WO2010071836A1

WO2010071836A1 - Il-15 mediated nk and t cell maturation

Info

Publication number: WO2010071836A1
Application number: PCT/US2009/068826
Authority: WO
Inventors: James Di Santo; Nicholas Huntington; Yannick Jacques
Original assignee: Inserm; Cnrs; Institut Pasteur
Priority date: 2008-12-19
Filing date: 2009-12-18
Publication date: 2010-06-24

Abstract

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 large numbers of innate lymphocytes such as mature human natural killer (NK) cells γδ 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 (inhibit or activate) the activity or maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes. Such compounds may be used in immunotherapies for treatment of pathogenic diseases, cancer, autoimmune, infectious and inflamatory diseases, immunodeficiency and for treatment of transplant patients. The present invention is based on the discovery that NK cell differentiation and expression of killer-like inhibitory receptors (KIRs) as well as homeostasis and development of adaptive human CD4 and CD8 T cells is regulated by IL-15 mediated signal transduction.

Description

KENYON & KENYON

ONE BROADWAY NEW YORK, NEW YORK 10004

IL-15 MEDIATED NK AND T CELL MATURATION

1. INTRODUCTION

[001] 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, γδ 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- 15Rα complexes. 2. BACKGROUND OF INVENTION 2.1. NATURAL KILLER CELLS

[002] 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^loCD16⁺) subset which possess abundant intracellular perforin and granzymes and display spontaneous cytotoxicity (2, 3). Li contrast, CD56^hlCD16^" NK cells rarely express KIRs (3) and since they are more prevalent in blood early post-bone-marrow transplant (4, 5), give rise to CD56^l0CDlό⁺ NK cells when transferred into NOD/SCID mice (6) and have longer telomeres than CD56^IoCD16⁺ NK cells (7), it is likely that CD56^h'CD16TsfK cells are (or contain within this population) precursors of CDS^⁰CDIo⁺ NK cells, however the tools to definitively prove this hypothesis are lacking.

[003] While inhibitory KIRs control reactivity of mature NK cells, their expression also influences the functional maturation of developing NK cells, as 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. Patients lacking the transporter associated with antigen processing (TAP) have dramatically reduced surface expression of MHC-I molecules and consequently, NK cells that are hypo-responsive to MHC-I deøcient cells (11). KIR⁺ NK cells are present in these patients indicating that normal MHC-I expression itself is not required for KIR expression. Given the importance of KIR expression in regulating NK cell function, knowledge of elements influencing KIR acquisition would improve the understanding and clinical approaches to diseases where KBR. and HLA haplotypes influence susceptibility, progression or outcome such as autoimmune/inflammatory disease, cancer, infections (HIV, HCV) and bone marrow transplants/graft versus leukemia effects (12, 13).

[004] Since KIRs are expressed on mature CD56^1oCD16+ NK cells, factors that influence NK cell homeostasis could potentially influence KIR acquisition in vivo. An elegant series of murine studies using gene targeting and bone marrow chimeras have reveled that NK cell development requires IL-lSRα 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- 15Rα "trans-presentation" (14, 15). Both WO2007/001677 and WO2007/046006 disclose that combinations of IL-15 and IL-lSRα, or complexes of fragments thereof, are capable of modulating an immune response in a host. When not bound to IL- 15Rα, IL-15 appears to have a minimal effect on NK cell homeostasis in vivo (16, 17). In man, mutations in IL- 15Rq have not been reported however NK cells are dramatically reduced in patients carrying mutations in the common gamma chain (γ_c) cytokine receptor (used in IL-15/-7/-4/-9/-2 and -21 signal transduction), Jak3 or the shared IL-2/15Rβ, while they are present in IL-7Rα-deficient patients suggesting that IL-15 may regulate human NK cell development (18, 19). [005] In vivo studies of NK cells have been largely restricted to mice and while this line of experimentation is valuable, some of this knowledge is not transferable to human NK cell biology. A clear example of this is NK cell development, where the kinetics, frequency and phenotype are clearly different between species (3). An intermediate between murine and human in vivo studies exists in the form of human immune system (HΪS)-mice. A recently developed HIS-mouse model is the engraftment of newborn Balb/c Rag2^~/"γ_c ^"/" mice with human hematopoietic stem cells (HSC) from fetal liver, cord blood or adult bone marrow (20, 21). Balb/c Rag2^"/"γ_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).

2.2. T-CELL HOMEOSTASIS

[006J The maintenance of peripheral T cell pools expressing highly diverse T cell receptors is essential for sterilizing immunity against viruses, bacteria and parasites capable of infecting humans. 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 naϊve to effector or memory [51]. Several signals have been implicated in controlling T cell homeostasis including those emanating from the T cell receptor (TCR) following interactions with self-peptide + MHC (pMHC) and those induced by growth factors including cytokines [51]. The common gamma chain (gc) family of cytokines (which comprise IL-2, IL-4, IL-7, IL-9, IL-13, IL- 15 and IL-21) in particularly have been demonstrated to play a role in T cell homeostasis in mammals [52]. [007] IL- 15 is a peculiar cytokine because it requires expression of the IL- 15Rα chain by the same cell in order to be functional. Thus cells expressing IL- 15 such as monocytes, dendritic cells and stromal cells must also express the IL-15Rα in order to trans-present IL- 15 to IL- 15 responsive cells (expressing the IL-2Rβ/γ_c complex), and both IL- 15 and IL-15Rα are up-regulated on myeloid cells following inflammation, thereby increasing IL- 15 bioavalability [63-65]. While humans carrying mutations in IL- 15 or IL-15Rα have not been reported, mice lacking these proteins have reduced numbers of memory CD8 T cells (most notably the CD122^hl subset), NK T cells and γ/δ T cells in the periphery [66_f 67]. Furthermore, 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].

[008] In vivo studies of T cell development and homeostasis have been largely restricted to mice and while this line of experimentation is valuable, 60 million years of evolution has resulted in many differences between murine and human immune systems, thus some of this knowledge may not be directly applicable to human T cell biology. An intermediate between murine and human in vivo studies exists in the form of human immune system (HΙS)-mice. A recently developed HIS-mouse model is the engraftment of newborn Balb/c Rag2^'A y_c ^''^' mice with human hematopoietic stem cells (HSC) from fetal liver or cord blood [69-71]. In this model, 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], However, the number of peripheral T cells observed in Balb/c Rag2^"A γ_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].

[009] As described herein, using HIS Balb/c Rag2^"y" γ_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 naϊve 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.

3. SUMMARY OF THE INVENTION

[010] 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, γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes. Specifically, 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. In a non-limiting embodiment of the invention, the transgenic mice may further be transgenic for expression of human HLA class I and/or HLA class II molecules. 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-15Rα 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.

[Oil] 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.

[012] The transgenic animal model system of the invention also provides a system for generating new therapeutics. In an embodiment of the invention, 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.

[013] The present invention is based on the discovery that IL- 15/IL- 15Rα 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-15Rα 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.

