WO2014207009A2

WO2014207009A2 - Methods and kits for determining whether a nk cell is activated and is able to proliferate

Info

Publication number: WO2014207009A2
Application number: PCT/EP2014/063329
Authority: WO
Inventors: Martin VILLALBA GONZALEZ; Amélie CORNILLON; Ewelina KRZYWINSKA; Jean-François ROSSI
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Université de Montpellier I; Centre Hospitalier Universitaire De Montpellier
Priority date: 2013-06-28
Filing date: 2014-06-25
Publication date: 2014-12-31
Also published as: WO2014207009A3

Abstract

The present invention relates to methods and kits for determining whether a NK cell is activated and is able to proliferate. In particular, the present invention relates to a method for determining whether a NK cell is activated and is able to proliferate comprising the steps consisting of i) determining the expression level of CD45RO at the cell surface of the NK cell, ii) comparing the expression level determined in step i) with a predetermined reference value and iii) concluding that the NK cell is activated and is able to proliferate when the expression level determined at step i) is higher than the predetermined reference value. The present invention also relates to a population of CD45RA+RO+ NK cells.

Description

METHODS AND KITS FOR DETERMINING WHETHER A NK CELL IS

ACTIVATED AND IS ABLE TO PROLIFERATE

FIELD OF THE INVENTION:

The present invention relates to methods and kits for determining whether a NK cell is activated and is able to proliferate.

BACKGROUND OF THE INVENTION:

Natural Killer (NK) cells are members of the lymphocyte lineage and belong to the innate immune system. They show natural cytotoxicity and produce cytokines ^l'². The majority of human NK cells in peripheral blood are CD3^~CD56^dim cells while the minority shows a CD3^" CD56^bngth phenotype. The last population shines at cytokine production whereas CD56^dim cells show mainly cytotoxic activity ³. In vitro evidence indicates that CD56^bnght cells are precursors of CD56^dim cells, which can also be the case in vivo . NK cells mostly target cells lacking major histocompatibility complex-I (MHC-I), which include transformed or virus-infected cells, which downregulate MHC-I expression to evade recognition by cytotoxic T lymphocytes (CTL). The "missing self hypothesis proposes that NK cells distinguish target cells from other healthy "self cells based on MHC-I expression. However, NK cell activation depends on a complex signaling mechanism mediated by both activating and inhibitory receptors. The main NK cell inhibitory receptors recognize MHC-I complexes and include NKG2A, which recognizes HLA-E, and Killer-cell Immunoglobulin- like Receptors (KIRs), which recognize the self classical class I molecules HLA-A, -B and -C. The first clinical trial using an anti-KIR that can block KIR-mediated inhibition of natural killer (NK) cells has recently been published ⁵. It shows absence of toxicity and favorably overall and relapse-free survival compared to reports in comparable patient populations of acute myeloid leukemia (AML). Activating NK cell receptors perceive stress and/or non-self ligands on cells, i.e. the stress-induced ligands UL16-binding protein (ULBP) and MHC class I polypeptide-related sequence (MIC) are recognized by the activating receptor NKG2D.

Long-term survival is limited in a significant number of patients with hematological cancers. NK cells are an interesting option to treat these patients because clinical-grade production of NK cells has proven efficient ⁶, and NK cell-mediated therapy after hematopoietic cell transplantation seems safe ^7"9. However, NK cells are not a homogenous population, there are different subsets keeping different physiological activities. It would be interesting to identify the populations with higher anti-tumor activity and select them for expansion and/or patient infusion.

CD45 is a protein tyrosine phosphatase (PTP) encoded by the PTPRC gene, which is specifically expressed in hematopoietic cells ¹⁰. PTPs regulate cellular processes including differentiation, mitotic cycle, cell growth and oncogenic transformation ⁿ. CD45 regulates receptor signaling by direct interaction with components of the receptor complexes or by activating and dephosphorylating various Src family kinases (SFK) i.e. Lck ¹². But it can inhibit cytokine receptor signaling by inhibiting JAK kinases ¹³ or by dephosphorylating the activating residues of Src ¹². Usually, CD45 levels increase with cell maturation ¹⁴. The CD45 family comprises several members derived of a single complex gene ¹⁴. Naive T lymphocytes are usually positive for the long CD45RA isoform. Activated and memory T cells express CD45RO, the shortest CD45 isoform by activation-induced alternative splicing of CD45 pre- mRNA ^14~17. Most studies in CD45 function have been developed in T cells. Much less is known about its function on NK cells, although it is commonly accepted that CD45 positively regulates the activation of these cells through its ability to dephosphorylate the inhibitory site of SFKs. This is particularly true for the activation that leads to the production of cytokines and chemokines, whereas cytotoxicity is only slightly impaired in NK cells derived from CD45- deficient mice ^18~20. However, in contrast to T and B cells, CD45 -deficient NK cells show increased basal phosphorylation of multiple phosphoproteins suggesting that CD45 may also dephosphorylate other substrates in NK cells, including the activating tyrosine residue of SFKs ²⁰. Hence, the role of CD45 in NK cells is an open issue, although it could depend on the type and strength of the activation. However, in vivo studies show that CD45 -deficient NK cells do not protect mice from cytomegalovirus infection due to impaired function of all immunoreceptor tyrosine-based activation motif (ITAM)-dependent NK cell functions, including degranulation ²¹. However, most of the results cited above were obtained in mouse and could not reflect the human sketch.

SUMMARY OF THE INVENTION:

DETAILED DESCRIPTION OF THE INVENTION:

The use of alloreactive NK cells seems a promising co-treatment or an alternative to allogenic HSCT, which has greatly increased the clinical interest for these lymphocytes. However, it is important to identify the different NK cell subpopulations, in particular those with superior antitumor activity. Here, we use the ubiquitous lymphocyte marker CD45 to identify different ex vivo and in vitro expanded NK cell populations from PBL and UCBL. NK cells from healthy donors express high CD45 levels that correlate with the expression of maturation markers. Ex vivo, healthy donor NK cells are exclusively CD45RA and in vitro IL- 2-mediated activation induces CD45RO expression with down regulation of CD45RA. This effect is enhanced when NK cells encounter their targets. After in vitro activation, NK cells increase in size and granularity, mainly in a newly identified CD45RA⁺RO⁺ population, which shows the higher CD 16 expression and degranulating activity. Some blood borne cancer patients and all tested bone marrow transplanted patients show CD45RO RA NK cells and a new population of CD45RA^dimRO⁺ NK cells. Some cancer patients with normal CD45RA expression in blood show CD45RA^dimRO^dim/+ and/or CD45RO⁺RA^~ NK cells. However, long term survivors of hematological malignancies that show abnormally elevated numbers of NK cells show almost exclusively CD45RA NK cells. Taking these data together suggest that CD45RO is mainly a marker of activation/proliferation and not a marker of memory NK cells; or that memory NK cells do not exist in vivo.

