, CD45RO, CD127, CD45RA, CD62L), maturation (CD122) or regulatory function (CD25). FACS plots are representative of 5 mice per group.
  • CD4+- and CD8+ human T cells (hCD45 + CD3 + ) from (A) spleen and (B) mLN were analysed by flow cytometry for surface antigen corresponding to activation status (CD45RA) and incorporation of BrdU. FACS plots are representative of 3 individual experiments. * P ⁇ 0.05. BrdU uptake by human NK cells in the same organs are shown as a positive control for IL- 15 responsiveness.
  • C Serum from mice at time of sacrifice was tested by Luminex® assay for the concentration of the indicated cytokines. Values represent mean ⁇ s.e.m of 3 mice per group except non- reconstituted mouse who served as a negative control.
  • FIG. 11 Trans-presented IL-15 augments thymopoiesis by targeting T cell progenitors in the thymus.
  • the frequency of (A) DP, iSP4 and DN thymocytes from HIS mice treated as in Fig 8 A were determined by flow cytometry according to the cell surface phenotype shown in (B) and cellularity enumerated. Values represent mean ⁇ s.e.m of 9 mice per group. ** P ⁇ 0.02.
  • B HIS-mice treated as in Fig IA were injected intra-peritoneally with 1 mg BrdU daily on the last 2 days before being sacrificed.
  • FIG. 12 Diverse TCR V ⁇ repertoire in Balb/c Rag2 ' ⁇ c A HIS mice is unaltered following IL-15 treatment in vivo.
  • A Thymocytes or
  • B Splenocytes from HIS mice treated as in Fig 8A were isolated and TCR CDR3 immunoscope analysis was performed for different V ⁇ families. Histograms are representative of results obtained from 5 mice of each group and display amino acid length of CDR3 regions (x-axis) and relative frequency (y-axis).
  • FIG. 13 Development and diversity of ⁇ / ⁇ TCR + T cells in Balb/c Rag2 " ' " ⁇ c "A HIS mice in vivo.
  • Histograms represent the mean percentage + SEM of 3 mice in each group (PBS and IL-15-IL-15R ⁇ ) or 1 human donor (hPBMCs).
  • D Splenocytes from HIS mice treated as in Fig 8 A or human PBMCs were isolated and TCR CDR3 immunoscope analysis was performed for different V ⁇ and V ⁇ families. Histograms are representative of results obtained from 3 mice of each group and display amino acid length of CDR3 regions (x-axis) and relative frequency (y-axis).
  • FIG. 14 IL-15 accelerates thymopoeisis and appearance of peripheral T cells when administered in vivo to Balb/c Rag2 ⁇ A ⁇ c "A HIS mice in the early post-HSC graft period.
  • A Thymus and spleen from HIS mice (5 - 6 weeks post-HSC engraftment) were analysed by flow cytometry for human T cell reconstitution by hCD45, CD56, CD3, CD4, CD8 and CD45RA.
  • HIS mice (5 - 6 weeks post-HSC engraftment) were treated every 3 days with with 2.5 ⁇ g IL-15-IL-15R ⁇ fusion protein (RLI) or PBS and sacrificed 12 days after the first injection.
  • RLI 2.5 ⁇ g IL-15-IL-15R ⁇ fusion protein
  • Human (hCD45 + ) lymphocytes from thymus, spleen and BM were analyzed for mature T cells (CD3 and CD45RA) and double positive thymocytes (CD4 and CD8) by flow cytometry.
  • HIS mice treated as in 7B were bled on days 0, 4, 7 and 12 after the first injection and the total number of human T cells (hCD45 + CD3 + ) per ml of blood was enumerated by flow cytometry.
  • Data represents the fold increase in T cell number in mice treated with IL-15-IL-15R ⁇ compared to PBS at each time point. Data is mean percentage + s.e.m of 3 mice.
  • C Almost a 10-fold increase in human T cells in the blood after the second injection (day 4) and increasing up to 20-fold more T cells at the end of the treatment regime was observed.
  • the present invention relates to a transgenic animal model system based on the development of transgenic mice bearing components of the human immune system, including innate lymphocytes such as mature human natural killer (NK) cells, ⁇ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes.
  • innate lymphocytes such as mature human natural killer (NK) cells, ⁇ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes.
  • the transgenic mice of the invention may be used to identify compounds that modulate the maturation or activity of innate human lymphocytes such as mature human natural killer (NK) cells, ⁇ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes and to further assess the efficacy, toxicity and side effects of any newly developed immunotherapies, including, for example, vaccine development.
  • Such immunotherapies are designed for treatment of pathogenic diseases, cancer, autoimmune, infectious and inflamatory diseases and for treatment of transplant patients.
  • the present invention is based on the discovery that innate human lymphocytes in general and human NK cell differentiation and expression of killer-like inhibitory receptors (KIRs) in particular as well as adaptive CD4 and CD8 T cells in humans are all regulated by IL- 15 that operates in a species-specific fashion where human IL-15-responsive cells require human IL-15/IL-15R ⁇ complexes.
  • KIRs killer-like inhibitory receptors
  • the present invention relates to a transgenic animal model system based on the devlopment of mice bearing components of the human immune system, including mature NK cells, ⁇ T cells, NK T cells, CD 4 and CD8 T cells and B cells.
  • a transgenic mouse is provided, characterized in that it has (i) a phenotye comprising a deficiency for murine T lymphocytes, B lymphocytes and NK cells; (ii) is engrafted with human hematopoietic cells; and (iii) contains human NK cells in the lymphoid tissues of said engrafted mouse.
  • the transgenic mouse further comprises human CD4 and CD8 peripheral T cells and human B cells.
  • the transgenic mice contain at least 1% mature human NK cells in the lymphoid tissues of said engrafted mouse.
  • the transgenic mouse further comprises a phenotype comprising a deficiency for murine MHC class I and/or MHC class II molecules and transgenic for the expression of human HLA class I and/or HLA class II molecules.
  • the transgenic mice according to the present invention which are deficient for murine T and B lymphocytes, and NK cells (immunologically deficient mice) may comprise two genes essential in T, B and/or NK cell development that are inactivated by a spontaneous mutation or a targeted mutation.
  • These mutations which are well- known to those of ordinary skill in the art include, for example; a first mutation which is the mouse scid mutation (Prkdc scid ; Bosma et al., Nature 183, 301, 527-530; Bosma et al., Curr. Top.
  • the transgenic mice of the invention may further comprise a phenotype having a deficiency for murine MHC class I and/or MHC class II molecules and transgenic for the expression of human HLA class I and/or HLA class II molecules.
  • Such mice may be engineered using the methods set forth in WO 2008/010100, which is incorporated by reference herein in its entirety.
  • the transgenic mice of the invention which are deficient for murine T and B lymphocytes, and NK cells, and which may also express human HLA Class I and Class II molecules, are further engrafted with human hematopoietic stem cells (HSCs).
  • HSCs may be derived from, for example, fetal liver, cord blood or adult bone marrow and are characterized by expression of CD34.