4. BRIEF DESCRIP TIOK OF THE FIGURES

[014] Figure 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 10⁴ 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 10⁵ K562 AML cells in the presence of IL-15 for 18h. Cells were stained with anti-CD 107a or fixed, permeabilized and stained with anti-IFN-γ or anti-granzyme-B and analyzed by flow cytometry. (F) 2 x 10⁵ CD56⁺ NK cells purified from human peripheral blood were labeled with CFSE and cultured in human IL- 15 (grey line) or murine IL- 15 (black line) for 120 hours. CFSE histograms of CD56^loCDlό⁺ (black line) and CD56^wCDlό^~ (grey line) cultured in human IL- 15 are also shown. (F) 1 x 10⁴ CD56⁺ NK cells purified from human peripheral blood were labeled with CFSE and cultured with the indicated combinations of human (h) and murine (m) IL-15 (15) and IL- 15Ra-Fc (15Ra) for 72 hours. Cells were the analyzed flow cytometry. Numbers indicate live cells (PI) per well. N.D (not determined - due to no live cells). Histograms are representative of 2 individual experiments using different donor blood. (G) 8 weeks following CD34⁺CD38^" HSC engraftment, FOXNl^+/"and FOXNl^+7" (Nude) HIS-mice were analyzed for human T and NK cell reconstitution by flow cytometry. FACS plots are gated on hCD45⁺ cells in spleen (top) and BM (bottom) and are representative of 3 mice of each genotype.

[015J Figure 2. Trans-presentation by human IL-15/IL-15Rα promotes human NK cell homeostasis in vivo. (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. Organ cellularities were counted and hCD45⁺NKp46⁺ cells were enumerated based on positive surface expression determined by flow cytometry. Data is mean ± s.e.m of 5 mice in each group generated from 2 different CD34⁺ HSC sources. P values are given when statistically significant. (D) NK cell subsets and BrdU incorporation in the bone marrow of HIS -mice treated as in (C) were analyzed by flow cytometry. (E) hCD45⁺NKp4ό⁺ cells from bone marrow were analyzed for intracellular proteins and incorporation of BrdU by flow cytometry. For (D) and (E) HIS -mice treated as in (C), however 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. (F) 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.

[016] Figure 3. Ectopic expression of Bcl-xL mildly augments human NK cell reconstitution in vivo. Human fetal liver HSCs were infected with retrovirus encoding hBcl-xL and GFP or GFP alone and used to generate HIS mice. (A) 8 weeks after engraftment thymus, spleen and bone marrow cellularities were counted and CD45⁺ cells were enumerated based on surface expression determined by flow cytometry. (B) GFP expression within hCD45⁺ thymocytes of HIS-mice 8 weeks after engraftment was detected by flow cytometry. (C) The percentages of GFP+ cells within the human graft were enumerated by flow cytometry. (D) NK cells were enumerated based on (A) and surface expression hCD45 and NKρ46 determined by flow cytometry. (D) 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.

[017] Figure 4. Human IL-15/IL-l 5Rα 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^!oCD16⁺ cells and are representative of 5 mice and 2 different CD34⁺ HSC sources. **P < 0.01, *P < 0.05. (B) The ratio of CD16⁺:CD16^'NK cells (hCD45⁺NKρ46⁺) in HIS-mice treated with RLI and PBS as in (2C) were determined. Data are mean ± s.e.m of 4 mice in each group. P values are given when statistically significant. (C) Expression of KIRs (OR-2DL2/3/1/-2DS1/2/4/-3DL1/S1) on CD56^loCD16^÷ in HIS-mice treated as in (2A) or engrafted with modified HSCs as in Fig. 3. Facs plots pre-gated on hCD45⁺ and are representative of at least 3 mice in each group. (D) Expression of KIR2DL1/2/3 was analyzed on KrR3DLl/DSl⁺ or KIR3DL1/DS1⁺ NK cells in the bone marrow of HIS-mice treated with RLI as in (2C). Facs plots are gated on hCD45⁺CD56⁺CD3^" and are representative of 3 mice.

[018] Figure 5. IL-15/IL-l 5Rα-dependent development of CD16⁺KIR⁺ NK cells from CDl 6^"KIR^" precursors in vivo, (A) NK cell subsets in the bone marrow of RLI treated HIS-mice from (2D) were analyzed for BrdU up-take using flow cytometry. Facs plots are representative of 2 individual experiments of 3-5 mice per treatment group engrafted with 2 different CD34⁺ HSC sources. (B) CD56^hiCD16^"KIRs^" or CD56ⁱ⁰CDl O⁺KIRs NK cells were FACS sorted from fetal spleen (CD45⁺CD3) and transferred i.h into 1 week old sub-lethally irradiated Rag2^"/7γc^"A recipients for 7 days. Mice were treated i.p with 2.5μg RLI or PBS on day 0 and day 4 post-transfer. Sorted KIRs^" NK cells lacked KIR-2DL2/3/1/-2DS1/2/4/-3DL1/S1 expression. Data is representative of 4 mice per NK subset transferred and 2 mice per treatment group.

[019] Figure 6. 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.

[020] Figure 7. In vitro NK cell cultures and in vivo IL-15 neutralization. (A)

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. (B) 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^"/"γc^"/" bone marrow). Myeloid cells were purified by aπti-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. (C) 16 week old HIS-mice were treated with anti-mouse IL- 15 or anti-human IL-15 neutralizing antibodies for 7 days and analyzed forNKρ46⁺ cells in the spleen. NK cellularity following treatment is expressed as a mean percentage + s.e.m of PBS treated age and donor HSCs matched HIS-mice. *P = 0.035.

[021] Figure 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^*Λ γ_c ^"A mice were irradiated with 3.3 Gy injected intra-hepalic (i.h.) with 5x10⁴ CD34⁺CD38^" human fetal liver cells. At 8,9,10 and 11 weeks of age, HIS mice were injected intra-peritoneally (i.p) with 2.5μg IL-15-ΪL-15Rα fusion protein (RLI) or PBS. Mice were sacrificed and analysed at 12 weeks. (B) Lymphoid organs from HIS mice were analysed for total human hematopoietic (human CD45; hCD45⁺)_? human T cells (hCD45⁺CD3⁺ and CD4⁺ or CD8⁺) and (C) human B cells (hCD45⁺CD19⁺) reconstitution by flow cytometry and cellularity of the indicated organs was enumerated. hCD45⁺ (%) is the percentage of total cells, whereas CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8^÷ (%) are the percentage within the hCD45⁺ population. Values represent mean ± s.e.m of 9 mice per group. P values are indicated when significant. (D) HBV immunization results in marked increases in IgG levels in HIS mice that had received trans-presented human IL-15.

[022] Figure 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. (C) 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 CD 161, but not CD56 were also induced or up-regulated on T cells from all organs analyzed from IL- 15 treated mice. [023] Figure 10. Trans-presented IL-15 enhances the proliferation of both naϊve and activated phenotype CD8 and CD4 T cells in HIS mice. HIS-mice treated as in Fig 8A were injected intra-peritoneally with 1 mg BrdU daily on the last 2 days before being sacrificed. 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.

[024] Figure 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. BrdU incorporation by the 6 thymocyte populations (DN, iSP4, DP, SP4, SP8 and NK) was determined by flow cytometry. Control mice were injected with an identical volume of PBS. * P < 0.05. (C) Thymic T cells and their progenitors were analysed for intracellular Bcl-xL expression by flow cytometry. Facs plots are pre- gated on hCD45⁺ cells and are representative of 3 mice for each group. (D) The indicated thymocyte subsets were electronically purified by flow sorting and cultured in media alone or media with IL-7 or IL-15. The frequency of viable cells after 30 h was determined by PI exclusion using flow cytometry. Data is representative of 2 experiments. (E) The indicated thymocyte subsets were electronically purified by flow sorting from PBS or IL- 15 treated HIS mice and cultured in media alone or media with IL-7 or IL-15. The frequency of viable cells was determined by PI exclusion using flow cytometry. Data is representative of 2 experiments.

[025) Figure 12. Diverse TCR Vβ repertoire in Balb/c Rag2 ' γ_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 Vβ 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).