Accordingly an aspect of the invention relates to a method for determining whether a NK cell is activated and is able to proliferate comprising the steps consisting of i) determining the expression level of CD45RO at the cell surface of the NK cell, ii) comparing the expression level determined in step i) with a predetermined reference value and iii) concluding that the NK cell is activated and is able to proliferate when the expression level determined at step i) is higher than the predetermined reference value. As used herein, the term "NK cell" has its general meaning in the art and refers to natural killer (NK) cell. One skilled in the art can easily identify NK cells by determining for instance the expression of specific phenotypic marker (e.g. CD56) and the ability to express different kind of cytokines or the ability to induce cytotoxicity. Typically a population of NK cells is generally prepared from a blood sample. The term "blood sample" means a whole blood sample obtained from a subject (e.g. an individual for which it is interesting whether a population of NK cells is activated and is able to proliferate). Preferably, the NK cell population is prepared from a PBMC sample. The term "PBMC" or "peripheral blood mononuclear cells" or "unfractionated PBMC", as used herein, refers to whole PBMC, i.e. to a population of white blood cells having a round nucleus, which has not been enriched for a given sub-population. Typically, the PBMC sample may have been subjected to a selection step to contain non-adherent PBMC (which contain T cells, B cells, natural killer (NK) cells, NK T cells and DC precursors). A PBMC sample according to the invention therefore contains lymphocytes (B cells, T cells, NK cells, NKT cells). Typically, these cells can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates layers of blood, with the PBMC forming a cell ring under a layer of plasma. Additionally, PBMC can be extracted from whole blood using a hypotonic lysis buffer, which will preferentially lyse red blood cells. Such procedures are known to the expert in the art. The NK cells can be prepared by Percoll density gradients, by negative depletion methods or by FACS sorting methods. These cells can also be isolated by column immunoadsorption using an avidine-biotin system or by immunoselection using microbeads grafted with antibodies. It is also possible to use combinations of these different techniques, optionally combined with plastic adherence methods. For example, the NK cells can be prepared by providing blood mononuclear cells depleted of T cells from the donor, activating said cells with phytohemagglutinin (PHA) and culturing said cells with interleukin (IL)-2 and irradiated feeder cells.

In a particular embodiment, the population of NK cells is prepared from a subject suffers from a disease, for example from a cancer or an infectious disease.

In a particular embodiment, the population of NK cells is prepared from a transplanted subject. Example of grafts include, but are not limited to transplanted heart, kidney, lung, liver, pancreas, pancreatic islets, brain tissue, stomach, large intestine, small intestine, cornea, skin, trachea, bone, bone marrow, muscle, or bladder.

As used herein the term "CD45" has its general meaning in the art and refers to the protein tyrosine phosphatase (PTP) encoded by the PTPRC gene, which is specifically expressed in hematopoietic cells ¹⁰. CD45 regulates receptor signalling by direct interaction with components of the receptor complexes or by activating and dephosphorylating various Src family kinases (SFK) i.e. Lck 12. But it can inhibit cytokine receptor signalling by inhibiting JAK kinases or by dephosphorylating the activating residues of Src ¹². Typically it is possible to distinguish two iso forms of CD45: CD45RA and CD45RO. Standard methods for detecting the expression of specific surface markers at cell surface (e.g. NK cell surface) are well known in the art. Typically, the step consisting of determining the expression levels of CD45RO at the NK cell surface may consist in collecting a NK cell population from a subject and using at least one differential binding partner directed against CD45RO, wherein said NK cells are bound by said binding partners to said CD45RO.

As used herein, the term "binding partner directed against the CD45RO" refers to any molecule (natural or not) that is able to bind the CD45RO with high affinity. Said binding partners include but are not limited to antibodies, aptamer, and peptides. The binding partners may be antibodies that may be polyclonal or monoclonal, preferably monoclonal, specifically directed against said CD45RO. In another embodiment, the binding partners may be a set of ap tamers.

Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.

Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally; the human B-cell hybridoma technique; and the EBV-hybridoma technique.

In another embodiment, the binding partners may be aptamers. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library. The random sequence library is obtainable by combinatorial chemical synthesis of DNA or RNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods.

The binding partners of the invention such as antibodies or aptamers may be labelled with a detectable molecule or substance, such as preferentially a fluorescent molecule, or a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.

As used herein, the term "labelled", with regard to the antibody or aptamer, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a fluorophore [e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)]) or a radioactive agent to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Inl 11, Rel86, Rel88.

Preferably, the antibodies against the CD45RO are already conjugated to a fluorophore (e.g. FITC-conjugated and/or PE-conjugated).

The aforementioned assays may involve the binding of the binding partners (ie. antibodies or aptamers) to a solid support. The solid surface could a microtitration plate coated with the binding partner. After incubation of the NK cell sample, NK cells specifically bound to the binding partner may be detected with an antibody to a common NK cell marker Alternatively, the solid surfaces may be beads, such as activated beads, magnetically responsive beads. Beads may be made of different materials, including but not limited to glass, plastic, polystyrene, and acrylic. In addition, the beads are preferably fluorescently labelled. In a preferred embodiment, fluorescent beads are those contained in TruCount(TM) tubes, available from Becton Dickinson Biosciences, (San Jose, California).

According to the invention, methods of flow cytometry are preferred methods for measuring the level of CD45RO at the NK cell surface. Said methods are well known in the art. For example, fluorescence activated cell sorting (FACS) may be therefore used. Typically, a FACS method such as described in Example here below may be used to measuring the level of CD45RO at the NK cell surface.