  • the cells may be cultured for an appropriate time before transplantation, to improve the engraftment rate of the hematopoietic progenitors into the transgenic mouse. The number of cells that are transplanted is determined so as to obtain optimal engraftment into the transgenic mouse.
  • human CD34 + cells from 10 4 to 10 6 cells
  • cord blood or fetal liver are transplanted intraperitoneally, intra-hepatically, or intraveniously, for example via a facial vein, into sub-lethally irradiated newborn transgenic mice.
  • the engraftment of cells into immunodeficient mice can be accomplished using methods well known to those of skill in the art (Traggiai et al., Science, 2004,304, 104-107; Ishikawa et al., Blood, 2005, 106, 1565-1573; Gimeno et al, Blood, 2004, 104, 3886-3893; Vodyanik el al., Blood, 2005, 105, 617-626).
  • the numbers of innate human lymphocytes in general and NK cells in particular and the maturation of NK cells as well as human adaptive CD4 and CDS T lymphocytes can be induced through administration of IL- 15 and EL- 15 Ra agonists, resulting in imraunodeficient mice containing large quantities of innate human lymphocytes and mature NK cells and adaptive CD4 and CD8 T cells.
  • agonists include, for example, IL-15/IL-15R ⁇ complexes, including for example IL-15/IL-ISRa, a complex of IL-15/IL-15R ⁇ fragments, hIL-15+IL-l 5Ra-Fc and RLI (Receptor-Linker-Interleukin).
  • the IL-15/IL-15R ⁇ complex could also be administered as an expression vector (lentivirus, adenovirus, retrovirus, AAV for example) or as a transgene.
  • an expression vector lentivirus, adenovirus, retrovirus, AAV for example
  • a transgene for expression in mammalian cells a codon optimized version of the IL- 15 and IL- 15Ra genes, as described in US209/0082299, may be used.
  • the engrafted transgenic mice of the invention as defined above may also be used for the production of humanized antibodies or fragments of these antibodies. Accordingly, the subject of the present invention is also a method for preparing humanized antibodies or fragments of these antibodies, comprising (i) the immunization of the engrafted transgenic mice with an antigen of interest; and (ii) the production of humanized antibodies or fragments of these antibodies, from serum or B lymphocytes of said transgenic mice. In a further embodiment of the invention, the engrafted transgenic mice are stimulated with IL- 15 or an IL- 15 or IL-15 Ra agonist such as an IL- 15/IL-ISRa complex prior to immunization.
  • the invention encompasses the production of polyclonal or monoclonal antibodies consisting of monomeric or dimeric antibodies, and fragments thereof, in particular the Fab, Fab'2 and Fc fragments.
  • the transgenic mice according to the invention have the advantage of allowing the production of monoclonal antibodies which are immediately humanized antibodies.
  • the humanized antibodies and fragments thereof are prepared by conventional techniques known to persons skilled in the art, such as those described in Antibodies: A Laboratory Manual, E. Howell and D. Lane, Cold Spring Harbor Laboratory, 1988.
  • the humanized antibodies according to the invention and fragments thereof as defined above are well tolerated in humans and have a prolonged half-life in humans, given that the constant region of the heavy chain and the entire light chain of these antibodies are of human origin.
  • the present invention is also directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a humanized antibody or a fragment of this antibody in conjunction with a carrier.
  • Such compositions, comprising antibodies or its fragment thereof, may be used for the prevention and treatment of an infectious disease or cancer.
  • the transgenic animals of the invention provide a model system to screen for, or identify, compounds that modulate (inhibit or activate) the activity or maturation of innate human lymphocytes in general and NK cells in particular, and adaptive CD4 and CD8 T lymphocytes. Accordingly, the present invention provides for methods for identifying a compound that induces NK cell maturation, comprising (i) contacting a transgenic mouse of the invention with a test compound and (ii) measuring the level of NK cell maturation; wherein an increased in the level of NK cell maturation in the presence of the test compound indicates that the test compound induces NK cell maturation.
  • the present invention provides for methods for identifying a compound that induces T cell maturation, comprising (i) contacting a transgenic mouse of the invention with a test compound and (ii) measuring the level of T cell maturation; wherein an increased in the level of T cell maturation in the presence of the test compound indicates that the test compound induces T cell maturation.
  • the present invention provides for methods for identifying a compound that modulates T cell maturation, comprising (i) contacting a transgenic mouse of the invention with a test compound and (ii) measuring the level of T cell maturation; wherein an increased in the level of T cell maturation in the presence of the test compound indicates that the test compound modulates cell maturation.
  • the T cells are CD4 or CD8 T cells.
  • the present invention also provides for methods for identifying a compound that inhibits NK cell maturation comprising (i) contacting a transgenic mouse of the invention with a test compound, in the presence of an IL-15/IL-15R ⁇ complex and measuring the level of NK cell maturation; (ii) in a separate experiment, contacting a transgenic mouse of the invention with an IL-15/IL-15R ⁇ complex and measuring the level of NK cell maturation, where the conditions are essentially the same as in part (i) and then (iii) comparing the level of NK cell maturation measured in part (i) with the level of NK cell maturation in part (ii), wherein a decrease level of NK cell maturation in (i) compared to (ii) indicates that the test compound is an inhibitor of NK cell maturation.
  • the present invention also provides for methods for identifying a compound that inhibits T cell maturation comprising (i) contacting a transgenic mouse of the invention with a test compound, in the presence of an IL-15/IL-15R ⁇ complex and measuring the level of T cell maturation; (ii) in a separate experiment, contacting a transgenic mouse of the invention with IL-15/IL-15R ⁇ complex and measuring the level of T cell maturation, where the conditions are essentially the same as in part (i) and then (iii) comparing the level of T cell maturation measured in part (i) with the level of T cell maturation in part (ii), wherein a decrease level of T cell maturation in (i) compared to (ii) indicates that the test compound is an inhibitor of T cell maturation.
  • the T cells are CD4 or CD 8 T cells.
  • the animal model system of the invention bearing components of the human immune system including expression of human NK cells, CD4 T cells and CD8 T cells further provides a means for assaying the efficacy, toxicity, or side effects of newly developed immunotherapies. Newly developed cancer treatments may also be tested for their efficacy, toxicity, and/or presence of side effects.
  • infectious disease treatments may be assayed using the transgenic mice of the invention.
  • infectious diseases include, for example, bacterial, viral, fungal or parasitic diseases.
  • treatments designed to inhibit transplant rejection may be assayed using the transgenic mice of the invention.
  • the transgenic animals of the invention also provide an animal model sytem for screening for compounds that modulate the activity, or level of expression, of IL- 15 and/or IL-lSR ⁇ , thereby regulating the maturation of NK cells, ⁇ T cells, CD4 T cells, and/or CD8 T cells.
  • Such compounds may be used in immunotherapies for treatment of pathogenic diseases, cancer, autoimmune and inflamatory diseases and for treatment of transplant patients.