[026] Figure 13. Development and diversity of γ/δ TCR⁺ T cells in Balb/c Rag2^"'^" γ_c ^"A HIS mice in vivo. (A) Human γ/δ TCR⁺ T cells from thymus and LN of HIS mice were analyzed by flow cytometry. Human T cells were separated into two subsets based on CD161 expression (CD3⁺CD161⁺ or CD3⁺CD161^") and the expression of Vγ9, Vδ2_> Vδl and CD8. (B) Total human γ/δ TCR⁺ T cells from thymus and LN from HIS mice treated as in Fig 8 A were analyzed by flow cytometry using a Pan γ/δ TCR antibody amongst total human T cells (hCD45+CD3+) in these organs. FACS plots are representative of (A) 4 and (B) 5 mice in each group. (C) The relative proportion of T cells expressing the indicated γ or δ TCR was determined by quantitative PCR for both control PBMC and HIS splenocytes from mice treated as in Fig 8 A. Percentages represent the frequency of T cells containing the indicated Vγ or Vδ PCR products out of the total Vγ or Vδ PCR products. Histograms represent the mean percentage + SEM of 3 mice in each group (PBS and IL-15-IL-15Rα) 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 Vγ and Vδ 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).

[027] Figure 14. IL-15 accelerates thymopoeisis and appearance of peripheral T cells when administered in vivo to Balb/c Rag2^~A γ_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. (B) HIS mice (5 - 6 weeks post-HSC engraftment) were treated every 3 days with with 2.5μg IL-15-IL-15Rα fusion protein (RLI) or PBS and sacrificed 12 days after the first injection. 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-15Rα 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.

5- DETAILED DESCRIPTION OF THE INVENTION

[028] As will be described in detail below, 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, γδ 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, γδ 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-15Rα complexes.

5.1. TRANSGENIC ANIMAL MODEL SYSTEM

[029] 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, γδ T cells, NK T cells, CD 4 and CD8 T cells and B cells. In a specific embodiment of the invention, 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. In another embodiment of the invention, the transgenic mouse further comprises human CD4 and CD8 peripheral T cells and human B cells. In a specific embodiment of the invention, the transgenic mice contain at least 1% mature human NK cells in the lymphoid tissues of said engrafted mouse. In another embodiment of the invention, 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.

1030] 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. Microbiol., Immunol., 1988, 137, 197-202) or the disruption of the recombination activating gene (Ragl^''^' or Rag2^"Λ; Mombaerts et al., Cell, 1992, 68, 869-877; Takeda eta L, Immunity, 1996, 5, 217-228), and a second mutation which is the beige mutation (Lystbg; MacDougall etal., Cell Immunol., 1990, 130, 106-117) or the disruption of the β2-microglobulm gene (β2m^"/"; Kollet et al., Blood, 200, 95, 3102-3105), the IL-2 receptor γ chain (or common cytokine receptor γ chain (γ_c)) gene (IL-2Rγ^~Λ or γ/; DiSanto et al., PNAS 1995, 92, 377-381), or the IL-2 receptorβ chain (IL-2R β) gene (IL-2Rβ^"Λ; Suzuki et al., J. Exp. Med., 19979, 185, 499-505). In a specific embodiment of the invention, Rag2^~/"γc^"/~ transgenic mice may be used in the practice of the invention.

[031] In a non-limiting embodiment of the invention, 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.

J032] Methods for generating transgenic animals with targeted mutations and/or transgene expression via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. No. 4,736,866 by Leder et al. and U.S. Pat. No. 4,870,009 Evans et al., U.S. Pat. No. 4,873,191 by Wagner et al., and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1986), incorporated herein by reference in their entirety.

[033] 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). Such 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. For example, human CD34⁺ cells (from 10⁴ to 10⁶ cells), isolated from 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).

[034] In said engrafted transgenic mice, 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. Such agonists include, for example, IL-15/IL-15Rα complexes,including for example IL-15/IL-ISRa, a complex of IL-15/IL-15Rα fragments, hIL-15+IL-l 5Ra-Fc and RLI (Receptor-Linker-Interleukin). See, for example, [029]. In an embodiment of the invention, the IL-15/IL-15Rα complex could also be administered as an expression vector (lentivirus, adenovirus, retrovirus, AAV for example) or as a transgene. Further, 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.

[035] 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. [036] 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.

[037] The present invention is also directed to 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.

5.3. SCREENING ASSAYS

[038] 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. Further, 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.

[039] 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. In specific embodiments of the invention the T cells are CD4 or CD8 T cells.

[040] 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-15Rα 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-15Rα 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.

[041] 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-15Rα 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-15Rα 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. In specific embodiments of the invention the T cells are CD4 or CD 8 T cells.

[042] 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.

[043] Infectious disease treatments may be assayed using the transgenic mice of the invention. Such infectious diseases include, for example, bacterial, viral, fungal or parasitic diseases. In addition, treatments designed to inhibit transplant rejection may be assayed using the transgenic mice of the invention.

[044] 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-lSRα, thereby regulating the maturation of NK cells, γδ 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. [045] 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.

[046] 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. In specific embodiments of the invention the T cells are CD4 or CD8 T cells.

[047] The present invention provides for methods for identifying a compound that increases IL-15Rα 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-15Rα expression or activity.

[048] The present invention provides for methods for identifying a compound that increases IL-15Rα 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-15Rα expression or activity. In specific embodiments of the invention the T cells are CD4 or CD8 T cells.

[049] The present invention also provides for methods for identifying a compound that inhibits IL- 15 or IL-lSRα 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-15Rα 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-15Rα 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.

[050] 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-15Rα 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-15Rα 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. In specific embodiments of the invention the T cells are CD4 or CD8 T cells.

[051] The ability of a test molecule to modulate the maturation of innate human lymphocytes such as mature human natural killer (NK) cells, γδ 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. In a specific embodiment of the invention, 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.

[052] In a specific embodiment of the invention, 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.

[053] 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. In addition, 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,

[054] A sample of a biological fluid or biological tissue to be assessed for levels of NK-ceils, CD4 T cells or CD8 T cells, such as blood or other biological tissue, is obtained from the test animal. 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. In a specific aspect, such binding of antibody, for example, 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.

[055] 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. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin and fluorescamine. Likewise, 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.

5.2.5. COMPOUNDS THAT CAN BE SCREENED

IN ACCORDANCE WITH THE INVENTION

[056) The assays described above can identify compounds which modulate the maturation or activity of innate human lymphocytes such as mature human natural killer (NK) cells, γδ T cells, NK-T cells, as well as adaptive CD4 and CD8 T cells. For example, compounds that affect IL- 15 and/or IL- 15R activity include but are not limited to compounds that bind to IL- 15 or the IL-15R₅ and either activate the ligand/receptor activity (agonists) or block the ligang/receptor activity (antagonists). Alternatively, 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.

[057] 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).

[058] 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, ^g₁, 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. et al., 1993, Cell 72:767-778), 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.

[059] 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.

5.3. COMPOSITIONS CONTAlMNG MODULATORS OF NK CELL, CD4 AND CD8 T CELL MATURATION AND THEIR USES [060] The present invention provides for compositions comprising an effective amount of a compound capable of increasing the number of mature NK cells, and a pharmaceutically acceptable carrier. The present invention further provides for 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. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "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.

[061] 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.

[062] 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.

[063] 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 .

[064] 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 .

[065] 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.

[066] 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.

[067] 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.

[068] In a specific embodiment, it 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.

[069] 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.

[070J 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 reøects approval by the agency of manufacture, use or sale for human administration.

EXAMPLES

[071] The invention is illustrated herein by the experiments described above and by the following examples, which should not be construed as limiting. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. Those skilled in the art will understand that this invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Many modifications and other embodiments of the invention will come to mind in one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing description. Although specific terms are employed, they are used as in the art unless otherwise indicated.