In a particular embodiment, the method further comprises the steps consisting of i) determining the expression level of CD45RA at the cell surface of the NK cell, ii) comparing the expression level determined in step i) with a predetermined reference value and iii) concluding that NK cell is activated and is able to proliferate when the expression level determined at step i) is lower than the predetermined reference value. The techniques described for determining the expression of CD45RO may be applied mutatis mutandis for determining the expression of CD45RA in the population of NK cells.

The present invention also relates to a method for isolating a population of NK cells capable of activation and proliferation comprising the steps consisting of i) providing a population of NK cells and ii) selecting the population of CD45RO⁺ NK cells.

The present invention also relates to a method for isolating a population of NK cells capable of activation, proliferation and high level of cytotoxicity comprising the steps consisting of i) providing a population of NK cells and ii) selecting the population of CD45RA⁺RO⁺ NK cells.

Typically the population of NK cells as above mentioned are isolated from a blood sample obtained from a subject.

The methods are particularly suitable to be implemented in in vitro protocols of expansion of NK cells. For example the protocol of expansion may consist in contacting a population of NK cells with an amount of IL-2. The protocol of expansion may also consist of the protocol described in WO2012146702. Briefly the protocol comprise the steps consisting of (i) contacting a population of NK cells with at least one accessory cell (i.e. a cell wherein the expression of one gene encoding for a Killer-Cell Immunoglobulin-like Receptor(s) (KIR) ligand is inhibited) under conditions and for a duration sufficient to induce activation of the population of NK cells; (ii) recovering said activated population NK cells. Then the method of the invention may be performed for determining whether the protocol leads to a population of NK cells that is activated and that is capable to proliferate, and/or for isolating at the end of the protocol the population of NK cells that that is activated and that is capable to proliferate.

The methods of the invention may also suitable for determining whether an agent is able to induce activation and proliferation of NK cells. For example said agent may represent a pharmaceutical agent for the treatment of a disease (e.g. a cancer or an infectious disease), and the methods of the invention may thus be useful for determining whether said pharmaceutical agent is able to induce the development of population of NK cells that is activated and that is able to proliferate. Typically, said pharmaceutical agent may be selected from the group of recombinant interleukins, recombinant cytokines, recombinant growth factors, and antibodies... Accordingly, an aspect of the invention relates to a method for monitoring the treatment of a subject with a pharmaceutical agent comprising the steps consisting of administering the subject with the pharmaceutical agent, providing a blood sample or PBMC sample from the subject after said treatment, and determining whether said blood or PBMC sample contain a population of CD45RO+ NK cells (or CD45RA+RO+ NK cells).

The methods of the invention are also particularly suitable for the immunopro filing of a subject, typically suffering from an infectious disease or a cancer. For example, if a subject harbours a population of CD45RO+ NK cells, more particularly a population of CD45RO+RA- NK cells or a population of CD45RA+RO+ NK cells, then it can be concluded that the patient harbours an efficient immune response and the subject can have a good prognostic. Thus, a further aspect of the invention relates to a method for determining the survival time of a subject suffering of a cancer or an infectious disease comprising the steps consisting of i) detecting in a blood sample obtained from the subject the presence of a population of CD45RO+ NK cells, more particularly a population of CD45RO+RA- NK cells or a population of CD45RA+RO+ NK cells and ii) providing a good prognosis when the population is detected at step i).

A further aspect of the invention also relates to a method for determining whether a transplanted subject is at risk of graft rejection comprising the steps consisting of i) detecting in a blood sample obtained from the subject the presence of a population of CD45RO+ NK cells, more particularly a population of CD45RO+RA- NK cells or a population of CD45RA+RO+ NK cells and ii) concluding that the transplanted subject is at risk of graft rejection when the population is detected at step i).

The method may be used to evaluate survival of a variety of different types of grafts. Grafts of interest include, but are not limited to: transplanted heart, kidney, lung, liver, pancreas, pancreatic islets, brain tissue, stomach, large intestine, small intestine, cornea, skin, trachea, bone, bone marrow, muscle, or bladder. The population of NK cells isolated by the method of the invention may be suitable for testing whether a therapeutic antibody is capable to induce an antibody dependent cellular cytotoxicity (ADCC). "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted antibodies bound onto Fc receptors (FcRs) present on NK cells enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell. Typically the antibody is incubated with a population of cells expressing the antigen (i.e. the population of "target cells") that is recognized by the antibody in presence of an isolated population of NK cells according to the invention. Then the cytoxicity on the population of target cells may be then evaluated.

The population of NK cells isolated by the method of the invention may be then used in therapeutic protocols such as methods for treating cancers. Typically the population that is more interesting, although not exclusively, for a therapeutic purpose is the population of CD45RA+RO+ NK cells.

For example, the populations of NK cells isolated according to the method of the invention are particularly suitable to enhance the efficacy and safety of allogeneic grafts. By a way of example, a method of transplanting allogeneic graft into a patient in need thereof may comprise the steps consisting of a) administering to said patient an effective amount of the activated NK cells as described above; and, b) transplanting the allogeneic graft into the recipient. This method of transplanting allogeneic graft, more particularly hematopoietic graft, can be applied for reducing the GVHD, for decreasing the intensity of the conditioning regimen, for treating a subject having hematologic disorder, more particularly leukemia, for treating or preventing an infection in a recipient of allogeneic graft, for enhancing immune reconstitution in an allogeneic graft recipient, for proceeding a hematopoietic graft with a greater T cell content, for increasing the engraftment, for reducing the graft rejection, for avoiding the tumor relapse and/or for conditioning a patient in need of a hematopoietic graft. Hematologic disorder includes neoplastic proliferation of hematopoietic cells. Optionally, said hematologic disorder is selected from the group consisting of lymphoblastic leukemia, acute or chronic myelogenous leukemia, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, myelodysplasia syndrome, multiple myeloma, and chronic lymphocytic leukemia. Preferably said hematologic disorder is a leukemia, more preferably myeloid leukemia. Hematologic disorder also includes non-malignant disorders such as an inherited erythrocyte abnormalities, an inherited immune system disorders or a hemoglobinopathy, e.g. sickle cell anemia, aplastic anemia or thalassemia. In a particular embodiment the isolated population of NK cells of the invention (e.g. CD45RA+RO+ NK cells) expressing only one type of KIR (Killer-Cell Immunoglobulin- like Receptor). For example, when shall be used in the treatment of hematological cancers, the physicians may perform HLA genotyping of patients. They could then order the isolated population of NK cells of the invention missing expression of the required KIRs (e.g. an AML patient has tumor cells that do not express ligands for KIR2DL1). NK cells expressing only one KIR (e.g. KIR2DL1) may then be provided and used in the therapeutic protocol.