  • the present invention provides for methods for identifying a compound that increases IL- 15 expression or activity, i.e., an agonist, thereby inducing NK cell maturation, comprising (i) contacting a transgenic mouse of the invention with a test compound in the presence of IL-15 and (ii) measuring the level of NK cell maturation; wherein an increased in the level of NK cell maturation in the presence of the test compound indicates that the test compound increases IL- 15 expression or activity.
  • the present invention provides for methods for identifying a compound that increases IL- 15 expression or activity, i.e., an agonist, thereby inducing T cell maturation, comprising (i) contacting a transgenic mouse of the invention with a test compound in the presence of IL- 15 and (ii) measuring the level of T cell maturation; wherein an increased in the level of T cell maturation in the presence of the test compound indicates that the test compound increases IL- 15 expression or activity.
  • the T cells are CD4 or CD8 T cells.
  • the present invention provides for methods for identifying a compound that increases IL-15R ⁇ expression or activity, i.e., an agonist, thereby inducing NK cell maturation, comprising (i) contacting a transgenic mouse of the invention with a test compound and (ii) measuring the level of NK cell maturation; wherein an increased in the level of NK cell maturation in the presence of the test compound indicates that the test compound increases IL-15R ⁇ expression or activity.
  • the present invention provides for methods for identifying a compound that increases IL-15R ⁇ expression or activity, i.e., an agonist, thereby inducing T cell maturation, comprising (i) contacting a transgenic mouse of the invention with a test compound and (ii) measuring the level of T cell maturation; wherein an increased in the level of T cell maturation in the presence of the test compound indicates that the test compound increases IL-15R ⁇ expression or activity.
  • the T cells are CD4 or CD8 T cells.
  • the present invention also provides for methods for identifying a compound that inhibits IL- 15 or IL-lSR ⁇ activity or expression, i.e., an antagonist, thereby inhibiting NK cell maturation comprising (i) contacting a transgenic mouse of the invention with a test compound, in the presence of an IL-15/IL-15R ⁇ complex and measuring the level of NK cell maturation; (ii) in a separate experiment, contacting a transgenic mouse of the invention with an IL-15/IL-15R ⁇ complex and measuring the level of NK cell maturation, where the conditions are essentially the same as in part (i) and then (iii) comparing the level of NK cell maturation measured in part (i) with the level of NK cell maturation in part (ii), wherein a decrease level of NK cell maturation in (i) compared to (ii) indicates that the test compound is a IL- 15 of IL- 15R inhibitor.
  • the present invention also provides for methods for identifying a compound that inhibits IL- 15 or IL- 15 Ra activity or expression, i.e., an antagonist, thereby inhibiting T cell maturation comprising (i) contacting a transgenic mouse of the invention with a test compound, in the presence of an IL-15/IL-15R ⁇ complex and measuring the level of T cell maturation; (ii) in a separate experiment, contacting a transgenic mouse of the invention with an IL-15/IL-15R ⁇ complex and measuring the level of T cell maturation, where the conditions are essentially the same as in part (i) and then (iii) comparing the level of T cell maturation measured in part (i) with the level of T cell maturation in part (ii), wherein a decrease level of T cell maturation in (i) compared to (ii) indicates that the test compound is a IL- 15 of IL-15R inhibitor.
  • the T cells are CD4 or CD8 T cells.
  • a test molecule to modulate the maturation of innate human lymphocytes such as mature human natural killer (NK) cells, ⁇ T cells s NK-T cells, as well as adaptive CD4 T cells and CD 8 T cells may be measured using standard biochemical and physiological techniques.
  • the maturation of NK cells can be measured through detection of specific cell surface markers that are expressed on the surface of NK cells as they mature and differentiate. For example, as NK cells differentiate the level of cell surface CD56 expression decreases and the levels of CD 16 and KIRs increases. NK cell differentiation can also be measured by NK cell effector functions including cell cytotoxicity and cytokine production.
  • the maturation of T cells can be measured through detection of specific cell surface markers that are expressed on the surface of T cells as they mature and differentiate. For example, as T cells differentiate the level of cell surface CD62L expression decreases and the levels of CD44 and KLRG-I increases. T cell differentiation can also be measured by T cell effector functions including cell cytotoxicity and cytokine production.
  • Preferred methods for the identification of such cell surface markers in the biological sample of a test animal can involve, for example, immunoassays wherein cell surface markers are detected by their interaction with a cell surface specific antibody.
  • Such antibodies include, but are not limited to anti ⁇ CD56, anti-CD 16 and anti-KIR, anti-CD62L, anti-CD44 and anti-KLRG-1 antibodies, to name a few.
  • Antibodies useful in the present invention can be used to quantitatively or qualitatively detect the presence of NK or T-cell surface markers.
  • reagents other than antibodies, such as, for example, polypeptides that bind specifically to the cell surface marker proteins can be used in assays to detect the level of protein expression.
  • Immunoassays useful in the practice of the invention include but are not limited to assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, imraunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few,
  • Immunoassays for detecting NK-ceils, CD4 T cells or CD8 T cells typically comprise contacting the biological sample, such as a blood or tissue sample derived from the test animal, with an anti-cell surface marker antibody under conditions such that an immunospecific antigen-antibody binding reaction can occur, and detecting or measuring the amount of any immunospecific binding by the antibody.
  • such binding of antibody can be used to detect the presence of on or more cell surface marker proteins specifically expressed on differentiated NK cells, or CD4 or CD8 T cells wherein the detection of said proteins is an indication of NK cell differentiation or T cell differentiation.
  • Detection of antibodies bound to NK or T cell surface specific markers may be accomplished using a variety of methods. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect cytidine deaminase protein expression through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986).
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • the antibody may also be labeled with a fluorescent compound.
  • fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin and fluorescamine.
  • a bioluminescent compound may be used to label the antibody. The presence of a bioluminescence protein is determined by detecting the presence of luminescence.
  • Important bioluminescence compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • IL- 15 and/or IL- 15R activity include but are not limited to compounds that bind to IL- 15 or the IL-15R 5 and either activate the ligand/receptor activity (agonists) or block the ligang/receptor activity (antagonists).
  • compounds (agonists or antagonists) may be identified that do not bind directly to IL- 15 or IL-15 R but which are capable of altering their activity by altering the activity of a protein involved in the IL-15 mediated signal transduction pathway.
  • the compounds which may be screened in accordance with the invention include, but are not limited to, small organic or inorganic compounds, peptides, antibodies and fragments thereof, and other organic compounds e.g., peptidomimetics) that bind to IL- 15 or IL- 15R and either mimic the activity triggered by ligand/receptor binding (i.e., agonists) or inhibit the activity triggered by ligand/receptor binding (i.e., antagonists).
  • Compounds may include, but are not limited to, peptides such as, for example, soluble peptides, including but not limited to members of random peptide libraries (see, ⁇ g 1 , Lam, K.S. et al, 1991, Nature 354:82-84; Houghten, R. et al, 1991, Nature 354:84-86); and combinatorial chemistry-derived molecular library made of D- and/or L- configuration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries; (see, e.g., Songyang, Z.