6. EXAMPLE

[072] The following example demonstrates that IL-15 trans-presentation regulates human NK cell homestatis in vivo,

6.1 MATERIALS AND METHODS [073] Mice. C57BL/6 mice were purchased from Harlan (France). Rag2^~/"γc^"/"mice

(47) were backcrossed onto the Balb/c background and Rag2^{" "}γc^{" "} 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^'/"γc^"/" mice received sub-lethal (3.3 Gy) total body irradiation from a Cs source, and were injected intra-hepatic (i.h.) with IxIO⁵ sorted CD34+CD38- or 5xlO⁵ CD34+ human fetal liver cells. All manipulations of HIS mice were performed under laminar flow.

[074] In vivo treatments. HIS-mice were injected intra-peritoneally with lOOul of either human IL- 15 (2.5μg), human IL- 15Ra-Fc (7.5μg) + human IL-15 (2.5μg) both from R&D Systems (Minneapolis, MN), 2.5μg RLI(29) or PBS alone commencing at a minimum of 6 weeks after reconstitution. lOOμl of 10mg/ml of BrdU from BrdU flow kit (BD Bioscience, CA) was injected IP daily 2 days prior to sacrificing mice. For NK cell transfer assays, 3 x 10⁴ CD56^hiCD16TOR^' or 2 x 10⁵ CD56^?0CD16⁺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^"/"γc^~/~ 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 2.5μg of RLI on day 0 and day 4 post- transfer. IL-15 in vivo neutralization was performed by i.p administration of 50μg of goat anti-human IL-15 or 25 μg of goat anti -mouse IL-15 both from R&D Systems (Minneapolis, MN) every second day for 7 days.

[075] Flow cytometry analysis for cell-surface and intracellular markers. Cell suspensions were labeled with mAb against the following human cell- surface markers: CD3 (SK7), CD4 (SK3), CD34 (581), CD8 (SKl), CD19 (HIB 19), CDlO (HIlOa), CD38 (HB7), NKG2D (IDl 1), NKp46 (9E2), CD16 (3G8), CD161 (DX12), CD56 (B159), HLA-DR (L243), HLA-A/B/C (G46-2.6), CD117 (YB5.B8), IgD (IA6-2), IgM (G20-127), CD14 (M5E2), CD122 (Mik-B3), TCR-α/β (T10B9.1A-31), CD127 (hIL-7R-M21), CDl Ic (B-ly6), CD7 (M-T701), CD107a (lD4B), CD45 (2Dl), CD69 <L78), Bcl-2 (Bcl-2/100), IFN-μ (XMGl.2), BrdU (B44), Ki67 (B56) from BD Bioscience (San Jose, CA), KIR2DL2/L3 (DX27), KIR3DL1 (DX9), CD94 (DX22), NKp44 (p44-8), CD27 (0323), CD62L (DREG-56), NKp30 (P30-15) from Biolegend (San Diego, CA), KIR2DS4 (FES172), KIR2DL1/DS1 (EB6B), KIR2DL2/L3/DS2 (GL183), KIR3DL1/DS1 (Z27.3.7), Bcl-xL (7B2.5), CD159a (NKG2A; Z199) from Beckman Coulter (Fullerton, CA), Granzyme-B (GBl 1) from CALTAG (Invitrogen, CA) and CDl Ib (ICRF44), CD25 (BC96), CDl 16 (4Hl), CD83 (HBlSe), anti-mouse NKLl, CDlIb, CDl Ic₅ F480 and DX5 from eBioscience (San Diego, CA). 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. For BrdU detection, cells were incubated for 1 h at 37⁰C with 30μg DNAse from BrdU flow kits (BD Bioscience, CA). All washings and reagent dilutions were done with PBS containing 2% 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).

[076] Cell preparation. Human fetal material was obtained from elective abortions, with gestational age ranging from 14 to 20 weeks. The use of this tissue was approved by the Medical Ethical Committee of the AMC-UvA and was contingent on informed consent. Single cell suspensions of human fetal organs were achieved by mechanical disruption using a Stomacher® Biomaster lab system (Seward, Hadleigh, UK).

[077] Magnetic enrichment of CD34⁺ cells (> 98% pure) was performed by using the CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech, Auburn, CA), after preparation of 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 (24).

[078] In vitro assays. Human 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 μM CFSE (Molecular Probes) and cultured at 2 x 10⁵ 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. Alternatively, 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. The final concentration of 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 2.5x10⁵ 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 2.5xlO⁶ K562 cells/ml (ATCC) for 18 hours with brefeldin A (Sigma) added for the last 4 hours of culture.

[079] 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). The following day, cells were incubated for 6 to 8 hours with control LZRS IRES-GFP or LZRS Bcl-xL-IRES-GFP virus supernatant in fibronectin-coated plates (30 μg/mL; Takara Biomedicals, Otsu, Shiga, Japan). The cell bulk was then inoculated i.h. to the newborn recipients. The cDNA sequence encoding Bcl-xL, initially provided by the laboratory of J. G. Collard (The Netherlands Cancer Institute, Amsterdam) was inserted into the multiple cloning sites of LZRS vector upstream of an internal ribosomal entry site and enhanced green fluorescent protein (GFP) (48). Control vectors were empty LZRS IRES. Retroviral supernatants were produced as described (49) using the 293T-based Phoenix packaging cell line (50).

6.2. RESULTS [080J Human hematopoietic chimerism and NK cell development in HIS-mice.

Human HSC engrafted into newborn Balb/c Rag2^"/~γc^~/~ mice develop into mature myeloid and lymphoid cells (20, 21, 23). This approach was made to investigate human NK cell development in vivo. 8 - 12 weeks after HSC engraftment, HIS-mice displayed human hematopoietic chimerism in all organs analyzed, with human thymopoiesis, B lymphopoiesis and myelopoiesis evident (Fig. 1 A, B and Fig. 6). Using a NK cell specific antibody (anti-NKρ46), 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. These CD5ό^l0 cells are NK cells as they express NKp46, NKG2D, CD94 and like their counterparts in man, express KIRs whereas CD56^hlCD16^" 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). In vivo generated human NK express high levels of intracellular granzyme-B and IFN-α 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).

[081] IL-15 is a pleiotropic cytokine essential for murine NK cell development.

Given the sparse numbers of human NK cells, it was hypothesized that IL-15 availability in this HIS model might be suboptimal. HIS-mice represent a hybrid human-murine system where cytokines receptors compatibilities between species may not exist. Specifically, while human IL-15 (ML- 15) induces survival and proliferation of murine NK cells (24), it is not clear whether the reverse is true. This is a critical question as it is expected most of the IL-15 in HIS»mice to be rnurine-derived given the low human myeloid (20) or absent epithelial/stromal cell chimerism, (cells known to be sources of IL-15) (15, 25). It was found that human NK cells cultured in vitro with hIL-15 proliferated extensively (predominately the CD56^blCD16^" 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). Given mIL-15 is trans- presented in vivo, the effect of culturing human NK cells with low concentrations of IL-15 alone or when trans-presentated with IL-15 to IL- 15Ra-Fc of the same or alternate species was investigated. Irrespective of which species of IL-15Rα was used, mIL-15 failed to induce human NK cell proliferation, although some cells survived when mIL-15 was combined with hIL-15Rα-Fc (Fig. 1 G). In contrast, hIL~15Rα was clearly superior in inducing NK cell proliferation compared to mIL-15Rα when combined with hIL-15 (Fig. 1 G). Similarly, 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). Lastly, human NK cells are observed in athymic HIS-mice (Rag2^"Aγc^"/"FOXNr^/") ruling out a major role for T cell-derived IL-2 driving NK cell development (Fig. 1 H).