Preferably, said allogeneic graft is a hematopoietic graft. Optionally, said hematopoietic graft is a bone marrow transplant. In a particularly interesting embodiment of the methods according to the present invention, said patient is treated for leukemia, more preferably myeloid leukemia, optionally an acute or chronic myeloid leukemia.

In one embodiment of the methods according to the present invention, the isolated population of NK cells according to the invention and allogeneic graft are administered simultaneously. In an alternative embodiment, the isolated population of NK cells of the invention are administered prior to the allogeneic graft.

The efficient amount of the isolated population of NK cells of the invention administered to the recipient can be between about 0.05 10⁶ and about 100 10⁶ cells/kg of recipient's body weight. The efficient amount of hematopoietic cells administered to the recipient can be between about 0.2 10⁶ and about 10 10⁶ CD34+ cells/kg of recipient's body weight, In a preferred embodiment, the graft comprises a maximum of 1 10⁵ CD3+ cells/kg of recipient's body weight.

The isolated population of NK cells of the invention and hematopoietic cells are typically administered to the recipient in a pharmaceutically acceptable carrier by intravenous infusion. Carriers for these cells can include but are not limited to solutions of phosphate buffered saline (PBS) containing a mixture of salts in physiologic concentrations.

The hematopoietic cells can be provided by bone marrow cells, mobilized peripheral blood cells or cord blood cells. The bone marrow cells can be obtained from the donor by standard done bone marrow aspiration techniques know in the art, for example by aspiration of marrow from the iliac crest. Peripheral blood stem cells are obtained after stimulation of the donor with a single or several doses of a suitable cytokine, such as granulocyte colony- stimulating factor (G-CSF), granulocyte/macrophage colony- stimulating factor (GM-CSF) and interleukin-3 (IL-3). In a preferred embodiment of the invention, the donor is stimulated with G-CSF. In order to harvest desirable amounts of stem cells from the peripheral blood cells, leukapheresis is performed by conventional techniques and the final product is tested for mononuclear cells. Optionally, the hematopoietic cells can be T-cell depleted. T-cell depletion of bone marrow or of peripheral blood cell may be carried out by any known technique, for example, by soybean agglutination and E-rosetting with sheep red blood cells as described.

According to the invention the host patient is conditioned prior to the transplantation of the allogeneic graft. Conditioning may be carried out under sublethal, lethal or supralethal conditions, for example by total body irradiation (TBI) and/or by treatment with myelo- reductive or myelo-ablative and immunosuppressive agents. According to standard protocols, a lethal dose of irradiation is within the range of 7-9,5 Gy TBI, a sublethal dose is within the range of 3-7 Gy TBI and a supralethal dose is within the range of 9,5-16 Gy TBI.

Any immunosuppressive agent used in transplantation to control the rejection, or a combination of such agents, can be used according to the invention, such as prednisone, methyl prednisolone, azathioprine, cyclophosphamide, cyclosporine, monoclonal antibodies against T-cells, e.g. OKT3, and antisera to human lymphocytes (antilymphocyte globulin - ALS) or to thymus cells (antithymocyte globulin - ATG). Examples of myelo-ablative agents that can be used according to the invention are busulphan, dimethyl myleran and thiotepa.

The advantage of the administration of the isolated population of NK cells of the invention is the possibility to reduce the intensity of the conditioning regimen. For example, the conditioning regimen can be reduced to the intensity of conditioning regimen adopted for matched human transplants. A reduced version of a high- intensity regimen according to the present invention includes Fludarabine at the total dose of 200 mg/M2, Thiotepa 5 mg/Kg, and Melphalan 70 mg/M2, plus anti-T cell antibodies such as ATG, 20 mg/Kg. Optionally, the doses of Thiotepa and Melphalan can be increased by 50%. Indeed, such conditioning regimen is highly toxic and some patients are unable to withstand such toxicity. Therefore, the present invention makes possible the allogeneic graft for these patients.

The isolated population of NK cells of the invention as prepared according to the invention may be used as first line of treatment in combination with standard total body irradiation (TBI) and/or by treatment with myelo -reductive or myelo-ablative and immunosuppressive agents; or used to control the residual disease after total body irradiation (TBI) and/or by treatment with myelo -reductive or myelo-ablative and immunosuppressive agents.

The isolated population of NK cells of the invention as prepared according to the invention may also be used for the treatment of other NK-sensitive malignancies including pediatric cancers such as the sarcomas Ewing sarcoma and rhabdomyosarcoma (Cho...Campana clinical cancer Research 16:3901 (2010), neuroblastoma, malignant glioma and, possibly, prostate cancer (Cho, D. & Campana, D. Expansion and activation of natural killer cells for cancer immunotherapy. Korean J Lab Med 29, 89-96 (2009).

The isolated population of NK cells of the invention as prepared according to the invention may also be used for the treatment of aggressive cancer with no major drugs in order to improve the response rate and PFS. Typical said cancers include but are not limited to pancreatic cancer, lung cancer, breast and colon cancer. In particular, the isolated population of NK cells according to the invention is used in combination with a therapeutic antibody, typically a therapeutic antibody capable of ADCC (e.g. a CD20 antibody).