  • antibodies including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab') 2 and Fab expression library fragments, and epitope binding fragments thereof), and small organic or inorganic molecules.
  • Other compounds which maybe screened in accordance with the invention include but are not limited to small organic molecules that affect the expression of the 11-15 or IL- 15R gene or some other gene involved in IL- 15 mediated signal transduction.
  • compositions comprising an effective amount of a compound capable of increasing the number of mature NK cells, and a pharmaceutically acceptable carrier.
  • compositions comprising an effective amount of a compound capable of increasing the number of mature T cells, including but not limited to CD4 and CD8 T cells, and a pharmaceutically acceptable carrier.
  • Such compounds include, but are not limited to IL- 15 and IL-15R agonists.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical sciences” by E. W. Martin.
  • the present invention relates to methods and compositions for preventing the occurance or progression of infectious diseases, including but not limited to those arising from infections with pathogens such as viruses, bacteria, fungi or parasites.
  • the method comprises administering to a mammal a compound that induces NK cell maturation, or T-cell development, in an amount effective to prevent the occurance of the infectious diesase, or to slow or halt the progression of said disease.
  • Such compounds include those that act as agonists of IL-15 or I1-15R.
  • the compounds can be administered as a therapeutic to treat an existing condition or as a prophylactic in advance of exposure to pathogen.
  • the present invention relates to methods and compositions for preventing the occurance or progression of a cancer or pre-cancerous condition.
  • the method comprises administering to a mammal a compound that induces NK cell maturation, or T-cell development, in an amount effective to prevent the occurance of the cancer, or to slow or halt the progression of said disease.
  • Such compounds include, but are not limited to, agonists of IL-15 or IL-15R.
  • the compounds can be administered as a therapeutic to treat an existing condition or as a prophylactic in advance of exposure to a carcinogenic compound or event.
  • the present invention also relates to methods and compositions for preventing transplant rejection in transplant patients.
  • the method comprises administering to a mammal a compound that induces NK cell maturation, or T-cell development, in an amount effective to prevent transplant rejection.
  • Such compounds include, but are not limited to, agonists of IL-15 or I1-15R .
  • the present invention relates to methods and compositions for preventing the occurance or progression of an autoimmune disorder.
  • the method comprises administering to a mammal a compound that inhibits NK cell maturation, or T-cell development, in an amount effective to prevent the occurance of the autoimmue disorder, or to slow or halt the progression of said disease.
  • Such compounds include, but are not limited to, compounds that act as antagonists of IL- 15 or I1-15R .
  • the present invention also relates to methods and compositions for preventing the occurance or progression of an inflammatory disorder.
  • the method comprises administering to a mammal a compound that inhibits NK cell maturation, or T-cell development, in an amount effective to prevent the occurance of the autoimmue disorder, or to slow or halt the progression of said disease.
  • Such compounds include, but are not limited to, antagonist of IL- 15 or IH 5 R.
  • Such anti-infiamatory conditions include, but are not limited to arthritis, asthma and allergies.
  • the present invention also relates to methods and compositions for preventing the occurance or progression of an immunodeficiency disorder.
  • the method comprises administering to a mammal a compound that modulates NK cell maturation, or T-cell development, in an amount effective to prevent the occurance of the immunodeficiency disorder, or to slow or halt the progression of said disease.
  • Such compounds include, but are not limited to, agonist of IL- 15 or I1-15R.
  • Various delivery systems are known and can be used to administer a compound capable of modulating NK cell maturation or T-cell development, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e ⁇ g,, Wu and Wu, 1987, J Biol. Chem. 262:4429-4432).
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. Pulmonary administration can also be employed, e ⁇ g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • Administration can be systemic or local.
  • Pulmonary administration can also be employed, e ⁇ g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • compositions of the invention may be desirable to administer the compositions of the invention locally to a specific area of the body; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e ⁇ g,, in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • the amount of the compound of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses maybe extrapolated from dose response curves derived from in vitro or animal model test systems. Additionally, the administration of the compound could be combined with other known efficacious drugs if the in vitro and in vivo studies indicate a synergistic or additive therapeutic effect when administered in combination.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention, optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice re ⁇ ects approval by the agency of manufacture, use or sale for human administration.
  • mice C57BL/6 mice were purchased from Harlan (France).
  • mice were backcrossed onto the Balb/c background and Rag2 " " ⁇ c " " mice on a Nude background were maintained in isolators with autoclaved food and water.
  • Mice with a human immune system (HIS) were generated as previously described (20, 21). Briefly, newborn (3 - 5 day old) Rag2 '/" ⁇ c "/” mice received sub-lethal (3.3 Gy) total body irradiation from a Cs source, and were injected intra-hepatic (i.h.) with IxIO 5 sorted CD34+CD38- or 5xlO 5 CD34+ human fetal liver cells. All manipulations of HIS mice were performed under laminar flow.
  • HIS-mice were injected intra-peritoneally with lOOul of either human IL- 15 (2.5 ⁇ g), human IL- 15Ra-Fc (7.5 ⁇ g) + human IL-15 (2.5 ⁇ g) both from R&D Systems (Minneapolis, MN), 2.5 ⁇ g RLI(29) or PBS alone commencing at a minimum of 6 weeks after reconstitution.
  • lOO ⁇ l of 10mg/ml of BrdU from BrdU flow kit (BD Bioscience, CA) was injected IP daily 2 days prior to sacrificing mice.
  • NK cell transfer assays 3 x 10 4 CD56 hi CD16TOR ' or 2 x 10 5 CD56 ?0 CD16 + KIRs NK cells were sorted from human CD45 + CD3 " fetal splenocytes (15 weeks gestation) and transferred i.h into 1 week old sub-lethally irradiated Balb/c Rag2 "/" ⁇ c ⁇ / ⁇ mice. A cocktail of KIR antibodies recognizing KJR-2DL2/3/1/-2DS1/2/4/-3DL1/S1 was used. Recipients were then treated i.p with PBS or 2.5 ⁇ g of RLI on day 0 and day 4 post- transfer.
  • IL-15 in vivo neutralization was performed by i.p administration of 50 ⁇ g of goat anti-human IL-15 or 25 ⁇ g of goat anti -mouse IL-15 both from R&D Systems (Minneapolis, MN) every second day for 7 days.
  • Intracellular staining was performed after fixation and permeabilization of the cellular suspensions using BD Perm/Wash and BD Cytofix/Cytoperm reagents from BD Bioscience (San Jose, CA) according to manufacturer instruction.
  • BrdU detection cells were incubated for 1 h at 37 0 C with 30 ⁇ g DNAse from BrdU flow kits (BD Bioscience, CA). All washings and reagent dilutions were done with PBS containing fetal calf serum (FCS). All acquisitions were performed using LSRII, Canto 1 or Canto 2 cylometers, cell sorting was performed using FACS ARIA, all machines were interfaced to the FACS-Diva software (BD Bioscience).