[082] Taken together, these data indicate that the few resident NK cells in HIS-mice are dependent on the available human IL-15. Indeed, the number of human NK cells in the spleen of HIS-mice could be further reduced following treatment with neutralizing antibody against human not murine IL-15 (Fig. 7). Human IL- 15 transcripts are found in the bone marrow of HIS-mice (27) as are IL-ISRa⁺ cells known to express IL-15 and support NK cell development such members of the myeloid linage (15, 20) (and our unpublished observation). Given this last point, it is possible that enhancing the chimerism of human IL-15/IL-15Rα expressing cells will improve human NK cell development in HIS-mice. This would also be important in the context of NK cell priming/activation/survival in vivo by IL- 15Ra⁺ myeloid cells in response to pathogen exposure (26, 28).

[083] Given the likely dependence of human NK cells on hϊL- 15, HIS-mice were treated with the same concentration of ML- 15 alone or hIL-15 pre~incubated with ML-15Ra-Fc to mimic IL- 15 trans-presentation in vivo. Consistent with murine studies (16, 17), a significant increase in NK cell frequency was observed when ML- 15 was complexed to IL- 15Ra-Fc but not when administered alone (Fig. 2 A). Increased NK lymphopoeisis by ML-15⁺IL-15R^α-Fc compared to IL- 15 alone was associated with increased Bcl-xL expression and cell proliferation as demonstrated by BrdU uptake and Ki-67 expression (Fig. 2 B). This data indicates that IL- 15R- mediated human NK cells responses are more readily evoked in vivo when IL- 15 is complexed to IL-15Ra-Fc.

[084 J Two different IL- 15R agonists were compared, both mimicking IL- 15 trans- presentation, hIL- 15 +IL- 15Ra-Fc and RLI (ML- 15 covalently linked to an ML-15Rα extended sushi domain, but lacking any Fc fragment) (16, 17, 29). Four injections of 2.5MLg ML-15R agonists (one per week) resulted in a significant increase in the number of NKp46⁺ cells in all organs (Fig. 2 C). Both these agonists markedly enhanced NK cell in vivo uptake of BrdU, expression of Bcl-xL, Ki-67 and CD69 and resulted in delayed apoptosis of NK cells when withdrawn from cytokines (Fig, 2 D, E) suggesting both proliferation and survival result from IL-15⁺IL-15Rα binding to NK cells in vivo. Human CD8 T cells are known to proliferate in response to IL- 15 in vitro (30) and were also significantly augmented in HIS-mice and had an obvious increase in BrdU uptake following treatment with RLI (Fig. 2 F). RLI that activates both the IL~15Rβ/α (trans-presentation) and IL-15Rα/β/y (cis-presentation) was consistently more effective in vivo than non-covalent association of hIL-15+hIL- 15Ra-Fc that activates IL-15Rβ/γ (trans-presentation) alone. This observation is in agreement with in vitro studies showing RLI functions more efficiently than the non- covalent associations of IL- 15 + sushi domain or other soluble forms of IL-15R (29, 31). The evident effect of RLI compared to IL-15+IL-15Rα-Fc also indicates that the Fc protein does not contribute to the augmented NK lymphopoeisis and rales out any activation of human NK cells through CDl 6.

[085] 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). 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. To address this, 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^"/"γc^"/" 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 A₅ 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). In addition, while 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^loCD16⁺). Lastly, the number of NK cells in HIS-mice engrafted with Bcl-xL infected HSCs in terms of absolute numbers and fold- difference compared to control were substantially lower than mice treated with trans- presented IL- 15. These findings indicate that IL-15/IL-15Rα effects extend beyond providing survival signals in promoting human NK cell development in vivo.

[086] A consistent observation in all lymphoid organs following treatment with trans-presented IL- 15 was the skewing of NK cell maturation towards the more differentiated CD56^loCD16⁺ phenotype. In particular, significant and specific increases in the frequency of CD16⁺ NK cells and the ratio of CD16⁺:CD16^"NK cells 7 days after the final treatment was observed (Fig. 4 A and B). This in vivo accumulation is consistent with model where CD56^loCD16⁺ NK cells represents the terminal stage of NK cell development (36) and suggests trans-presented IL-15 promotes this differentiation. CD 16 expression is associated with human NK cell differentiation and increased cytotoxicity by means of increased intracellular effector granules and the ability to perform antibody dependent cell cytotoxicity. IL- 15 itself is known to enhance NK cell cytotoxicity via up-regulating effector molecules such as IFN-α, 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%). This may result from greater IL- 15 concentrations in the model compared to humans, however the frequency OfKIR⁺ NK cells in human thymus has not been reported using all existing commercial antibodies to KIRs. In contrast to IL- 15 trans-presentation, no enhanced NK cell differentiation and induction of KIR expression was observed in HIS-mice treated with IL- 15 alone, nor those engrafted with Bcl-xL expressing HSCs (Fig. 4 C). Furthermore, the increase in KIR⁺NK cells did not represent an expansion of one CD56^!oCDl O⁺KIR⁺ NK clone as CD56^1oCDlό⁺ NK cells expressing a combination of 1 to 5 different KIR members were present in this population (Fig. 4 D).

[087] It is unlikely the accumulation of CD56^loCD 16⁺ NK cells does results from a preferential expansion of this subset in response to hIL-15 trans-presentation as both CD56^l0CD16+ and CD56^h!CD16^" 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^l0CD16⁺ NK cells are refractory to IL- 15 stimulation in vitro (40, 41), especially when compared to CD56^hlCD16^~ NK cells (Fig. 1 E). To test this hypothesis, highly purified CD56^hiCD16 KIR s or CD56^ioCD16⁺KIR^"sNK cells from fetal spleen into Balb/c Rag2^{" "}γc^^* in the presence or absence of RLI treatment were sorted and transferred. Although unable to recover the donor cells in mice receiving PBS alone, clear populations of donor cells were recovered from RLI treated mice 7 days after transfer (Fig- 5 B). Trans-presented IL-15 induced the differentiation of CD56¹¹¹CD 16^* NK cells with between 34 ~ 40% of recovered NK cells becoming CD16⁺ (Fig. 5 B). In addition a fraction of the CDIo⁺NK cells also now expressed KIRs. Similarly, while CDSo¹⁰CDIo⁺NK cells retained this phenotype in the presence of RLI following transfer, expression of KIRs were acquired by a fraction of these CDSo¹⁰CDIo⁺NK cells that lacked KIR expression prior to transfer (Fig. 5 B). The absence of NK cells in non-RLI treated recipients clearly shows the available murine IL- 15 is inadequate to support human NK cell survival and highlights the importance of a source of human IL- 15 in HIS-mice in promoting NK cell differentiation.

[088] Binding of IL- 15 to NK cells ultimately activates signaling pathways stemming from STATS phosphorylation such as activation of NP-αB, 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. Coordination between granzyme/perforin induction and KIR expression would be one means to limit NK cell activity during differentiation and while most NK cells express other inhibitory receptors for MHC-I (CD94/NKG2 A and LILR) throughout development, it appears acquisition of KIRs is a late event and likely unique compared to other forms of MHC-I-mediated inhibition. Taken together, this data provides the first in vivo demonstration that trans-presented IL- 15 is essential for NK cell survival and subsequent differentiation and that KIR expression on mature NK cells is dynamically regulated by IL- 15 signaling.

[089] Murine studies show that 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-lSRα on the same cell (14, 15). The clear effect of ML-lSRα 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.