The isolated population of NK cells of the invention as prepared according to the invention may also useful for the treatment of infectious diseases or dysimmune diseases.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

EXAMPLE: Material & Methods PBMC and UCBMC purification

PBMC and UCB mononuclear cells (UCBMC) were respectively collected from peripheral blood samples and UCB units using Histopaque (Invitrogen). Briefly, thirteen ml Histopaque were put in 50 ml centrifugation tubes and 20-30 ml of 1/2 diluted blood in RPMI, (Invitrogen) were slowly added at the top. Tubes were centrifugated 30 minutes at 1600 rpm and 20 °C without break. Mononuclear cells were collected from the interlayer white ring. After washing in RPMI, cells were suspended in complete RPMI medium supplemented with 10% FBS (Invitrogen).

FACS analysis

For phenotype analysis, cells were stained with 7AAD (Beckman) to identify viable cells and antibodies against surface markers, CD25-FITC, CD45RO-FITC, CD69-PE, CD62L- PE, CD19-PE, CD3-PE, CD19-ECD, CD56-PECy7, CD56-APC, CD3-APC, CD45- APCAlexaFluor750, CD45RA-APCAlexaFluor750, CD16-PacificBlue, CD57-PacificBlue, CD45-KromeOrange, CD16-KromeOrange (Beckman), CD158b-FITC, CD158a-PE, CD107a- HV500 (BD Biosciences), CD158e-Vioblue (Miltenyi), by incubating Ixl0⁵-3xl0⁵ cells with different antibodies in PBS 2,5% FBS for 20-30 minutes at 37 °C. Cells were then washed and suspended in 200-250 μΐ PBS 2,5% FBS. Staining was analyzed on a flow cytometer Gallios (Beckman) using the Kaluza software.

Alive lymphocytes were gated using FS/SS and 7AAD staining. B lymphocytes

(CD 19+), T lymphocytes (CD3+CD56-) and NK cells (CD56+CD3-) were distinguished using respectively CD 19, CD3 and CD56 antibodies. For special NK analysis depending CD45RA/RO phenotypes, NK cells were separated in 3 distinct populations CD45RA+RO-, CD45RA-RO+ and CD45RA+RO+. These different populations were then analyzed for CD 16, CD69, CD62L, CD57, IFNy and CD 107a expression or size/granularity (FSC/SSC) parameters.

Stimulation protocol

PBMC and UCBMC were stimulated for NK amplification and activation for 10 to 20 days. Two different protocols were compared, starting with PBMC/UCBMC 1.10⁶ cells/ml. Firstly, stimulation with high dose of IL-2 (1000 U/ml, eBiosciences) added to complete medium. Secondly, co-stimulation with the NK cell target EBV cell line C30, developed in our laboratory, together with IL-2 100 U/ml + IL-15 (5 ng/ml, Miltenyi).

Cytotoxicity assay

Ex vivo or stimulated UCBMC cytotoxic capacities were tested against the target cell line K562 by using a degranulation assay based on CD 107a labelling. Briefly, 50 thousand target cells per well were put in RPMI 10%FBS IL-2 lOOU/ml with monensin (BD Biosciences) in a 96-V bottom-plate. Effector cells (UCBMC) were added at different effectontarget ratios.

Cells were incubated over night at 37°C 5%C02. After incubation, living cells in the wells were counted using a cytometer Muse (Millipore) with the count and viability kit (Millipore). Cells were analyzed by FACS using 7AAD, CD45RO-FITC, CD19-PE, CD56- PECy7, CD3-APC, CD45RA-APCAlexaFluor750, and CD16-PacificBlue for cytotoxicity assay; or 7AAD, CD45RO-FITC, CD19-PE, CD56-PECy7, CD3-APC, CD45RA- APCAlexaFluor750, CD16-KromeOrange and CD107a-HV500 (BD Biosciences) for degranulation assay. FACS results were analyzed using the Kaluza software. For cytotoxicity assay, target cells were gated in living cells (7AAD-) using FS/CD45 plots and FS/CD3 (Jurkat) or FS/CD19 plots. The numbers of living target cells after incubation with effector cells were subtracted to the number of living target cells when incubated alone to give the percentage of target cell lysis. For degranulation assay, the percentages of CD107a⁺ NK cells were analyzed. Statistics

All the experiments were performed at least three times with similar results.

Results NK cell expansion is required for clinical means. In general, the source of these cells can be PBL or UCBL. It is expected that lymphocytes for the second source would be na^'ive and/or less committed into differentiation. We compared the expression of CD45, a marker of maturation ¹⁴, in the three main types of human lymphocytes: T, B and NK cells derived from PBL and UCBL. We show that B and NK cells express similar CD45 levels in PBL and UCBL. NK cell population can be divided in two regarding CD56 expression. CD56^bnght are relatively immature, with low cytotoxic activity and high cytokine production and CD56^dim, which are more mature with high cytolytic activity and lower cytokine production ³. CD56^bnght cells showed lower CD45 expression in NK cells, according to the observation that CD45 levels increased with cell maturation ¹⁴. However, the median percentage of CD56^bnght was similar in UCBL and PBL²². T cells derived from UCBL showed lower CD45 expression than other lymphocytes and than T cells derived from PBL. The lower CD45 expression was probably linked to the stage of maturity because we showed that T cells derived from UCBL expressed lower levels of CD45RO and higher levels of CD45RA , a sign of immaturity ^14~17. In contrast, B and NK cells were basically equivalent in PBL and UCBL regarding expression of the CD45 isoforms.

Next, we analyzed if total CD45 was associated to the expression of NK cell activation or maturation markers. Killer-Inhibitory Receptors (KIRs) and CD 16 expression are markers of maturation ²³'²⁴, and during in vitro expansion of NK cells, proliferation is associated to CD25 expression whereas cytolytic activity is associated to CD69 expression ²⁵. CD45 showed a positive correlation with CD 16 and CD69, although in the last the differences were very small. There were a negative correlation between CD45 and CD25 and CD56 expression. Regarding expression of the KIRs, we observed that there was a positive relationship of CD45 with CD158a and CD158b. These results showed that total CD45 expression mainly correlated with maturation markers such as high CD 16, CD69 and KIRs and low CD56 and CD25 in NK cells derived from both UCBL and PBL.

Finally, independently of their origin and CD56 level, naive NK cells expressed almost exclusively the isoform CD45RA, although the CD56^bnght NK cells at lower level than other NK cells.