  • NK cells were purified from donor blood buffy coat prepared by density gradient centrifugation over Ficoll-Hypaque (Nycomed Pharma, Roskilde, Denmark) using anti-CD56 magnetic beads (Miltenyi Biotech, Auburn, CA). C57BL/6 splenic NK cells were purified by anti-DX5 magnetic beads (Miltenyi Biotech, Auburn, CA).
  • Purified cells were loaded with 5 ⁇ M CFSE (Molecular Probes) and cultured at 2 x 10 5 cells/ml in RPMI with 10% FCS and lOng/ml rhIL-15 (rhIL-15; R&D Systems, Minneapolis, MN) or 30ng/ml rmIL-15 (Peprotech) for 3 or 5 days.
  • 1 x 104 CD56+ NK cells purified from human peripheral blood were labeled with CFSE and cultured for 72 hours in combinations of human or murine IL-15 which had been pre-incubated with human or murine IL-15Ra-Fc for 1 hour at 4°C.
  • IL- 15 and IL- 15Ra-Fc were 5ng/ml and 20ng/ml respectively.
  • In vitro re-stimulation of HIS-derived NK cells was performed using CD56 + NK cells purified by magnetic beads (Miltenyi Biotech, Auburn, CA) from a cell suspension of spleen and bone marrow from 7 HIS-mice pooled together.
  • NK cells were cultured at 2.5x10 5 cells/ml in RPMI supplemented with 10% FCS, rhIL-15 (5 ng/ml) and either rhIL-12 (5 ng/ml) + rhIL-18 (20 ng/ml, R & D Systems) or 2.5xlO 6 K562 cells/ml (ATCC) for 18 hours with brefeldin A (Sigma) added for the last 4 hours of culture.
  • Retroviral Bcl-xL expression Human fetal liver cells were prepared and modified by retroviral transduction as follows. The Sorted CD34 + CD38 " fetal liver cells were cultured overnight in IMDM (Invitrogen) supplemented with Yssel's medium, 5% normal human serum, 20ng/mL human stem cell factor, 20ng/mL human thrombopoietin and 20ng/mL human interleukin-7 (PeproTech, Rocky Hill, NJ).
  • NK cell specific antibody anti-NK ⁇ 46
  • human NK cells were identified in all lymphoid organs of HIS-mice although at low frequencies, typically between 0.3% to 1.5% human lymphocytes (Fig. 1 A). Both CD56hiCD16- and CD561oCD16+ NK cells subsets are present, with the majority of NK cells having the latter phenotype (Fig. 1 C). Some CDIo + cells expressed CD56 at levels indistinguishable from non- NK cells.
  • CD5 ⁇ l0 cells are NK cells as they express NKp46, NKG2D, CD94 and like their counterparts in man, express KIRs whereas CD56 hl CD16 " cells are rarely KIRs + (Fig. 1 C).
  • NKp46 + cells present a phenotype consistent with peripheral NK cells in man including expression of CD 122 (IL-2RJJ), NKG2A, CD 161, but not CD3 (Fig. 1 D).
  • IL-2RJJ CD 122
  • NKG2A CD 161, but not CD3
  • Fig. 1 D In vivo generated human NK express high levels of intracellular granzyme-B and IFN- ⁇ when stimulated with IL- 12 and IL- 18 ex vivo, and de- granulate when co-cultured with K562 human leukemia cells as determined by expression of CD 107a (Fig. 1 E).
  • IL-15 is a pleiotropic cytokine essential for murine NK cell development.
  • HIS-mice represent a hybrid human-murine system where cytokines receptors compatibilities between species may not exist.
  • human IL-15 ML- 15
  • ML- 15 induces survival and proliferation of murine NK cells (24)
  • IL-15 induces survival and proliferation of murine NK cells (24)
  • NK cells cultured in vitro with hIL-15 proliferated extensively (predominately the CD56 bl CD16 " subset) which was in contrast to those cultured in murine IL-15 (mIL-15) which itself sufficiently induced proliferation of murine NK cells (Fig. 1 F and Fig. 7).
  • mIL-15 murine IL-15
  • Fig. 1 F and Fig. 7 murine IL-15
  • mIL-15 failed to induce human NK cell proliferation, although some cells survived when mIL-15 was combined with hIL-15R ⁇ -Fc (Fig. 1 G).
  • hIL ⁇ 15R ⁇ was clearly superior in inducing NK cell proliferation compared to mIL-15R ⁇ when combined with hIL-15 (Fig. 1 G).
  • activated human but not murine myeloid cells were able to induce human NK cell proliferation in vitro (Fig. 7), which is consistent with previous studies(26).
  • IL- 15 While IL- 15 is known to heighten NK cell cytotoxicity and induce proliferation, it also protects cells from apoptosis (the latter possible at lower concentrations) by suppressing pro-apoptotic Bim and elevating Bcl-2 family members such as Bcl-xL and McH (32-35).
  • Bcl-2 family members such as Bcl-xL and McH (32-35).
  • a clear in vivo effect of RLI and IL- 15+IL-l 5Ra-Fc was the up-regulation of Bcl-xL in NK cells (Fig. 2 B and E). It was next asked if enhanced survival could improve NK cell reconstitution in the limiting ML- 15 environment of HIS-mice.
  • HSCs were infected with a bicistronic retrovirus encoding the pro-survival protein Bcl-xL and GFP (to detect infected cells) in vitro prior to engrafting newborn Balb/c Rag2 "/" ⁇ c "/” mice.
  • Ectopic expression of Bcl-xL in human HSCs resulted in a significant increase in thymocyte and splenocyte cellularity eight weeks after engraftment, with Bcl-xL transduced cells (GFP+) representing a greater proportion of hCD45 + cells compared to control transduced cells in all organs (Fig. 3 A 5 B and C).
  • NKp46 + cells were significantly increased in thymus and spleen of Bcl-xL HIS-mice compared to control HIS-mice, however this appeared to be primarily a result of increased cellularity in these organs as the percentage of NK cells largely unchanged (Fig. 3 D and E).
  • a greater proportion of NK cells were GFP+ in Bcl-xL infected mice compared to controls, no accumulation of Bcl-xL expressing NK cells was observed amongst the most mature subset (CD56 lo CD16 + ).
  • IL- 15 itself is known to enhance NK cell cytotoxicity via up-regulating effector molecules such as IFN- ⁇ , perform and granzymes at the level of transcription and/or translation and by increased surface expression of LFA-I and IL-12RB1 (37-39). Strikingly, amongst the enhanced CD16 + population following IL- 15 trans-presentation treatment, the percentage of NK cells expressing KJRs was also significantly elevated resulting in a large increase in the KIR + NK cell pool, an effect again more prominent with RLI (Fig. 4 A). The fraction of KIR + NK cells following exogenous IL- 15 trans- presentation was typically highest in the thymus and elevated compared to normal frequencies in human blood (typically 50-60%).