[090] The present study provides the first evidence that trans-presented IL- 15 induces KIR expression on CD56^l0CDlό⁺ 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. Interestingly, 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. The finding that this efficiently occurs in vivo in the absence of non-haematopoietic self MHC-I expression indicates that treatment of patients recovering from haematopoietic cell transfer (who have reduced KIR expression compared to healthy adults) (44, 45) with trans-presented IL- 15 may improve clinical outcomes. Use of WL-15R agonists such as RLI could also benefit mis-matched haematopoietic cell transfers, transporter associated with antigen processing (TAP) deficient patients (who have a reduced population of CD 56¹⁰CDl 6⁺ NK cells) (46) and cancer immuno-therapy. The efficiency of 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 α/α T cell) responses to human pathogens and disease in vivo. 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. Taken together the results described herein define an essential role for IL- 15 in human NK cell development in vivo and demonstrate the efficacy of IL-15/TL-15Rα complexes in promoting human NK cell homeostasis suggesting human IL- 15 is likely bound and trans-presented by IL-15Rα expressing cells in man.

7. EXAMPLE

[091] The following example demonstrates that IL- 15 trans-presentation regulates human T cell (CD4 AND CD8 T cell) homestasis in vivo.

7-1 MATERIALS AMD METHODS

[092] Mice. Rag2^"/"γ_c ^"/~ mice on the Balb/c background 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^~/"γ_c ^~/~ mice received sub-lethal (3.3 Gy) total body irradiation from a Cs source, and were injected intra-hepatic (i.h.) with 5xlO⁴ 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.

[093] In vivo treatments. HIS mice were injected intra-peritoneally with lOOμl of 2.5μg RLI or PBS alone commencing at a either 5-6 weeks after reconstitution or 8 weeks after reconstitution. lOOμl of lOmg/ml of BrdU from BrdU flow kit (BD Bioscience, CA) 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 μl 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. Two weeks after the last immunization, HIS mice were sacrificed for analysis. [094) Flow cytometry analysis for cell-surface and intracellular markers. Cell suspensions were labeled with mAb against the following human cell-surface markers: CD3 (SK7)_} CD4 (SK3), CD34 (581), V52 (B6), CDS (SKl), CD19 (HIB 19), NKp4ό (9E2) CD38 (HB7), CD 16 (3G8), CD45RO (UCLHl), CD56 (B159), CD122 (Mik-β3), TCR-α/β (T10B9.1A-31), CD127 (ML-7R-M21), CD45 (2Dl), CD69 (L78), Bcl-2 (Bcl-2/100), IFNPy (XMGl.2), BrdU (B44) from BD Bioscience (San Jose, CA), KIR2DL2/L3 (DX27), KIR3DL1 (DX9), CD27 (O323), CD62L (DREG-56) from Biolegend (San Diego, CA), KIR2DS4 (FES 172), KIR2DL1/DS1 (EB6B), KIR2DL2/L3/DS2 (GL183), KIR3DL1/DS1 (Z27.3.7), Vγ9 (MMU 360), Bcl-xL (7B2.5) from Beckman Coulter (Fullerton, CA), Granzyme-B (GBl 1) from CALTAG (Invitrogen, CA) and CD25 (BC96), γ/δTCR (BLl) and CD45RA (HIlOO) from eBioscience (San Diego, CA). 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. For BrdU detection, cells were incubated for 1 h at 37°C with 30μg DNAse from BrdU flow kits (BD Bioscience, CA). All washings and reagent dilutions were done with PBS containing 2% 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-Dϊva software (BD Bioscience).

[095] Cell preparation. Human fetal liver was obtained from elective abortions, with gestational age ranging from 14 to 20 weeks. Experiments using human fetal liver cells were approved by the Medical and Ethical Committees at the Institut Pasteur and AMC-UvA and performed in fall compliance with French law. Single cell suspensions of fetal material was achieved by mechanical disruption using a Stomacher^® Biomaster lab system (Seward, Hadleigh, UK). Magnetic enrichment of CD34⁺ cells (> 98% pure) was performed by using the CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech, Auburn, CA), after preparation of 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].

[096] in vitro assays. Thymocyte populations from HIS mice treated with PBS or RLI were electronically sorted using FACSARIA (BD Bioscience) and were cultured at 1 x 10⁴ 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.

[097] TCR Vβγ6 and CDR3 immunoscope analysis. 12 weeks after CD34⁺CD38^" HSC engraftment, HIS mice were killed and single cell suspensions of splenocytes were prepared. Red cells lysis was performed in 1 ml of red cell lysis buffer (Sigma) for 10 min. Splenocytes were washed, resuspended in 600 μl of RLT lysis buffer (Qiagen) and homogenized by passing through a 21 -gauge needle several times using RNase free syringes. RNA was prepared using RNeasy mini kits (Qiagen) according to manufactures instructions. TCR Vβ immunoscope was performed as previously described [100], Briefly, cDNA was prepared and real-time PCR performed by combining primers for the different Vβγδ chains (Vβl-24, Vγ2-9 and Vδl-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).

[099] 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 lOμg/ml goat anti-human IgM or 10 μg/ml goat anti-human IgG (Jackson ImmunoResearch Laboratories) in PBS for lhr at 37°C or o/n at 4⁰C. 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).

7-2 RESULTS

[0100] Improved development of human CD4 and CD8 T cells in HIS mice receiving trans-presented human IL- 15 results in improved humoral responses following immunization. It has been reported that human fetal liver HSC (CD34^÷CD38^~) engrafted into newborn Balb/c Rag2^{" "} γ_c ^{~ "} mice develop into mature myeloid and lymphoid cells [69-73]. This approach was used to investigate the effect of trans- presented human IL- 15 on human T cell development in vivo, since it had previously been shown that murine IL- 15 has poor activity on human NK cells [71]. 8 weeks after HSC engraftment, HIS mice were injected intra-peritoneally weekly for 4 weeks with 2.5μg of a potent human IL-15R agonist known as RLI consisting of human IL- 15 covalently linked to an extended human IL-15Rα 'sushi' domain [66, 74, 75] and therefore capable of mimicking IL- 15 trans-presentation (Fig. 8A). Administration of hIL-15+IL-15Rα (RLI) results in a significant improvement in the number of human hematopoetic cells in the thymus and the spleen of recipient mice (Fig. 8B). CD8⁺ T cells were significantly augmented in the spleen (P = 0.025) and thymus (P - 0.033) following IL- 15 trans-presentation in humanized mice whereas CD4⁺ T cells where also augmented in spleen (P = 0.017) and lymph node (P = 0.025) following IL-15 treatment. Thus the increase in total human hematopoetic cells in the thymus (P = 0.0005) and spleen (P = 0.002) following IL-15 treatment is attributed to significant increases in total T cell numbers (Fig. 8B) in addition the increase in NK cells previously reported [71], whereas B cell numbers remain constant (Fig. 8C).

[0101] To assess the impact of improved human T cell homeostasis in IL-15 'boosted' HIS mice, cohorts of HIS mice were immunized with the commercially available HBV vaccine. While overall serum IgM levels were not appreciably modified following vaccination (Fig. 8D), a slight increase in IgG levels was found in HB V- vaccinated mice compared to non-vaccinated HIS mice. In contrast, HBV immunization resulted in marked increases in IgG levels in HIS mice that had received trans-presented human IL-15 (Fig. 8D). Thus, IL-15 'boosting' resulted in improved humoral responses that correlated with improved human T cell homeostasis.