After 10 days of in vitro activation with a high dose of IL-2, NK cells of both origins increased CD45RO expression and decreased CD45RA expression. UCBL T cells also showed the same changes although less pronounced than PBL T cells. In UCBL T cells, these changes were mainly observed at day 10, whereas 3 days after stimulation the changes were minimum and a 20-day stimulation did not increase further the CD45 changes. In contrast, these changes started 3 days after activation in UCBL NK cells. In agreement with previous descriptions ¹⁵, 20 days after stimulation some NK cells recovered CD45RA expression without loosing CD45RO expression. Hence, a new population CD45RA RO⁺ (from now on called CD45RARO) appeared that was mainly absent in T cells.

To study more in detail the expression of CD45 isoforms during in vitro activation, we compared IL-2 and co-stimulation conditions. The latter reflected incubation with a prototypical NK cell target, in this case an EBV cell line, together with low concentrations of two NK cell activating cytokines: IL-2 and IL-15 ²⁶. After 10 days of co-stimulation NK cells expressed higher CD45RO and lower CD45RA levels compared to IL-2 stimulation. In addition, CD45 isoforms changes appeared faster during co-stimulation although the CD45RARO population was similar after 20-day stimulation.

This co-stimulation was not optimized for T cell activation; however we still observed an increase of CD45RO compared to cells treated with a high dose of IL-2. These results showed that UCBL T cells responded to this co-stimulation, which induced a faster change from CD45RA to CD45RO isoform. However, the CD45RARO population was mainly absent at day 20.

CD69 is used as a general marker of NK cell activation including in vivo ⁵. We observed that in vitro activation with IL-2 induced a larger increase in CD69 than co- stimulation. In contrast co-stimulation induced a larger decrease in CD45RA expression that IL-2. These data suggested that both protocols, CD69 up regulation and CD45RA down regulation, were compatible to study NK cell activation and could be used depending of the NK cell activation stimulus. In our opinion, the CD45RA/RO detection method has the advantage of couple two phenomena: CD45RA down regulation and CD45RO up regulation. This should decrease the number of false positives and the artifacts. A more detailed time course of in vitro NK cell stimulation showed that IL-2 treatment induced a large declined of CD45RA RO^" (from herein called CD45RA) population with increase of CD45RA^~RO⁺ (from now on called CD45RO) population. In contrast, CD45RARO showed a biphasic curve with a first peak around day 3 and a second increase around day 12. Co-stimulation induces a faster and more complete decline of CD45 cells and increase in CD45RO cells. The biphasic curve of CD45RARO was more apparent. Whereas we cannot prove it, we believe that the first peak is related to acquisition of CD45RO expression without totally loosing CD45RA expression. The CD56^bngth probably respond faster to stimulus i.e. cytokines could be responsible of this peak. However, we could not study this fact, because NK cell activation increased CD56 levels, and therefore we could not distinguish anymore the two populations CD56^bnght and CD56^dim. The second peak is probably due to expression of CD45RA in the CD45RO population generated after in vitro activation.

Resting CD45RA NK cells were mainly small cells with low granularity. After activation, NK cells increased in size and granularity. This was particularly true for CD45RO and, mainly, CD45RARO populations. These results were in agreement with the notion that CD45RO expression was associated to stimulation and that re-acquisition of CD45RA could be an additional sign of activation.

During in vivo maturation CD56^bright cells originates transitory CD56^dimCD62L⁺CD57^~ cells that started generating higher levels of perforin while keeping high IFN-γ production in response to cytokines ³'²⁷. In the next step, CD56^dimCD62L^~CD57⁺ cells show low response to cytokines but increased cytotoxic capacity ³'²⁸. UCBL NK cells expressed low CD62L levels and almost no expression of CD57 compared to PBL NK cells in agreement with previous reports ^{2 '}. In vitro activated UCBL NK cells increased CD62L expression in all CD45 subpopulations; however, the expression decreased with time and even disappeared by day 20 in cells activated by co-stimulation. Regarding CD57, in vitro NK cell activation induced a significantly increase only in the CD45RARO population. PBL NK cells expressed higher levels of both CD62L and CD57. After activation, CD62L barely increased whereas CD57 decreased. In summary, in vitro activation of UCBL NK cells did not induce an in vivo model of NK cell differentiation; probably the CD56^dim cells are already fully differentiated even in UCBL.

Hence, we investigated which population expressed the maturation marker CD 16 after in vitro UCBL activation. As an additional marker of activation, we studied the degranulating capacity of NK cells on K562 targets by analyzing CD 107a expression, which is commonly used as a marker of degranulation. During in vitro expansion, the pool of CD45RA cells partially lost CD 16 expression. This was less evident in CD45RO cells, whereas CD45RARO cells showed the higher expression. Regarding degranulation, CD45RARO cells displayed the higher levels, with CD45RA and CD45RO showing similar levels. In summary we identified in in vitro expanded NK cells the population with the higher degranulation activity and CD 16 expression.

However, our in vitro studies could not reflect the real populations in vivo because in PBL and UCBL from healthy donors, we found almost exclusively CD45RA expression. We hypothesized that the other two populations CD45RO and CD45RARO could be present in a framework of NK cell expansion and/or activation. As a model of NK cell activation we chose patients of hematological cancers where NK cells could be in contact with their target tumor cells ²⁹. As a model of NK cell proliferation we chose patients who have received a bone marrow transplant and show a rapid reconstitution of their NK cell pool ³⁰.

Transplanted patients showed both CD45RA down regulation and CD45RO up regulation, generating a comet-like figure, similar to our observation during in vitro NK cells activation, although less pronounced. We identified a new population in vivo, the CD45RA^dim cells, which were the first step for the lost of CD45RA before acquisition of CD45RO, and represented the start of the comet tail. We considered these cells as "activated" cells and were not included as part of the resting CD45RA population. We show that the number of CD45RA resting NK cells clearly diminished in these patients. These patients expressed higher percentages of CD56^bnght cells, which were CD 16^" suggesting that these were de novo produced NK cells and not CD56^dim cells that gain CD56 expression. We next analyzed the maturation state of these cells. Independently of CD 16 expression, CD56^bnght cells were CD62L⁺CD57^~ and showed a more immature phenotype than the CD56^dim cells, which were CD62L^~CD57⁺.