  • NK cell subsets had a similar level of incorporated BrdU during treatment (Fig, 5 A). While one cannot distinguish between CD16 + NK cells that have incorporated BrdU and BrdU+ cells that have up-regulated CD 16, it is most likely that the latter accounts for most of the BMU + CDIo + NK cells as CD56 l0 CD16 + NK cells are refractory to IL- 15 stimulation in vitro (40, 41), especially when compared to CD56 hl CD16 ⁇ NK cells (Fig.
  • Binding of IL- 15 to NK cells ultimately activates signaling pathways stemming from STATS phosphorylation such as activation of NP- ⁇ B, induction of cyclin D, down-regulation of pro-apoptotic proteins and up-regulation of Bcl-2 family members, cytolytic granules and the transcription factors Ets-1 and T-bet (34, 37, 42, 43). While it is clear how these signaling events promote cell division, survival and effector functions it is not known how these pathways regulate acquisition of KIRs.
  • IL- 15 functions as a membrane bound cytokine that can only be present at the cell surface and support NK cell development when bound to IL-lSR ⁇ on the same cell (14, 15).
  • the clear effect of ML-lSR ⁇ to enhance hIL-15 activity in vivo suggests this is also likely in man.
  • the findings using a novel HIS- mouse approach demonstrate that hIL-15 trans-presentation is necessary to promote human NK cell development and differentiation in vivo.
  • the failure to observe accumulation of CDIo + KIR + NK cells in HIS-mice with HSCs ectopically expressing Bcl-xL suggests that NK cell survival is alone not sufficient to promote differentiation and a source of human IL-15 is essential for this process.
  • trans-presented IL- 15 induces KIR expression on CD56 l0 CDl ⁇ + NK cells in vivo and suggests that KTRs are expressed after CD 16 and that acquisition of KTRs represents a further step in NK cell differentiation.
  • expression of human self-MHC-I on a fraction of the haematopoietic cells in HIS-mice appears sufficient in generating phenotypically mature KIR + NK cells.
  • IL-15R agonists particular RLI in augmenting human NK cell development in HIS-mice will enable us to more readily dissect the role of IL- 15 dependent lymphocytes (NK cell, memory CD8 T cell, NK T cell and ⁇ / ⁇ T cell) responses to human pathogens and disease in vivo.
  • IL- 15 dependent lymphocytes NK cell, memory CD8 T cell, NK T cell and ⁇ / ⁇ T cell
  • Given obvious crosstalk between innate and adaptive immune cells having robust reconstitution of IL-15 -dependent cells in vivo improves the accuracy and application of HIS-mice for studying human immune responses.
  • mice were maintained in isolators with autoclaved food and water. Mice with a human immune system (HIS) were generated as previously described [69-71]. Briefly, newborn (3 - 5 day old) Rag2 ⁇ /" ⁇ c ⁇ / ⁇ mice received sub-lethal (3.3 Gy) total body irradiation from a Cs source, and were injected intra-hepatic (i.h.) with 5xlO 4 sorted CD34 + CD38 ' human fetal liver cells. All manipulations of HIS mice were performed under laminar flow. Mouse experiments were approved by an institutional committee at the Institut Pasteur and validated by the French Ministry of Agriculture.
  • HIS human immune system
  • HIS mice were injected intra-peritoneally with lOO ⁇ l of 2.5 ⁇ g RLI or PBS alone commencing at a either 5-6 weeks after reconstitution or 8 weeks after reconstitution.
  • lOO ⁇ l of lOmg/ml of BrdU from BrdU flow kit was injected IP daily 2 days prior to analysis.
  • HIS mice were immunized by intramuscular route (biceps femoris) using a 29G needle, three times on weeks 12, 14 and 16 with 100 ⁇ l of the HBV vaccine (Engerix-B, Glaxo SmithKline). These amounts correspond to 1/10 of the normal human dose.
  • Negative controls received the same volume of PBS buffer.
  • Intracellular staining was performed after fixation and permeabilization of the cellular suspensions using BD Perm/Wash and BD Cytofix/Cytoperm reagents from BD Bioscience (San Jose, CA) according to manufacturer instruction.
  • BrdU detection cells were incubated for 1 h at 37°C with 30 ⁇ g DNAse from BrdU flow kits (BD Bioscience, CA). All washings and reagent dilutions were done with PBS containing fetal calf serum (FCS). All acquisitions were performed using LSRII, Canto 1 or Canto 2 cytometers, cell sorting was perform using FACS ARIA, all machines were interfaced to the FACS-D ⁇ va software (BD Bioscience).
  • CD34 Progenitor Cell Isolation Kit Miltenyi Biotech, Auburn, CA
  • Single-cell suspension and isolation of mononuclear cells by density gradient centrifugation over Ficoll-Hypaque (Nycomed Pharma, Roskilde, Denmark).
  • Cell suspensions were prepared in RPMI medium with 2% fetal calf serum.
  • Single cell suspensions of murine organs were prepared as previously described [99].
  • Thymocyte populations from HIS mice treated with PBS or RLI were electronically sorted using FACSARIA (BD Bioscience) and were cultured at 1 x 10 4 cells/ml in RPMI with 10% FCS or either lOng/ml rhIL-15 (rhIL-15; R&D Systems, Minneapolis, MN) or 10ng/ml rhIL-7 (Peprotech) for 30 hours. Viable cells were determined by PI exclusion using flow cytometry.
  • TCR V ⁇ immunoscope was performed as previously described [100], Briefly, cDNA was prepared and real-time PCR performed by combining primers for the different V ⁇ chains (V ⁇ l-24, V ⁇ 2-9 and V ⁇ l-8). Fluorescent products were separated on ABI-Prism 3730 DNA analyzer to determine CDR3 lengths. Analysis of five individual HIS-mice from each group containing greater than 30% human chimerism in the spleen was performed. [098] Luminex® cytokine detection. Sera from HIS mice were prepared from total blood at time of sacrifice by centrifugation at 13,000 rpm for 10 minutes at 4 degrees. Cytokine human 25-plex panal Luminex ® assays were performed according to manufactures instructions (Invitrogen).
  • ELISA The plasma harvested from HIS mice was screened by ELISA for the presence of antigen-specific and total antibodies. For this purpose, 96-well plates were coated either with Engerix B (glaxoSmithKline BV, Zeist) (1Ox diluted in PBS) or lO ⁇ g/ml goat anti-human IgM or 10 ⁇ g/ml goat anti-human IgG (Jackson ImmunoResearch Laboratories) in PBS for lhr at 37°C or o/n at 4 0 C. After coating, the plates were washed in PBS with 0.5% Tween-20.
  • a PBS solution containing 4% of milk was used as a blocking agent, before serial dilution of HIS mouse plasma or cell culture supernatants.
  • ELISA was revealed with horseradish peroxidase-labeled goat anti-human IgM and IgG antibodies (Jackson ImmunoResearch Laboratories) followed by TMB substrate/stop solution (Biosource, Carlsbad, CA, USA).
  • HIS mice 8 weeks after HSC engraftment, HIS mice were injected intra-peritoneally weekly for 4 weeks with 2.5 ⁇ g of a potent human IL-15R agonist known as RLI consisting of human IL- 15 covalently linked to an extended human IL-15R ⁇ 'sushi' domain [66, 74, 75] and therefore capable of mimicking IL- 15 trans-presentation (Fig. 8A).