[0102] Characterization of human CD4⁺ and CD8⁺ T cells in HIS mice receiving trans-presented human IL-15. Since IL-15 was effective in increasing both CD4^÷ and CD8^H" T cells, it was investigated whether any phenotypic changes or signs of toxicity such as overt activation were induced on expanded T cell population (Fig. 9A). No differences in classic T cell markers or any change in the proportion of naϊve (CD62L⁺CD45RA⁺) CD4⁺ and CD8⁺ T cells in the periphery following the 4 week IL-15 treatment regime was observed (Fig. 9B). With respect to T cell activation, an occasional increase in the CD45RO⁺CD45RA^~ population was observed within the splenic CD8⁺ T cell pool but not in other lymphoid organs nor amongst CD4^÷ T cells. In line with this more splenic CD8⁺ T cells expressing the activation marker CD69 were observed. The expression of receptors for γ_c cytokines were also unchanged following treatment including CD 122, CD 127 and CD25 as were the populations of regulatory T cells (CD25⁺CD4⁺CD127^~) (Fig 9B).

[0103] It was previously shown that trans-presented IL- 15 induces the expression of killer-Ig-like inhibitory receptors for MHC-I (KIRs) on NK cells in vivo [71]. Interesting, a similar phenomenon was observed on a subset of human T cells following staining with a pool of 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).

[0104] 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). Furthermore, 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 naϊve and activated T cells. To test this, humanized mice treated with RLI were injected IP with lOμg of BrdU 24 and 12 hours before being sacrificed and T cell populations analysed for BrdU incorporation. Both CD8⁺ and CD4^÷ naϊve (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'ϊve phenotype showed the greatest fold increase in proliferation in both the spleen and the mesenteric lymph node after IL- 15 treatment followed by naϊve CD4⁺ T cells, activated CD8⁺ T cells and lastly activated CD4⁺ T cells (Fig 10 A, B). 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 γ_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. Noteworthy, large increases in NK cell-derived cytokines in hIL-15+IL-15Rα treated mice were not observed, suggesting the large increase in NK cells following this treatment regime is unlikely to contribute to toxicity via hyper-production of pro-inflammatory cytokines (Fig 10 C).

[0105] Effects of trans-presented human IL-15 on human thymopoiesis in HIS mice. The increase in total thymocytes far outnumbered the contribution from the increase in single positive CD4⁺ (SP4) and CD8⁺ (SP8) T cells (Fig 8 B) so the IL-15 effect on the CD3^' thymocyte populations was not examined. Both double positive (DP) and double negative (DN) (with NK cells being excluded), but not immature single positive CD4 (ΪSP4) thymocytes were significantly increased following IL- 15 treatment (**P < 0.02; Fig 11 A). Thymocytes subsets were grouped according to the phenotype in (Fig 11 B). Thymocyte turnover was analysed in the same manner as previously mentioned by injecting mice with lOμg of BrdU 24 and 12 hours before being sacrificed. Interestingly, while 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. Of all the thymocyte populations, ΪSP4 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. As this is the first report that trans-presented IL-15 is functional on human DN and DP thymocytes, other parameters were next examined such as cell survival. Intracellular staining for pro-survival proteins revealed enhanced expression of Bcl-xL in DN and SP4 thymocytes of IL-15 treated mice but not in DP, CD8 or iSP4 thymocytes, whereas Bcl-2 protein levels were unchanged in each population following IL- 15 treatment (Fig 11 C). Futhermore, it is clearly evident that Bcl-xL expression is up- regulated during human thymocyte maturation in HIS mice, Bcl-xL being most highly expressed in DP thymocytes before being down-regulated in positively selected SP4 and SP8 T cells. [0106] Interestingly, 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). In light of this in vitro finding, 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.

[0107] TCR repertoires in HIS mice receiving trans-presented human IL-15.

The maintenance of a highly diverse T cell receptor repertoire (TCR) is essential for efficient immune responses against the extensive number of foreign antigens encountered during our lifetime. We next examined the effect of IL-15 treatment on the human TCR repertoire by performing immunoscope profiling on the β, γ and δ variable chains of the TCR expressed by splenic and thymic T cells. This analysis is important as it is plausible that the T cell expansion we observed following IL-15 treatment arises from clonal proliferation of a relatively restricted population of IL-15 responsiveness T cells such as NKT (Vα24/Vβl 1) or γ/δ TCR⁺ T cells which in turn could compromise the homeostasis of α/β T cells reducing overall T cell diversity.

[0108] 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 Vβ 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 Vβ chains used except for the occasional over-representation of clones within the Vβl 1 and Vβ7. 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).

[0109] The degree of γ/δ TCR⁺ T cell development in humanized mice has not been characterized. Using commercially available antibodies a clear population of Vγ9⁺Vδ2⁺ T cells in the thymus and LN of humanized mice was observed and these γ/δ T cells were largely restricted to the CD161⁺CD3⁺ T cell subset (Fig 13 A). In the thymus, a clear Vγ9^~Vδ2⁺ and a minor Vγ9⁺Vδ2^~ T cell population was detected that appeared absent or at least diminished in LN. Following IL- 15 treatment, it became obvious that the γ/δ 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, γ/δ T cells are not more responsive to IL- 15 than α/β T cells.

[0110] CDR3 immunoscope analysis of γ and δ variable chain genes was next used to identify all γ and δ usage in HIS mice (since monoclonal antibodies against each unique γ and δ 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 γ/δ T cells found in the periphery of HIS mice use Vδ2 and this is similar to human PBMCs and unaffected by IL-15 treatment (Fig 13 C). Interestingly, a very small population of Vγ5⁺ T cells and two large populations of Vγ8⁺ and Vγ9⁺ T cells were observed which each represent between 40 - 60% of total γ/δ T cells and who obviously pair almost exclusively with Vδ2, although occasional usage of V63, 5 and 8 was observed (Fig 13 C). Interestingly, significantly greater use of Vγ8 TCR in humanized mice was observed compared to what was detected in healthy human PBMC, although the significance of this in terms of T cell development or potential immune response is not evident (Fig 13 C). In vivo administration of IL-15 did not influence the ratio of the different γ and δ chain usage nor the variability in CDR3 length for any given Vγ or Vδ chains. Thus the modest increase in γ/δ XCR⁺ T cells following IL- 15 treatment likely results from an increase in all γ/δ TCR⁺ T cells subsets generated (Fig 13 C).

[0111] 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. In order to perform this study, the 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). These HIS mice were then treated with a more intense IL- 15 treatment regime that entailed 2.5μg of IL-15R agonist, IL-15+IL-15Rα (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 naϊve mature T cells (hCD45⁺CD45RA⁺CD3⁺; Fig 14 B). Furthermore, this effect could be monitored over time in the blood of HIS mice receiving IL-15+IL- 15Rα where the we detected 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 (Fig 14 C).

[0112] The experiments described herein demonstrate that the homeostasis of both CD4⁺ and CD8⁺ human peripheral T cells and some immature human thymocyte populations is markedly improved in HIS mice when human IL- 15 is trans-presented (IL-15/IL-15Rα). The findings differ from a similar murine study reporting increased CDS⁺ T cell numbers following treatment with trans-presented mouse IL- 15 [74, 76]. The results further underscore the fact that human and murine immune systems are not identical, and highlight the value of the HIS model to study human immunology in vivo. During the past 30 years 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]. Given that the immune system in HIS mice is heavily skewed towards B cell development (with around 90% of peripheral blood or splenic human hematopoietic cells being B cells), there is a clear need to improve T lymphopoeisis in these models [69, 70], It has been recently reported that a beneficial effect is observed on human thymopoeisis following human JL-I treatment in the HIS model, however peripheral T cell numbers remained unchanged despite transient increases in thymocytes suggesting that other factors are involved [73], The beneficial effect of human 'trans-presented' IL-15/IL-15Ra resulted in increased T cell numbers that also impacted on the ability to generate humoral immune responses with increased immunoglobulin levels following immunization. These results further enlarge the spectrum of potential applications of the HIS model for human vaccine testing.