In transplanted patients and independently of CD 16 expression, CD56^bnght cells were mainly CD45RO cells, whereas CD56^dim cells were mainly CD45RA. These data showed that the more differentiated cells displayed a lower level of CD45RO than the immature cells. A possible explanation is that in bone marrow transplanted patients there is a boost of cytokines that preferably activate CD56^bnght cells, which are more immature ³ and that are shown in bigger numbers in transplanted patients than in healthy donors. Activation and/or proliferation of these cells would provide them with the CD45RO marker. The CD45RARO population was mainly absent in most of these individuals, although 2 out of 6 patients showed around 5% of their NK cells with this phenotype. In summary, the CD45RO population was mainly CD56^brightCD62L⁺CD57^", in contrast to the CD45RA population that shows a CD56^dimCD62L^" CD57⁺ phenotype. In this context, it should be noted that CD56^dim cells are usually CD57⁺ whereas CD56^bnght cells usually express CD62L ²⁷'²⁸. The results strongly supported that the _CD56bright _{cells that appeared in these} patients were initially CD56^bright cells and not CD56^dim cells that could increase CD56 expression. The increase in CD56^bnght cells compared to healthy donors was probably due to recovery after lymphopenia, which induced a large production of na^'ive cells.

In the second model we investigated NK cell subpopulations in patients with hematological cancers including B-CLL and MM. Five out of 12 of these patients showed down regulation of the CD45RA population and CD45^dim and/or CD45RO up regulation.

We were surprised for the lack of activated NK cells in 7 of 12 cancer patients. Recently, it has been shown that tumor infiltrating NK cells show a specific phenotype that distinguish them from circulating NK cells ²⁹'³¹. We hypothesized that the activated, CD45RA^dim and/or CD45RO, NK cells could be diluted in the blood of hematological cancer patients preventing their detection. Hence, we investigated NK cells that were in contact with tumor cells in patients with low numbers of activated NK cells in blood. We focused in patients with bone marrow disorders where it was easy to identify the NK cell population in contact with tumor cells i.e. multiple myeloma (MM). We observed that several patients that did not show activated NK cells in blood showed activated cells in the tumor site, in our case in the bone marrow. The pC9 expressed a low reduction of CD45RA cells. This patient showed a gammopathy and NK cells in the bone marrow showed a higher activation with reduction of CD45RA levels and increase in CD45RO. We next analyzed pC2, suffering from multiple myeloma (MM), and with CD45RA levels similar to healthy donors. NK cells in the bone marrow show a decline in the number of CD45RA cells and increased in CD45RA^dim and CD45RO cells. In contrast, two patients suffering from a B-CLL did not show a decrease in CD45RA number in his bone marrow, suggesting that CD45RO expression depended on the contact with tumor cells, at least in certain patients. We next analyzed expression of CD45 isoforms in the tumor ganglion of 2 B-CLL patients. We found a large decrease in CD45RA expression in the tumor site. In summary, expression of different CD45 isoforms in vivo can identify NK cell activation and/or proliferation.

We have identified a new population of NK cells identified by co-expression of CD45RO and CD45RA molecules. This CD45RARO population was a minority and we wondered if in certain individuals this population could be enlarged after activation. We analyzed 14 UCB and found one, UCB439, which after activation with IL-2 or co-stimulation, showed the atypical phenotype of keeping CD45RA after CD45RO expression, generating a large CD45RARO population. This was more remarkable after co-stimulation. We identified a new CD45RA^hlgh/RO⁺ population, which in the other UCBs, corresponded to common CD45RARO population in FSC/SSC behavior. Gain of CD45RO expression is similar in time and intensity to other UCBs. However, NK cell amplification was significantly larger in this UCB compared to other UCBs. These results suggested that individuals keeping CD45RA in their CD45RO NK cell compartment would develop a larger number of NK cells after sensing "danger" and could show improved anti-tumor activity.

We next try identifying other individuals with distinct CD45 expression after activation. We focused on patients with long-term survival after an hematological cancer and with elevated NK cell levels. We hypothesized that in these patients there was a direct relationship between their survival and high NK cell levels, which could be due to a specific NK cell phenotype. We found 3 patients with these characteristics (pCSNK), which express very low levels of _CD56bri_gth _{and the CD56}dim _{cells were} predominantly CD57⁺/CD16⁺. Although CD57 expressing cells increased with age, they rarely overpassed 50% even in very old donors ²². We did not observe this high numbers in cancer patients of similar age, suggesting that high CD57+ population could be related to cancer survival. However, ex vivo, we did not find differences in CD45RA expression.

One of these patients suffered of cancer and has survived more than 20 years and is considered now cured. This individual showed high NK cell numbers during a long period of years and when we finally analyzed him, he showed 15.2 % of NK cell lymphocytes, more than double than a healthy donor. His NK cells showed a resting phenotype similar to healthy donors regarding CD25, CD 16 and CD69 expression. Conversely, these cells showed the classical phenotype of healthy donors: absence of CD45RO expression. However, they expressed higher levels of CD57 than healthy donors. After stimulation, NK cells from this patient showed and increase of CD45RARO cells. However, the amplification and the cytotoxic activity of the amplified NK cells were similar to a healthy donor.