  • RLI a potent human IL-15R agonist
  • RLI a potent human IL-15R agonist
  • RLI a potent human IL-15R agonist
  • RLI a potent human IL-15R agonist
  • IL- 15 induces the expression of killer-Ig-like inhibitory receptors for MHC-I (KIRs) on NK cells in vivo [71].
  • KIRs killer-Ig-like inhibitory receptors for MHC-I
  • Fig 9C KIR antibodies recognizing KIR2DL2/3/1, KIR2DS1/2/4 and KIR3DL1/S1
  • Fig 9C A significant increase was observed in the percentage and total number of KIR + T cells, which included an expansion of T cells expressing either one or both of the KIR2DL2/3 and KIR3DL1 receptors.
  • Other NK- associated cell surface markers such as CD 16 and CDl 61, but not CD 56 were also induced or up-regulated on T cells from all organs analyzed from IL-15 treated mice (Fig 9C).
  • HIS mice receiving trans-presented human IL-IS show increased proliferation of peripheral human CD4 and CD8 T cells.
  • the ratio of CD8 + to CD4 + T cells following IL- 15 treatment was only mildly increased in the spleen and unchanged in other lymphoid organs of recipient mice (Fig 9B).
  • the clear augmentation in T cell numbers but no diminution in the relative size of the naive T cell pool compared to control mice indicates that IL-15 trans-presentation in vivo targets the expansion of both na ⁇ ve and activated T cells.
  • humanized mice treated with RLI were injected IP with lO ⁇ g of BrdU 24 and 12 hours before being sacrificed and T cell populations analysed for BrdU incorporation.
  • CD8 + and CD4 ⁇ na ⁇ ve (CD45RA + ) and activated (CD45RA " ) T cell proliferation in the spleen and mesenteric lymph node were significantly increased following IL- 15 trans- presentation in vivo (Fig 10A, B).
  • CD8 + T cells, in particularly those with a na' ⁇ ve phenotype showed the greatest fold increase in proliferation in both the spleen and the mesenteric lymph node after IL- 15 treatment followed by na ⁇ ve CD4 + T cells, activated CD8 + T cells and lastly activated CD4 + T cells (Fig 10 A, B).
  • IL-15 The efficiency of IL-15 treatment and relative responsiveness of T cell populations can be compared to that of NK cells who are highly dependent on IL-15 (Fig 10 A, B).
  • Other ⁇ c cytokines such as IL-2 and IL-7 could also influence the observed increase in human T cells by inducing proliferation and enhancing survival.
  • the serum concentration of these and other cytokines that are produced by IL- 15 responsive lymphocytes such as NK cells was not analysed.
  • the serum concentration of IL-2 and IL-7 were unchanged in mice treated with trans-presented IL- 15 and were only slightly above the level of detection using Luminex bead array assays (Fig 10 C), with the IL-2 result being confirmed by intracellular staining of T cells from these mice showing no difference between treatment groups.
  • Thymocyte turnover was analysed in the same manner as previously mentioned by injecting mice with lO ⁇ g of BrdU 24 and 12 hours before being sacrificed.
  • thymic NK cell turnover is doubled following IL- 15 treatment in vivo
  • SP4 and SP 8 T cell turnover is unchanged, which is in sharp contrast to what was observe in the periphery (Fig 11 B).
  • the increase in DN and DP thymocytes likely arises from the significant increase (*P ⁇ 0.05) in proliferation of these two thymocyte populations in IL- 15 treated mice (Fig 11 B). While both the number and turnover of iSP4 thymocytes were always increased in. IL- 15 treated mice, this increase was not statistically significant.
  • ⁇ SP4 thymocytes presented the highest rate of proliferation with around 75% of these cells dividing within 24 hours, thus if IL- 15 is capable of enhancing the division of this population it is less likely to be observed given the high baseline turnover.
  • IL- 15 trans-presented IL-15 is functional on human DN and DP thymocytes, other parameters were next examined such as cell survival.
  • IL-15 offered very little survival advantage to purified DN thymocytes compared to media or IL-7, whereas both SP4 and SP8 thymocyte populations displayed a clear increased survival when cultured in IL- 15 for 30 hours (Fig 11 D).
  • DP thymocytes however only displayed improved survival when cultured in IL-7, but not IL- 15 (Fig 11 D).
  • Fig 11 E an improved ex vivo survival of thymocyte populations sorted from IL- 15 treated humanized mice compared to those sorted from control mice was not observed (Fig 11 E) indicating that increased proliferation as opposed to improved survival is the major contributing factor for the increased number of immature thymocytes following this treatment regime.
  • TCR T cell receptor repertoire
  • the peripheral T cell pool generated in our model of humanized mice appears extremely diverse especially considering the limited number of peripheral T cells that are generated in this model. Indeed, T cells bearing TCRs from all V ⁇ family members with numerous CDR3 lengths used amongst each family member were detected (Fig 12 A, B). This is a valuable piece of data considering that the model of humanized mice is becoming more popular for the study of T cell immune responses and indicates at least that an extensive TCR repertoire exists and could react against a large number of peptide antigens. A similar diversity was observed in the CDR3 length amongst all V ⁇ chains used except for the occasional over-representation of clones within the V ⁇ l 1 and V ⁇ 7. This was not consistent observed in all mice following IL- 15 treatment and was far less evident amongst thymic T cells from the same mice (Fig 12 A, B).
  • ⁇ / ⁇ T cell population was increased in frequency and number (P ⁇ 0.05; Fig 13 B), however this was inline with the general augmentation in total T cells and was much less impressive than the increase we previously reported for NK cells using a identical treatment regime [71] and indicates in the HIS model, ⁇ / ⁇ T cells are not more responsive to IL- 15 than ⁇ / ⁇ T cells.
  • CDR3 immunoscope analysis of ⁇ and ⁇ variable chain genes was next used to identify all ⁇ and ⁇ usage in HIS mice (since monoclonal antibodies against each unique ⁇ and ⁇ are not commercially available). These type of analyses on spleen- derived human T cells were used as they are more numerous than those derived from LN in the HIS model. Consistent with the flow cytometry data, most human ⁇ / ⁇ T cells found in the periphery of HIS mice use V ⁇ 2 and this is similar to human PBMCs and unaffected by IL-15 treatment (Fig 13 C).
  • Trans-presented human IL-15 accelerates T cell development in HIS mice. Since trans-presented IL- 15 was extremely efficient in increasing total human T cell numbers in HIS mice and displayed a clear effect in the thymus, it was next determined whether IL- 15 immunotherapy could promote early thymopoeisis, before mature T cells are generated, thus accelerating mature T cell production.
  • thymus and spleen from HIS mice were analysed at various ages and it was found that between 5 - 6 weeks after HSC engraftment represented the ideal window to commence the IL- 15 immunotherapy as the thymus was seeded with immature thymocytes including DPs, DNs and iSP4s and almost no mature T cells, the latter also being the case for the spleen (Fig 14 A).