[0113] The observation that both naϊve and activated phenotype CD4⁺ T cells proliferate in response to IL- 15 receptor ligation in vivo differs from in vitro studies where only memory but not naϊve CD4⁺ T cells respond to IL-15 [81], Moreover, this in vitro study observed that IL- 15 promoted the generation of effector memory phenotype CD4⁺ T cells from central memory phenotype CD4⁺ T cells. While impact of trans-presented IL- 15 on non-naϊve CD4⁺ T cells was not dissected in the study, the collective data suggests that in vivo IL-15 could assist in the generation or maintenance of both antigen-dependent and -independent memory CD4 T cells. In vitro studies also suggested that IL- 15 can skew activated T cells towards a ThI phenotype as determined by an increased IFN-γ/IL-4 protein ratio. Tehre was a failure to detect any difference in serum or intracellular IFN-γ 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.

[0114] Several in vitro reports suggest that 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 CD8αα and Vα24⁺Vβl 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, γ/δ T cells were only increased to a similar level as α/β T cells, which is in contrast to data suggesting that human γ/δ T cells are more responsive on IL- 15 in vitro. It is possible that other factors (such as the selecting ligands for human γ/δ T cells) are limiting in HIS mice. Indeed, a recent report using IL- 15 supplementation in Macaques demonstrated a rare but severe preferential expansion of CD4 CD8^" γ/δ T cells that comprised 70% of the T cell compartment in the blood of one subject [85]. IL-15 treated Macaques displayed increases in CD8 T cells, NK cells and to a lesser extent CD4 T cells that was associated with increased turnover in vivo. Still, IL- 15 toxicity in these macaques was also associated with hypocellularity, decline in hemoglobin, weight loss, low-grade fever and non-specific dermatitis, that was avoided when IL- 15 was given intermittently [85]. hi contrast, we failed to detect any obvious signs of IL- 15 toxicity in HIS mice using our treatment regime.

[0115] It was previously reported that a likely reason for the poor human NK cell development in HIS mice was a result of the poor responsiveness of human NK cells to murine IL-15 [71]. Indeed, exogenous trans-presented human IL-15/IL-15Rα (RLI) induced a large increase in human NK cells in HIS mice [71]. It is speculated that a similar mechanism may explain the improved T cell homeostasis that is observed. It is possible that trans-presented IL- 15 together with signals delivered through peptide/MHC complexes may be critically involved in maintaining human T cell homeostasis in vivo. An additional factor may result from the expansion and potential activation of NK cells and T cell following trans-presented IL- 15 treatment that induces production of other cytokines and growth factor which provide bystander effects. 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 γ_c receptor, thus all of the observed effects involve IL- 15 receptor triggering on human cells.

[0116] The ability of 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]. Furthermore, IL-15 has been shown to induce antigen independent expansion of naϊve, 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 naϊve 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.

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Claims

What is claimed:

1. A transgenic mouse, 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 innate lymphocytes such as mature human natural killer (NK), cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes.

2. The transgenic mouse of claim 1 , wherein the innate human lymphocytes in general and NK cells in particular and the maturation of NK cells as well as the human adaptive CD4 and CD8 T lymphocytes are induced through administration of an IL- 15 or IL- 15Rα agonist.

3. The transgenic mouse of claim 2, wherein said IL- 15 or IL- 15Rα agonist is an IL-15/IL-15Rα complex.

4. The transgenic mouse of claim 1 further comprising a phenotype comprising a deficienccy 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.

5. The transgenic mouse of claim 1 , wherein the mouse is Ragl^^'γc^"'^" transgenic mice.

6. The transgenic mouse of claim 1 wherein the human hematopoietic cells are derived from cord blood or fetal liver.

7. A method for increasing the number of innate lymphocytes such as mature human natural killer (NK), cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes in a subject, comprising the administration of an IL- 15 agonist.

8. A method for increasing the number of innate lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes in a subject, comprising the administration of an IL-15R agonist.

9. A method for increasing the number of innate lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes in a subject, comprising the administration of an IL-15/IL- 15Rα complex.

10. The method of claim 7 to 9, wherein the subject is a transplant patient.

11. The method of 7 to 9, wherein the subject is a cancer patient.

12. The method of claim 7 to 9, wherein the subject is infected with a pathogen.

13. The method of claim 7 to 9, wherein the subject is an immunodeficient patient.

14. The method of claim 7 to 9, wherein the mature NK cells are CD16+Kir+.

15. A method for identifying a compound that induces maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CDS T lymphocytes, comprising

(i) contacting a transgenic mouse characterized in that it has

(a) a phenotye comprising a deficiency for murine T lymphocytes, B lymphocytes and NK cells;

(b) is engrafted with human hematopoietic cells with a test compound and

(ii) measuring the level of maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes; wherein an increased in the level of maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes in the presence of the test compound indicates that the test compound induces cell maturation.

16. The method of claim 15, wherein the compound is tested in the presence of an IL-15/IL-15Rα complex.

17. A method for identifying a compound that inhibits maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD 8 T lymphocytes comprising

(i) contacting a transgenic mouse characterized in that it has

(b) is engrafted with human hematopoietic cells

with a test compound, in the presence of an IL-15/IL-15Rα 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-15Rα complex and measuring the level of maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes, where the conditions are essentially the same as in part (i) and then

(iii) comparing the level of maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes measured in part (i) with the level of maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes in part (ii),

wherein a decrease level of maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes in (i) compared to (ii) indicates that the test compound is an inhibitor of maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes.

18. A method for identifying a compound that increases IL-15 expression or activity, i.e., an agonist, thereby inducing maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes, 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 maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CDS T lymphocytes; wherein an increased in the level of maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes in the presence of the test compound indicates that the test compound increases IL- 15 expression or activity.

19. A method for identifying a compound that increases IL- 15R expression or activity, i.e., an agonist, thereby inducing maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes, comprising (i) contacting a transgenic mouse of the invention with a test compound and (ii) measuring the level of maturation of innate human lymphocytes such as mature human natural killer (NK cells γδ T cells, and NK-T cells as well as adaptive CD4 and CDS T lymphocytes; wherein an increased in the level of maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes in the presence of the test compound indicates that the test compound increases IL-15R expression or activity.

20. A method for identifying a compound that inhibits IL- 15 or IL- 15R expression or activity, i.e., an antagonist, thereby inhibiting maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes comprising (i) contacting a transgenic mouse of the invention with a test compound, in the presence of an IL-15/IL-I5Rα complex and measuring the level of maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes; (ii) in a separate experiment, contacting a transgenic mouse of the invention with an IL-15/IL-15Rα complex and measuring the level of maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes, where the conditions are essentially the same as in part (i) and then (iii) comparing the level of maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes measured in part (i) with the level of maturation of innate human lymphocytes such as mature human natural killer (NK) cells γδ T cells, and NK-T cells as well as adaptive CD4 and CD8 T lymphocytes in part (ii), wherein a decrease level of maturation of innate human lymphocytes such as mature human natural killer (NK cells γδ T cells, and NK-T cells as well as adaptive CD4 and CDS T lymphocytes in (i) compared to (ii) indicates that the test compound is an inhibitor of IL- 15 of IL-15R expression or activity.

21. A method for preparing humanized antibodies, or fragments thereof, comprising (i) immunizing the transgenic mouse of claim 1 with an antigen of interest; and (ii) producing humanized antibodies, or fragments thereof, from the serum or B lymphocytes of said transgenic mice.

22. The method of claim 17 wherein the engrafted transgenic mouse is stimulated with IL- 15 or an IL- 15 agonist such as an IL-15/IL-15Rα complex prior to immunization.

23. An antibody, or fragment thereof, produced using the method of claim 21.