Discussion:

Current clinical protocols fail inducing long-term survival in a significant number of patients with hematological cancers. Those refractory to standard treatment i.e. radio- and/or chemotherapy are often subjected to a myeloablative regimen followed by allogenic hematopoietic stem cell transplantation (HSCT). However, the mortality linked to this treatment is closed to 20%. Moreover, Graft versus Host (GvH) and HvG diseases and opportunistic infections hamper this procedure. Relapse has become the leading cause of death following allogeneic HSCT: the relapse rate has not decrease over the past 20 years ³². In general, prognosis is poor for patients who relapsed to an allograft since effective treatment options are limited. These include donor lymphocyte infusions, withdrawal of immunosuppressive medication and second allogeneic HSCT. However, new specific cellular approaches are under investigation. In particular, the use of alloreactive natural killer (NK) cells after umbilical cord blood transplantation (UCBT) seems promising. Killer cell immunoglobulin-like receptors ( 7i?)-ligand incompatibility in the GvH direction improves outcomes after UCBT in the clinic ³³'³⁴. Moreover, NK cells: 1) are not responsible of GvHD; 2) can be injected as "differentiated" cells without the need of long time survival on patient's body; 3) protect from opportunist infections ³³, probably through their immunoregulatory effects on B cells, T cells, macrophages, and more importantly on polymorphonuclear cells (PMNs ³⁵). We, and others, have shown the requirement of NK cells to eradicate tumors in several mouse models ²⁶'³⁶. NK cell infiltration gives a good prognostic in several cancers

29 31 37

( ' ' . However, some unresolved problems remain before the standard use of allogeneic NK cells in the clinic. The most important is engraftment of an adequate number of cells and that these cells show clinically efficient anti-tumor activity. For the last purpose, it is basic to identify the different NK cell populations and their cytolytic activity, which is believed to be the most essential component on the anti-tumor NK cell function ³. Here, we identify that the expression of the CD45 isoforms CD45RA and RO distinguishes NK cell populations with diverse cytolytic activity, in particular the CD45RARO population shows improved cytotoxicity. Hence, expansion protocols keeping this population could have a better clinical effect.

CD45 activity is regulated by dimerization and CD45 spontaneously homodimerizes at the plasma membrane, which inhibits its activity ³⁸. The size of the CD45 extracellular domain is inversely proportional to the extent of CD45 dimerization and auto inhibition ³⁸. Larger CD45 isoforms such as CD45RA dimerize less efficiently and, accordingly, and perhaps they are more efficient at promoting TCR signaling than are smaller isoforms such as CD45RO ¹². However, CD45 activity also depends on its plasma membrane localization that depends on the extracellular domain ⁿ'¹². At least in T cells, too high CD45 activity leads to dephosphorylation of the activating residues in the Src kinases, whereas too low would leave phosphorylated the inhibitory residues. Therefore, it is important for efficient activation that CD45 activity is between this window ¹⁴, and the amount and the specific CD45 isoform would regulate the final activity. NK cell decision to kill depends on activating and inhibitory signals. We have found that CD45RA⁺/RO⁺ NK cells show the maximal cytolytic activity. The presence of both CD45RA and RO could give the NK cells the appropriate level of CD45 activity for efficient signaling to boost the cytolytic activity.

It is important to note that regulation of CD45 activity is critical for an efficient immune response, because its deficiency results in a severe combined immunodeficiency (SCID) phenotype in both mice ^{39 41} and humans ⁴²'⁴³. This could explain the high expression of this phosphatase and its complex regulation. In agreement to previous results ¹⁶ , IL-2 stimulation induces CD45RA down regulation and CD45RO up regulation in PBL and this occurs first in NK cells. We show here that UCBL show a similar behavior. It was proposed that IL-2 was the molecule that drives those changes ¹⁶. In contrast, we have found that other signals independent to IL-2 contribute to CD45RO up regulation and CD45RA down regulation because high IL-2 doses do not reach the same level of changes than co-stimulation with target cells.

Ex vivo T cells derived from PBL show more CD45RO expression than T cells derived from UCBL. This suggests that in PBL there are more activated and/or memory cells than in UCBL, what is intuitive. It is fair to assume that in these situations CD45RO cells are memory cells. However, an extremely low number of NK cells express CD45RO ex vivo in both PBL and UCBL. At least in vitro, NK cell activation clearly induces CD45RO expression. Hence, it is surprising that ex vivo we find so low numbers of NK cells expressing CD45RO in PBL. Several non-exhaustive explanations are that the amount of memory NK cells is extremely low, CD45RO is not a marker of memory NK cells or CD45RO is specifically lost during ex vivo manipulations in NK cells, although the last possibility is unlikely because NK cells express slightly higher levels of total CD45 than other lymphocyte. In vivo we found a significant CD45RO population in bone marrow transplanted patients. These cells that are CD57^" are probably proliferating cells and are predominantly CD56^bnght. Hence, our results suggest that CD45RO is mainly a marker of NK cell proliferation in this specific setting and degranulation activity is mostly related to CD45RARO cells, which are however, a minority population. This is in agreement with results in mice where CD45 is related to ITAM-dependent cytokine production, but is not related to ITAM-dependent cytotoxicity in vitro ^18~20, although it is required for full cytotoxic NK cell activity in vivo ²¹. This suggest that in NK cells the expression of different CD45 isoforms plays a different role that in T cells where it plays a main role in activity. In this context, long-term cancer survival patients with high percentages of NK cells do not show activated, or memory, NK cells, at least regarding to CD45 RA/RO expression. This could reflect the difference between the innate immune system that is based in the number of cells protecting the host; and the adaptive immune system where a small population of memory cells can originate a strong response by clonal expansion of the selected TCR-expressing cells.

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Claims

CLAIMS:

1. A method for isolating a population of NK cells capable of activation, proliferation and high level of cytotoxicity comprising the steps consisting of i) providing a population of NK cells and ii) selecting the population of CD45RA+RO+ NK cells.

2. The method of claim 1 wherein the population of NK cells is isolated from a blood sample obtained from a subject.

3. An isolated population of CD45RA+RO+ NK cells obtainable by the method of claim 1.

4. A method for monitoring the treatment of a subject with a pharmaceutical agent comprising the steps consisting of administering the subject with the pharmaceutical agent, providing a blood sample or PBMC sample from the subject after said treatment, and determining whether said blood or PBMC sample contain a population of CD45RA+RO+ NK cells.

5. The method of claim 4 wherein the pharmaceutical agent is be selected from the group of recombinant interleukins, recombinant cytokines, recombinant growth factors, and antibodies.

6. A method for determining the survival time of a subject suffering of a cancer or an infectious disease comprising the steps consisting of i) detecting in a blood sample obtained from the subject the presence of a population of CD45RA+RO+ NK cells and ii) providing a good prognosis when the population is detected at step i).

7. The isolated population of NK cells isolated by the method of claim 1 for use in a method for treating cancer or an infectious disease.

8. A pharmaceutical composition comprising a population of NK cells isolated by the method of claim 1.