  • HIS mice were then treated with a more intense IL- 15 treatment regime that entailed 2.5 ⁇ g of IL-15R agonist, IL-15+IL-15R ⁇ (RLI) every three days (day 0, 3, 6 and 9) and HIS mice were then sacrificed and analyzed on day 12.
  • Thymus, spleen and BM from IL- 15 immunotherapy HIS mice and PBS controls were then analyzed for human chimerism (hCD45 + ) and mature T cell development (hCD45 + CD3 + ).
  • IL- 15 trans-presentation resulted in significant increase in human T cells in all organs analysed, promoting both SP4 and SP 8 development in the thymus resulting in a significantly increased pool of na ⁇ ve mature T cells (hCD45 + CD45RA + CD3 + ; Fig 14 B).
  • HIS mice models have been continually improved and are now at a stage where immune responses can be elicited by the engrafted human lymphoid system (reviewed in [77]). Indeed both antibody and cellular immune responses against vaccines such as tetanus toxoid and viruses such as Epstein Barr virus have been detected in various HIS models [78-80].
  • IL- 15 can skew activated T cells towards a ThI phenotype as determined by an increased IFN- ⁇ /IL-4 protein ratio. Tehre was a failure to detect any difference in serum or intracellular IFN- ⁇ levels in CD4 + and CD 8* T cells of IL- 15 treated mice compared to controls. Furthermore, intracellular granzyme-B levels in CD4 + and CDS + T cells were also unchanged.
  • IL- 15 induces anNK-like phenotype (with CD56 expression) on thymocytes or preferentially expands the existing CD56 + T cell pool [82-84]. It was shown that CD56 " T cells from PBMC do not proliferate to IL-15, whereas CD8 ⁇ and V ⁇ 24 + V ⁇ l I + robustly proliferated to IL-15 in vitro [82]. We failed to detect CD56 expression on T cells in HIS mice following treatment with trans-presented human IL- 15 treatment. In addition, ⁇ / ⁇ T cells were only increased to a similar level as ⁇ / ⁇ T cells, which is in contrast to data suggesting that human ⁇ / ⁇ T cells are more responsive on IL- 15 in vitro.
  • IL-2 is a likely candidate as it is produced by antigen activated T cells and promotes T cell division. There was a failure to detect any difference in the serum level of IL-2 between treated and control mice, nor in the intracellular concentration of IL-2 in T cells from both groups of mice. While human IL-15 is functional on mouse hematopoeitic cells, HIS mice lack their endogenous ⁇ c receptor, thus all of the observed effects involve IL- 15 receptor triggering on human cells.
  • trans-presented IL-15 to promote human thymopoeisis offers a unique new therapeutic approach to augment human T cell numbers during immunotherapy to cancer or to reduce the period of immunodeficiency following radiotherapy/chemotherapy and HSC transplant by promoting NK and T lymphopoeisis. It has been previously shown that trans-presented IL- 15 (RLI) is effective in an NK cell-dependent murine metastatic melanoma and colorectal cancer model [86], while others have reported similar effects with CD8 + T cell-dependent systems [87, 88].
  • IL-15 has been shown to induce antigen independent expansion of na ⁇ ve, virus specific and tumor specific cytotoxic CD8 + T cells in vitro [74, 89-92] and rescue tolerant CD8 + T cells for use in adoptive immunotherapy of established tumors [93].
  • IL-15 has been shown to be more potent than IL-2 in inhibiting apoptosis of memory CD4 + T cells in vitro as well as of na ⁇ ve phenotype CD4 + T cells from HIV infected individuals in both examples by up-regulating Bcl-2 [94-96].
  • IL-15 has also been shown to enhance number, function and survival of HTV- specific CD8 + T cells [97,98] indicating that trans-presented IL-15 treatment could improve the clinical condition of diverse disease states by not only boosting T cell numbers but by augmenting antigen specific T cell responses.
  • the findings using human immune system mice demonstrates that human IL-15 trans-presentation is effective in promoting human T cell development and homeostasis in vivo and in the absence of any obvious toxicity.
  • IL- 15 boosted HIS mice provide an improved humanized mouse model for evaluating pre-clinical immunotherapies and for deriving and screening novel immunomodulatory drugs.
  • Hierarchy of the human natural killer cell response is determined by class and quantity of inhibitory receptors for self-HLA-B and HLA-C ligands. J Immunol 179:5977-5989.
  • Dendritic cells prime natural killer cells by trans-presenting interleukin 15. Immunity 26:503- 517.
  • Soluble interleukin- 15 receptor alpha (IL- 15R atpha)-sushi as a selective and potent agonist of IL-15 action through IL- 15R beta/gamma.
  • IL- 15R atpha Soluble interleukin- 15 receptor alpha
  • Glucocorticoid-induced tumor necrosis factor receptor negatively regulates activation of human primary natural killer (NK) cells by blocking proliferative signals and increasing NK cell apoptosis, J Biol Chem 283:8202-8210.
  • Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. J Exp Med 180:1395-1403.
  • Interleukin-15 is a potent survival factor in the prevention of spontaneous but not CD95-induced apoptosis in CD4 and CD8 T lymphocytes of HIV-infected individuals. Correlation with its ability to increase BCL-2 expression. Cell Death Differ 1999. 6: 1002-1011.

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Abstract

La présente invention concerne un système de modèle d'animal transgénique, fondé sur le développement de souris transgéniques portant des composants du système immunitaire humain, comprenant un grand nombre de lymphocytes innés tels que des cellules tueuses naturelles (NK) humaines matures, des cellules T γδ et des cellules NK-T, ainsi que des lymphocytes T CD4 et CD8 adaptatifs. L'invention concerne de plus l'utilisation dudit système de modèle pour évaluer, ou pour identifier, des composés qui modulent (inhibent ou activent) l'activité ou la maturation de lymphocytes humains innés, tels que des cellules tueuses naturelles (NK) humaines matures, des cellules T γδ, et des cellules NK-T, ainsi que de lymphocytes T CD4 et CD8 adaptatifs. Ces composés peuvent être utilisés dans des immunothérapies pour le traitement de maladies pathogènes, du cancer, de maladies auto-immunes, infectieuses et inflammatoires, de l'immunodéficience, et pour le traitement de patients ayant subi une greffe. La présente invention est fondée sur la découverte du fait que la différenciation et l'expression de cellules tueuses naturelles de récepteurs d'inhibiteur du type tueur (KIR), ainsi que l'homéostasie et le développement de cellules T CD4 et CD8 humaines adaptatives, sont régulés par la transduction de signal à médiation par IL-15.
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CN113786416A (zh) * 2021-09-22 2021-12-14 北京创世客生物技术有限公司 转基因的nk细胞在治疗癌症中的用途
US11365236B2 (en) 2017-03-27 2022-06-21 Nkarta, Inc. Truncated NKG2D chimeric receptors and uses thereof in natural killer cell immunotherapy
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